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European Biopharmaceutical Review

Hitting the Right Target

David Koos, Steven F Josephs and Ewa Carrier at Entest BioMedical, and Thomas E Ichim at Medistem Inc explain the significance of the cancer stem cell to immune therapy

With the exception of Dendreon’s recent approval for the Provenge cancer vaccines, immunotherapy of cancer has been a dismal failure overall. In addition to the obvious explanations such as antigen loss, tumour immune evasion, and mutation of targeted epitopes, one possibility is that tumour vaccines have been designed for the wrong cell. The original discovery of leukemic stem cells has led to the identification of stem cells in a variety of solid tumours. Tumour stem cells are analogous to resident organ stem cells in that they reside in a quiescent state, express drug resistance proteins, and enter the cell cycle in response to local tissue injury. Given that for the past century tumour vaccines have focused on cell lines or autologous tumour tissue, which contain little if any stem cell components, we believe it is time to re-examine the previous approach in light of the emerging tumour stem cell paradigm.

The paradigm shift currently occurring in cancer research is the result of the realisation that there is extreme heterogeneity of tumour composition in terms of cellular proliferative potential. In the same way that the full haematopoietic system can be reconstituted by a very small number of haematopoietic stem cells administered to an irradiated host, a belief is being established in the field that, within a tumour mass, there resides a rare cell population with extreme potential for self-renewal driving tumorigenesis (1). It is this rare cell type, referred to as the ‘tumour stem cell’ by some, that contributes to the maintenance of the mass of the tumour. Until recently, most preclinical models of cancer have studied the tumour mass or cell lines derived from the tumour mass. Accordingly, in a preclinical model, agents would demonstrate significant reduction in the mass of the tumour, but these agents were not actually targeting the ‘tumour initiating cell’ or the ‘cancer stem cell’. Therefore, these approaches, despite demonstrating good preclinical data, are bound to fail clinically, as has been the case with many therapeutics. In fact, this is what prompted some cancer researchers to state that the ‘war on cancer’ had failed (2). By failing to target the quiescent tumour stem cell, drugs or vaccines may actually accelerate the tumour. In the same manner, tissue injury in the form of an infarction stimulates proliferation of resident cardiac specific stem cells. It is likely that by killing the rapidly proliferating cells in a tumour mass using conventional approaches, the tumour stem cells exit quiescence and start cycling (3). This will contribute to the demise of the patient in the form of a relapse or an expanded number of cells with a drug resistant phenotype.

LEUKAEMIA STEM CELLS: THE FIRST TUMOUR STEM CELL IDENTIFIED

One way to identify tumour stem cells involves isolation of a patient primary tumour sample, generating mononuclear single cell preparations, observing the ability of various cell types within the sample to form tumours in an animal model. The commonly used animal model for this type of investigation is the non-obese diabetic (lacks NK activity) crossed onto a severe combined immunodeficient mouse (lacks T and B cells). This strain of mice, called NODSCID mice, readily accept tumour cells of human origin and have been used extensively as a ‘semi-human’ model of cancer, although other immune-deficient models such as the nude mouse have also been used.

Initial investigations into human tumour stem cells were performed on leukaemia. In 1994, Lapidot et al sought to identify the ‘leukaemia initiating cell’ from leukaemic blasts isolated from patients with acute myelocytic leukaemia (AML) (4). Given that the non-malignant bloodmaking (haematopoietic) stem cells express the markers CD34 and lack expression of CD38, the investigators reasoned that the cell initiating leukaemia may have the same phenotypic markers. It is important to keep in mind that the normal haematopoietic stem cell comprises less than one in 1 million blood cells. The investigators used magnetic separation techniques and purified cells from AML patients into CD34+, CD34+ CD38+, CD38+, and CD34+ CD38-. These groups of cells were injected into immunocompromised mice and assessed for the ability to produce leukaemic colony forming units. Colony forming units are the human leukaemic cells that enter the mouse and grow colonies on the spleen of the mouse. The larger the number of colony forming units there are, the more aggressive the leukaemia. Previous studies have demonstrated correlation between colony forming units in the spleen of mice and poor patient prognosis (5). The investigators found that only the CD34+ CD38- subgroup of leukaemic cells had the ability to generate substantially more leukaemic colonies in the mouse. While at first glance these findings may seem obvious (in that the haematopoietic stem cell is CD34+ CD38-, so it would make sense that the leukaemic stem cell has the same characteristics), it is important to point out that all of the drug discovery efforts targeting leukaemia before this study were using either cell lines or leukaemic blasts which do not represent the leukaemic stem cell phenotype. It should be stated here that both malignant and non-malignant stem cells are extremely difficult to expand in tissue culture, perhaps explaining the lack of drug development efforts targeting these cells.

SOLID TUMOUR STEM CELLS

In the situation of solid tumours, identification of breast cancer stem cells was reported in 2003 by Al-Hajj et al (6). The investigators obtained patients’ samples of primary and metastatic infiltrating ductal carcinoma, adenocarcinoma, invasive lobular carcinoma and inflammatory breast carcinoma. All metastatic tumours were obtained from plural effusions. The investigators observed that the cells had the ability to grow in immunodeficient mice. To identify markers found on tumour cells with the ability to form tumours, cells were dissociated into single cell suspensions and purified based on the expression of several markers. The adhesion molecules CD24 and CD44 were assessed, as well as B38.1, a breast cancer-specific marker. It was found that an injection of two to eight million cells were capable of inducing tumour growth in all animals when the cells were selected for; CD44+ (8/8), B38.1+ (8/8), and CD24/low cells (12/12) all gave rise to visible tumours within the twelfth week of injection, but none of the CD44- cells (0/8), or B38.1- cells (0/8) formed detectable tumours. In a representative example, as little as 20,000 CD44+ CD24- cells induced in vivo tumour formation in the mouse, whereas CD44+ CD24+ did not. These data support the notion that within the breast cancer tumour mass, numerous subpopulations of cells exist. With some subpopulations having a higher tumourinitiating potential than others, the existence of specific markers that allow for determination of tumour-causing potential provides the first step for obtaining cells with ‘tumour-initiating’ or ‘stem cell’ qualities.

The concept that tumour stem cells have similar phenotypic and functional properties to their non-malignant counterparts was not only demonstrated in the leukaemia model discussed above but also in numerous models of solid tumours. For example, the marker CD133 is known to reside on non-malignant tissue resident stem cells. CD133+ stem cells have been described in liver stem cells (oval cells), prostate stem cells, muscle stem cells, haematopoietic stem cells, and stem cells of the small intestine (7). Accordingly, it is reasonable to believe that tumour stem cells would also express this marker. Indeed, CD133 has been detected on tumour stem cells in the colon, ovarian, liver, brain, prostate, head and neck cancers (8,9). Numerous other markers and properties have been associated with cancer stem cells, for example, expression of CD44, c-kit, multiple drug resistance protein (MDR), and DAF (10). Interestingly, all of these proteins are also present in tissue specific stem cells that are non-malignant.

WHY IS KILLING TUMOUR STEM CELLS DIFFICULT?

It is important to note that cancer stem cells have unique properties from other tumour cells that renders them particularly difficult to destroy. For example, the cancer stem cell is normally regarded as a noncycling cell thus making it resistant to chemotherapy and radiotherapy that targets the cycling cells. Additionally, cancer stem cells express, high concentration of MDR, an ABC efflux protein that pumps out chemotherapy so as to make the cells resistant to small molecule agents (11,12). Immunologically speaking, tumour stem cells express higher levels of complement inhibitors, theoretically to block antibody mediated cytolysis (13). Additionally, microarray studies have demonstrated expression of CD200 on tumour stem cells (14). CD200 is known to inhibit maturation of dendritic cells, as well as being involved in direct suppression of Th1 immunity through the stimulation of T regulatory cells (15,16).

IMMUNE STRATEGIES FOR KILLING CANCER STEM CELLS

One of the major problems in development of any immune-based strategy is selection of ideal antigens. The issue may be exemplified in the major debate on whether it is more beneficial to use whole cell lysates versus protein antigens or specific peptides. The use of whole cell lysates allows for a wide number of antigens to be present in the vaccine, but this also creates a potential for autoimmunity and, perhaps more importantly, a lack of efficacy. On the other extreme, when peptides are used, the tumour has the possibility of mutating so as to lose expression of the peptide that is being targeted. The other issue is that tumour cells in general, and tumour stem cells specifically, express high levels of immune suppressive molecules, such as DAF and CD200 among others (13,14). The third issue with generating immunity to tumour stem cells is actually having the immune effector cells enter the tumour and penetrate into the stem cells. Tumour stem cells are known to reside in hypoxic environments within the tumour that are acidified and contain high interstitial pressures (17). These environments are not conducive for immune effector attack. For example, localised tumour acidity selectively kills effector cells while sparing T regulatory cells (18), thus amplifying existing local immune suppression.

A biotechnology company in the US is working to address these issues in a systematic manner, focusing primarily on the canine model as a point of first entry (19). Current approaches under investigation include:

  • Generation of CD133 selected primary cells as an autologous vaccine using encapsulation technology
  • Identification of cancer stem cell specific peptides using bioinformatics-based approaches
  • siRNA silencing of tumour associated immune inhibitory factors

The first approach revolves around the finding that irradiated tumour cells placed in a three-dimensional environment in the presence of various cytokines can elicit immune responses at a systemic level. Although this technology has previously been used in the context of solid tumours, the purification of tumour stem cells and increasing their immunogenicity by cytokine treatment is expected to increase specificity of induced responses to the proper target.

Bioinformatics approaches have been previously used for identifying MHC I bound peptides. Databases are currently being generated based on proteomic expression of de novo peptides on tumour stem cells versus control tumour cells. Several potential hits have been identified, with immunogenicity to be tested using ex vivo human dendritic cell/T-cell cultures. Gene silencing has been used with success to inhibit expression of the immune suppressive IDO gene. These techniques are now being used to inhibit tumour stem cell specific genes such as CD200. By combining these approaches, we may have a shot at targeting the main cause of cancer: the tumour stem cell.

References

  1. Reya T, Morrison SJ, Clarke MF and Weissman IL, Stem cells, cancer and cancer stem cells, Nature 414: pp105-111, 2001
  2. Sporn MB, The war on cancer: a review, Ann N Y Acad Sci 833: pp137-146, 1997
  3. Liang SX, Tan TY, Gaudry L and Chong B, Differentiation and migration of Sca1+/CD31- cardiac side population cells in a murine myocardial ischemic model, Int J Cardiol 138: pp40-49, 2010
  4. Lapidot T, Sirard C, Vormoor J, Murdoch B, Hoang T, Caceres-Cortes J, Minden M, Paterson B, Caligiuri MA and Dick JE, A cell initiating human acute myeloid leukaemia after transplantation into SCID mice, Nature 367: pp645-648, 1994
  5. Jacobs P and Wood L, Clonogenic growth patterns correlate with chemotherapy response in acute myeloid leukaemia, Hematology 10: pp321-326, 2005
  6. Al-Hajj M, Wicha MS, Benito- Hernandez A, Morrison SJ and Clarke MF, Prospective identification of tumorigenic breast cancer cells, Proc Natl Acad Sci USA 100: pp3,983-3,988, 2003
  7. Katoh Y and Katoh M, Comparative genomics on PROM1 gene encoding stem cell marker CD133, Int J Mol Med 19: pp967-970, 2007
  8. Harper LJ, Piper K, Common J, Fortune F and Mackenzie IC, Stem cell patterns in cell lines derived from head and neck squamous cell carcinoma, J Oral Pathol Med 36: pp594-603, 2007
  9. Wei C, Guomin W, Yujun L and Ruizhe Q, Cancer Stem-like Cells in Human Prostate Carcinoma Cells DU145: The Seeds of the Cell Line? Cancer Biol Ther 6: pp763-768, 2007
  10. Xu JX, Morii E, Liu Y, Nakamichi N, Ikeda J, Kimura H and Aozasa K, High tolerance to apoptotic stimuli induced by serum depletion and ceramide in side-population cells: high expression of CD55 as a novel character for side-population, Exp Cell Res 313: pp1,877-1,885, 2007
  11. Hirschmann-Jax C, Foster AE, Wulf GG, Goodell MA and Brenner MK, A distinct ‘side population’ of cells in human tumour cells: implications for tumour biology and therapy, Cell Cycle 4: pp203-205, 2005
  12. Hirschmann-Jax C, Foster AE, Wulf GG, Nuchtern JG, Jax TW, Gobel U, Goodell MA and Brenner MK, A distinct ‘side population’ of cells with high drug efflux capacity in human tumour cells, Proc Natl Acad Sci USA 101: pp14,228-14,233, 2004
  13. Wognum AW, Eaves AC and Thomas TE, Identification and isolation of hematopoietic stem cells, Arch Med Res 34: pp461-475, 2003
  14. Ieta K, Tanaka F, Haraguchi N, Kita Y, Sakashita H, Mimori K, Matsumoto T, Inoue H, Kuwano H and Mori M, Biological and Genetic Characteristics of Tumour- Initiating Cells in Colon Cancer, Ann Surg Oncol 15(2): pp638-648, 2007
  15. Cameron CM, Barrett JW, Liu L, Lucas AR and McFadden G, Myxoma virus M141R expresses a viral CD200 (vOX-2) that is responsible for down-regulation of macrophage and T-cell activation in vivo,J Virol 79: pp6,052-6,067, 2005
  16. Gorczynski RM, Lee L and Boudakov I, Augmented induction of CD4+CD25+ Treg using monoclonal antibodies to CD200R, Transplantation 79: pp488-491, 2005
  17. Yoshii Y, Furukawa T, Kiyono Y, Watanabe R, Waki A, Mori T, Yoshii H, Oh M, Asai T, Okazawa H et al, Copper-64-diacetyl-bis (N4-methylthiosemicarbazone) accumulates in rich regions of CD133+ highly tumorigenic cells in mouse colon carcinoma, Nucl Med Biol 37: pp395- 404, 2010
  18. Liao YP, Schaue D and McBride WH, Modification of the tumour microenvironment to enhance immunity, Front Biosci 12: pp3,576-3,600, 2007
  19. Koos D, Josephs SF, Alexandrescu DT, Chan RC, Ramos F, Bogin V, Gammill V, Dasanu CA, Necochea-Campion RD, Riordan NH and Carrier E, Tumour vaccines in 2010: Need for integration, Cell Immunol, 3rd April 2010

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David Koos is the Founder, Chairman and CEO of Entest BioMedical Inc, a publicly traded biotechnology company currently developing a stem cell/laser regenerative therapy for COPD and an immunotherapeutic cancer vaccine for canines. David has over 26 years of investment banking and venture capital experience with a primary focus on medical and biotechnology ventures. He holds a PhD degree in Sociology and a Doctor of Business Administration in Finance. Additionally, he has authored and co-authored several peer reviewed journal articles primarily on biotechnology related subjects.

Steven F Josephs was with the NIH’s Laboratory of Tumor Cell Biology for more than 15 years researching oncogenes and viruses. He is credited with co-discovery of human herpesvirus-6. At Baxter Healthcare he was involved with developing a gene therapy vector for the treatment of haemophilia A. Currently, Steven is Founder and CSO of Therinject LLC, a company that is focusing on developing cancer immunotherapeutics in a joint venture with Entest BioMedical, Inc. He has a PhD in Chemistry from American University (Washington DC, US).

Ewa Carrier is Associate Professor of Clinical Medicine and Pediatrics at the University of California San Diego, Blood and Marrow Transplant Program. She finished her Paediatric Residency at Stanford University and stem cell transplant fellowship at the University of California San Francisco. She is actively involved in research related to embryonic and adult stem cells and cancer vaccines. She participates in Phase II clinical trials of the lung cancer vaccine Lucanix, and has extensive experience in tumour immunology and stem cell therapies.

Thomas E Ichim is an accomplished immunologist, drug developer with numerous contributions to the biomedical sciences spanning over 16 years. He has authored dozens of patents and has developed numerous novel therapeutics solutions primarily in oncology. To date he has published 61 peer-reviewed papers and is a reviewer for several journals. He is author of the textbook RNA Interference: From Bench to Clinical Implementation. Thomas has served as the Chief Scientific Officer at such biotech firms as MedVax and MarrowTech Pharma. He is a co-founder of bioRASI LLC and is CEO of Medistem Inc.

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David Koos
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Steven F Josephs
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Ewa Carrier
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Thomas E Ichim
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