In the 1971 State of the Union address President Richard Nixon ‘declared’ war on cancer.1 Sadly despite a painstaking effort by many intelligent researchers, the war continues to drag on with no clear end in sight. Throughout the last forty years new strategies and new technologies have broadened the ability to attack cancer, but despite these advances, from a standpoint of long-term survival rates positive change has fallen to the old adage ‘walk a mile just to move an inch’. However, the advent of genetic engineering and nanotechnology has once again stimulated excitement in the cancer community driving the belief that genetic engineering eliminates the anonymity of a particular cancer and nanotechnology allows for more efficient combat. This excitement must be tempered with rationality and caution though for while new cancer-fighting strategies have flooded the research environment the methodologies which make up these strategies have yet to be fully assessed.
Scientific involvement in cancer can be divided among three different avenues: prevention, detection/diagnosis and treatment. For most of modern medicine the focus has be on that third avenue, treatment of cancer. Unfortunately most of the time in the past cancer treatment began at a much later time than desired because of limited diagnostic methodologies. As technology has advanced, there is hope for the development of better diagnostic strategies which will not only detect cancer earlier in its development, but also identify the type of cancer, information that can be used to better predict cancer prognosis and its development. With this additional information better treatments can then be applied significantly increasing survival rates.
However, the first prong of cancer, prevention, still remains slow developing because of a lack of genuine understanding regarding the origin(s) of cancer. While some claim eating food x or taking drug y should potentially reduce the probability of cancer occurrence, there is little to no scientific statistical evidence that have validated any of these claims. Some believe that certain anti-cancer drugs can be taken in a preventative manner, but these drugs also carry significantly detrimental side effects which reduces their attractiveness largely due to the low absolute percentage drops in cancer development in their application (chance of developing cancer type x goes from 1.3% to 0.9%). Based on this uncertainty this blog post will largely explore the strategies that are currently being used to combat cancer both from a standpoint of treatment and diagnosis and those that some hope to utilize in the future.
There are a number of individuals who would argue that diagnosis is the most important element in cancer treatment because very few individuals die from single organ/primary cancer, a notable exception being pancreatic cancer. Instead mortality much more frequently comes after metastasis. Another way to look at the diagnosis philosophy is that even an inefficient cancer treatment can manage cancer localized to a small region of the body, but even the most effective existing cancer treatments have a difficult time eliminating metastasized cancer. As previously mentioned this reasoning underscores one of the principle reasons cancer survival rates have increased at such a low rate over the last decades, earlier diagnosis of cancer has been methodologically difficult.
One major reason for this difficulty is due to the exhausting, side effect riddled and potentially toxic nature of cancer treatment. Due to the expense and strain required when treating a cancer case, physicians favor definitive diagnosis to confirm cancer which involves histologic examination of a specimen derived from a potential developing tumor. This entire process is commonly referred to as a biopsy. Due to the invasive nature of biopsies, physicians need some form of probable cause before considering one, probable cause which is typically acquired through some form of symptomatic, radiographic or visual abnormality. Unfortunately recognition of any of these abnormalities is rare in the very early stages of cancer development and by the time they emerge the probability for metastasis has significantly increased. Early diagnosis would be limited in importance if a very specific (no side-effects) and inexpensive cancer treatment were developed because then probable cancer cases that turned out to be false would not be detrimentally served by treatment, but such a treatment is not realistically viable at this point in time.
Currently biopsy is the only genuinely accurate means to determine whether or not an individual has cancer. The usefulness of some diagnostic techniques like mammograms have lingering questions over their statistical accuracy and whether or not they are useful to administer in the first place whereas other like Pap smears have demonstrated more reliability.2,3,4 However, biopsies have also come into question, not for their accuracy, but for whether or not they can inadvertently aid in metastasis.
The process of a biopsy involves inserting a needle into a cluster of cells thought to be cancerous and drawing off some of those cells. Through this process some have raised concerns that the penetration of the needle into the cluster of cells may dislodge some of these cells allowing them to be caught up in the bloodstream and carried to other parts of the body creating secondary cancers, in short accelerating metastasis. Some empirical evidence5 does exist to support this hypothesis, but fortunately new sterilization instruments have been developed within the injecting needle to minimize concern; unfortunately these new instrument have yet to be put into widespread use.
While biopsies are accurate one of the big goals in the cancer community is to develop a technique that rivals in accuracy, but is also much less invasive. Among the new diagnostic strategies being developed to avoid the invasiveness of a biopsy, cancer identifying biomarker are clearly gaining the most attention. Note that in biology the term ‘biomarker’ is used to describe two different types of elements.
First, biomarkers can be chemical molecules that are attached to other components in the body to identify them. For example various forms of fluorescent molecules, like luciferase, are commonly used to track the travel and/or presence of molecules in the bloodstream or observe some other biological function. Second, biomarkers can be DNA, RNA or protein that belies an abnormality within the body typically indicating some form of disease or deleterious condition.6 Basically biomarkers are biological characteristics, typically associated with DNA, RNA or protein expression, which are used to develop probability profiles regarding disease acquisition or probability of successful treatment with a given therapy.
The second definition for biomarker can be broken down further into diagnostic biomarkers and prognostic biomarkers where prognostic describes a biomarker that when present either increases or decreases the probability of a given condition over a given baseline average probability. A diagnostic biomarker confirms the existence of a given condition. In cancer treatment while significant publicity and research has gone into developing prognostic biomarkers, having gene x over gene y means greater chance for lung cancer, etc. the ‘black box’ nature of cancer negatively impacts the overall importance of those prognostic biomarkers.
The general idea with prognostic biomarkers in cancer should be to create two tiers of individuals: those without higher probabilities for cancer who continue to undertake standard screenings and those with certain prognostic biomarkers who undertake more specific and focused cancer screening in order to detect any higher probability cancers at an earlier stage in their development.
Diagnostic biomarkers have made significant gains in importance and viability over the last decade under the guidance of an improved understanding of genetics and the dream of personalized medical treatments. In order to replace biopsies, diagnostic biomarkers must be less invasive, preferably non-invasive, and accurate. The ideal for a diagnostic biomarker is an easily identifiable element which changes dramatically in concentration during the development of cancer vs. non-cancer and can be extracted through the examination of a blood, urine or saliva sample. In addition uniformity in the analysis is also necessary; it should not matter if the analysis is performed at laboratory A or laboratory B. Although speed of analysis is also viewed as important by some it is significantly less important than accuracy and invasiveness because the probabilistic mortality rate for cancer does not typically change significantly on a day-by-day basis so it should make little difference if the analysis takes 1 minute, 1 hour or 1 day to complete once an accurate biomarker has been established.
Biomarker developmental intent can take one of two paths: general or genetically specific. General uses viruses which only invade cells overexpressing a certain antigen or receptor, usually attributable to some form of cancer. The major advantage for the general method is an easier means to identify cancer, while the major disadvantage is it is unable to determine the location or the type of cancer. Not surprisingly these strengths/weaknesses are basically reversed for more genetically specific methods where one can better identify the type and possibly even the specific mutation of cancer. However, due to a wide variety of cancers among the same general diagnostic category (for example there are 15-16 different types of breast cancer, which are typically simply referred to as breast cancer in lay speak)1 there is a chance to miss cancers with small mutational differences. Due to the importance of biomarkers in the future of cancer treatment a wide variety of techniques are being explored with some promising ones highlighted below.
One of the challenges of creating personalized cancer treatments is that genetically specific biomarkers first have to be individually synthesized from each patient because of a lack of personal genetic information from that patient. Fortunately almost all cancer cells experience both DNA repair mechanisms breakdown and a widespread rearrangement of chromosomes due to chromosomal instability.7,8 In addition this near certainty of genetic disruption appears to occur near the earliest stages of tumor formation (tumorigenesis) and is thought to persist throughout tumor development.7 Therefore, cancer cells develop a form of uniqueness apart from the original cells they mutated from, yet maintain a form of homogeneity in their development between different patients. Through this pathway different cancers would develop different rearrangements which could be used to not only differentiate between cancerous cells and normal cells, but between different types of cancer.
In effort to take advantage of this form of cancer development some researchers have turned to high-throughput screening to identify these genetic changes and create biomarkers which could identify those changes.7 One technique to create these biomarkers is to use recurrent somatic structural (usually single-base) alterations, especially for hematopoietic (blood) malignancies9-12, but these structural alterations do not persists in most solid tumors limiting the techniques usefulness for total detection. Also analysis of somatic single-base alterations have polymerase error rates derived from the specific bases of interest, which can produce false positives.7
The use of Personalized Analysis of Rearranged Ends (PARE) has demonstrated that specific identified breakpoints can detect tumor DNA at very small sensitivities (small amount of tumor DNA among large quantities of non-tumor DNA) within the blood or plasma.7 One of the reasons PARE works is that like all cells tumors secrete parts of their DNA into the bloodstream, a sample of blood can then be taken and these DNA pieces amplified through polymerase chain reaction (PCR) and later matched to previously developed biomarkers. Using PARE is thought to result in almost zero chance for false positives because the diversity of the combinations that are amplified through PCR should not appear in non-tumor cells.7 This process is more accurate than examining somatic single-base alterations because of the aforementioned false positives drawn from some instances when cancer cells have similar somatic single-base alterations which mimic normal non-cancerous cells.
Unfortunately PARE analysis is still in early stages of development and significant questions still remain. The biggest question for PARE as well as all somatic single-base alteration techniques is whether or not these somatic DNA alterations persist throughout tumor development. There are two possible design methods to address this problem: first, identify a large number of PARE biomarkers for a particular type of tumor and test for all of them using the reasoning that not all of those biomarkers would be lost over progressive tumor development. Second, identify the various PARE biomarkers that exist throughout the lifecycle of a tumor tracking when certain biomarkers appear and disappear in relation to the stage of development. One useful feature with regards to the second method is that if researchers are lucky unique PARE biomarkers will exist for specific time designations in tumor development so not only could PARE biomarkers be used to identify whether or not a patient has cancer, but also its stage of development, which could demand a specific treatment regimen.
Other problems revolve around the costs associated with developing these PARE biomarker libraries. The initial sample studies which were used to generate a proof-of-concept for PARE biomarker viability utilized 12 tumor variations (6 colorectal and 6 breast) where each required an average of 194.7 million reads per patient creating an assay cost of approximately $5,000, which is very high for clinical repetitive use.7 Fortunately because the initial proof-of-concept study utilized a new technique that has not been fully brought to scale proponents believe that significant opportunities exist for cost reduction both in general sequencing demands as well as PARE specifics like less stringent mapping criteria and lower physical coverage.
The final major problem that exists in the development and use of PARE biomarker analysis is although PARE should not produce false positives a non-zero probability exists for the generation of false negatives.7 At this point PARE requires at least one rearrangement template molecule to be in the analyzed plasma sample to develop a positive result. In the very early stages of cancer development these required templates may not be available for a given sample. While not a crippling concern in the methodology, if correct it does limit the overall diagnostic potential of PARE, especially because the gap is on the front end not the back end of the cancer lifecycle.
The majority of new cancer detection techniques focus on general detection through blood sampling, but others have been developing techniques for regional detection of specific cancers. A possible advantage to these more regional strategies, despite their cancer type detection limitations, is the potential to demonstrate broad biomarker association with certain cancers creating another base point for potential treatments. One of the more promising detection methodologies involves examining saliva for specific biomarkers.
Not surprisingly the focus when testing saliva for cancer is to detect oral squamous cell carcinoma (OSCC) and other similar type cancers. While biopsies generate worthwhile results, a more specific method appears to be on the horizon. Tapping into the same type broad strategy that is used in PARE biomarker development, saliva contains a multitude of mRNA where non-cancerous mRNA and cancerous mRNA have different sequence structures.13 The origins of these mRNA samples tend to be the three major saliva glands, gingival crevice fluid and desquamated oral epithelial cells.14
In addition to mRNA, saliva also contains microRNA (miRNA) which more accurately cluster in various forms of solid tumors than mRNA creating a better opportunity for detection, thus increasing testing accuracy over mRNA in detecting cancer.15 miRNA are created through transcription by RNA polymerase II or III because they initially represent a portion of an intron of mRNA and typically act as post-transcriptional regulators.14,16 However, for miRNA to be a useful biomarker for cancer detection a significant difference in the concentration of at least one miRNA between cancer and non-cancerous states must be identified where it is attributable to the cancer itself.
A possible marker to reflect the difference between miRNA in those with OSCC and those without OSCC has been isolated to two specific forms of miRNA. In both whole and supernatant saliva two salivary miRNAs, miR-200a and miR-125a, had different concentrations relative to whether the patient suffered from OSCC or not.13 Interestingly the concentrations of these two miRNAs were lower in the OSCC patients than the non-OSCC patients.13 In most situations biomarkers are selected based on their increased concentrations for deleterious conditions not decreased due to easier detection parameters (larger concentrations over smaller concentrations). There is also evidence to support miR-200a playing a role in other head and neck cancers, so this method could be expanded from OSCC patients to other cancers.17-19 While at the moment the use of miRNA diagnostics have focused on head, neck and oral cancers, it stands to reason that expansion of this technique may provide a means to general cancer diagnosis for other types of cancers as well if more miRNA associations can be identified. Signs do look promising in this avenue of detection.
The two previous techniques, PACE and miRNA concentration changes, focused on using inherent biological elements associated with the tumor and its lifecycle as biomarkers. The advantage to this system is specificity and a general level of certainty. However, there are no guarantees regarding the successful creation of a PARE library, which even if successful is still years away. Also there is no further information regarding the probability that other cancers demonstrate miRNA concentration shifts and if they do determining what type of shift will be difficult. Therefore, other researchers have sought a more general method of detecting cancer by foregoing identifying a specific type in lieu of simply identifying any type.
The application of exogenously distributed biomarkers requires a delivery mechanism that has acute specificity towards tumors over non-tumors. There are typically four practiced techniques used to achieve this goal: ligand-guided active targeting20-22, passive Enhanced Permeability and Retention (EPR)23,24, tumor microenvironment-dependent targeting25, 26 and viral injection.27 Regarding the fourth element, viruses are sometimes referred to as ‘nature’s nanoparticles’ and due to their behavior and target functionality, that are useful for delivering genetic information to target cells. Not only are viruses effective targeting devices, but their inherent replication abilities upon reaching their targets also add a form of positive autonomy with regards to molecular expression.
Taking advantage of these elements one of the chief and more versatile tools in battling cancer is the oncolytic virus. Oncolytic viruses are viruses which are typically engineered to have dramatically higher infection rates for cancer cells over non-cancer cells. In fact the fewer non-cancer cells a particular oncolytic virus infects the better. This infection disparity is developed one of two ways: first transductional targeting which involves changing the docking antigens on the viral coat to respond to a protein that is expressed at a much higher rate in cancer cells vs. non-cancer cells.28 Second, non-transductional targeting which involves genetically engineering the virus so that it only replicates in response to cues that are limited in non-cancerous cells, but are of a higher frequency in cancer cells.28 Specific tumor-only promoters are usually the cue utilized in these types of oncolytic virus. Attenuation is also used where specific genetic deletions are applied to the viral DNA, which then progressively neutralize cancer-required proteins.
There are natural oncolytic viruses28, but engineering viruses is viewed as a more effective way to address the wide demand for oncolytic viral use due to the diversity of cancer cells. Among the types of viruses that are converted into oncolytic viruses adenoviruses and herpes simplex virus (HSV) are the most effective due to their double-stranded DNA providing greater storage stability (reducing mutation probability), consistence tolerance of high production titres, transduce both dividing and quiescent cells efficiently, express transgene products and are scalable.27
One of the chief uses of oncolytic viruses is in the associated production of an artificial biomarker for general detection of cancer. The general method of oncolytic viruses involves an association with a detection element, normally some form of fluorescent marker. The general idea is that because the vast percentage of cells infected by the oncolytic virus will be cancerous, after cell lysis due to infection or induced secretion the concentration of fluorescent marker in the blood stream should increase significantly generating a high probability that the individual has some form of cancer. Also there is the potential that the overall differences in concentration could provide a basic means to track the progression of cancer development.
One of the more promising general detection techniques involves the use of HSV with a controlled mutation of the ICP6/UL39 and late viral protein ICP34.5 to limit viral replication and amplify dependence on late gene expression.27 Upon entering a cancer cell the modified HSV virus induces the cancer cell to secrete a specific biomarker, which would then be measured from the bloodstream to determine a basic pattern of cancer progression. One group of researchers who tested this concept selected gaussia luciferase (GLuc) as a high quality biomarker for use in this particular experiment because of ease of secretion, extent of lumination (1000 times brighter than other luciferases), detectable in both blood and urine in vivo and greater sensitivity than other popular biomarkers like alkaline phosphatase.29,30
The use of GLuc as a biomarker was explored using various tumor locations such as: intra-peritoneal, subcutaneous, intra-muscular and orthotropic intra-renal tumors.27 Regardless of tumor location, GLuc results were consistent with more than 90% of the tumor mice presenting higher concentrations of GLuc than non-tumor mice.27 One concern from these experiments was whether or not smaller tumors would produce the necessary vascular surface area required for HSV to infect the tumor. A lack of sufficient HSV infection would reduce the sensitivity of the overall test. While a relevant concern, it is not surprising as the most common concern with cancer diagnostic techniques is the question of what is the minimum detection limit for the given technique.
A more important concern involves the immune response limiting the overall effectiveness of the biomarker because since approximately 80% of the population has been exposed to HSV at one point in their lifetimes there will be a significant immune response.27 The problem from the immune response is that the baseline for no cancer is no significant concentrations of biomarker in the bloodstream because HSV is only infecting a small amount of cells due to the lack of cancer cells to drive infection. An immune response can reduce the number of HSV in the bloodstream after injection, which will in turn reduce the total ceiling for biomarker secretion making it more difficult to determine whether or not the patient has cancer with appropriate confidence. This problem underscores another important requirement for such a technique, the establishment of a ratio or scale to judge tumor progression relative to biomarker concentration. Clearly such a ratio would develop small differences between patients, but a general range scale could be developed as a universal measure for broad cancer progression designation.
Another concern regarding potential immune response is elevated levels of macrophages having a detrimental effect on tumor prognosis due to recruitment of these new macrophages into the tumor microenvironment, which benefits the tumor. One possible strategy to reduce problems with the immune system would be treating the patient with immunosuppressant drugs during the treatment protocol before infection with HSV and for a short time afterwards. Some may argue that the use of immunosuppressant drugs create a problem as studies have shown that extended use of immunosuppressant drugs increase the probability of developing cancer.
Such a concern does not appear applicable in such a scenario for two reasons. First, the application of any immunosuppressant drugs would only be over a very short time frame almost eliminating any legitimate increase in probability for new cancer development. Second, the methodology of cancer development may moot this point. Not surprisingly the immune system appears to be best at neutralizing tumors in their beginning stages and significantly lose effectiveness more than likely due to the formation of the tumor microenvironment as the tumor develops. Therefore, unless the oncolytic virus detection procedure is undertaken during the very initial stages of cancer development any risk associated with the application of immunosuppressant drugs should be minimal.
In a similar vein to the idea of using oncolytic viruses to release a fluorescent molecule into the bloodstream to identify cancer, some researchers want to use oncolytic viruses to deliver fluorescent molecules that instead of amplifying in cancer cells bind to exclusive cancer cell receptors to identify specific locations of tumors. The preferred protein comes from the crystal jelly and the viruses utilized operate similar to other oncolytic viruses except these viruses do not actually lyses the cancer cells.31 Instead their amplification leads to the further amplification of these crystal jelly attached proteins which then fluoresce the cancer cells creating a visual differentiational characteristic from non-cancer cells. Developing this technique is thought to not only better determine whether or not an individual has cancer, but in the future may even assist in a more accurate surgical removal of smaller tumors that could escape detection through sight alone.
Unfortunately there are two significant problems in developing this technique. The first problem is that very few colors can penetrate tissue due to frequency mismatch. Early testing identified that low frequency red is the most successful lumination color, but is still somewhat weak.31 The second problem is overall cost. Even with successful fluorescence, accurate assessment of the fluorescence currently requires a special camera which scans the body slice by slice and typically cost $750,000 dollars.31 The concern with this problem is that there is little potential due to physical tissue barriers of developing a stronger fluorescence which will not require this type of expensive camera. Thus, the chief hope for proponents is that direct costs associated with the camera will drop during the development of the technique. While this technique may not pan out to be successful it represents an important characteristic in cancer diagnostics and treatment: focusing on a wide variety of detection methodologies using logical theory in order to determine what works and what doesn’t to develop the best approaches.
In addition to new biomarkers, an older piece of technology has been augmented to aid in the diagnosis of cancer, especially with regards to metastasis. Normally when a patient is diagnosed with cancer he/she undergoes a sentinel lymph node biopsy to determine if the cancer has metastasized. In the body a clear fluid called lymph, which is part of the interstitial fluid, moves through lymph vessels eventually leading to a lymph node.32 From a technical standpoint the interstitial fluid becomes lymph after entering the lymph capillary and mixes back into the blood after exiting through the left or right subclavian vein.32 Lymph nodes are small round organs which filter out harmful substances from the lymph like bacteria. The most common lymph nodes are found in the neck, underarms, chest, abdomen and groin. The sentinel lymph node is viewed as: the first lymph node which become cancerous after taking in cancer cells which originated from the principle tumor (the reason for this definition is because in some cases by the time the biopsy is carried out multiple lymph nodes could be cancerous).33
The biopsy involves removing the lymph node closest to the identified principle tumor using the logical reasoning that most metastasized cancer will spread in an orderly and organized manner as the blood moves away from the tumor infecting nodes closer to the principle tumor rather than randomizing infections. Traditionally the lymph node is identified by injecting a specialized radioactive dye (usually filtered sulfur colloid tagged with Technetium-99m) near the principle tumor.33 After allowing for sufficient time for migration, the dye is tracked through a scanner to determine if any lymph nodes are stained with the dye. If significant amounts of dye are in a lymph node and respond to a certain gamma probe range then that node is excised and examined further under a microscope looking for cancer appropriate histological features. If the cancer has metastasized then a number of patients (some estimate up to 35%) have to undergo additional biopsies to confirm and to further evaluate how far the cancer has metastasized.34
Depending on the number of lymph nodes removed side effects for the procedure will vary.33 For instance if only a small number of nodes are removed then the most common side effects are associated simply with those of normal minor surgery (pain and/or bruising at the site of incision) and slight difficulty moving the affected body area. When more nodes are removed then the side effects increase to the potential for swelling due to excess fluid build-up (lymphedema), numbness, infection and a burning sensation. Most physicians believe that these side effects are justified for the advantage of having a better idea to how aggressive a treatment will be required for the patient based on the prevalence of the cancer spread. However, there is argument over whether the early removal of the SLN improves survival rates.33 SLN biopsies are most commonly used for breast cancer and malignant melanoma patients.
A new technique which appears to be more effective than SLN in both reducing side effects and accuracy in determining the SNL incorporates microbubbles into an ultrasound to aid detection.34,35 Initial experiments utilizing microbubbles as an additional enhancement agent in an ultrasound has accurately identified the sentinel lymph node in 89% of eighty breast cancer patients studied.34 For this process the microbubbles are small (1-4 micrometer diameter), constructed of a lipid or albumin shell, filled with a biologically inert gas (usually perfluoropropane) and typically behave like red blood cells from a hemodynamical perspective, thus they can be modeled and accounted for during the ultrasound in a predictable fashion.35
Microbubble augmented ultrasound has also been explored as a means to monitor changes in tumor vascular which could aid study of how anti-angiogenesis agents fail by tracking the changes in tumor vascular development.35,36 Some initial studies have in fact used this technique for such a purpose. One of the major advantages to this technique is that microbubbles can be selectively applied to specific regions through conjugation with certain ligands.35-37 The application of the angiogenesis mapping technique involves targeting microbubbles to specific vascular markers such as VEGFR1 or VEGFR2. Observing changes in the expression of these receptors is a viable means of tracking vascular changes due to expression patterns largely correlating with molecular demand and availability.
Unfortunately the promise of biomarkers have lead some to place more emphasis on their importance and accuracy vs. what they genuinely deserve. The perceived usefulness and power of the biomarker has spawned a form of bias where some researchers continue to believe that a given biomarker possesses a high association with a given condition even after large meta-analyses have demonstrated much less significance to that given biomarker.38,39 Basically researchers will still cite an initial analysis of a biomarker which has a stronger positive effect than a later more thorough analysis. For example researchers would cite a paper that calculated a 25 times greater association over a later paper calculating a 4 times greater association from a meta-analysis.38
This bias is not surprising, other studies have demonstrated a positive correlating bias towards other aspects of important/popular research, however, it can be quite detrimental to determining viable treatments. The continuing lack of definitive answers regarding the origins of cancer and its progression means that refusal to abandon once promising biomarkers could create more confusion and inaccuracy in the pursuit of both cancer diagnosis and treatments. The difficultly in giving up on what once appeared to be a significant step forward in understanding and/or treating cancer is understandable, but unless biomarkers are looked upon accurately, addressing cancer appropriately will be all the more difficult.
Diagnosis is only part of the battle against cancer. Streamlining treatment efficiency through exploration of different strategies is also an essential part to the future development of a cure. While lower costs and efficiency are useful features to treatment, the primary goal needs to focus on certainty of tumor death in a way that can be properly confirmed. Presently there are three mainstream treatments of cancer: chemotherapy, surgery and radiotherapy.
Among these three treatments surgery is still the preferred choice because of its overall success rate due to the direct elimination of cancer with associated visual conformation. Unfortunately surgery is not a universal treatment. The highest probability of success for surgery logically occurs in the median lifespan of a tumor when the tumor is large enough to identify and isolate from other healthy cells, but before the tumor metastasizes. After the tumor metastasizes surgery is no longer an attractive option because of its invasiveness and the general trauma it inflicts upon the body. If the tumor is too small surgery is more risky because it is difficult to confirm whether or not the entire tumor has been removed due to lack of tumor size. Fortunately chemotherapy is widely paired with surgery as a combination treatment strategy: eliminate a significant amount of the tumor via surgery and destroy the remaining weakened cancer cells via chemotherapy.
The term ‘chemotherapy’ now describes a methodology of cancer treatment that involves the use of anti-neoplastic drugs most commonly delivered intravenously. Development of the chemotherapy strategy evolved in the very early stages of the biochemical revolution before any specific biochemical markers and genetic information was uncovered about various cancers. Without the knowledge of nanotechnology or even very specific differentiating biochemical signals or proteins, cancer treatment demanded a much broader strategy. Anti-neoplastic drugs operate by interfering with mitosis; therefore they are most effective against cells which have a fast rate of mitosis, a common feature for most cancer cells. Using this anti-mitosis strategy chemotherapy techniques could attack cancer without having specific information on the location of the cancer (necessary for surgical strategies) or the specific type of cancer (necessary for individualized treatments).
However, there are other cells in the body that have a fast rate of mitosis as well, most notably associated with bone marrow (red and white blood cells), digestive tract (stomach) and hair follicles. Note that other healthy cells could be damaged by chemotherapy as well, but with a lower probability due to their slower rates of mitosis. Due to potential collateral cell damage the most common side effects from chemotherapy include myelosuppression leading to anemia and immunosuppressant, mucositis increasing ulcer probability and alopecia almost guaranteeing the most telltale sign of chemotherapy treatment, hair loss.
Despite decades of experimenting with chemotherapeutic applications resulting in an effective treatment methodology, chemotherapy still has problems. First, due to the collateral damage nature of chemotherapy once the cancer metastasizes the effectiveness of chemotherapy significantly drops not in its ability to kill tumors, but in its ability to kill tumors without killing the patient in the process because so much more of the drug is needed.
Second, tumor identification still functions principally on a visual scale which makes it difficult to know when to stop a chemotherapy regiment. Basically the tumor shrinks to a size where it is difficult to know whether or not the patient still has cancerous tissue, thus whether or not further chemotherapy treatments should be administered under threat of causing unnecessary healthy cell loss.
Third, biology does not typically remain static, thus cancers can, and have, evolve mechanisms of resistance against various chemotherapy drugs; therefore, chemotherapy regiments that seek to significantly increase patient lifespan apply numerous drugs to reduce the probability of tumor survival and associated development of resistance. However, the application of numerous drugs per treatment can significantly increase cost of treatment. Overall chemotherapy has actually been a very effective cancer treatment when treatment could actually make a difference, but it real effectiveness has been marred by the lack of quality logistics and diagnosis.
While first and second-generation chemotherapy treatments have proven somewhat effective at managing cancer because biology does not remain static and new chemotherapy strategies are being developed. One may ask the question of why new chemotherapy treatments are needed when nanotechnology and better understanding of genetics are available to develop more target-specific treatments? Chemotherapy treatments have been the most successful non-invasive therapies and although the promise of more target-specific treatments is worthwhile, over the last few decades these treatments have not amounted to any new breakthrough treatment which can rival the effectiveness of chemotherapy at the human level. Therefore, it is important to continue to focus on developing new chemotherapy treatments until new target-specific treatments are ready.
New application techniques have been applied in chemotherapy treatment such as isolated infusion or perfusion which modify rate of delivery and total amount of chemo agent delivered. Identification of appropriate dosage through either an infusion or perfusion methodology can limit the amount of site-nonspecific collateral damage as well as potentially penetrate more solid mature tumors at the site of application.
The third commonly used method of treatment is radiation therapy which involves the application of ionizing radiation in effort to damage the DNA of cancerous cells to the point where either the cell dies straightaway or triggers apoptosis. The DNA damage is caused by either photons or charged particles applied directly or indirectly. Direct action targets the cancer cells themselves whereas indirect action occurs through ionization of water leading to the formation of free radicals which precede to damage cells within the localized radius. Photons typically produce an indirect effect where charged particles are used directly.40 One chief advantage to using charged particles is that it frequently results in double-strand breaks over single-strand breaks.40 DNA repair mechanisms are designed to address single-stranded DNA damage, thus cannot effectively manage double-stranded breaks. However, because DNA repair mechanisms are typically compromised in cancer cells, single strand breaks are also significantly dangerous to cancer cells.
Most radiation therapy requires transition from an external source to the internal tumor, thus there is a high probability that the radiation will pass through healthy tissue. A common strategy used to reduce the damage potential to this ‘obstructing’ tissue is applying beams originating from multiple angles all with an endpoint focused on the tumor. Basically multiple beams are used at an individual radiation intensity which heavily limits permanent damage potential to the contacted healthy tissue, but with all of these beams meeting at a single point (the tumor) the collective radiation intensity can hopefully destroy the tumor. The lack of lateral side scatter from the charged particles also helps limit the healthy tissue collateral damage.40
While photons comprised the first major radiation therapy, they could be falling out of favor (proton proponents have high hopes for their revival) due to two chief problems. First, oxygen is a potent radiosensitizer, thus the higher the oxygen concentrations in the localized region the higher the generated free radical concentrations and the associated cancer damage probability. Unfortunately for photon radiation therapy most solid tumors create an inflammatory microenvironment that is generally hypoxic in nature. The reduction in available oxygen reduces potential free radical concentration reducing treatment effectiveness. Second, hypoxic states may be detrimental to normal cells and tissues, but cancer cells and tumors appear to be more resilient in such an environment, possibly even more dangerous. Thus as photon radiation therapy consumes oxygen to make free radicals it not only becomes less effective, but also may make the remaining cancer cells more deadly.
Unlike photons which have a set energy value, charged particles can more precisely target a tumor incorporating the Bragg peak effect as well as be charged to different peak amounts for optimal decay upon reach the tumor.40 For example as photons move through the tumor environment they pass through healthy tissue when exiting the body. The multiple to single to multiple beam design attempt to reduce damage, but does not entirely eliminate the probability, thus any additional damage when exiting is entirely collateral because the tumor has already been targeted. In the case of charged particles the particle charge can be designed so that the bulk of the charge is expended at the tumor and the exiting particle has no damage potential.
A newer radiation therapy, brachytherapy, involves using sealed radioactive sources that are implanted into the body at precise locations. Brachytherapy can provide a larger level of radiation over a much more controlled and localized area near the tumor eliminating any collateral damage away from the tumor site.41 However, one of the chief drawbacks to brachytherapy is the surgical requirement. Adding an additional invasive step to a cancer treatment is always troublesome. Some argue that surgery as well as brachytherapy can be combined into a single surgical event, but whether or not surgeries are combined is determined on a case-by-case basis and is typically not as successful as most hope.
Overall the usefulness of radiation therapy is defined by the radiosensitivity for a given cancer. Leukemias, lymphomas and germ cell tumors are the more sensitive to radiation followed by epithelial cancers whereas melanoma and most forms of renal cell cancer are radio-resistant.40 However, while leukemia is sensitive to radiation, their widespread dissemination through the body makes treatment more complicated. Therefore, radiation therapy is mostly useful on smaller tumors that have yet to metastasis.
The above three therapies have been useful in the past and continue to be useful in the present, but a better understanding of genetics and the advent of nanotechnology have opened the door for the development of new cancer treatments which are less broad-based than chemotherapy and more site and target-specific offering the hope of greater treatment probability with reduced side effects. The two major new categories of treatment evolving from these new understandings and technologies are agents that specifically target a particular protein and delivery mechanisms which specifically target cancer cells themselves.
In the first half of the 2000s, one of the most promising new ideas for cancer treatment was derived from the chemotherapy distinctive question of ‘what is a cancer cell doing at a greater rate than normal cells other than dividing?’ One clear answer was forming new blood vessels (angiogenesis) to catalyze and maintain rapid cell division and tumor development. When cancer cells are underdeveloped and simply beginning to form solid tumors those cells are supplied with nutrients through diffusion from adjacent blood vessels. However, the rapid uncontrolled cellular replication of cancer cells demands new blood vessels to ensure proper oxygenation. Angiogenesis is commonly viewed as a fundamental step between the evolution of cancer cells from a benign state to a malignant one.
The process of tumor associated angiogenesis proceeds in a series of controlled steps, similar to sprouting angiogenesis; first endothelial cells from pre-existing blood vessels are transported and accumulate nearby in order to form the base matrix for the formation of the new blood vessel. This step requires angiogenesis factors, most notably fibroblast growth factor (FGF-1) and vascular endothelial growth factor (VEGF), to stimulate the existing endothelial cells to produce proteases to break them from their pre-existing matrices allowing for migration towards the cancer cells.42-44 The next step involves the proliferation of the endothelial cells and the extension of the solid sprouts to connect the new vessels to the existing neighboring vessels. After the sprouts extend towards the source of the angiogenic stimulus (the cancer cells) the endothelial cells migrate using integrins.42,43 Finally the sprouts begin to loop becoming fully operational vessels.
Based on the above understanding of angiogenesis and the fact that tumor size typically ceilings at 1-2 mm with little chance for metastasis when only having access to diffused oxygen and nutrients (no new blood vessels), most anti-angiogenesis drugs were designed to repress vascular endothelial growth factor (VEGF) thereby reducing the number of blood vessels that would be developed to support tumor growth.42
The hope surrounding anti-angiogenesis was because angiogenesis and tumor growth were not directly cooperatively regulated elements (one did not directly influence the other) tumor growth would remain unchanged relative to the number of blood vessels until the tumor grew to a size that could not be supported due to a lack of nutrients, a direct results of fewer blood vessels, eventually leading to the death of a vast majority, if not all, of the cancer cells. There was and still is significant aplomb regarding anti-angiogenesis drugs because of the perceived universal nature in their ability to treat cancer regardless of type (in the initial stages of development all tumors need a number of blood vessels) and with limited collateral damage.
Unfortunately some studies have demonstrated that while the theory behind eliminating blood vessels leading to tumor death makes sense, the empirical results do not strongly support the theory. While knocking out VEGF does result in fewer blood vessels those vessels that form are better organized, for when VEGF is not disrupted, blood vessels are numerous, but ‘leaky and chaotic’.42,45-46 It has been hypothesized that this better organization could provide even more nutrients to a tumor, despite the fewer number of vessels, because typically VEGF knockouts resulted in the formation of larger tumors than non-VEGF knockouts.45
In the short-term this result seems to eliminate the usefulness of VEGF blocking anti-angiogenesis drugs, but there may be a silver lining in that while fewer numbers of blood vessels may allow for better nutrient transport, they would also allow for better chemotherapy agent transport as well increasing the probability of drug delivery and tumor death.42 Unfortunately this result has not been supported by empirical evidence provided by patient use of Avastin.45,46 One explanation for this lack of coordination may simply be resistance to the chemotherapeutic agent. In fact Avastin, the most noteworthy anti-angiogenesis drug, which received FDA approval in 2004 just recently had its short-term approval as a preventative treatment for breast cancer expire with no further extension by the FDA.
One question is why do VEGF knockouts create stronger (less leaky) more organized blood vessels. Two explanations come to mind. The more obvious explanation is just like with almost everything else cancer cells heavily overexpresses VEGF creating a signal pull on the endothelial cells in a large number of different and contrasting directions instead of having one or two uniform signals directing strong migration and amalgamation of endothelial cells which would create a strong more extensive blood vessel. Also VEGF plays a role in vessel permeability, thus an excessive concentration of VEGF could create a ‘leaky’ type vessel behavior. The second explanation is that the cancer cells express VEGF and maybe another unidentified signaling molecule and while this unidentified molecule is better able to induce vessel formation, VEGF is produced at greater concentration which masks the effect of this unidentified molecule; a masking that no longer exists when VEGF is blocked by knockout or certain drugs.
Another study has demonstrated that the use of anti-angiogenesis drugs also appears to increase the invasiveness of cancer cells and the associated probability of metastasis.47 This increased probability seems to occur due to an increase in hypoxia due to the fewer blood vessels relative to tumor size which results in the cancer cells synthesizing a greater amount of Met and hepatocyte growth factor (HGF).47 HGF could act as a molecular trigger which induces cell migration. Therefore, for any form of anti-angiogenesis strategy to work it appears that Met and/or HGF need to be inhibited as well.
Another strategy has been to target the endothelial cells which create the new blood vessels themselves. One reason this method is supported is because the endothelial cells that are recruited to form these new blood vessels are thought to be much more genetically stable than the associated cancer cells. However, other studies48 support a loss of genetic stability in endothelial cells which are recruited by cancer cells. Some argue that this lack of stability could allow cancer-derived vessels to develop a higher probability for drug resistance similar to cancer cells; however, the lack of stability may be more controlled than that of the original cancer cells directed by the cancer cells themselves (1 or 2 mutations vs. a cascade), thus eliminating any serious detrimental resistance potential. In fact if this assertion is accurate, the endothelial mutation may actually provide a new means to attack cancer which will not affect non-cancerous systems.
Other elements aside from VEGF have also been identified as potential therapeutic targets for cancer treatment. Similar to the aforementioned biomarkers explored for diagnostic purposes, manipulating the changes in concentration of various elements can be used as a means to control or even eliminate cancer expansion. One of the principle advantages of targeting VEGF for cancer treatment was that the role of VEGF was known and increases significantly with the progressive advancement of cancer. Unfortunately these two principles are lacking in a lot of the other promising targets for either there is a lack of certainty in function or a lack of identifiable exclusive regional change.
The lack of certainty in function is a problem because although concentrations of a given element may change significantly between cancer and non-cancer patients, but whether or not the element acts in some measure of positive/negative feedback or is simply a byproduct of tumor growth is unclear until specific information pertaining to the function of the element is determined. For example from above one useful biomarker for head, neck and throat is miRNA 200a. However, further research is needed before miRNA 200a can be thought of as a therapeutic target or just a byproduct from a form of transcription mismatch caused by a somatic mutation transforming a normal cell into the type of cancerous cell which typifies these types of cancers.
Lack of exclusive regional change is also a problem because if the element is determined to play a feedback role of sorts in the progression of cancer it would initially look like an appealing therapeutic target. However, if that element also plays a critical role in the normal cell growth and/or function then targeting that element will more than likely make cancer treatment difficult at best with lots of side effects and collateral healthy cell damage and pointless at worse as it ends up killing the patient due to too much healthy cell damage. A good empirical example for these two principles is the relationship between tumor development and tumor necrosis factor alpha (TNFa).
New York physician William Coley first deduced the existence of TNFa in 1890 after discovering a former cancer sufferer, Fred Stein, had his cancer ‘cured’ after a post-operative bacterial infection. After talking with the patient and much research Coley developed his ‘Coley’s mixed toxins’ therapy which seemed to have some success treating tumors.49 However, recreation of Coley’s successes were few and far between. In 1962 O’Malley used endotoxin-derived serum to initiate tumor necroses in mice drawing the conclusion that the serum contained some form of ‘tumor necrotizing factor’.50 The term ‘tumor necrosis factor’ was officially coined by Carswell later in 1975 by determining that the necrosis was caused by a factor made by the host cells in response to the endotoxin, not directly through the endotoxin itself.51 Even later it was determined that TNFa and a molecule previously known as lymphotoxin bound themselves to the same receptor and were part of the same genetic family. Lymphotoxin is now commonly known as TNF-beta.52
In the immediate aftermath after confirming the existence of TNFa it appeared that TNFa would be a powerful candidate in the treatment of cancer. Early on this belief was supported by evidence demonstrating high doses of human recombinant TNF inducing necrosis in both syngeneic and xenografted tumors.53,54 At lower doses in combination with melphalan or doxorubicin chemotherapy treatments, TNFa appeared to increase tumor blood vessel permeability and aided in the delivery of the chemotherapy drug in addition to destroying tumor vasculature.55,56
Unfortunately the belief in TNFa as a cancer treatment agent was short-lived. In 1984 Wallach determined that TNF is only weakly cytotoxic to malignant cells when used alone.65 Apoptosis activated with high probability only when inactivating certain downstream TNF signaling using a combination of metabolic inhibitors.52 Later investigators discovered TNF mRNA and TNF itself in malignant and stromal cells during biopsies along with a somewhat proportional relationship between higher concentrations of TNF and lower survival rates.52 The TNF-cancer relationship finished flipping in 1996 when Kollias developed the first TNF-knockout mouse and using this model in 1998 demonstrated that TNF-knockout mice developed fewer skin tumors when treated with a skin carcinogen relative to non-TNF-knockout mice.66,67 Basically the less TNF the animal had the fewer number of tumors it developed, TNF acted as a tumor promoter.
Now instead of trying to treat cancer by increasing TNF concentration the strategy shifted to treating cancer by decreasing TNF concentration. Unfortunately TNF plays an important role in the inflammation response. Changing the inflammatory response at a general level in the body has demonstrated mixed results as TNF inhibitors have generated positive results in treatment of rheumatoid arthritis, but in other inflammatory disorders TNF inhibitors have been linked to tuberculosis along with other infections and even lymphoma in some cases.59
One interesting result with regards to expression of TNF is that its signaling pathway components appear to have widely variable concentrations between individuals.52 Such a result may demonstrate that the overall role played by TNF in cancer development is heavily variable and dependent on the type of somatic mutation that induces cancer initiation in the first place. This result may explain why in recent Phase I and Phase II cancer trials TNF antagonists have resulted in disease stabilization in only 20% of patients.60-62 Anti-TNF therapies may not be a universal treatment for all patients or even a majority of patients, but instead a controlled specific personalized treatment after identifying average TNF concentrations in the blood stream.
While TNF treatments may not be the ‘silver bullet’ some hope exists for cancer treatment, research into TNF did identify another promising target for cancer treatment, nuclear factor kappa B (NF-kB). NF-kB lies at the end of one of the multiple TNF pathways that is triggered when TNF binds to either of its receptors (TNFr-1 or TNFr-2) and is partly responsible for triggering the inflammation pathway.
Beyond the pathway connection between NF-kB and TNF there appears to be a large amount of evidence that suggests sustained activation of NF-kB is rampant in various solid tumors and cancer types. For example major components of the NF-kB pathway are activated in human lymphomas,63 pancreatic cancer,64 cervical cancer,65 carcinomas,66 prostate cancer,67 colon cancer,68 head and neck cancer,69 esophageal cancer70 and lung cancer71. While the pathway is active in all of these cancers, the methodology of this activation and its specific components differ.
Five different molecules make up the nuclear factor kappa beta (NF-kB) transcription factor family: NF-kB1 (p105/p50), NF-kB2 (p100/p52), Rel A (p65), c-Rel, and Rel B.72 These different molecules commonly associate with one another to form various heterodimeric and homodimeric elements. The formation of these hetero and homodimeric elements is normally required to induce receptor activation as the NF-kb molecules typically remaining inactive in the cytoplasm before compound formation sometimes through additional interaction of ankyrin-containing inhibitor-kBs (I-kB).72 The I-kB inhibition complex for NF-kB members is typically made up of three different inhibitor elements (IKKa, IKKb, and IKKg).72 Also note that only the Rel members of the family (Rel A, Rel B and c-Rel) have transactivation domains which can activate transcription.72,73
The most common compound element among the NF-kB family is NF-kB1/REL A or (p50/p65), which is commonly regarded as the ‘classic pathway’ and is activated by TNFa.73,74 This classic pathway has also been identified in having a role in the promotion and pathogenesis of cancer where activation is largely induced by cytotoxic agents and maintained by oncogenic activation of various tyrosine kinases. An alternative to the classic pathway is induced by binding of other TNF family members and processes p100/RelB to p52/RelB.73,74 The alternative pathway components, which includes IKKa/IKKa homodimers, largely regulate survival of premature B lymphocytes and development of peripheral lymphoid tissues.72 Both pathways are complex with various cofactors such as CK2 or Akt influencing whether NF-kB compounds will have a gene inducing or gene suppressing effect including multiple overlaps.72 This complexity makes a straight application of ‘agent that inhibits element x in the NF-kB pathway’ as a treatment difficult because in some cancers it will promote survival and in other cancers the same element will promote death.
Of the five members of the NF-kB family, fully processed NF-kB1 (p50) seems to be the most important. Not only can it form a heterodimer with p65 which can then activate the classic pathway it appears that its formation of a homodimer (p50/p50) can actually inhibit classic pathway activation.76 While the formation of homodimer (p50/p50) can still bind at the kB binding sites, recall that only those Rel family members have the necessary transactivation domains to begin gene transcription. Therefore, homodimer (p50/p50) binding instead of heterodimer (p65/p50) prevents gene transcription and the activation of the classic pathway.76,77
While cancer cells themselves initiate the initial stages of forming a microenvironment, full expansion and functionality of the microenvironment seems to require the recruitment of macrophages; a recruitment tied to NF-kB. Macrophages are multifunctional and highly versatile cells which can engulf microbes or cell debris from appropriate, usually injured, sites secrete a variety of cytokines, present T cell antigens or aid in the activation of lymphocytes.76 Not surprisingly this behavior is governed through a phenotype alteration brought on by environmental molecular cues.
These behaviors are typically categorized into two subdivisions of macrophage activation: M1 or M2. M1 activation is classified as classically activated where the macrophage exhibits a pro-inflammatory phenotype largely resulting in secretion of bactericidal factors and T-helper-1 (TH1) promotion and typically occurs through activation by lipopolysaccharides (LPS) and interferon gamma (IFNg).76,78 M2 activation is classified as alternatively activated where the macrophage exhibits an immunosuppressive phenotype largely resulting in secretion of cytokines and the promotion of the T-helper-2 response (TH2) and typically occurs through activation by IL-4, IL-10 or IL-13.76,79,80 In either case macrophages that possess a phenotype are also sometimes referred to as tissue macrophages.
Note that it appears the initial recruitment of macrophages is due to the presence of cancer, not due to molecules released by cancer. This initial recruitment of macrophages can actually lead to cancer destruction because they enter the local tumor environment with a M1 phenotype. In some situations it is rational to conclude that these macrophages actually do successfully eradicate the cancer, otherwise average cancer rates would probably be much higher than in actuality.43,81 However, in some situations the cancer cells are able to survive the initial onslaught. The means of survival probably involve one of two methodologies. First, the attrition methodology in that cancer replication outpaces the ability of the macrophages to kill cancer cells. Second, mutations in some of the cancer cell population allow these specific cells to resist the bactericidal factors released by the M1 macrophages and/or evade engulfment.
Macrophages that stay within or around the localized tumor environment for a certain, unknown, period of time interact with cancer-associated molecules which change their M1 phenotype to an M2-type phenotype.43,82,83 The converted phenotype seems to occur through a different pathway than genuine M2 phenotype activation, but behaves very similar to the M2 phenotype, thus it is called an M2-type phenotype.43 In fact this M2-type phenotype has been subdivided into 3 different classes (M2a, M2b and M2c).43 M2a is induced through IL-4 or IL-13 activation, M2b is induced through combined exposure to immune complexes and Toll-like receptor or IL-1R agonists and M3c is induced by IL-10 activation. Both M2a and M2b exert immunoregulatory functions, whereas M2c tend to suppress immune responses and induce tissue repair.43
These converted macrophages are commonly referred to as tumor-associated macrophages (TAM). Therefore, what could occur in the tumor microenvironment is that after this first ‘wave’ of macrophages fails to destroy the tumor, tumors alter that first wave into TAMs and then begin to recruit monocytes through various chemoattractants like CCL2 and VEGF.84-86 These monocytes typically extravasate across the tumor vasculature then later differentiate into TAMs.
The recruitment of TAMs starts the next stage of tumor microenvironment formation. TAMs are thought to be responsible for a large percentage of the VEGF released in the newly formed microenvironment.87 As previously discussed VEGF is one of the key components to initiating angiogenesis. In addition to VEGF, TAM also appear to up-regulate another pro-angiogenic protein, matrix metalloproteinase (MMP7) most notably in hypoxic regions of the tumor.43 MMP7 cleaves an active form of RANKL from the cell surface88 which increases RANKL concentration in the tumor microenvironment. TAMs also can provide a form of positive feedback by secreting additional amounts of IL-10 converting more macrophages into M2c types.76
RANKL is normally expressed by T-helper cells as a means to accelerate dendritic cell maturation as well as aids survival. The increase of RANKL concentration brought on by MMP7 in a tumor microenvironment could also serve the same role accelerating dendritic cell maturation which would accelerate the expansion of TAM concentration in the microenvironment. Interestingly enough there is some evidence to suggest that deletion of RANKL reduces the probability of developing breast cancer induced by medroxyprogesterone acetate (MPA).89 RANKL also appears to play some role in regulation of the T cell-dependent response. This element is interesting because a potential high concentration of RANKL in the tumor microenvironment may interfere with any coordinated immune response driven by T-helper cells by masking the tumor using certain antigen presenting dendritic cells.
Recall that one of the hypothesized reasons for the failure of Avastin was that the remaining blood vessels were able to better deliver nutrients to the tumors stimulating more effective growth than when VEGF is not blocked. There may be another explanation for the increased aggressiveness of tumors which survive Avastin treatment. Reducing the number of blood vessels could also create hypoxic conditions, which under most circumstances would be welcome because it would increase the probability of tumor death. However, recruited TAMs appear to significantly bolster the survival ability of tumor cells in hypoxic conditions.43
First, TAMs have a tendency to migrate towards hypoxic regions in the tumor microenvironment, at least in breast, endometrium, ovary, bladder, colon and oral cancers.90-93 In these hypoxic regions TAMs also up-regulate NF-kB and HIF1.94 The positive influence on tumors of HIF1 has been previously discussed and ATF4 and EGR1 have perceived positive unknown effects, thus higher expression rates should stimulate tumor growth and metastasis. Also basal NF-kB is necessary to drive significant HIF1a protein synthesis.78,90 Now it can be said that even without treatment of Avastin or another anti-angiogenesis drug that tumor microenvironments will develop hypoxic regions, but these regions appear to be more widespread after treatment with Avastin over no treatment because of the reduced number of blood vessels. Basically the tumors that are connected to blood vessels get stronger due to the greater level of nutrients provided by those vessels and those that lack vessels either die or survive in a hypoxic region due to the release of these various factors. Note that creating hypoxic regions is still thought to have more positives than negatives when treating cancer.
TAM behavior is largely controlled through the NF-kB pathway. Inhibition of IKKbeta results in a phenotype shift from M2-type to M1.78,95 In addition recall that the formation of homodimer (p50/p50) can actually inhibit classic pathway activation. It could be argued that these (p50/p50) homodimer interactions chiefly interfere with the activation of the NF-kB pathway in LPS.96 This ‘inhibition’ of LPS-derived NF-kB action reduces the probability of LPS influencing M1 phenotype shifts in macrophages thus increasing the probability for M2 phenotype shifts. While still not understood the relationship of (p50/p50) in the NF-kB pathway appears to be important. So one reason why neutralizing NF-kB behavior may demonstrate positive results in cancer treatment is the reduction of TAM in the tumor microenvironment reducing the ability of tumors to survive hypoxic conditions.
TAMs are not the only monocyte that is recruited by tumors to aid in their development. TIE2-expressing monocytes (TEMs) are monocytes which interact with angiopoietins 1-4.43 Interestingly TEMs do not express chemokine receptor 2 (CCR2) thus they are unable to bind CCL2 which means they require another recruitment factor than the CCL2 which recruits monocytes that will eventually become TAMs.43 One study suggests that ANGPT2 is the molecule chiefly responsible for recruitment of TEMs into tumors.97 In addition ANGPT2 also could regulate the rate at which TEMs release angiogenesis promoting cytokines through the suppression of IL12.97
The specific role of TEMs in tumor facilitation is unknown, but in experiments when TEM concentrations were eliminated in tumors a statistical reduction in angiogenesis and tumor growth was observed.98 However, while TEM was removed, the loss of TEMs did not influence TAM or neutrophil concentrations further supporting the notion that TEMs and TAMs are independent elements in tumor promotion. Also even though the TAM concentration was not influenced by the loss of TEM, angiogenesis loss was significant which seems to suggest that TEMs have more influence on angiogenesis than TAMs.
Tumors also recruit mast cells largely using members of the stem cell factor (SCF) family.99,100 Like TAMs and TEMs mast cells appear to have the principle purpose of aiding tumor angiogenesis for inhibition of SCF expression in rat tumors reduced tumor vascularity and angiogenic responses.101,102 However, the angiogenesis influence of mast cells may be weighted towards the initial stages of the angiogenesis over the later stages meaning blocking mast cells may be more important than blocking TAMs.103,104 The reason for this behavior could come from mast cell interaction with endothelial cells lining the vascular lumina of multiple myeloma.105 Mast cells also appear to de-granulate and release angiogenesis factors in hypoxic conditions largely directed through HIF interaction.106,107
Angiogenesis is an important element of tumor progression, but tumors recruit additional molecules which provide other tumor promoting effects. One of these molecules are Myeloid-derived suppressor cells (MDSCs). MDSCs is a broader term for a heterogeneous population of cells primarily consisting of immature myeloid progenitors for neutrophils, monocytes and dentritic cells.108 While mature neutrophils typically act as immuno-promoting, MDSCs seem to act as immuno-suppressive partially due to their competitive nature with neutrophils and corresponding low levels of major histocompatiblity complex (MHC) class II and CD80 expression.109-111 The other method MDSCs use to suppress T and Natural Killer cell activity against tumors is the release of arginase 1 and iNOS (a.k.a. NOS2A).112-114 Somewhat ironically the most accurate way to identify a group of MDSCs is to look for their immunosuppressive ability. Tumors appear to recruit MDSCs from the bone marrow into their microenvironment through cytokine BV8 (a.k.a. PROK2).115,116
While immunosuppression is the principle tumor promotion action taken by recruited MDSCs, in murine models they appear to aid in angiogenesis.108,115 However, there does not appear to be any definitive evidence that MDSCs release pro-angiogenesis factors. Their support methodology may be through their immunosuppressive behavior which allows the tumor greater opportunity to release pro-angiogenesis factors from other cells. The latter explanation may be the more accurate one because MDSCs can secrete IL-10 which increases the probability for higher concentrations of TAMs which aid in angiogenesis through the release of VEGF.117 Another explanation is that BV8 helps recruit other elements which aid angiogenesis apart from MDSCs. There is some evidence to suggest that one of these ‘other elements’ are neutrophils.118,119
Based on the duel role of BV8 as a pro-angiogenesis agent as well as an indirect immunosuppressive agent, looking for a way to neutralize the BV8 pathway could be a useful strategy in cancer treatment and even prevention. The focus on prevention could be even more appropriate because the more prominent effects of BV8 seem to occur in the earlier stages of cancer progression. One advantage in creating BV8 based treatments may be that most of the identified functions for BV8 seem to occur in development and childhood. One disadvantage, beyond the general lingering unknown, is a possible connection between BV8 and circadian rhythms.
Cell surface markers in human MDSCs are somewhat inconsistent, but support CD11b and CD33 expression, but no CD14 or Lin.120,121 One of the reasons cell surface markers are not valid identifiers for MDSCs is that there is some question to whether or not certain unknown characteristics can lead MDSCs to differentiate into mature macrophages, dentritic cells or granulocytes110,122 thus there is a wide variances of cell surface markers in MDSC clusters due to various cell types that man inhabit these clusters.
Earlier the mixed results of Avastin were discussed as a representation of addressing angiogenesis by neutralizing the signaling factors. However, others believe the best way to treat tumors is to prevent angiogenesis which would prevent tumor growth and dramatically reduce metastasis probability, but not through neutralization of angiogenesis factors, but neutralization of the cells which provide these factors. As discussed above there are four major targets to preventing angiogenesis from an origin perspective: TAMs, TEMs, mast cells and (not discussed neutrophils). Some researchers have already used specific DNA vaccines to enable cytotoxic T cells to attack TAMs resulting in significant reduction in population in pre-clinical murine models with associated reductions in tumor angiogenesis, growth and metastasis.123
Despite the success in targeting TAMs with this strategy, little information exists regarding whether or not such a strategy increases lifespan or will in the long term have results similar to Avastin administration. One of the bigger questions is that does the removal of TAMs even matter that much when TEMs appear to have a more pronounced influence on angiogenesis on an overall level and mast cells apparently driving a lot of the initial angiogenesis action. Either way more study regarding this anti-TAMs strategy needs to be done.
One mechanistic problem with limiting macrophages may be neutrophils picking up the slack. When macrophages were stripped of CCR2 (receptors for CCL2 a major recruitment molecule), neutrophils were recruited in much larger numbers than normal and augment angiogenesis maintenance.124 Such a contingency action by tumors seems likely for other long-term anti-macrophage treatment strategies as well (antibodies); therefore, somehow addressing neutrophil recruitment may also be necessary.
Another issue with attacking angiogenesis signaling molecules is that the two most prominent elements driving angiogenesis are VEGF and hypoxic conditions with both appearing to be non-starters at this moment. The disappointment in Avastin has raised questions regarding the usefulness of blocking VEGF alone. Also the hypoxic element could cause problems because a non-decisive reduction in angiogenesis through one pathway could result in the expansion of angiogenesis via hypoxic-induced agents. However, an important element to recruitment that could be fortunate is that pro-angiogenic factors do not appear to be released by recruited cells until they are actually in the tumor microenvironment. Thus tumor specific targeting elements that are properly designed could address a vast majority of the angiogenesis potential if applied early enough or in pre-emptive preparation for metastasis.
Unlike monocytes, B cells do not infiltrate into the tumor environment, thus inflammatory-based functions from innate immune cells were thought to operate through a more remote signal pathway.78,125 In the K14-HPV16 murine model B cells were able to activate Fcg receptors on myeloid cell.126 Also B1 cells could suppress LPS-induced genes (those which would drive M1 phenotype expression), possibly through the expression of IL-10, which then increase the probability that macrophages in undertake a M2-type phenotype.127 Inhibition of IL-10 switched infiltrated macrophages from M2 to M1 and stimulated a more aggressive innate immune response against the tumor.128 This result implies that IL-10 may be required for the maintenance of a tumor-induced M2 phenotype for recruited macrophages. Therefore, long-term inhibition of IL-10 may be a therapeutic strategy worth exploring.
Dendritic cells regulate adaptive immune responses and are the principle antigen-presenting cells (APC), which can induce primary and secondary T and B-cell responses.43,130 Dendritic cells are divided into two types of classes: myeloid (MDC) and plasmacytoid (PDC).130 Both are commonly identified through their unique cell surface markers (myeloid: CD11c, CD33 and Lin and plasmacytoid: CD45RA, CD4 and ILT3).131 Under normal circumstances MDCs originate in the bone marrow as immature cells which mature after acquiring any foreign antigen. After maturation the mature DC migrates to lymphoid tissue where they initiate activation of antigen-specific T cells.43 With respects to tumors, MDCs are less likely to initiate maturity reducing the number of mature DCs within cancerous tissue relative to healthy tissue.132 Instead immature MDCs are recruited into the tumor microenvironment by members of the CXCL family (notably 8 and 12), VEGF and HGF.133-135
In the tumor microenvironment immature MDCs function in two major roles. Immature MDCs aid in angiogenesis not only by releasing pro-angiogenesis cytokines, but also through their ability to be used as endothelial progenitors.134,136 There is also some question to whether or not immature MDCs can secrete members of the CXCL family to further recruit other cells into the microenvironment.137 Also some evidence exists to support dendritic cells initiating an anti-immune response via a TLR4-MyD88 pathway in the presence of dying tumor cells.138 Finally hypoxic conditions appear to reduce the probability of dendritic cell maturation perpetuating their immature, pro-tumor state.135,139 Despite all of the pro-tumor roles that dendritic cells activate, their overall role in tumor promotion is currently regarded as minimal; however that minimal effect may be amplified by the sheer number of dendritic cells in the microenvironment.
The prominent role that both TNFa and NF-kB have in tumor development was a major factor in the development of the theory that inflammation is a centerpiece in tumorigenesis. A number of individuals agree that inflammatory factors do play a role in the development of a clump of cancer cells into a full-blown tumor as the tumor microenvironment is aided in development by secretion of inflammatory promoters.140 However, there is some argument regarding whether or not inflammation actually can drive the necessary cellular changes to induce non-cancerous cells to become cancerous in the first place. Proponents of inflammation as cancer triggers have two points of argument: chronic inflammation eventually induces cancerous cells and viral induction. Viral induction, where tumorigenic pathogens such as Human Pamplona Virus (HPV) evade the immune system and establish persistent infections associated with low grade, but chronic inflammation, seems on surer footing than non-viral induced chronic inflammation.
One important side note is that when references are made to pathogen x, like HPV, causes cancer y the pathway of cancer inducement commonly comes from the NF-kB pathway.141 In large part the reason some of these pathogenic agents can enhance the development of cancer is that they typically have transactional domains which mimic NF-kB family members or NF-kB activation receptor sites.141 in the local environment where the virus persists. This interaction allows these specific pathogens the ability to enhance the developmental probability of cancer. For example individuals can still get cervical cancer even if vaccinated against HPV; however, the vaccination is thought to significantly reduce the probability of acquiring cervical cancer because it is thought that the most common pathway to developing cervical cancer seems to stem from NF-kB mimicry by HPV.
It is more difficult to link up non-viral chronic inflammation as the cause of the cancer. One method of action could be that over time the inflammation leads to the creation of a significant quantity of free radicals. These free radicals are then able to induce enough genetic damage to nearby cells localized within the region of inflammation that they eventually mutate and become cancerous. The major empirical problem with this theory is that individuals who suffer from conditions, which have origins from chronic inflammation such as rheumatoid arthritis or psoriasis, do not seem to develop cancer at any higher rates than those that do not suffer from these conditions.
Currently no quality explanation exists for these results relative to the above theory. Some proposes that other dietary or environmental carcinogens are not able to interact with the inflammation catalyzing the cancer development, but if such catalysts are required to produce cancer from chronic inflammation then it is difficult to argue that chronic inflammation gives rise to a significant number of cancer cases. In fact recent evidence demonstrates that mediators and most signaling pathways for inflammation are downstream of oncogenic mutations supporting a conclusion that cancer causes inflammation not inflammation causes cancer.140
Perhaps the best theory behind why chronic inflammatory conditions like psoriasis do not develop into cancer, as most inflammatory – cancer proponents would theorize, is that there is some evidence to suggest most of the macrophages in psoriasis express the M1 phenotype. This excessive expression of M1 phenotypes more than likely is derived from psoriasis being a T helper 1 mediated disease causing large accumulation of neutrophils and monocytes in the skin. The large-scale expression of M1 phenotype should increase the probability that those macrophages would attack cells expressing initial stages of tumor development. This increased probability of cancer termination could then mask the cancer inducing effects of chronic inflammation in chronic inflammatory conditions like psoriasis.
Recalling for a moment that one of the original ideas behind the importance of studying angiogenesis was to develop a means to design treatments which would force higher levels of oxidative stress on a tumor or pre-tumor due to a lack of nutrients and oxygen from the lack of blood vessels. Unfortunately not only has anti-angiogenesis treatments proven less effective than previously thought, the base idea of inducing high oxidative stress environments may have been in error as well. To understand whether or not this is the case it is important to investigate how tumors behave in these oxidative environments as opposed to normal cells.
Environments of high oxidative stress are created when the large increases in concentrations of reactive oxidative species (ROS) occur. Among those ROS elements the most dangerous are free radicals (O-) and super oxides which are commonly formed when electrons from the electron transport chain operate in error and interact with oxygen. In localized regions which cover tumor cells there is a higher probability for the formation of a high concentration ROS environment due to oncogenic transformation and alterations in metabolic activity (including angiogenesis processing vs. cellular growth rates).142,143 The result should not be surprising simply based on the premise that with less restraint on growth cells will undergo greater metabolic activity in order to expand their growth which will result in a greater concentration of ROS due to a lack of corresponding increase in antioxidants.
Normally an increase in ROS within the localized environment should be viewed as a positive in the context of addressing cancer because a higher ROS concentration leads to a higher mortality rate. However, in the case of cancer, there are questions about how ROS may change the nature of translation for various signaling molecules, some which stimulate cell proliferation and promoting cell motility.144,145 Initially an increase in concentration for molecules governing both proliferation and motility seem to make some sense on a logical level. A cell under high levels of oxidative stress three potential responses could be expected. First, cells would take an attrition response where they start multiplying at a much higher than normal rate with the ‘intent’ to out-propagate the threat (in this case the ROS agents), an attempt to sacrifice enough cells to either neutralize or mitigate the significance of the ROS agents in the oxidative environment. Second, cells would take a ‘bunker down’ response, similar to that of a bacterial spore, where all resources are devoted to survival of the given cell, no communication, no growth, just survival. Third, cells would simply try to move away from that high stress environment by increasing their motility. For cancer cells the first and third reaction appear predominant.
The most important consideration for addressing tumors is that their functionality commonly behaves like a double-edged sword in that they frequently develop alternative methods for accomplishing a given function. While these alternative methods can be frustrating through the complication or reduction of more common pathways, these new methods also provide possible new attack strategies if they can be successfully identified. One particular pathway which could involve a useful therapy is how tumor-based mitochondria behave in high ROS environments.
The chief safeguard to the mitochondria is that a double membrane with selective protein transport complexes protects it. However, if a detrimental agent, normally superoxides, compromises those membranes the raw mitochondrial DNA (mtDNA) is more susceptible to damage because of a lack of histones.145 Combine the lack of histones with the limited DNA repair capacity in the mitochondria and excessive ROS concentrations and mtDNA tends to be damaged more easily in cancer cells than other types of DNA. When this DNA is damaged it can compromise proper functioning of the mitochondria and result in the greater synthesis of ROS.
The release of excess ROS for the maintenance and stabilization of high ROS concentration environments is also important with respects to metastasis. Recall that above it was briefly discussed that one possible response to a high ROS environment was increased probability for motility and cellular migration. This response pathway seems to persist in cancer cells as well as normal cells. Some evidence already exists which suggests the mitochondria dysfunction and the corresponding increase in ROS production can enhance metastatic probability.145,146
The reason for this increase in metastatic probability appears to come from various ROS activating activator protein 1 (AP-1).145 AP-1 plays a major role in a number of critical biological processes including cell proliferation, apoptosis and differentiation. Among these processes AP-1 appears to also be able to bind CXCL14 (a.k.a. BRAK or MIP-2g), a chemokine of unconfirmed functionality.145 Unfortunately despite its ability to interact with AP-1 the function(s) of CXCL14 remains controversial due to conflicting empirical data.146,147 One possible function appears to be increasing the release of calcium ions in to the cytosol. It is hypothesized that this excess calcium is released from isolated ER microsomes when CXCL14 binds to IP3R.145
If this hypothesis is correct then there may be an interesting connection between CXCL14 and cytochrome C. Cytochrome C is one of the more common methods for the initiation of apoptosis. When cytochrome C is released into the cytoplasm, usually due to mitochondrial response to apoptotic precursors, it binds apoptotic protease activating factor149 and later binds to IP3R on the ER facilitating calcium release, similar to a theory regarding how CXCL14 is thought to behave. This calcium release acts as a positive feedback mechanism, creating cytotoxic levels of calcium, which results in the further release of more cytochrome C both into the cytoplasm and into the mitochondria. The release into the mitochondria leads to the activation of caspase 9 which begins the caspase cascade initiating apoptosis. So does CXCL14 create some form of cytochrome C mimic response or does it actually trigger the release of cytochrome C which is the agent which creates the cytoplasmic calcium concentration increase?
In addition this release of calcium into the cytosol may also support the notion of higher glycolysis activation in cancer cells. Most enzymes which function in glycolysis bind to the cytoskeleton and cytosolic calcium promotes assembly of actin filaments along with cytoskeleton re-organization which could in turn promote greater than normal activation of glycolysis dependent enzymes, thus increasing glycolytic activity. Intuitively such a thought process makes sense because under conditions of stressed and dysfunctional mitochondria there exists a real probability that these dysfunctional mitochondria will be unable to produce sufficient amounts of ATP through the Krebs Cycle and electron transport chain. Therefore, under periods of dysfunction the mitochondria could release molecules which indicate a dysfunction stimulating non-mitochondrial based ATP generating pathways, the most prominent one being glycolysis.
However, such a system would probably only be able to stem the ATP loss from the mitochondria for a short period of time largely because of a lack of required ATP concentration as well as eventual mitochondrial driven apoptosis. It is important note that clearly some cancer cells probably overexpress some type of anti-capsasin molecules which block mitochondrial driven apoptosis. Also there may only be a small window for metastasis through this mechanism as there have been observations that cancer cells with high ROS generations eventually reduce this excess generation falling back into a rebalanced dynamic equilibrium of ROS generation and ROS elimination, which aids overall sustained proliferation.145
One aspect of treating cancer that has not been addressed to this point is the scenario of relapse. There are two principle rationalities to explain the reemergence of cancer once a patient is thought to be in remission beyond an incredibly unlucky second separate and independent occurrence. First, that the patient was never fully in remission, some of the cancer become resistant to the applied treatment, incomplete surgical removal or an inability to target all cancer cells with chemo or radiotherapy. This explanation is understandable because there is no existing definite cancer detection technique, thus smaller cancer cells (typical post-treatment cells) are more difficult to detect. Second, during the development of the tumor cancer stem cells (CSC or cancer-initiating cells (CICs)) are produced, so even after the main tumor is eliminated these CSCs reinitiate cancer development.
Two different theories emerged to explain the functional heterogeneity (different proliferation ability and differentiated states) in cancer: small population of self-renewable cells (cancerous stem cells) that created all other cancer cells in the development of the tumor or clonal evolution where all cells have similar tumorigenic capacity.150-153 Early on there were serious questions regarding whether or not CSCs actually existed.154,155 However, while an expansion of empirical evidence has limited the concern regarding the existence of CSCs, it has not created a single predominant theory regarding their role in the development and progression of tumors. Also an increase in the supporting evidence for CSCs does not eliminate clonal evolution as a valid theory for cancer evolution as serial transplantations typically result in more aggressive tumors,156 CSC theory is just the primary cancer causing agent not the only one.
CSCs are loosely defined as stem cells that induce a cancerous state. There is no general acceptance to the origins of CSCs, do they arise from cancer cells or are do they mutate from normal stem cells. Devising these origins is difficult because CSCs have similar properties to normal stem cells like self-sustained replication, pluripotency and drug resistance genes like ABCG2.157 However, most cancer cells share most of these properties and with the genetic variances in cancer cells it cannot be dismissed that certain mutations could restore pluripotency to some cancer cells making them CSCs. It is difficult to conclude that normal tissue could become both cancerous and pluripotent in a small replicative time period, thus it makes sense to conclude that CSCs must be derived from a group of cells that already has one of those two traits, either tumors or normal stem cells. Basically the chief question of CSC origin come down to: do CSCs develop first which lead to tumor development or do tumors develop first with some of the cancerous cells mutating into CSCs?
Some evidence that supports CSCs originating from tumors is the enhanced chemotherapy resistance attributed to most CSCs seems to come more from previously exposed cancer cells over normal stem cells. Also haematopoietic malignancies moZ-TIF2, mil-AF9 and mll-enl16 all appear to grant stem-like properties on committed progenitor cells putting them on the pathway towards becoming CSCs.158-160
However, other evidence points to more CSCs coming from normal stem cell corruption. For example carcinomas seem to target epithelial stem cells and CSCs derived from carcinomas express similar molecules like Oct4 and Bmi1, have similar size, greater adhesiveness and similar patterns of gene expression.158,160,161 While one could argue that these features are common among stem cells in general, so CSCs derived from differentiated tumors would have them as well not just those derived from normal stem cells, the lack of a ‘missing link’ cell makes it difficult to decisively support that conclusion. Overall it may be just a situation where CSCs can develop from both potential pathways.
Despite the need for more information to determine a better treatment strategy, another reason behind determining the origins of CSCs is that some theorize that CSCs arising from transformation of a normal stem cell are more aggressive than those derived from a more differentiated progenitor cell.158 One association with this theory has been melanoma and its connection to epithelial stem cells. The theory tends to make more sense because it stands to reason intuitively that conversion of normal stem cells would result in faster tumor progression through greater cell division than conversion of a previously committal progenitor cell.
Some of the first pieces of evidence which supported the existence of CSCs was through fragmentation of human breast cancers into single cell suspension and isolation of subpopulations.162,163 This experiment found that tumor-initiating ability was almost exclusive to a small fraction of the cancer cells all which expressed CD44.163 Quick reminder that the term CD describes a particular cell-surface glycoprotein which involves cell-cell interaction. Other studies confirmed this CD44 expression as well as adding CD133 expression.164-166 After these studies it was concluded that CSCs either expressed CD44 and/or CD133.158 Unfortunately at the moment the expression patterns of type specific cancer seem to be either capricious or unknown as CD44 expression is found in head and neck cancers, pancreatic and breast cancer where as CD133 expression is found in brain cancer, liver and colon cancer.165-170 Also there does not appear to be a universal marker which identifies CSCs, but various groups of markers that hint at CSC characteristics. In fact some believe that CD133 is not a CSC maker, but a stem cell maker instead because it is also expressed in normal stem cells, but comparison of expression levels varies.163
The conservation of CD44 and CD133 expression through a wide variety of cancers has logically lead researchers to question whether these two elements confirm stem-cell characteristics. Work has been done to investigate the role of CD44 on CSCs. Application of a universal (works against all isoforms) CD44 antibody was thought to eliminate transplanted leukemic stem cells while a humanized monoclonal antibody for CD44 demonstrated tumor reduction in advanced oral cancer.171 Therefore it appears that CD44 does play some important role in tumor growth and/or maintenance. However, there are some concerns as administration of the CD44 ligand, hyaluronan,172 seemed to reduce breast cancer propagation, which is contradictory to what is expected. Also the biggest concern at the moment with using a CD44 antibody strategy is its major skin toxicity due to the presence of CD44 on normal skin stem cells.171 There is still hope that CD44 can be used as a therapeutic target in the right dosage because CSCs seem to express more CD44 than normal haematopoietic stem cells.171,173
Another question with regards to CSCs is their highly sporadic variable frequency relative to non-CSCs. Instead of most tumors consisting of relatively conserved flat ratios of CSCs the percentage of CSCs vary wildly relative to tumor type. For example CSCs in colorectal carcinomas have ranged between 1.8 to 24.5% and CSCs in melanoma have ranged between 1.6 and 20%.162,169 One reason behind this variance may be a non-uniform sampling or distribution of CSCs leading to inaccurate results (impurity in sample collection). However, the number of studies that report this wild fluctuation in CSC ratio seem to rule that conclusion out.
Some argue that the hypoxic, pH and nutrient variant microenvironment with necrotic elements create a form of stochastic aspect to the number of CSC within a given tumor formation.174 Therefore, it is difficult to assign a rationality to why one tumor formation may have x% of CSCs versus another tumor of same type that only has y% of CSCs. Another explanation could be that CSCs increase depending on a given event such as metastasis in order to replenish any losses from the principle tumor. Therefore, elements like CXCL14, which could promote metastasis, could also initiate the production of more CSCs.
Is there any evidence to support a suggestion that CSC ratio changes in tumors in preparation for metastasis? To address that question one must ask what role, if any, CSCs may play in metastasis. Most believe that the process of metastasis is preceded by an epithelial mesenchymal transition (EMT) at the invading edge of the tumor which leads to cellular detachment increasing the probability that the cell in question is caught up in the blood stream and can then move to a different place in the body.159,175 There is evidence to suggest that canonical pathways initiate EMT. Some have concluded that because canonical pathways can be activated during neoplasia that the reversal of differentiation that may convert progenitor cells into CSCs may also detach those cells from the tumor matrix.176 Basically this ‘accidental’ activation of the canonical pathway kills two birds with one stone with regards to cancer aggressiveness, more CSCs and increased probability of metastasis.
A more specific example of metastasis behavior was demonstrated when researchers found a distinct group of CD133+ CXCR4+ cells which exhibited stronger migratory activity in vitro over CD133+ CXCR4- cells.177 Both cell types exhibited similar tumor progression rates. In addition the CD133+ CXCR4- cells did not demonstrate metastatic activity in the liver where as CD133+ CXCR4+ cells did.177 Furthermore inhibition of the CXCR4 receptor significantly reduced metastatic potential in pancreatic tumors with no significant change to localized tumor progression.159 One outcome connecting metastasis and CSCs is that there could be two distinct types of CSCs. One that is developed for the purpose of metastasis (the more resistant nature of the CSC would be better suited for successful metastasis) and one that is developed for localized tumor progression which ‘hides’ inside a solid tumor.
On a side note the nature of metastasis also draws a question of whether or not macrophage fusion is required. Some believe that when cancer cells reach high grades of malignancy (solid tumor formation) the high plasticity of the cells along with the high inflammatory environment some of the cancer cells themselves progressively develop endothelial and immunity phenotypes. These new phenotypes allow these cancer cells to more freely transport within the blood stream increasing probability for metastasis. In contrast John Pawelek and his supporters believe that fusion between macrophages (most likely these TAMs that congregate near the tumor formation) and cancer cells fuse forming a macrophage/cancer cell hybrid cell that is capable of traveling through the blood stream.178
Both explanations have their problems. While the cancer plasticity theory makes sense, especially when considering the role of cancer stem cells in cancer progression, very little supportive evidence has been demonstrated for the ability of the inflammatory environment to induce immunity characteristics. The same story exists for the hybrid macrophage-cancer cell as while numerous studies have been done that supposedly produced hybrids, there have been no solid confirmations that those new hybrids actually were hybrids.179 The two reasons for the difficulty in detecting hybrids are that both the cancer cells and macrophages are almost virtually identical genetically and very narrow differences between genuine hybrids and two cells simply adhering to each other.
There is evidence to suggest that EMT induction is reversible which could revert converted epithelial-based CSCs back into epithelial cells.174,180 Such a strategy could be an anti-metastasis treatment option. The ability to reverse CSCs into non-CSC epithelial cells may also provide an explanation for the great variance in CSC ratio in various tumors. The undifferentiated and differentiated states of cancer cells may not be as permanent as previously thought and depending on given conditions cells may move between CSC and non-CSC states. Most of the existing CSC models do not incorporate a dynamic differentiated to undifferentiated state because of limited empirical evidence.
While little direct empirical evidence exists for this reversible differentiation ability, strong anecdotal evidence does exist. Take this scenario for example… a group of 100 cells exist where 7 are stem cells and 93 are non-stem cells. If one of the non-stem cells undergoes a genetic mutation which would induce malignancy under a non-reversible differentiation model such a mutation would be irrelevant because that mutated cell does not self-renewable capabilities, thus cannot develop into a full-growth tumor unless it also developed a type of telomerase (self-renewal) mutation; as previously noted such dual mutations seems unlikely. Therefore, such a cancerous mutation would need to occur in one of the stem cells. However, such a restriction does not appear viable to correlate known mutation probability rates vs. general cancer incidence in the general population (mutation rates would need to be much higher).
With regards to CSC chemo-resistance the more illuminating studies seems to involve brain cancer and CD133+ CSCs24. One theory behind why CD133+ cells survive over CD133- cells when exposed to a chemotherapeutic agent is that CD133+ overactivate the DNA damage checkpoint response system.159 The checkpoint system is designed to limit the ability of cells to enter mitosis with damaged DNA. Based on the overactivity of the checkpoint, CSCs may overexpress wee1 to ensure entrance into the checkpoint to repair single stand break damage. Also while CSCs are through able to survive chemotherapeutic agents than non-CSCs through drug pumps like ABCG2 another advantage CSCs have is the ability to enter the quiescent state. There is reason to believe that CSCs in the G0 phase gain a greater resistance to chemotherapy agents.181 So perhaps CSCs can linger longer in both G0 and G2 phases increasing survivability.
CSC location is also problematic when addressing treatment possibilities. CSCs are typically localized in the center of a solid tumor mass. The CSCs act as a keystone to the tumor continuing to expand the tumor from the inside in an outward fashion. Therefore, to neutralize these CSCs the treatment regiment must ‘bore a hole’ of sorts through the solid tumor to reach the center and the CSCs. However, boring that hole is difficult because as the treatment is destroying the more differentiated cancer cells that make up the outer portion of the tumor, the CSCs are continuing to synthesize new cancer cells which substitute for the dead cells in the tumor itself.
The lack of understanding surrounding CSC origin makes developing a treatment strategy complicated. Some argue that due to the general resistances against chemotherapy oncolytic viruses are the best option because of their cytotoxic and non-targeting nature. The problem with this strategy is that, more than likely due to their lack of definite differentiation, CSCs are could be resistant to infection with certain oncolytic viruses, like HSV-2 mutant, that can normally infect differentiated cancer cells.182 This problem can be overcome by adding certain specific elements like histone deacetylase inhibitor, trichostatin A, as it did for one type of breast CSC.182 However, because of incomplete information and possible heterogeneity surrounding CSCs it is difficult to confirm whether or not such a strategy can be universally applied or if it was simply valid for that specific experiment. For example Jordan identified parthenolide as a molecule that can selectively target human leukemia stem cells over normal stem or progenitor cells, but other cancerous non-leukaemia targets are unconfirmed.183 One element which demands further study is the question of whether or not intact IFN response is a defining characteristic of CSCs.
Another treatment strategy in its early stages is inducing CSC differentiation through exposure to bone morphogenetic proteins (BMPs).184 This result makes more sense when correlating the evidence that a reduction in BMP concentrations increases the probability for colon cancer.185 Forcing CSCs to differentiate could be the better treatment strategy than trying to kill them because there appear to be fewer, if any, CSC safeguards to preventing induced differentiation vs. toxic agents. Once differentiated the CSCs lose their ability to replenish cancer cells reducing the rate of tumor growth. This strategy seems to be supported by experiments which down regulated BMP resulting in more tumor aggression via alleged greater CSC proliferation.186 However, because BMPs could also cause normal stem cells to differentiate, the application of this type of therapy would need specific cancer targeting to ensure limited BMP exposure to non-CSCs. Despite these difficulties there is hope that a novel treatment can be developed to attack CSCs and undermine the continued progression and maintenance of the tumor collapsing it from the inside out.
While oncolytic viruses have been explored as a means for specific targeting it is not the only strategy. A new technological strategy attaches nanoparticles made from diamonds to chemotherapeutic agents to increase their effectiveness.187,188 The advantages of this new nanodiamond-chemo compound is that the nanodiamond is made from carbon, is non-toxic and does not drive an immune response.187 Also the addition of the nanodiamond increases the size of the chemo agent is thought to dramatically reduce the ability of tumors cells to use anti-drug pumps to eliminate the chemo agent out of the cytoplasm before it does any mortal damage to the cancer cells.187 Formation of the compound also seems to reduce chemotoxicity which should reduce detrimental side effects associated with such treatment.
Finally the nanodiamond can also act as a linker molecule bridging the chemo agent and another molecule, perhaps an antibiotic, which can increase tumor targeting efficiency. Nanodiamonds and similar elements like gold may also be an effective delivery mechanism for other agents like BMPs as well. At the moment there could be two drawbacks to adding nanodiamonds to the treatment. First, the additional cost associated with their manufacture and formation and second while initial tests have demonstrated reasonable biocompatibility there have been no real long-term studies to determine long-term side effects.
One of the trickiest issues in treating cancer is identifying sustainable treatments. As seen with Avastin above with respects to failed attempts to control tumor angiogenesis, researchers frequently discover drugs which perform well against cancer in the short-term, but when the treatment scope is expanded the effectiveness of these drugs falter making them almost no more valuable than existing treatments. One example of the ability of cancer to thwart these new drugs is in the new chemotherapy drug, vemurafenib, used to treat metastatic melanoma. In Phase III trials vemurafenib shrank tumors by almost 10 times more than dacarbazine (the standard treatment for metastatic melanoma at the time) [48% vs. 5%] and also improve six-month survival rate from 64% to 84%.189 Unfortunately the successes of vemurafenib were only short-lived as tumors switched from the BRAF triggered pathway to an alternative pathway, essentially becoming immune to the effects of vemurafenib.189 After factoring out the six-month survival rate improvement, the long-term survival rate did not change significantly. Ipilimumab, another melanoma drug, performs similar to vemurafenib in that it marginally increases short-term survival rate, but does not significantly change long-term survival rate.190
Cancer prevention has always been a questionable issue because, as demonstrated in this blog post, it is difficult to narrow down an effective measure of prevention due to the intricate nature in which cancer develops. However, for some specific cancers certain prevention therapies exist, most of them involving breast cancer because of the association between breast cancer development and oestrogen/estrogen. Early preventative treatments were Tamoxifen and Raloxifene which acted as competitive inhibitors with oestrogen. However, these two drugs did carry small risks of increasing the probability of womb lining cancer and blood clots which could increase the risk of heart attacks. The severity of these side effects were so great that most worried about using either as a preventative agent.
Some researchers believe that a new drug, Exemestane, provides a much better option due to its lack of side effects in initial clinical studies. Exemestane is an irreversible, steroidal aromatase inactivator which when bound to aromatase is converted to an intermediate that irreversibly binds to the active site on aromatase eliminating both elements.191 This type of inhibition is commonly known as ‘suicide inhibition’. Eliminating the available concentration of aromatase reduces the concentration of estrogen as aromatase is chiefly responsible for converting testosterone to estradiol. The reason for this lower estrogen concentration is that in post-menopausal women the ovaries stop producing estrogen, so estrogen production is largely reliant on the conversion of androgens. Of course exemestane is only effective against estrogen receptor positive breast cancer.191
Unfortunately there may be a drawback in using exemestane that has not yet been seen. Previous studies have demonstrated that animals exposed to aromatase inhibitors exhibit substantial gains in overall adiposity due to increased adipocyte hypertrophy and hyperplasia.192 Therefore, if individuals are placed on exemestane as a preventative treatment, especially post-menopausal women, there may be a significant increase in weight gain. At the moment there is no definitive information regarding whether or not this inhibition is developmental or time sensitive. Therefore, whether or not this weight gain side effect will affect those older women taking it for prevention is unknown.
Overall cancer research and therapy has come a long way in the last few decades, especially with regards to its functionality. Unfortunately while this additional knowledge has opened the door to new treatment options those options have yet to materialize into a cure. Fortunately promising attack avenues are still available and newer strategies are being explored each day. For example while VEGF neutralization strategies for angiogenesis may not be successful, focusing on the more overlooked TEMs than the TAMs could provide a better basis for attacking angiogenesis. Attacking BV8 may also prove to be useful not only as an anti-angiogenesis treatment, but also for anti-metastasis strategies.
Furthermore application of antibodies and/or nanomaterials to better target cancer cells could be aided by exploring RANKL inhibitory compounds which could reduce the potential of tumors to ‘hide’ from immune cells. CSCs could be attacked not directly through cytotoxic elements, but indirectly by BMP and its associated compounds to induce differentiation reducing tumor malignancy. Finally advances in diagnostic elements from PARE, miRNA screenings and microbubble ultrasounds to expansion of genetic information should improve diagnosis speed and associated survival rates. Overall the most important element to addressing cancer in the future is to continue to take positive steps forward and ensure that evidence and logic guide the way.
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