Health  Vol.6 No.12 , June 2014
Growth of Circulating Tumor Cell-Derived Colonies from Peripheral Blood of Melanoma Patients: Preliminary Characterization of Colony Composition
Abstract: Circulating tumor cells (CTC) are rarely detected in the blood of cancer patients, even though they are a direct harbinger of eventual patient demise. We developed an innovative CTC culture technology to allow more sensitive isolation, expansion, and characterization of viable colonies from patient blood. In this assay, the entire leukocyte fraction from 10 ml of anticoagulated patient blood is placed into culture medium without any pre-selection. After 16 days in culture, CTC derived colonies are counted. As a proof-of-principle, blood samples from 58 Stage IIa-IV melanoma patients were tested. Ninety percent of these samples grew colonies. The colony numbers ranged from 0 - 308 (mean 63 ± 9.5 SEM). Ten normal volunteers had virtually no growth (mean 0.5 ± 1.4 colonies). Colonies were harvested using a micropipette for characterization. Tumor-cell containing spheroids were embedded in paraffin, sectioned, and stained with melanoma-specific mAb for histologic characterization. MITF proved to be the most consistent immunostain that identified melanoma cells in these colonies. A host-cell component in colonies was also identified using CD68 and CD43 mAb staining. Following enzymatic dissociation of colonies, a variety of immunostains were tested. Papanicolau staining proved most useful for identifying the abnormal nuclei of tumor cells. Flow cytometry could readily distinguish host and tumor cell populations based on DNA content and forward/side scatter in dissociated colonies. The stem cell marker ALDH1A1 associated with the aneuploid population, but CD45 was expressed on both diploid and aneuploid cells. The ability to repeatedly isolate CTC derived colonies from cancer patient blood samples opens the door to a novel type of long-term clinical monitoring. This novel CTC culture technology may prove useful to perform molecular characterization, assessment of treatment response, and testing of drug sensitivity and resistance in patients during treatment.
Cite this paper: Samlowski, W. , McGregor, J. , Samlowski, S. , Tharkar, S. , Shen, S. and Bentz, J. (2014) Growth of Circulating Tumor Cell-Derived Colonies from Peripheral Blood of Melanoma Patients: Preliminary Characterization of Colony Composition. Health, 6, 1467-1481. doi: 10.4236/health.2014.612181.

[1]   Bernards, R. and Weinberg, R.A. (2002) A Progression Puzzle. Nature, 418, 823.

[2]   Jemal, A., Siegel, R., Ward, E., Hao, Y., Xu, J. and Thun, M.J. (2009) Cancer Statistics, 2009. CA: A Cancer Journal for Clinicians, 59, 225-249.

[3]   Pantel, K. and Alix-Panabieres, C. (2007) The Clinical Significance of Circulating Tumor Cells. Nature Clinical Practice Oncology, 4, 62-63.

[4]   Alunni-Fabbroni, M. and Sandri, M.T. (2010) Circulating Tumour Cells in Clinical Practice: Methods of Detection and Possible Characterization. Methods, 50, 289-297.

[5]   Balzar, M., Winter, M.J., de Boer, C.J. and Litvinov, S.V. (1999) The Biology of the 17-1A Antigen (Ep-CAM). Journal of Molecular Medicine, 77, 699-712.

[6]   Bertazza, L., Mocellin, S. and Nitti, D. (2008) Circulating Tumor Cells in Solid Cancer: Tumor Marker of Clinical Relevance? Current Oncology Reports, 10, 137-146.

[7]   Allard, W.J., Matera, J., Miller, M.C., Repollet, M., Connelly, M.C., Rao, C., et al. (2004) Tumor Cells Circulate in the Peripheral Blood of All Major Carcinomas but Not in Healthy Subjects or Patients with Nonmalignant Diseases. Clinical Cancer Research, 10, 6897-6904.

[8]   Cristofanilli, M., Budd, G.T., Ellis, M.J., Stopeck, A., Matera, J., Miller, M.C., et al. (2004) Circulating Tumor Cells, Disease Progression, and Survival in Metastatic Breast Cancer. The New England Journal of Medicine, 351, 781-791.

[9]   de Bono, J.S., Scher, H.I., Montgomery, R.B., Parker, C., Miller, M.C., Tissing, H., et al. (2008) Circulating Tumor Cells Predict Survival Benefit from Treatment in Metastatic Castration-Resistant Prostate Cancer. Clinical Cancer Research, 14, 6302-6309.

[10]   Cohen, S.J., Punt, C.J., Iannotti, N., Saidman, B.H., Sabbath, K.D., Gabrail, N.Y., et al. (2008) Relationship of Circulating Tumor Cells to Tumor Response, Progression-Free Survival, and Overall Survival in Patients with Metastatic Colorectal Cancer. Journal of Clinical Oncology, 26, 3213-3221.

[11]   Goodman Jr., O.B., Fink, L.M., Symanowski, J.T., Wong, B., Grobaski, B., Pomerantz, D., et al. (2009) Circulating Tumor Cells in Patients with Castration-Resistant Prostate Cancer Baseline Values and Correlation with Prognostic Factors. Cancer Epidemiology, Biomarkers & Prevention, 18, 1904-1913.

[12]   Allen-Mersh, T.G., McCullough, T.K., Patel, H., Wharton, R.Q., Glover, C. and Jonas, S.K. (2007) Role of Circulating Tumour Cells in Predicting Recurrence after Excision of Primary Colorectal Carcinoma. British Journal of Surgery, 94, 96-105.

[13]   Hayes, D.F., Cristofanilli, M., Budd, G.T., Ellis, M.J., Stopeck, A., Miller, M.C., et al. (2006) Circulating Tumor Cells at Each Follow-Up Time Point during Therapy of Metastatic Breast Cancer Patients Predict Progression-Free and Overall Survival. Clinical Cancer Research, 12, 4218-4224.

[14]   Danila, D.C., Heller, G., Gignac, G.A., Gonzalez-Espinoza, R., Anand, A., Tanaka, E., et al. (2007) Circulating Tumor Cell Number and Prognosis in Progressive Castration-Resistant Prostate Cancer. Clinical Cancer Research, 13, 7053-7058.

[15]   Stott, S.L., Hsu, C.H., Tsukrov, D.I., Yu, M., Miyamoto, D.T., Waltman, B.A., et al. (2010) Isolation of Circulating Tumor Cells Using a Microvortex-Generating Herringbone-Chip. Proceedings of the National Academy of Sciences of the United States of America, 107, 18392-18397.

[16]   Zheng, S., Lin, H.K., Lu, B., Williams, A., Datar, R., Cote, R.J., et al. (2011) 3D Microfilter Device for Viable Circulating Tumor Cell (CTC) Enrichment from Blood. Biomedical Microdevices, 13, 203-213.

[17]   Hsieh, H.B., Marrinucci, D., Bethel, K., Curry, D.N., Humphrey, M., Krivacic, R.T., et al. (2006) High Speed Detection of Circulating Tumor Cells. Biosensors and Bioelectronics, 21, 1893-1899.

[18]   Rao, C.G., Chianese, D., Doyle, G.V., Miller, M.C., Russell, T., Sanders Jr., R.A., et al. (2005) Expression of Epithelial Cell Adhesion Molecule in Carcinoma Cells Present in Blood and Primary and Metastatic Tumors. International Journal of Oncology, 27, 49-57.

[19]   Sieuwerts, A.M., Kraan, J., Bolt, J., van der Spoel, P., Elstrodt, F., Schutte, M., et al. (2009) Anti-Epithelial Cell Adhesion Molecule Antibodies and the Detection of Circulating Normal-Like Breast Tumor Cells. Journal of the National Cancer Institute, 101, 61-66.

[20]   Königsberg, R., Obermayr, E., Bises, G., Pfeiler, G., Gneist, M., Wrba, F., et al. (2011) Detection of EpCAM Positive and Negative Circulating Tumor Cells in Metastatic Breast Cancer Patients. Acta Oncologica, 50, 700-710.

[21]   Yu, M., Stott, S., Toner, M., Maheswaran, S. and Haber, D.A. (2011) Circulating Tumor Cells: Approaches to Isolation and Characterization. Journal of Cell Biology, 192, 373-382.

[22]   Yang, G.C. and Alvarez, I.I. (1995) Ultrafast Papanicolaou Stain. An Alternative Preparation for Fine Needle Aspiration Cytology. Acta Cytologica, 39, 55-60.

[23]   Prieto, V.G. and Shea, C.R. (2011) Immuno-histochemistry of Melanocytic Proliferations. Archives of Pathology & Laboratory Medicine, 135, 853-859.

[24]   Lang, D., Lu, M.M., Huang, L., Engleka, K.A., Zhang, M., Chu, E.Y., et al. (2005) Pax3 Functions at a Nodal Point in Melanocyte Stem Cell Differentiation. Nature, 433, 884-887.

[25]   Lister, J.A., Capper, A., Zeng, Z., Mathers, M.E., Richardson, J., Paranthaman, K., et al. (2013) A Conditional Zebrafish MITF Mutation Reveals MITF Levels Are Critical for Melanoma Promotion vs. Regression In Vivo. Journal of Investigative Dermatology, 134, 133-140.

[26]   Eccles, M.R., He, S., Ahn, A., Slobbe, L.J., Jeffs, A.R., Yoon, H.S., et al. (2013) MITF and PAX3 Play Distinct Roles in Melanoma Cell Migration; Outline of a “Genetic Switch” Theory Involving MITF and PAX3 in Proliferative and Invasive Phenotypes of Melanoma. Frontiers in Oncology, 3, 229.

[27]   Koyanagi, K., O’Day, S.J., Gonzalez, R., Lewis, K., Robinson, W.A., Amatruda, T.T., et al. (2006) Microphthalmia Transcription Factor as a Molecular Marker for Circulating Tumor Cell Detection in Blood of Melanoma Patients. Clinical Cancer Research, 12, 1137-1143.

[28]   Gil-Bernabé, A.M., Ferjancic, S., Tlalka, M., Zhao, L., Allen, P.D., Im, J.H., et al. (2012) Recruitment of Monocytes/ Macrophages by Tissue Factor-Mediated Coagulation Is Essential for Metastatic Cell Survival and Premetastatic Niche Establishment in Mice. Blood, 119, 3164-3175.

[29]   Eljaszewicz, A., Wiese, M., Helmin-Basa, A., Jankowski, M., Gackow-ska, L., Kubiszewska, I., et al. (2013) Collaborating with the Enemy: Function of Macrophages in the Development of Neoplastic Disease. Mediators of Inflammation, 2013, Article ID 831387.

[30]   Donepudi, S., Reisinger, S.A., McGregor, J.R., Tharkar, S., Samlowski, S., Ostler, D., et al. (2013) Circulating Tumor Cell Cultures as a Predictive Marker during Salvage Therapy of Refractory Merkel Cell Carcinoma with Chemotherapy and Electron Beam Radiation. Journal of Cancer Therapy, 4, 1162-1166.

[31]   Clawson, G.A., Kimchi, E., Patrick, S.D., Xin, P., Harouaka, R., Zheng, S., et al. (2012) Circulating Tumor Cells in Melanoma Patients. PLoS ONE, 7, e41052.

[32]   Zhang, L., Ridgway, L.D., Wetzel, M.D., Ngo, J., Yin, W., Kumar, D., et al. (2013) The Identification and Characterization of Breast Cancer CTCs Competent for Brain Metastasis. Science Translational Medicine, 5, p. 180ra148.

[33]   Quintana, E., Shackleton, M., Foster, H.R., Fullen, D.R., Sabel, M.S., Johnson, T.M., et al. (2010) Phenotypic Heterogeneity among Tumorigenic Melanoma Cells from Patients That Is Reversible and Not Hierarchically Organized. Cancer Cell, 18, 510-523.

[34]   Santini, R., Vinci, M.C., Pandolfi, S., Penachioni, J.Y., Montagnani, V., Olivito, B., et al. (2012) HEDGEHOG-GLI Signaling Drives Self-Renewal and Tumorigenicity of Human Melanoma-Initiating Cells. Stem Cells, 30, 1808-1818.

[35]   Ni, C. and Huang, J. (2012) Dynamic Regulation of Cancer Stem Cells and Clinical Challenges. Clinical and Translational Oncology, 15, 253-258.

[36]   Scheel, C. and Weinberg, R.A. (2011) Phenotypic Plasticity and Epithelial-Mesenchymal Transitions in Cancer and Normal Stem Cells? International Journal of Cancer, 129, 2310-2314.

[37]   Joosse, S.A. and Pantel, K. (2013) Biologic Challenges in the Detection of Circulating Tumor Cells. Cancer Research, 73, 8-11.