R&D 3.0: The Return of the Patient

For years, patients and their caregivers were mostly uninformed outsiders as pharmaceutical companies and research institutes developed new drugs and strategies for future growth.

Now, however, in order to increase brand awareness or accelerate predictive medicine development, more and more pharmaceutical and biotechnology companies realize the importance of patient engagement, starting at the pre-clinical R&D process all the way through clinical trials and the commercialization phase.

The Problem:

Despite all the ongoing efforts to engage patients, Big Pharma ranks near bottom in patient attitudes”. The FiercePharma article from January 18, 2013 briefly describes the outcome of a survey of 500 international, national, and regional patient groups, and their views of the different stages of the drug/device development as well as the paying and distribution process. As expected, high drug pricing was ranked on top of the list as the main cause for negative attitudes towards Big Pharma. The survey also indicates that patients found drug companies “too secretive, doing a poor job of letting patients know about adverse drug event news”.  Consequently, many patients have issues with the industry’s integrity.

describe the imageOverall, I think companies are slowly getting better in bridging the gap between industry and patients. As we all know, change – whether it needs to occur within a company or the mind of a patient – takes time. Gaining patients’ trust will be a long-term task, an infinite circle of strategizing, implementing, observing, and analyzing. Generally speaking, most companies could still need a healthy portion of empathy. If they really care about patients, they would need to develop a real understanding of patients’ or caregivers’ needs, fears, and reasoning of current attitudes.

The good news is: There are several ways to connect with patients on a deeper level.

Depending on a) purpose, b) stage of drug or biomarker development, and c) patient or, eventually, caregiver demographics, one has to develop appropriate recruitment/engagement strategies.

Below, I am outlining some of my thoughts that are based on my own experiences as well as reading many articles and blog posts. It is not a complete list of current status/issues/solutions, and is written to rather start a discussion.

Nevertheless, here are my 2 cents about current developments:

 

A) Pre-clinical Drug Development

The status quo:

For drug target/validation studies or biomarker development researchers oftentimes needdescribe the image larger quantities of patient samples (often, from hundreds of patients) in order to obtain statistically significant data. Hospital-based biobanks and, eventually, independent biobanks, are organizations that usually have direct access to patients. Although some US-based hospitals have established patient-friendly/engaging websites, those are not used to source samples “on demand”. As a result, it becomes quite time-consuming to source needed samples to start or continue certain research projects.

 

The Problem:

As Matt Jones, staff reporter for GenomeWeb Daily News, pointed out in his most recent article:

  • Two-thirds of the nation’s biobanks were established over the past decade – it is estimated that there are about 800 biobanks (with 90% embedded within institutions) within the US.
  • The increase of genomics and related large-scale studies have led biobanks to play an increasingly central part in biomedical R&D – genomic research appears as if it is a key driver in the “biobank explosion”.

In short, based on increasing demand, competition is increasing, and given the fact that sequencing costs are rapidly decreasing, one can imagine that this trend will continue. But as biobanks increase and specific patient populations stay the same (especially rare diseases), sample/data sourcing becomes a big issue.

Here is another issue to think about:

Matt Jones also wrote: Several issues that have been flagged by the National Human Genome Research Institute’s Ethical, Legal, and Social Implications Research program are stirred up by the expansion of biobanks, such as questions about policies governing data sharing and security, privacy and the identifiability of genomic information, how and when to return research results and incidental findings, how governance structures function at genomic repositories, and informed consent issues caused by the multiple uses for samples by genome researchers.”

In short, with all the data being generated, certain new regulations might “kick in” within the next few years. Privacy protection always was, and increasingly will be, a topic of many future discussions – especially after the Yaniv Ehrlich’s recent Science article: Identifying Personal Genomes by Surname Inference”. No matter what your attitude (as a researcher) towards these findings is, patients that become aware of these potential “threats” to privacy will increasingly fear breach of “protected” private data and therefore probably be less willing to donate and share their valuable biosamples and medical information. (If interested, further comments can be read in a New York Times article from January 17, 2013: Web Hunt for DNA Sequences Leaves Privacy Compromised”).

 

Why engage with patients?

describe the imageIt is necessary to connect and build trust. Real trust, however, can only be established through an open, genuine 2-way communication. Since there are already many articles written about this topic, I will not go too far into any details. However, patients/caregivers could potentially be engaged through social media (despite IRB regulations and/or oftentimes long turnaround times) and/or patient portals: for instance, at the biobank Sanguine Biosciences’ patient blog, patients are writing about their experiences, their fears and how they overcame those. At Sanguine, we realize that it is not only important to educate (potential) customers (as Sanguine does too: here is our researcher blog) but even more important to actually work with patients to learn about and educate them – learning, listening and educating needs to go both ways.

Furthermore, increased transparency is another way to develop trust. In the near future, Sanguine Biosciences will notify patients about the impact their samples made by informing them about where they have been sent to and/or for what purpose they were used for.  For instance: “Today, we sent some of your serum and CSF to a pharmaceutical company located on the East Coast. This company is seeking to develop a new biomarker that will help detect Alzheimer’s disease at a very early stage. We thank you for helping us accelerate research for Alzheimer’s!”

Sure, some companies or research institutes will not allow us to communicate any information at all; however, others are very interested in working with us to help them bridge the company-patient-gap.

Additional engagement strategies include partnerships/collaborations with non-profit organizations to provide patients the right education and to learn first-hand about the difficulties patients are dealing with on a daily basis.  Engaged patients are more likely to share their experiences with others.  This leads to a word-of-mouth campaign, which can help increase participation in research studies.

Ultimately, if there is already an existing hospital/biobank-patients-bond, sourcing samples and data will be faster, and less costly, and longitudinal collection studies will become easier.  Recruiting donors for clinical trials (i.e. based on their genetic profile) will become easier too.

 

B) Clinical Trials Phase

describe the imageOftentimes, pharmas, CROs, clinics/hospitals are faced with relatively high patient recruitment costs (about $2000/patient) and/or lengthy recruitment periods. Having engaged patients already “on-hand” might therefore lead to reduced recruitment cost and time. Besides, and as mentioned above, a 2-way dialogue (as much as possible) will help companies build better reputations.

The Pfizer-way:

As a former Pfizer R&D executive LaMattina suggested, big pharma should consider providing more transparency regarding payments to physicians and data derived from clinical trials. In addition, LaMattina advised “to stop pushing drugs for unapproved uses and give up television advertising”. Despite some strong criticism, several pharmaceutical companies already started following these recommendations.

However, regulations will always be a barrier for an open 2-way dialogue. As Todd Kolm, Director, Emerging Channel Strategy at Pfizer pointed out: “Because of the regulatory environment, we’re not able to engage in a true dialogue”.

Furthermore, what makes patient engagement even more difficult is the relatively poor understanding of new media channels, a lack of FDA guidance, a lack of pharmaceutical executives promoting it, the scarcity of demonstrated success stories, the often inadequate resources allocated to new patient engagement strategies, and the simple fact that it is often not really perceived as a must-have, value-added strategy.

Nonetheless, Pfizer’s REMOTE (Research on Electronic Monitoring of OAB (overactive bladder) Treatment Experience) trial proved that through innovative thinking, courage and implementation of completely new strategies, hurdles can slowly be overcome.

REMOTE, which ended in mid-2012, was the first randomized virtual clinical trial conducted under an IND application.

The main problem, however, was recruiting the right patients needed for the study. This was mainly caused by being very regulatory-cautious so no wrong patients would enter the virtual trial. Ultimately, it became very difficult for patients to actually get into the study.

To quote Craig Lipset, Worldwide Head of Clinical Innovation at Pfizer: “As an industry, we will continue to fail to recruit patients in our studies if we cannot create an ecosystem of patients already engaged and aware about research studies and research participation”.

The good news: Having a web-based recruiting site simplified patient recruitment (including consenting) and allowed patients to participate from their own home – web-based and mobile platforms were used to capture self-reported data from those patients. In addition, primary care physicians engaged with patients by screening and caring for them during the trial. Furthermore, patients received their data at the end of the trial period and were able to even share those with their physician.

 

C) Post-approval/Commercialization Phase

Pharmaceutical biotechnology companies are slowly becoming more patient-centric. Belgium-based UCB, for example, developed a very patient-focused website, engaging patients through education and disease-specific patient-support programs.

Other companies are also trying to bridge the gap to their patients. Major firms such as Biogen Idec , Pfizer, and French pharmaceutical giant Sanofi, the parent of Genzyme, are partnering on a number of initiatives with patient groups such as The Michael J. Fox Foundation for Parkinson’s Research.

 

Taken together, it is key to provide top-quality, objective, reliable, unbiased education, training and information to patient organizations and patients at large on all aspects of R&D, in order for patients to get involved and become empowered players in the medical drug development process.

Companies that stay on top of this and develop new strategies will ultimately create the necessary paradigm shift, making sure that patients’ needs and insights are at the center in all relevant areas of medical R&D.

 

Further Reading:

Big Pharma ranks near bottom in patient attitudes – FiercePharma  http://www.fiercepharma.com/story/big-pharma-ranks-near-bottom-patient-attitudes/2013-01-18#ixzz2JEbcBxOV

US Sees Boom in Diverse Range of Biobanks, But Regulations are Lacking – GEN http://www.genomeweb.com/us-sees-boom-diverse-range-biobanks-regulations-are-lacking

Identifying Personal Genomes by Surname Inference – Science http://www.sciencemag.org/content/339/6117/321.abstract

Web Hunt for DNA Sequences Leaves Privacy Compromised – New York Times  http://www.nytimes.com/2013/01/18/health/search-of-dna-sequences-reveals-full-identities.html?pagewanted=1&_r=0

Between patients and pharmas online, a disconnect – Medical Marketing & Media  http://www.mmm-online.com/between-patients-and-pharmas-online-a-disconnect/article/260723/

Pfizer Perseveres In Pioneering Virtual Clinical Trials – Life Science Leader  http://lifescienceleader.com/blogs/contributing-editors-2/item/4363-pfizer-perseveres-in-pioneering-virtual-clinical-trials

New role for p53 in regulating the inflammatory tumor microenvironment

small 3316209998p53 (TP53), a transcription factor activated by various types of cellular stress, is one of the most well studied tumor suppressor proteins.  Stress signals such as various types of DNA damage lead to p53 activation and subsequent induction of genes involved in cell cycle arrest, DNA repair, and apoptosis.

In many types of cancers including colorectal cancer, loss of function mutations in p53 commonly occur, particularly in the DNA-binding domain.  Thus, transcriptional activity of p53 is altered or lost, and cells with damaged genomes maintain viability.  In colorectal cancer, p53 mutations commonly occur at early stages and are thought to allow for accumulation of DNA damage and survival of mutated cells during tumorigenesis.

In the January issue of Cancer Cell, Schwitalla et. al, demonstrate that p53 plays a role in not only its classically associated functions as a critical DNA-damage checkpoint in early stages of tumorigenesis, but during the progression stage, p53 plays an additional significant role in regulating the inflammatory tumor microenvironment.

To study the role of p53 during various stages of carcinogenesis, the authors established a colon tumor model in which azoxymethane (AOM), a promoter of Wnt-driven tumorigenesis that induces stabilizing mutations in β-catenin, was administered to mice with a p53-deficiency in intestinal epithelial cells.  A gene expression analysis of AOM-derived tumors from wild-type versus p53-deficient mice, found that p53 deficiency led to upregulation of genes involved in inflammation and epithelial-mesenchymal transition (EMT).  A large number of genes upregulated in p53-deficient tumors were found to be regulated by the inflammatory transcription factor NF-ĸB, and phosphorylated p65, a major transcriptional co-factor in the classical NF-ĸB pathway, was enhanced in p53-deficient tumors.  NF-ĸB regulated genes included the EMT gene Twist as well as myeloid cell chemokines CXCL1, CXCL2, and CCL2, indicating that loss of p53 may lead to EMT and recruitment of inflammatory myeloid cells via upregulation of the NF-ĸB pathway.

Reciprocal regulation of the NF-ĸB and p53 pathways has long been known, and these pathways play opposing roles in cell fate in response to stress. However, in this study, the authors demonstrated that p53-deficient intestinal epithelial cells were impaired in their epithelial barrier function: leakage of intestinal bacteria and LPS resulted in the observed NF-ĸB activation and NF-ĸB-regulated gene expression.  Interestingly, NF-ĸB signaling was required in both tumor cells and the myeloid cells for maximal tumor progression.  NF-ĸB signaling in intestinal epithelial cells was required for recruitment of myeloid cells to the tumor microenvironment and EMT induction. NF-ĸB signaling in hematopoietic cells was required for tumor cell proliferation, survival, and metastasis, by inducing expression of cytokines that activate STAT3 in the tumor cells.

Thus, in this model, p53 plays an additional role during tumor progression by regulating inflammation in the tumor microenvironment, via its effects on intestinal barrier function.  In my last blog post, I discussed another recent study demonstrating links between the TNFα/NF-ĸB, IL-6/STAT3, and SphK1/S1P pathways in chronic inflammation driven colorectal cancer.  Thus, this study adds another layer of links to consider in the complicated interactions between inflammatory cells and tumor cells in the tumor microenvironment that drive colorectal cancer progression: p53, NF-ĸB, and STAT3.

 

Further Reading:

Loss of p53 in Enterocytes Generates an Inflammatory Microenvironment Enabling Invasion and Lymph Node Metastasis of Carcinogen-Induced Colorectal Tumors.  Schwitalla S, Ziegler PK, Horst D, Becker V, Kerle I, Begus-Nahrmann Y, Lechel A, Rudolph KL, Langer R, Slotta-Huspenina J, Bader FG, Prazeres da Costa O, Neurath MF, Meining A, Kirchner T, Greten FR. Cancer Cell. 2013 Jan 14;23(1):93-106.

p53 and NF-κB: different strategies for responding to stress lead to a functional antagonism.  Ak P, Levine AJ. FASEB J. 2010 Oct;24(10):3643-52.

Alterations of the TP53 gene in gastric and esophageal carcinogenesis.  Bellini MF, Cadamuro AC, Succi M, Proença MA, Silva AE. J Biomed Biotechnol. 2012;2012:891961.

Transcriptional control of human p53-regulated genes. T. Riley, E. Sontag, P. Chen, A. Levine.  Nat. Rev. Mol. Cell Biol., 9 (2008), pp. 402–412.

Immunity, inflammation, and cancer.  S.I. Grivennikov, F.R. Greten, M. Karin.  Cell, 140 (2010), pp. 883–899.

photo credit: sc63 via photopin cc

Mesenchymal Stem Cells in the Brain

Mesenchymal stem cells (MSCs) are multipotent cells present in, and can be isolated from a variety of adult tissues, such as bone marrow, umbilical cord blood or adipose tissue. MSCs have a number of advantageous characteristics that have made them a promising candidate for use in the new generation of cell-replacement therapy (CRT), even for central nervous system (CNS) disorders. Major obstacles to CRT in CNS disorders include successful delivery of therapeutic/stem cells to the damaged area/lesions within the CNS, host’s immune response against the allogenic cells and the possibility of ectopic tissue formation. MSCs exhibit unique characteristics which can overcome these obstacles, such as MSCs’ capacity to differentiate into multiple tissue-specific lineage cells, their role as progenitor-cell bioreactors of soluble factors that promote tissue regeneration from the damaged tissue and their immunomodulation capacity. Moreover, MSCs are considered immunoprivileged, meaning they have low expression of class II Major Histocompatibilty Complex (MHC-II) and other immune-stimulatory molecules on their cell surface. The focus of this discussion is on the utilization of MSCs in CNS-disease therapy, in particular multiple sclerosis (MS) and ischemic stroke (IS) .

Bone marrow derived (BM-MSCs) and adipose derived mesenchymal stem cells (AD-MSCs) have shown promising efficacy in an experimental autoimmune encephalomyelitis (EAE) preclinical model of MS, as well as the permanent middle cerebral artery occlusion (pMCAO) model of IS, respectively.

IS occurs as a result of an obstruction within a blood vessel supplying blood to a particular region of the brain, causing neuronal and astroglial damage within the immediate region. Replacement of these cells along with repairing the damaged tissue has been of great interest, turning IS clinical research towards stem cell therapy.

Stroke,stem cell therapy,regenerative therapy,cell replacement,CNS

According to the recent study conducted by Gutierrez-Fernandez’s group, AD-MSCs are as restorative as BM-MSC in promoting recovery, repair and brain protection in IS rat models. They showed significant functional recovery, decreased apoptosis and increased expression of neurogenesis, synaptogenesis, angiogenesis and oligodendrogenesis markers, following the intravenous administration of allogenic AD-MSC and BM-MSC. Although these results suggest a less invasive route for administration of therapeutic cells, further studies addressing the fate of the administered cells are required, prior to clinical consideration of this method.

MS is a chronic, immune-mediated demyelinating disease of the CNS, characterized by demyelinated plaques within the brain and spinal cord. MS plaque formation consists of immune-cell infiltration, damage to oligodendrocytes and their subsequent failure to remyelinate, degeneration of axons, and ultimately astrocytosis. Up to date, there is no cure for MS; the current disease modifying therapies utilized to reduce the frequency/severity of relapses are limited to immunomodulatoion and are only partially effective. Thus, MS researchers have turned their attention to discovering potential therapeutics that will not only stop the autoimmune attacks, but also replace the destroyed CNS cells with properly functioning ones, through CRT.

Nonetheless, several recent studies have reported promising results regarding autologous, culture-expanded MSC transplantation in MS models. Based on Harris’s publication in Stem Cells Translational Medicine, intrathecal delivery of Bone marrow mesenchymal stem cell-derived neural progenitors (MSC-NPs) is a promising strategy for cell-based therapy in MS. They show that MSC-NPs derived from both, MS patients and healthy controls, uniformly displayed properties that support MSCs’ therapeutic potential in the CNS, regardless of the donor disease status. Like MSCs, MSC-NPs secrete immunomodulatory factors (such as cytokines and growth factors, including TGF-β, IL-6, IL-10, HGF, heme oxygenase-1, and nitric oxide) which inhibited T-cell proliferation and promoted naïve CD4+ T-cell polarization into FoxP3+ T cells. MSC-NPs also exhibit trophic effects similar to MSCs, by secreting trophic factors that promote oligodendroglial differentiation of neural stem cells (HGF, IGF-1, SDF1α, and VEGF). Furthermore, it was reported that MSC-NPs are neuroectodermally committed and have reduced capacity for mesodermal differentiation (reduced risk of abnormal tissue formation), which makes them a more suitable candidate for cell-based therapy in CNS injury.

Stem Cell Therapy,Multiple sclerosis,MS,Cell replacement therapy

One of the significant aspects of this study is the possibility of utilizing MS-patient’s own cells as the therapeutic cell source; not only because of the reduced immune response, but also due to the evidence of genetic stability of the adult stem cells present in these patients. This suggests several promising genetic and cellular therapeutic strategies to be investigated in the near future.

Further readings:

Characterization of autologous mesenchymal stem cell-derived neural progenitors as a feasible source of stemcells for central nervous system applications in multiple sclerosis.

Effects of intravenous administration of allogenic bone marrow- and adipose

tissue-derived mesenchymal stem cells on functional recovery and brain repair
markers in experimental ischemic stroke

Mesenchymal stem cell transplantation in multiple sclerosis.

Immunosuppressive properties of mesenchymal stem cells: advances and applications.

Cancer Stem Cell Hypothesis: Proceed with Caution

CSC picture

In 1937, the cancer stem cell hypothesis was proposed to explain the concept of tumor heterogeneity (Clevers, 2011). In the mid 1990s, alongside the boom of stem cell biology, the theory that subpopulations of leukemia with stem cell-like properties was reintroduced with seminal work from John Dick (Clevers, 2011). These subpopulations were named “cancer stem cells” (although many today prefer the term “tumor-initiating cells”) due to their tumorigenicity and apparent self-renewal, thus mimicking the adult stem cell properties of multipotency and self-renewal. Today, cancer stem cell populations have been identified for cancers of the brain, pancreas, ovary, colon, liver, as well as leukemia (Magee et al, 2012). However, while confirmation of tumor-initiating cells in all tumors cannot be proven (Magee et al, 2012), the study of cancer stem cells remains important due to its possible impact on current cancer therapy.

In many tumors, cancer cell subpopulations are believed by many to be resistant to chemotherapy and radiation therapy (Clevers, 2011) (Magee et al, 2012). The importance becomes clearer when one looks at the possible outcome of not taking into account targeting cancer stem cells when developing cancer therapy. Computer simulations have shown that use of therapy that only targets non-tumorigenic cancer cells would enrich for the tumorigenic tumor-initiating cells, exacerbating the malignancy of many cancers (Vermeulen et al, 2012). This would explain why many cancers are more malignant after treatment with current therapy. In addition, the current intricacy of dealing with heterogeneity of the tumor is an issue since many different cancerous cell types respond differently to current therapies (Vermeulen et al, 2012) thus making ideal therapy difficult.

The cancer stem cell hypothesis is not the only theory to be brought forth to explain tumor heterogeneity.  One belief is in the stochastic model of tumor heterogeneity, where variances in genetics and epigenetics cause the heterogeneity of tumor. Due to selection of more robust subpopulations, clonal evolution causes cell populations to proliferative non-uniformly in a tumor (Magee et al, 2012). Another proposed theory of tumor heterogeneity is the belief in the variation of extrinsic factors caused by the changes in the tumor microenvironment (Magee et al, 2012). In this model, cells that are closer to areas, such as the vasculature, form a niche that change the properties of tumor cells in a temporary, or permanent, manner (Magee et al, 2012). The cancer stem cell model states that a distinct subset of a tumor is tumorigenic and has the ability to self-renew (form more tumorigenic cells) or differentiate into the bulk of the non-tumorigenic cells of the tumor (Magee et al, 2012).  Within the tumor-initiating cell community, there has been increasing support for a non-mutually exclusive model which has a combination of the hypotheses listed above that may contribute to tumor heterogeneity (Clevers, 2011)(Magee et al, 2012). One must account for all of these factors as possibilities when studying populations that may be tumorigenic inside a tumor model. For instance, attempting to study a certain population of ovarian cancer stem cells in vitro does not recapitulate the microenvironment and may negatively affect observation outcomes.

There have been many proposed mechanisms for identifying and studying cancer stem cells. These include the isolation of specific surface marker phenotypes, the use of cultures that are thought to favor the clonogenicity of the cancer stem cell population (such as the sphere forming cultures), serial transplantations of certain populations into immunocompromised mice to check tumorigenicity, and microscopic analysis of tumor heterogeneity through markers. However, there are limitations and caveats that one must consider when using these techniques to study cancer stem cell biology. First, studies have indicated that the cancer stem cell phenotype may be a context-specific event, showing up only in certain patient samples at certain ages (Magee et al, 2012). Furthermore, it is still unknown whether non-tumorigenic cells may become (through spontaneous formation or de-differentiation) tumorigenic cancer stem cells.  This phenotypic plasticity calls into question the isolation of certain populations and the validity of ex-post facto tumor heterogeneity since confirmatory data to the initial isolation of a cancer stem cell population and subsequent studies would be lacking.

Therefore, if one were to study cancer stem cells, the key is to not rely too heavily on one assay, but to interweave all of the assays for bona fide tumor-initiating cell experimentation.  For instance, one should study different populations of brain tumors, keeping in mind the limitations of their results, and be able to recapitulate their findings in a properly formed sphere formation assay as well as in an in vivo limited dilution model of tumorgenecity.  Even to this end, the expression of cancer stem cells, both in number and property, may be extremely patient specific and rigorous testing of individual cases must be performed before any basic-science concepts are used for treatment.

Things to keep in mind when studying cancer stem cells:

  • Although controversial, Cell surface markers have been correlated with a cancer stem cell phenotype, these include:
  • Glioma: CD133, SSEA1, CD49f, Musashi-1, and Nestin
  • Breast: BMI-1, CD44, CD24, CD49f, ALDHA1, and EpCAM
  • Lung: ALDHA1, CD90, CD117, and EpCAM
  • Upregulation of certain stem cell associated genes, such as Nestin, Oct4, Sox2, Nanog, Mushashi1, Notch1, and Notch4 have also been traditionally used to identify cancer stem cell subpopulations
  • Multiple primary tumors tend to be better specimens to study compared to immortalized cancer cell lines, which have undergone many mutations through passages that may affect the representative phenotype of tumor-initiating cells.
  • Many labs studying cancer stem cells agree that lineage tracing and side-by-side fate mapping of tumor subpopulations is essential for proper tumor-initiating cell studies.
  • Single-cell, serial transplantation into immunocompromised mice, if feasible in your system, is an adequate assay to test for cancer stem cell phenotype. However, there are still possible issues of minor immunoediting in immunocompromised mice. If one is dealing with murine specimens, the use of a syngeneic mouse line may limit this issue.
  • The possibility of quiescent cancer stem cells must be taken into account
    • This can be studied looking at the cell population of interest and performing a western blot analysis on stem cell associated proteins (such as Sox2, Nestin, Oct 4, Nanog, etc.) with cell proliferation markers (increase of cell cycle regulators, such as p21, Cyclin D2, TP53 and a downregulation of cell cyclin proliferative markers such as Cyclin B1, cdc20, and Myc)(Moore and Lyle, 2011).
    • Use label-retention/chase experiments, such as tritiated thymidine (3H-TdR) or 5-bromo-2-deoxy-uridine (BrdU), on your cell of interest is also a good technique and an in vivo alternative a (Moore and Lyle, 2011).
  • Genetically engineered mouse models that spontaneously form tumors are tools that allow for the study of tumor-initiating cells while controlling for most artificial biases seen in engraftment of xenospecies cells and/or high-passage cancer cells. There is some concern whether artificial plastic culture conditions may affect the in vitro study of cell populations due to the lack of mechanical sensitivity found in the actual tumor microenvironment.  This may be controlled for by assaying clonal analysis on a 3D scaffold system that is representative of the primary location of the tumor (Pastrana et al, 2011).
    • Sphere formation assays may select against tumor initiating cells that do not form spheres (Read  and Wechsler-Reya,  2012).

Suggested Reading/ References:

The cancer stem cell: premises, promises, and challenges. Clevers H. Nature Medicine 2011 Mar 7:17(3).

Cancer Stem Cells: Impact, Heterogeneity, and Uncertainty. Magee JA, Piskounova E, Morrison SJ. Cancer Cell 2012 Mar 20:(21).

The developing cancer stem-cell model: clinical challenges and opportunities. Vermeulen L, De Sousa e Melo F, Richel DJ, Medema JP. Lancet Oncology Feb: (13):e83-89.

Quiescent, Slow –Cycling Stem Cell Populations in Cancer: A Review of Evidence and Discussion of Significance. Moore N and Lyle S. Journal of Oncology, 2011.

Spheres without Influence: Dissociationg In Vitro Self-Renewal from Tumorigenic Potential in Glioma. Read TA, Wechsler-Reya RJ. Cancer Cell, 2012 Jan 17: (21).

Eyes Wide Open: A Critical Review of Sphere-Formation as an Assay for Stem Cells. Pastrana E, Silva-Vargas V, Doetsch F. Cell Stem Cell May6:(8).

Signal amplification loops in chronic inflammation and Colon Cancer

The inflammatory gastrointestinal diseases Crohn’s disease, inflammatory bowel diseases (IBD), and ulcerative colitis (UC), are all known to be key risk factors in the development of colorectal cancer.  TNFα and IL-6 are two pro-inflammatory cytokines that signal to activate the NF-ĸB and STAT3 transcription factors, respectively.  Both pathways are known to play a role in the persistent inflammation that drives development of colitis-associated colorectal cancer.

Sphingosine-1-phosphate (S1P) is a lysophospholipid that is metabolized from phosphorylation of sphingosine by the kinases, SphK1 and SphK2, and signals through the G-protein coupled receptor sphingosine-1-phosphate receptor-1 (S1PR1).  The S1P-S1PR1 signaling pathway has been shown to activate STAT3 and induce expression of both IL-6 and S1PR1, driving a positive feedback loop leading to persistent STAT3 activation in tumor cells (Lee et. al., 2010).  S1P and SphK1 have also been shown to play a critical role in the NF-ĸB signaling pathway.  S1P was required for TRAF2 E3 ubiquitin ligase activity and activation of NF-ĸB by TNFα independently from the S1PR1 receptor pathway (Alvarez et. al., 2010).  SphK1 has also been linked to development of colorectal cancer.  SphK1 was highly expressed in human colons cancers as compared with normal mucosa, and SphK1-/- mice were less susceptible to azoxymethane induction of colon cancer (Kawamori et.al., 2009).

In a study by Liang et al. in this month’s issue of Cancer Cell, the authors close these links between the TNFα/NF-ĸB, IL-6/STAT3, and SphK1/S1P pathways in chronic inflammation driven colorectal cancer.  In the dextran sodium sulfate (DSS) murine model of colitis driven tumorigenesis, deficiency of SphK2 led to a compensatory upregulation of SphK1 and S1P production, and exacerbated DSS driven colitis and subsequent tumorigenesis.  S1P-driven NF-ĸB activation was enhanced, as was production of TNFα, IL-6, and STAT3 activation in SphK2-/- mice, and the SphK1/S1P pathway primarily exerted its activities in colonic mucosa infiltrating immune cells.  FTY720, an antagonist of S1PR1 and SphK1, was able to inhibit the S1P positive-feedback loop and block persistent activation of STAT3 and NF-ĸB and production of IL-6.  Additionally, FTY720 inhibited the development of tumors in this colitis driven tumorigenesis murine model.

In summary, these studies show that SphK1 drives a feed-forward signal amplification loop that exacerbates inflammation and promotes tumorigenesis by production of S1P.  S1P then drives S1PR1 activation of STAT3 leading to further production of S1PR1, as well as activates NF-ĸB and induction of TNFα and IL-6 transcription, which further feed back on the NF-ĸB and STAT3 pathways, respectively.  Blocking this amplification loop by inhibiting S1PR1 and SphK1 may thus be a promising treatment strategy for colitis-associated colorectal cancer.

 

 

Further Reading:

Sphingosine-1-Phosphate Links Persistent STAT3 Activation, Chronic Intestinal Inflammation, and Development of Colitis-Associated Cancer.  Liang J, Nagahashi M, Kim EY, Harikumar KB, Yamada A, Huang WC, Hait NC, Allegood JC, Price MM, Avni D, Takabe K, Kordula T, Milstien S, Spiegel S. Cancer Cell. 2013 Jan 14;23(1):107-20.

Sphingosine 1-Phosphate Is a Missing Link between Chronic Inflammation and Colon Cancer.  Pyne NJ, Pyne S. Cancer Cell. 2013 Jan 14;23(1):5-7.

STAT3-induced S1PR1 expression is crucial for persistent STAT3 activation in tumors.  H. Lee H, Deng J, Kujawski M, Yang C, Liu Y, Herrmann A, Kortylewski M, Horne D, Somlo G, Forman S, Jove R, Yu H.. Nat. Med., 16 (2010), pp. 1421–1428.

Sphingosine-1-phosphate is a missing cofactor for the E3 ubiquitin ligase TRAF2.  Alvarez SE, Harikumar KB, Hait NC, Allegood J, Strub GM, Kim EY, Maceyka M, Jiang H, Luo C, Kordula T, Milstien S, Spiegel S. Nature. 2010 Jun 24;465(7301):1084-8.

Role for sphingosine kinase 1 in colon carcinogenesis. T. Kawamori, T. Kaneshiro, M. Okumura, S. Maalouf, A. Uflacker, J. Bielawski, Y.A. Hannun, L.M. Obeid.  FASEB J., 23 (2009), pp. 405–414.

Sphingosine 1-phosphate and its receptors: an autocrine and paracrine network.  Rosen H. and Goetzl EJ.   Nature Reviews Immunology 5, 560-570, July 2005.

 Immunity, inflammation, and cancer.  S.I. Grivennikov, F.R. Greten, M. Karin.  Cell, 140 (2010), pp. 883–899.

Intestinal inflammation and cancer.  T.A. Ullman, S.H. Itzkowitz.  Gastroenterology, 140 (2011), pp. 1807–1816.

Weird but common T cell populations in human PBMC

Human T cells are generally analyzed for expression of CD4 and CD8 to classify them as either of these two major classes of effector T cells.  But flow cytometry analysis of PBMCs stained with antibodies targeting CD3, CD4, and CD8 reveals several other populations with varying expression of these three markers.  So what are they?

For the sake of this discussion, I will refer to the largest typical single positive populations of CD3+CD4+ and CD3+CD8+ T cells as CD4high and CD8 high, respectively.

These are some other T cell populations that have been observed in human PBMC:

CD8lowCD4high (1): Populations of CD4 T cells that express CD8.  This population is likely heterogeneous, and compared with the CD4high population, includes a higher proportion of effector memory and terminally differentiated effector CD4 T cells that have re-expressed CD8.  CD8 is expressed as a heterodimer of either α/α, α/β, or β/β, and this population has been noted to be primarily CD8α/α.  Work by Zloza et. al, identified that up to 50% of these cells can be NKT cells, including invariant CD3+6B11+ NKT cells and non-invariant CD3+CD16/56+ NKT cells.  Of note, NKT cells may also be present at low frequencies in CD4+CD8+, CD4CD8, CD4 or CD8 single positive populations.

CD4lowCD8high (2): Populations of CD8+ T cells, of the primarily CD8α/β type that express CD4.   This population can be further subdivided into two groups:  CD4dimCD8high and CD4medCD8high.  Studies have shown that expression of CD4 on these CD8+ T cells is functional and inducible by stimulations such as anti-CD3/CD28.   These cells express markers of activated T cells and exhibit a higher frequency of memory cells (CD45RA) as compared with typical CD8high cells.

CD8low (3): These cells express CD8 at lower levels compared with CD8 high populations, are negative for CD4 expression, and can express higher levels of CD3.  Trautmann et. al, describe the frequency of CD8 low cells as being from 0.2%-7% of CD8 T cells in healthy donors and described these cells as populations of oligoclonal cytotoxic terminally differentiated effector CD8 T cells (CD45RA+CD62L).

WeirdCD3CD4CD8 Tcell Populations resized 600

CD4neg CD8neg CD3high (4):  CD4 and CD8 double negative cells that express high levels of CD3 compared with CD4 high and CD8 high populations.  This fraction has been shown to contain largely the TCRγ/δ T cell subset although γ/δ T cells can express and the CD8α and/or the CD8β chains.

CD4neg CD8neg CD3pos (5): This fraction has been shown to largely contain heterogeneously differentiated TCRα/β T cell subsets including regulatory T cells.  The expression of CD3 on this subset is lower than that of the CD4neg CD8neg CD3high subset containing γ/δ T cells, although γ/δ T cells may be present in this population as well.

An important thing to note is that characterizations of these populations are generalizations and individuals have been shown to have aberrant profiles compared with these.  Other populations have been described such as CD4high CD8high double positive cells which may be primarily effector memory T cells but here I have focused on those populations I see most frequently.   In summary, careful gating and analyses of each of these populations is necessary, as these are not only functionally unique subsets, but each population appears to be heterogeneous and also contain varying percentages of NKT cells.

 

Further Reading:

CD4(+)CD8(dim) T lymphocytes exhibit enhanced cytokine expression, proliferation and cytotoxic activity in response to HCMV and HIV-1 antigens.  Suni MA, Ghanekar SA, Houck DW, Maecker HT, Wormsley SB, Picker LJ, Moss RB, Maino VC. Eur J Immunol. 2001 Aug;31(8):2512-20.

Multiple populations of T lymphocytes are distinguished by the level of CD4 and CD8 coexpression and require individual consideration.  Zloza A. and Al-Harthi, L. Journal of Leukocyte BiologyJ Leukoc Biol. 2006 Jan;79(1):4-6.

Characterization of circulating CD4+ CD8+ lymphocytes in healthy individuals prompted by identification of a blood donor with a markedly elevated level of CD4+ CD8+ lymphocytes.  Prince HE, Golding J, York J. Clin Diagn Lab Immunol. 1994 Sep;1(5):597-605.

Upregulation of CD4 on CD8+ T cells: CD4dimCD8bright T cells constitute an activated phenotype of CD8+ T cells. Sullivan YB, Landay AL, Zack JA, Kitchen SG, Al-Harthi L. Immunology. 2001;103: 270-280.

Human CD8 T cells of the peripheral blood contain a low CD8 expressing cytotoxic/effector subpopulation.  Trautmann A, Rückert B, Schmid-Grendelmeier P, Niederer E, Bröcker EB, Blaser K, Akdis CA. Immunology. 2003 Mar;108(3):305-12.

CD3 bright lymphocyte population reveal gammadelta T cells.  Lambert C, Genin C. Cytometry B Clin Cytom. 2004 Sep;61(1):45-53.

Isolation and characterization of human antigen-specific TCR alpha beta+ CD4(-)CD8- double-negative regulatory T cells.  Fischer K, Voelkl S, Heymann J, Przybylski GK, Mondal K, Laumer M, Kunz-Schughart L, Schmidt CA, Andreesen R, Mackensen A. Blood. 2005 Apr 1;105(7):2828-35.

Distinct CD4+ CD8+ double-positive T cells in the blood and liver of patients during chronic hepatitis B and C. Nascimbeni M, Pol S, Saunier B. PLoS One. 2011;6(5):e20145.

CD4+ CD8+ double positive (DP) T cells in health and disease.  Parel Y, Chizzolini C. Autoimmun Rev. 2004 Mar;3(3):215-20.

In vitro modeling of hematopoiesis: from pluripotency to blood

Pluripotent stem cells (PSCs) derived from the inner part of a blastocyst (embryonic stem cells, ESCs) or through reprogramming of terminally differentiated adult cells (induced pluripotent stem cells, iPSCs) are capable of self-renewal and differentiation into almost all cell types in the human body. Their differentiation capacities and proliferation potential make pluripotent stem cells a promising source of cells for various clinical applications including regenerative medicine.

fetal red blood cells resized 600Blood is considered to be a connective tissue both functionally and embryologically. It originates from the mesodermal layer, the same germ layer that gives rise to the other connective tissues such as bone, cartilage and muscle. Blood cells and blood vessels develop in parallel and form a functional circulatory system. Various studies have shown that hematopoietic differentiation of PSCs in vitro closely resembles early steps of blood development in the embryo and induces blood forming cell populations with mesodermal and hemato-endothelial properties [1]. Different types of mature blood cells were successfully generated from murine, primate and human pluripotent stem cells. Here, we will briefly review the major in vitro systems of hematopoietic differentiation from PSCs.

Embryoid Body formation

Hematopoietic differentiation of PSCs can be carried out in either a two-dimensional system (2D), where cells are attached to the plate during differentiation, or in a three-dimensional system (3D), where isolated cells are dispersed into a liquid or a semisolid medium to form embryoid bodies (EBs).

Embryoid bodies are spherical structures that are formed by embryonic bodies resized 600pluripotent stem cells grown in non-adherent culture conditions (3D system). Differentiation of PSCs in aggregates mimics three-dimensional embryonic development and yields the establishment of cell adhesion, paracrine signaling and a microenvironment similar to native tissue structures. Thus, EB formation is often used as a method for initiating spontaneous differentiation of PSCs towards all three germ lines.

Differentiation in the presence of growth factors specific for mesoderm (BMP4, FGF, activin A) and blood formation (VEGF, SCF, Flt3, IL-3, IL-6, G-SCF, TPO) promotes hematopoiesis within embryoid aggregates and may result in the appearance of tissue-like structures such as blood islands and early blood vessels. The combination of BMP4 with hematopoietic cytokines yields up to 20% of CD34+CD45+ cells that will give rise to erythroid, macrophage, granulocytic and megakaryocytic colonies [2].

To produce EBs of equal size and standardize differentiation, a certain number of cells can be used to form aggregates by a spin technique (centrifugation) or in a hanging drops method. Hanging drops are single 10-20μl droplets with known cell densities that are placed on a glass surface or into hanging drop plates. Several studies have shown improvements of this method that would allow it in practical application.

Coculture with stromal cells

This two-dimensional differentiation system is based on induction of hematopoiesis upon exposure to extrinsic signals from the feeder cells that underlie the PSCs in coculture. Stromal cells with the capacity to induce and support hematopoiesis can be isolated from a variety of anatomical sites associated with the hematopoietic development in vivo. A number of cell lines were established from mouse bone marrow (OP9, MS5 and S17), yolk sac endothelium (C166), fetal liver (mFLSC, EL08) and other sources. The genetically modified stromal cells, immortalized or expressing specific growth factors and signaling molecules, are widely used in hematopoietic coculture.

The standard coculture conditions comprise prolonged, up to 4 weeks, incubation of undifferentiated pluripotent stem cells on top of the stromal cells in the presence of fetal bovine serum (FBS) and/or hematopoietic cytokines. Both mouse and human pluripotent cells can be successfully differentiated into CD34+ multi-lineage blood progenitors in a coculture, though the efficiency of hematopoietic differentiation significantly varies between different stromal cell lines and compositions of differentiation media [3].

Defined feeder-free, serum-free systems

These systems are designed to avoid the use of undefined, animal-origin components such as FBS and stroma cells to achieve highly reproducible and efficient outputs. Thus, PSCs can be plated on matrix protein collagen IV and differentiated into primitive CD34+CD43+ hematopoietic progenitors by exposure to BMP4, bFGF and VEGF. This initial differentiation is more efficient when accompanied with the hypoxic conditions (5% oxygen tension) that resemble the environment of a developing embryo. A further incubation of blood progenitors with the various combinations of cytokines yields maturation of CD71+CD235a+ erythroid cells, CD41a+ CD42b+ megakaryocytes, HLA-DR+CD1a+ dendritic cells, CD14+CD68+ macrophages, CD45+CD117+ mast cells and CD15+CD66+ neutrophils [4].

Despite the great progress achieved in the in vitro modeling of hematopoiesis, blood production from PSCs is still a variable process.  The final goal of intensive research in this area – a consistent production of engraftable cells, capable of reconstituting all blood lineages in the body, remains a major challenge. Finding critical intrinsic and extrinsic factors that can recreate the unique properties of a hematopoietic stem cell niche in vitro could advance the generation and expansion of PSCs-derived hematopoietic stem cells in the future.

 

References:

1. Moreno-Gimeno I, Ledran MH, Lako M. Hematopoietic differentiation from human ESCs as a model for developmental studies and future clinical translations. FEBS J. 2010 Dec;277(24):5014-25.Review.

2. Chadwick K, Wang L, Li L, Menendez P, Murdoch B, Rouleau A, Bhatia M.Cytokines and BMP-4 promote hematopoietic differentiation of human embryonic stem cells. Blood. 2003 Aug 1;102(3):906-15.

3. Vodyanik MA, Bork JA, Thomson JA, Slukvin II. Human embryonic stem cell-derived CD34+ cells: efficient production in the coculture with OP9 stromal cells and analysis of lymphohematopoietic potential. Blood. 2005 Jan 15;105(2):617-26.

4. Salvagiotto G, Burton S, Daigh CA, Rajesh D, Slukvin II, Seay NJ. A defined, feeder-free, serum-free system to generate in vitro hematopoietic progenitors and differentiated blood cells from hESCs and hiPSCs. PLoS One. 2011 Mar 18;6(3):e17829.

Histone Deacetylase Inhibitors: A New Treatment Option in Cancer

Even though cancer is considered as a disease of genetic defects, various studies have shown that epigenetic changes also play an important role in the onset and progression of cancer. Histone acetylation is one of the important epigenetic modifications, and is controlled by two enzymes: histone acetyltransferases (HATs) and histone deacetylases (HDACs).  HAT transfers the acetyl group from the acetyl co-enzyme A to lysine residues of the histones comprising the core. This is thought to loosen DNA resulting in greater access to DNA for transcription factors and RNA polymerase. HDAC on the other hand, removes the acetyl groups, resulting in the compaction of chromatin thus narrowing access to DNA. Aberrant acetylation of the histone tail by these enzymes is associated with carcinogenesis. Expression patterns of various genes may become changed due to the altered activities of these enzymes.

Histone Deacetylases (HDACs)

HDACs cause transcriptional repression of genes by deacetylating lysine residues on describe the imagehistone tails. HDACs also cause deacetylation of non-histone proteins thus altering the transcriptional activity of p53 (tumor suppressor gene), E2F (transcription factor), c-Myc (transcription factor), nuclear factor kB (NF-kB), hypoxia inducible factor 1α (HIF-1 α), estrogen receptor α, and androgen receptor complexes.

HDACs in Cancer

HDACs are important enzymes in regulating various cellular processes. However, over-expression and abnormal recruitment of HDACs to the promoter region of various tumor suppressor genes may cause tumor initiation and progression. A number of studies have reported a high level of expression of HDACs in various tumors compared to normal cells. Increased expression of HDAC1 was reported in gastric, prostate, colon, and breast carcinomas. Elevated expression of HDAC2 was found in colon cancer.  High levels of expression of HDAC6 were reported in breast cancer. In addition to the over-expression, aberrant recruitment of this enzyme to specific promoter regions may also promote tumor invasion and metastasis. For example, E-cadherin is a transmembrane protein that is found in epithelial cells and plays an important role in cell adhesion. Invasive carcinomas exhibit reduced expression or loss of function of E-cadherin. Recruitment of HDAC1 and HDAC2 to the promoter region of E-cadherin by transcription factor Snail caused reduced expression of E-cadherin.

In addition to histone deacetylation, HDACs also deacetylate non-histone proteins.  For example, mammalian HDAC1, 2, and 3 impair the function of tumor suppressor gene p53. HDACs also alter the transcriptional activity of transcription factor E2F, c-Myc, nuclear factor kB, and HIF-1 α. The chaperone activity of the heat shock protein Hsp90 is regulated by HDAC6. Most of the client proteins of Hsp90 are proteins kinases (c-Raf, MEK, Akt, HER-2) or transcription factors (androgen receptor, progesterone receptor, estrogen receptor) associated with cell proliferation, survival, and signaling.

Histone Deacetylase Inhibitors (HDIs)

With the increasing knowledge of the roles of the HDACs in cancer, efforts have been made to identify potent inhibitors. HDIs identified so far have been shown to induce growth arrest, differentiation, and apoptosis in tumor cells. These inhibitors were found to induce cell cycle regulatory protein p21, apoptotic proteins Bax, and PUMA. HDIs were also able to down-regulate various survival signaling pathways and were able to disrupt the cellular redox state. Therefore, in recent years HDIs have drawn interest as anti-cancer agents. Several HDIs aredescribe the image currently in clinical trials both in monotherapy and in combination therapy with other anti-tumor drugs. A review by Tan et al. (2010) reported that at least 80 clinical trials are underway, testing more than 11 different HDIs in hematologic and solid tumors, including leukemias, lymphomas, and multiple myeloma, lung, breast, pancreas, renal, and bladder cancers, melanoma, glioblastoma. To date, most of the responses using HDIs as single agents were observed in advanced hematologic tumors and few were observed in solid tumors. In 2006, HDI vorinostat (suberoylanilide hydroxamic acid, SAHA) was approved by the Food and Drug Administration (FDA, USA) for the treatment of relapsed and refractory cutaneous T-cell lymphoma CTCL. In November, 2009, the FDA also approved another HDI romidepsin (depsipeptide) for the treatment of CTCL, and in 2011 for the treatment of peripheral T-cell lymphoma patients who have already received prior therapy.

Even though HDIs have shown anti-tumor activity across a broad variety of hematologic and solid tumors in the clinical trials, only a portion of patients with a given diagnosis showed therapeutic response. Therefore, a detailed understanding of the mechanisms of action, as well as mechanisms of resistance, of HDIs would help to identify markers and formulate strategies which may enhance the efficacy of HDIs in the clinic.

Further reading:

1. Johnstone RW. Histone-deacetylase inhibitors: novel drugs for the treatment of cancer. Nat Rev Drug Discov. 2002;1(4):287-299.

2. Lane AA, Chabner BA. Histone deacetylase inhibitors in cancer therapy. J Clin Oncol. 2009;27(32):5459-5468.

3. Ropero S, Esteller M. The role of histone deacetylases (HDACs) in human cancer. Mol Oncol. 2007;1(1):19-25.

4. Shankar S, Srivastava RK. Histone deacetylase inhibitors: mechanisms and clinical significance in cancer: HDAC inhibitor-induced apoptosis. Adv Exp Med Biol. 2008;615:261-298.

5. Tan J, Cang S, Ma Y, Petrillo RL, Liu D. Novel histone deacetylase inhibitors in clinical trials as anti-cancer agents. J Hematol Oncol. 2010;3:5.

Cancer Biomarkers: How they are used for personalized medicine

As promising cancer treatments emerge the need for improved detection and characterization methods are still evident.  Identification of novel biomarkers is a promising area of cancer research and development but because of the high complexity and heterogeneity of tumors much remains to be learned.

What is a cancer biomarker? A biomarker is a biological molecule that can be found in the blood, bodily fluids or tissue of interest (i.e. tumor) that can give information about the molecular characteristics of a tumor.

Specimens for biomarker discovery

Potential biomarker biological molecules

  • DNA (copy number, methylation states, mutations)
  • RNA (mRNA, microRNA)
  • Protein (phosphorylation, post-translational modifications)
  • Metabolic products

Tools for cancer biomarker identification

Biomarkers can be used as tools for diagnosis (detect the presence of cancer), prognosis, tracking cancer progression, and assessing treatment efficacy.

In cancer, a biomarker is often a protein that is mutated or is expressed at higher levels in the cancerous cells compared to the normal tissue.  There are various proteins whose mutated status is shared by multiple types of cancers these include inactivating mutations of tumor suppressor proteins such as the cell cycle regulators p53, PTEN, and retinoblastoma protein (RB) and activating mutations of proto-oncogenes such as Ras and Myc.  Another prominent cancer biomarker is the cell proliferation marker, Ki67 that can be used not only as a prognostic indicator but also to assess the efficacy of a treatment where reduction in Ki67 expression indicates reduced cellular proliferation.

Types of biomarkers and their uses

types_of_biomarkers_uses.jpg

  1. Prognostic biomarker: Knowing the key molecular changes in a patient’s cancer allows a doctor to determine whether the patient is likely to have a poor outcome and thus more aggressive treatment is necessary.
  2. Predictive biomarker: Understanding the molecular characteristics about a patients cancer can lead to tailoring of drug treatments with a higher likelihood of efficacy.  For example, patients with certain kinase domain mutations on EGFR would possibly not respond to EGFR targeted treatments such as erlotinib. Additionally this gives the added benefit reducing a patients exposure to possible toxic side effects from a drug they may not have benefited from.
  3. Pharmacodynamic biomarker: Using biomarkers drug dosing could be tailored to each patient. Dosing a drug that has a specific molecular target can be decided based on its ability to decrease the activity of its biological target. For example, if a patient shows high activity for a particular kinase a targeted drug could be dosed up for that patients specific needs.

biomarker_drug_selection

With the advent of genomic and proteomic technology and improved data mining algorithms, it is now easier and faster to identify biomarkers.  Unfortunately, a major limitation in biomarker research and discovery is the need for biopsy samples and their limited availability for research. Another issue is that most biopsy samples available are taken from a patient during the initial diagnosis. Less available are samples from patients post treatment initiation or with advanced disease where the molecular characteristics of their cancer may have very likely changed.

Luckily, there is great interest in developing and improving current technologies to utilize blood specimens for protein, metabolic products, CTCs and circulating DNA as alternative non-invasive sources to identify and screen for cancer biomarkers.

Further reading:

Mining the plasma proteome for cancer biomarkers

The cancer biomarker problem

Taming the dragon: genomic biomarkers to individualize the treatment of cancer

Proteomics for Cancer Biomarker Discovery

 

Antagonism of Tumor-Immunity by Chemotherapeutics

dendritic cellSignificant steps forward are being made in immunotherapeutic approaches for treatment of cancer.  Over the past few years, two cancer immunotherapeutics were FDA approved.  In 2010, Dendreon Corporation’s Provenge, an autologous cellular vaccine, was approved for hormone refractory metastatic prostate cancer.  In 2011, Bristol-Myers Squibb’s anti-CTLA4 antibody Ipilimumab, was FDA approved for late stage melanoma.  Recent promising clinical trial results indicate several additional immune modulating therapies are likely to join this prestigious list in the coming years.  In addition, combinations of immune therapies with chemotherapeutics are being tested in clinical trials.  In light of this, it is important to know how chemotherapies interact with the immune system, in order to best generate synergistic effects.

There are multiple ways that chemotherapies may modulate anti-tumor immunity.  Some therapeutics such as anthracyclines induce an immunogenic cell death characterized by release of endogenous danger signals such as HMGB1, that activate antigen presenting cells (APCs) to elicit anti-tumor T cell responses.  Standard apoptosis elicited by a range of other therapeutics however, is often non-immunogenic.  Chemotherapies can induce lymphopaenia which may have both negative and positive effects on anti-tumor immunity, including loss of anti-tumor effector T cells as well as negative regulatory T cells and myeloid derived suppressor cells (MDSC).

In the recent January issue of Nature Medicine, Bruchard et. al., demonstrate that the widely prescribed chemotherapeutic agents, gemcitabine and 5-fluorouracil (5-FU) activated the NLRP3 (NOD-like receptor family, pyrin domain containing-3) inflammasome complex in MDSCs, leading to perturbed anti-tumor immunity and reduced therapeutic effect of these drugs.

The NLRP3 inflammasome is activated in response to damage associated molecular patterns (DAMPs) released during infection with a plethora of pathogens.  NLRP3 activation leads to formation of the multi-protein inflammasome complex that activates caspase-1.  IL-1b is a pro-inflammatory cytokine that is transcribed as an inactive pro-peptide and requires processing by caspase-1 into its active secreted form, and is a major inflammasome effector molecule.

In the study by Bruchard et. al., gemcitabine and 5-FU activated the NLRP3 inflammasome complex in MDSCs, and led to characteristic activation of caspase-1 and IL-1b.   In contrast, the chemotherapeutics Deticene, taxol, oxaliplatin, mitomycin C, and doxorubicin did not activate this pathway.  Cathepsin B release from damaged lysosomes into the cytosol was shown as the trigger of NLRP3 activation by gemcitabine and 5-FU.  Importantly, increased serum concentrations of IL-1b, and enhanced caspase-1 and cathepsin B activity in circulating MDSCs were found colorectal cancer patients one day after 5-FU treatment, validating the relevance of these observations.

Studies on the subset of CD4+ T helper cells that produce IL-17 (TH17) have demonstrated both positive and negative roles for these cells in cancer pathogenesis, and the contexts by which TH17 cells can play opposing roles is unclear.  In this study, IL-1b released by MDSCs treated with 5-FU promoted differentiation of CD4+ T cells into TH17 cells.  5-FU treatment promoted IL-17 production in PBMCs from colorectal cancer human patients as well.  Mice lacking inflammasome components or IL-17 demonstrated enhanced survival when treated with 5-FU.  Thus, in mice, 5-FU elicited TH17 cells play a pro-tumorigenic role.  Whether or not 5-FU elicited TH17 cells also promote tumor growth in human patients is a critical question to be addressed.

Finally, treatment of mice with the soluble form of IL-1Ra blocked the effects of IL-1b and promoted the anti-tumor effects of 5-FU.  Therefore, IL-1b blockade represents a rational immunotherapeutic strategy to enhance the effects of 5-FU and gemcitabine chemotherapies.

In conclusion, this study identified key mechanisms of immune modulation by gemcitabine and 5-fluorouracil in murine models and human cancer patients.  Chemotherapeutics elicit cellular damage by many different mechanisms, and understanding how each drug interacts with the immune system will be important for promoting critical synergy between chemotherapies and anti-tumor immunity.

Further Reading:

Chemotherapy-triggered cathepsin B release in myeloid-derived suppressor cells activates the Nlrp3 inflammasome and promotes tumor growth.  Bruchard M, Mignot G, Derangère V, Chalmin F, Chevriaux A, Végran F, Boireau W, Simon B, Ryffel B, Connat JL, Kanellopoulos J, Martin F, Rébé C, Apetoh L, Ghiringhelli F. Nat Med. 2013 Jan;19(1):57-64.

Immunological aspects of cancer chemotherapy. Zitvogel, L., Apetoh, L., Ghiringhelli, F. & Kroemer, G. Nat. Rev. Immunol. 8, 59–73 (2008).

Dual role of immunomodulation by anticancer chemotherapy.  Shurin MR.  Nat Med. 2013 Jan;19(1):20-2.

Inflammasomes and their roles in health and disease.  Lamkanfi M, Dixit VM. Annu Rev Cell Dev Biol. 2012;28:137-61.

5-Fluorouracil selectively kills tumor-associated myeloid-derived suppressor cells resulting in enhanced T cell–dependent antitumor immunity. Vincent, J. et al. Cancer Res. 70, 3052–3061 (2010).

Gemcitabine selectively eliminates splenic Gr-1+/CD11b+ myeloid suppressor cells in tumor-bearing animals and enhances antitumor immune activity. Suzuki, E., Kapoor, V., Jassar, A.S., Kaiser, L.R. & Albelda, S.M. Clin. Cancer Res. 11, 6713–6721 (2005).

Restoration of antitumor immunity through selective inhibition of myeloid derived suppressor cells by anticancer therapies. Apetoh, L., Vegran, F., Ladoire, S. & Ghiringhelli, F. Curr. Mol. Med. 11, 365–372 (2011).