Melanoma Initiating Cells and Circulating Melanoma Cells: a Blurred Picture
Whether a subset of tumorigenic cancer stem cells* exist in human melanomas remains a controversial issue though it is of critical importance in order since treatments which may eliminate the undifferentiated subset of cancer cells may cure patients (1,2). Therefore, it will be critical to identify cancer cells that have the tumorogenic potential in patients, in order to develop more effective targeting therapies.
Taking patient melanoma tumors from a broad spectrum of sites and stages, Boiko et al. (3) showed that in melanomas, tumor stem cells could be isolated as a highly enriched CD271+ population. Indeed, transplantation of highly viable FACS isolated melanoma cells resuspended in a Matrigel® into T, B, and NK deficient Rag2−/− γc−/− mice, resulted in melanoma from CD271+ but not CD271− cells. The authors also showed that tumors transplanted by CD271+ patient cells were capable of metastasis in-vivo; these cells lacked expression of TYR, MART and MAGE in 86%, 69% and 68% of melanoma patients respectively.
Schatton et al. (4) found that 1 in 120,735 (0.00083%) ABCB5+ metastatic melanoma cells formed tumours in NOD/SCID mice, a tenfold enrichment over unfractionated cells. In mice grafted with human cells, the researchers found that melanoma cells expressing ABCB5 [ATP-binding cassette, sub-family B (MDR/TAP), member 5, which mediates melanoma doxorubicin resistance via its function as a doxorubicin efflux transporter], had much greater tumorigenicity than the more abundant ABCB5– cells. ABCB5+ cells were capable of self-renewal and differentiation, giving rise to both ABCB5+ and ABCB5– cells. ABCB5– cells, by contrast, only generated more ABCB5– cells. Treatment of grafted mice with an anti-ABCB5 monoclonal antibody inducing cell-mediated cytotoxicity against ABCB5+ cells inhibited tumor growth.
Such results were not confirmed by Quintana et al. (5). The authors investigated whether melanoma is hierarchically organized into phenotypically distinct subpopulations of tumorigenic and non-tumorigenic cells or whether most melanoma cells retain tumorigenic capacity, irrespective of their phenotype. They found 28% of single melanoma cells obtained directly from patients of all stages formed tumors in NOD/SCID IL2Rγ(null) mice (lacking the interleukin-2 gamma receptor as wall as natural killer cells which may eliminate many of the transplanted cancer cells in the standard NOD/SCID mice). All tumorigenic cells appeared to have unlimited tumorigenic capacity on serial transplantation, and the authors were unable to find any large subpopulation of melanoma cells that lacked tumorigenic potential. None of 22 heterogeneously expressed markers, including CD271 and ABCB5, enriched tumorigenic cells. Some melanomas metastasized in mice, irrespective of whether they arose from CD271– or CD271+ cells. Many markers appeared to be reversibly expressed by tumorigenic melanoma cells.
To assess whether tumorigenic melanoma cells are phenotypically distinct from melanoma cells that fail to form tumours, the authors examined the expression of more than 50 surface markers on melanomas, including A2B5, c-kit, CD44, CD49B, CD49D, CD49f, CD54, CD133, CD166, E-cadherin, HNK-1, L1CAM, MCAM, N-cadherin and p75 which were heterogeneously expressed by melanoma cells from multiple patients and were tested for the ability to distinguish tumorigenic from non-tumorigenic melanoma cells in vivo. In every case, tumours arose from all fractions of cells. The authors found no marker that distinguished tumorigenic from non-tumorigenic cells
Adapting xenotransplantation protocols ?
Most human cancers have only rare (< 0.1%) cancer-initiating cells when transplanted into NOD/SCID or other highly immunocompromised mice (4, 6-14).Therefore, a critical question is whether optimization of xenotransplantation assays could reveal that some human cancers actually have very common cells with tumorigenic potential despite only having rare tumorigenic cells in NOD/SCID mice.
Though ABCB5 expression has been shown to correlate with the expression of CD166 and CD133 (15), Quintana et al. (16) found that CD166 or CD133 could not enrich tumorigenic melanoma cells in the modified xenotransplantation assay, raising the possibility that markers that enrich rare cells with tumorigenic potential in NOD/SCID mice may fail to distinguish tumorigenic from nontumorigenic cells in NOD/SCID Il2rg2/2 mice assays that detect much higher frequencies of tumorigenic cells. More generally, these authors showed that some cancers that appear to have rare tumorigenic cells in NOD/SCID mice actually have very common cells with tumorigenic capacity under modifications of xenotransplantation assays. Though, the authors observed a high percentage of melanoma cells that have the potential to proliferate extensively and form new tumours, it is possible that an even greater, or a much smaller, fraction of melanoma cells actually contributes to disease progression in patients.
Looking for Tumour Initiating Cells among Circulating Melanoma Cells (CMCs)?
In many neoplastic diseases, the presence of CTCs attracts considerable attention as a potential non-invasive approach prognostic and potentially predictive marker of treatment efficiency and monitoring. Though CTCs have been included into the international tumor staging systems for several carcinomas, it is not as yet the case for melanoma (17,18). Circulating Tumor Cells have been described with phenotypic and genotypic similarities to cancer stem cells (19-26). It is likely that the presence of tumor initiating cells among CMCs would have a significant impact on prognosis.
Three studies addressed the possible impact of the number of enriched CMCs on prognosis. In a prospective study by Ulmer et al. (27), 50 ml blood samples were drawn from 164 patients affected by cutaneous melanoma (29 stage I, 30 stage II, 42 stage III, 63 stage IV). CMCs were enriched by immunomagnetic cell sorting using a murine monoclonal antibody against the melanoma-associated chondroitin sulfate proteoglycan. Positivity for immunomagnetic melanoma cell enrichment correlated with the presence of detectable tumor, and detection of more than one melanoma cell in stage III or IV patients was associated with a significantly decreased overall survival. On the contrary, another study from the same group showed no significant difference between number of melanoma cells and established prognostic parameters in a cohort of patients
De Giorgi et al. (28) and Xu et al. (29) showed that analysis of circulating tumor cells (CTC) in the peripheral blood of cutaneous melanoma patients provides information on the metastatic process. Indeed, CTCs were shown in 29% of patients with primary invasive melanoma and in 62.5% of metastatic melanoma patients.
Rao et al. (30) enumerated CMCs in patients with metastatic melanoma. For that, circulating melanoma cells were immunomagnetically enriched from 7.5 ml of blood, fluorescently labelled with DAPI and identified as CD146+, HMW-MAA+ (High-molecular weight melanoma-associated antigen), CD45- and CD34-. Thirty to one hundred of CMCs expressed the proliferation marker Ki67. Such cells ranged from 0 to 8,042 per 7.5 ml, and patients with 2 CMCs per 7.5 ml of whole blood had a shorter overall survival as compared with the group with <2 CMCs.
Whether some CTCs subpopulation(s) have tumorogenic capacity is of critical importance since cancer treatments may be unsuccessful if they fail to target the specific minority subpopulation of tumour cells that have tumour initiation capacity (31-34).
*‘Cancer Stem Cells’ (35,36) are tumor cells with stem cell characteristics responsible for tumor progression; they are also known as Tumor Initiating Cells (TICs) (37).
- Horwich, A., Shipley, J. & Huddart, R. Testicular germ-cell cancer. Lancet 367, 754–765 (2006).
- Kleinsmith, L. J. & Pierce, G. B. Multipotentiality of single embryonal carcinoma cells. Cancer Res. 24, 1544–1551 (1964).
- Boiko AD, Razorenova OV, van de Rijn M, Swetter SM, Johnson DL, Ly DP, Butler, PD, Yang GP, Joshua B, Kaplan MJ, Longaker MT, and Weissman IL. Human Melanoma Initiating Cells Express Neural Crest Nerve Growth Factor Receptor CD271. Nature. 466: 133–137 (2010).
- Schatton, T. et al. Identification of cells initiating human melanomas. Nature 451:345-349 (2008).
- Quintana E, Shackleton M, Foster HR, Fullen DR, Sabel MS, Johnson TM, Morrison SJ. Phenotypic heterogeneity among tumorigenic melanoma cells from patients that is reversible and not hierarchically organized. Cancer Cell. 18: 510-523 (2010).
- Prince, M. E. et al. Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma. Proc. Natl Acad. Sci. USA. 104: 973–978 (2007).
- Wu, C. et al. Side population cells isolated from mesenchymal neoplasms have tumor initiating potential. Cancer Res. 67: 8216–8222 (2007).
- Wang, J. C. et al. High level engraftment of NOD/SCID mice by primitive normal and leukemic hematopoietic cells from patients with chronic myeloid leukemia in chronic phase. Blood. 91: 2406–2414 (1998).
- Al-Hajj, M. et al. Prospective identification of tumorigenic breast cancer cells. Proc. Natl Acad. Sci. USA. 100: 3983–3988 (2003).
- Cox, C. V. et al. Characterization of acute lymphoblastic leukemia progenitor cells. Blood. 104: 2919–2925 (2004).
- Hope, K. J., Jin, L. & Dick, J. E. Acute myeloid leukemia originates from a hierarchy of leukemic stem cell classes that differ in self-renewal capacity. Nature Immunol. 5: 738–743 (2004).
- Dalerba, P. et al. Phenotypic characterization of human colorectal cancer stem cells. Proc. Natl Acad. Sci. USA. 104: 10158–10163 (2007).
- O’Brien, C. A. et al. A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature. 445: 106–110 (2007).
- Ricci-Vitiani, L. et al. Identification and expansion of human colon-cancer initiating cells. Nature. 445: 111–115 (2007).
- Frank, N. Y. et al. ABCB5-mediated doxorubicin transport and chemoresistance in human malignant melanoma. Cancer Res. 65: 4320–4333 (2005).
- Quintana E, Shackleton M, Sabel MS, Fullen DR, Johnson TM, and Morrison SJ Efficient tumour formation by single human melanoma cells. Nature. 456:593-598 (2008).
- Singletary SE, Greene FL, and Sobin LH. Classification of isolated tumor cells Clarification of the 6th edition of the American Joint Committee on Cancer Staging Manual. Cancer. 98 : 2740–2741 (2003).
- Harris L, Fritsche H, Mennel R, Norton L, Ravdin P, Taube S, Somerfield MR, Hayes DF, Bast RC Jr; American Society of Clinical Oncology 2007 update of recommendations for the use of tumor markers in breast cancer. J Clin Oncol. 25:5287-312 (2007).
- Pantel, K. and S. Riethdorf, Pathology: are circulating tumor cells predictive of overall survival? Nat Rev Clin Oncol. 6: 190-191. (2009).
- Panteleakou, Z., et al., Detection of circulating tumor cells in prostate cancer patients: methodological pitfalls and clinical relevance. Mol Med. 15: 101-114 (2009).
- Ross, J.S. and E.A. Slodkowska, Circulating and disseminated tumor cells in the management of breast cancer. Am J Clin Pathol. 132:237-245 (2009).
- Mukai, M., Occult neoplastic cells and malignant micro-aggregates in lymph node sinuses: review and hypothesis. Oncol Rep. 14:173-175. (2005).
- Pestrin, M., et al., Correlation of HER2 status between primary tumors and corresponding circulating tumor cells in advanced breast cancer patients. Breast Cancer Research and Treatment. 118: 523 (2009).
- Muller, V., D.F. Hayes, and K. Pantel, Recent translational research: circulating tumor cells in breast cancer patients. Breast Cancer Res. 8:110. (2006).
- Riethdorf, S., H. Wikman, and K. Pantel, Review: Biological relevance of disseminated tumor cells in cancer patients. Int J Cancer. 123:1991-2006 (2008).
- Tomaskovic-Crook, E., E. Thompson, and J. Thiery, Epithelial to mesenchymal transition and breast cancer. Breast Cancer Research. 11:213 (2009).
- Ulmer A, Schmidt-Kittler O, Fischer J, Ellwanger U, Rassner G, Riethmüller G, Fierlbeck G, Klein CA. mmunomagnetic enrichment, genomic characterization, and prognostic impact of circulating melanoma cells. Clin Cancer Res.10:531-517 (2004).
- De Giorgi V, Pinzani P, Salvianti F, Panelos J, Paglierani M, Janowska A, Grazzini M, Wechsler J, Orlando C, Santucci M, Lotti T, Pazzagli M, Massi D. Application of a filtration- and isolation-by-size technique for the detection of circulating tumor cells in cutaneous melanoma. J Invest Dermatol.130: 2440-2447 (2010).
- Xu X, Zhong JF. Circulating tumor cells and melanoma progression. J Invest Dermatol.130: 2349-2351 (2010).
- Rao C, Bui T, Connelly M, Doyle G, Karydis I, Middleton MR, Clack G, Malone M, Coumans FA, Terstappen LW. Circulating melanoma cells and survival in metastatic melanoma. Int J Oncol. 2011 Mar;38(3):755-760.
- Morrison, B.J., et al., Future use of mitocans against tumour-initiating cells? Mol Nutr Food Res. 53:147-153 (2009).
- Schatton T, Frank NY, and Frank MH.Identification and targeting of cancer stem cells. Bioessays. 31: 1038–1049 (2009).
- Park CY, Tseng D, and Weissman IL. Cancer Stem Cell–Directed Therapies: Recent Data From the Laboratory and Clinic. Molecular Therapy. 17: 219–230 (2009).
- Gupta PB, Onder TT, Jiang G, Tao K, Kuperwasser C, Weinberg RA, and Lander ES. Identification of selective inhibitors of cancer stem cells by high-throughput screening. Cell.138:645-659 (2009).
- Ross, J.S., et al., The HER-2 receptor and breast cancer: ten years of targeted anti-HER-2 therapy and personalized medicine. Oncologist. 14: 320-368 (2009).
- Zhou, B.B., et al., Tumour-initiating cells: challenges and opportunities for anticancer drug discovery. Nat Rev Drug Discov. 8: 806-823 (2009).
- NCI.Executive Summary of the Tumour Stem Cell & Self-Renewal Genes Think Tank. 2009 [cited 12 December 2009]; Available from:http://dcb.nci.nih.gov/thinktank /Executive Summary of the Tumor Stem Cell.cfm.)