Methodologies in Assaying Prostate Cancer Stem Cells

  • Hangwen Li
  • Ming Jiang
  • Sofia Honorio
  • Lubna Patrawala
  • Collene R. Jeter
  • Tammy Calhoun-Davis
  • Simon W. Hayward
  • Dean G. Tang
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 568)

Summary

The cancer stem cell (CSC) theory posits that only a small population of tumor cells within the tumor has the ability to reinitiate tumor development and is responsible for tumor homeostasis and progression. Tumor initiation is a defining property of putative CSCs, which have been reported in both blood malignancies and solid tumors. In order to test whether any given human tumor cell population has CSC properties, the relatively enriched single cells have to be put into a foreign microenvironment in a recipient animal to test their tumorigenic potential. Furthermore, various in vitro assays need be performed to demonstrate that the presumed CSCs have certain biological properties normally associated with the stem cells (SCs). Herein, we present a comprehensive review of the experimental methodologies that our lab has been using in assaying putative prostate cancer (PCa) SCs in culture, xenograft tumors, and primary tumor samples.

Key words

Cancer stem cells (CSCs) Prostate cancer Clonal and clonogenic assays Side population Self-renewal Transplantation sites Sphere-formation assays 

Notes

Acknowledgments

We thank Mr. Kent Claypool for assistance in FACS, Histology and Animal Facility Cores for technical assistance, and other members (past and present) of the Tang lab for discussion and support. This work was supported in part by grants from NIH (R01-AG023374, R01-ES015888, and R21-ES015893-01A1), American Cancer Society (RSG MGO-105961), Department of Defense (W81XWH-07-1-0616 and PC073751), Prostate Cancer Foundation, and Elsa Pardee Foundation (D.G.T), by DOD-PCRP grant W81XWH-04-1-0867, and by two Center grants (CCSG-5 P30 CA166672 and ES07784). H. Li was supported in part by a predoctoral fellowship from DOD (W81XWH-07-1-0132). L. Patrawala was supported in part by a predoctoral fellowship from Department of Defense (W81XWH-06-1-023). C. Jeter was supported in part by a postdoctoral fellowship from American Urological Association.

References

  1. 1.
    Lapidot T, Sirard C, Vormoor J, et al. (1994) A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature 367, 645–648.PubMedCrossRefGoogle Scholar
  2. 2.
    Singh SK, Hawkins C, Clarke ID, et al. (2004) Identification of human brain tumor initiating cells. Nature 432, 396–401.PubMedCrossRefGoogle Scholar
  3. 3.
    Schatton T, Murphy GF, Frank NY, et al. (2008) Identification of cells initiating human melanomas. Nature 451, 345–349.PubMedCrossRefGoogle Scholar
  4. 4.
    Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. (2003) Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA 100, 3983–3988.PubMedCrossRefGoogle Scholar
  5. 5.
    Ginestier C, Hur MH, Charafe-Jauffret E, et al. (2007) ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell 1, 555–567.PubMedCrossRefGoogle Scholar
  6. 6.
    O’Brien CA, Pollett A, Gallinger S, Dick JE. (2007) A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature 445, 106–110.PubMedCrossRefGoogle Scholar
  7. 7.
    Ricci-Vitiani L, Lombardi DG, Pilozzi E, et al. (2007) Identification and expansion of human colon-cancer-initiating cells. Nature 445, 111–115.PubMedCrossRefGoogle Scholar
  8. 8.
    Todaro M, Alea MP, Di Stefano AB, et al. (2007) Colon cancer stem cells dictate tumor growth and resist cell death by production of interleukin-4. Cell Stem Cell 1, 389–402.PubMedCrossRefGoogle Scholar
  9. 9.
    Dalerba P, Dylla SJ, Park IK, et al. (2007) Phenotypic characterization of human colorectal cancer stem cells. Proc Natl Acad Sci USA 104, 10158–10163.PubMedCrossRefGoogle Scholar
  10. 10.
    Li C, Heidt DG, Dalerba P, et al. (2007) Identification of pancreatic cancer stem cells. Cancer Res 67, 1030–1037.PubMedCrossRefGoogle Scholar
  11. 11.
    Hermann PC, Huber SL, Herrler T, et al. (2007) Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer. Cell Stem Cell 1, 313–323.PubMedCrossRefGoogle Scholar
  12. 12.
    Yang ZF, Ho DW, Ng MN, et al. (2008) Significance of CD90(+) cancer stem cells in human liver cancer. Cancer Cell 13, 153–166.PubMedCrossRefGoogle Scholar
  13. 13.
    Eramo A, Lotti F, Sette G, et al. (2008) Identification and expansion of the tumorigenic lung cancer stem cell population. Cell Death Differ. 15, 504–514.PubMedCrossRefGoogle Scholar
  14. 14.
    Prince ME, Sivanandan R, Kaczorowski A, et al. (2007) 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.PubMedCrossRefGoogle Scholar
  15. 15.
    Hope KJ, Jin L, Dick JE. (2004) Acute myeloid leukemia originates from a hierarchy of leukemic stem cell classes that differ in self-renewal capacity. Nat Immunol 5, 738–743.PubMedCrossRefGoogle Scholar
  16. 16.
    Clarke MF, Dick JE, Dirks PB, et al. (2006) Cancer stem cells – Perspectives on current status and future directions: AACR workshop on cancer stem cells. Cancer Res 66, 9339–9344.PubMedCrossRefGoogle Scholar
  17. 17.
    Tang DG, Patrawala L, Calhoun T, et al. (2007) Prostate cancer stem/progenitor cells: Identification, characterization, and implications. Mol Carcinog 46, 1–14.PubMedCrossRefGoogle Scholar
  18. 18.
    Hill, RP (2006) Identifying cancer stem cells in solid tumors: case not proven. Cancer Res 66, 1891–1895.PubMedCrossRefGoogle Scholar
  19. 19.
    Yu F, Yao H, Zhu P, et al. (2007) let-7 regulates self renewal and tumorigenicity of breast cancer cells. Cell 131, 1109–1123.PubMedCrossRefGoogle Scholar
  20. 20.
    Bao S, Wu Q, McLendon RE, et al. (2006) Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 444, 756–760.PubMedCrossRefGoogle Scholar
  21. 21.
    Wang JCY. (2007) Evaluating therapeutic efficacy against cancer stem cells: New challenges posed by a new paradigm. Cell Stem Cell 1, 497–501.CrossRefGoogle Scholar
  22. 22.
    Isaacs, JT (1985) Control of cell proliferation and death in normal and neoplastic prostate: A stem cell model. In: Benign Prostate Hyperplasia. Rogers, CH, and Cunha, GR (Eds). Bethesda. Springer-Verlag, pp85–94.Google Scholar
  23. 23.
    Wang Y, Hayward SW, Cao M, Thayer KA, Cunha GR. (2001) Cell differentiation lineage in the prostate. Differentiation 68, 270–279.PubMedCrossRefGoogle Scholar
  24. 24.
    Signoretti S, Loda M. (2007) Prostate stem cells: from development to cancer. Semin Cancer Biol. 17, 219–224.PubMedCrossRefGoogle Scholar
  25. 25.
    Lawson, DA, Witte, ON (2007) Stem cells in prostate cancer initiation and progression. J Clin Invest 117, 2044–2050.PubMedCrossRefGoogle Scholar
  26. 26.
    Collins AT, Habib FK, Maitland NJ, Neal DE. (2001) Identification and isolation of human prostate epithelial stem cells based on α2β1-integrin expression. J Cell Sci 114, 3865–3872.PubMedGoogle Scholar
  27. 27.
    Richardson GD, Robson CN, Lang SH, et al. (2004) CD133, a novel marker for human prostatic epithelial stem cells. J Cell Sci 117, 3539–3545.PubMedCrossRefGoogle Scholar
  28. 28.
    Collins AT, Berry PA, Hyde C, Stower MJ, Maitland NJ. (2005) Prospective identification of tumorigenic prostate cancer stem cells. Cancer Res 65, 10946–10951.PubMedCrossRefGoogle Scholar
  29. 29.
    Patrawala L, Calhoun T, Schneider-Broussard R, Zhou J, Claypool K, Tang DG. (2005) Side population is enriched in tumorigenic, stem-like cancer cells, whereas ABCG2+ and ABCG2- cancer cells are similarly tumorigenic. Cancer Res 65, 6207–6219.PubMedCrossRefGoogle Scholar
  30. 30.
    Patrawala L, Calhoun T, Schneider-Broussard R, et al. (2006) Highly purified CD44+ prostate cancer cells from xenograft human tumors are enriched in tumorigenic and metastatic progenitor cells. Oncogene 25, 1696–1708.PubMedCrossRefGoogle Scholar
  31. 31.
    Patrawala L, Calhoun-Davis T, Schneider-Broussard R, Tang DG. (2007) Hierarchical organization of prostate cancer cells in xenograft tumors: the CD44+ alpha2beta1+ cell population is enriched in tumor-initiating cells. Cancer Res 67, 6796–805.PubMedCrossRefGoogle Scholar
  32. 32.
    Li HW, Chen X, Calhoun-Davis T, Claypool K, Tang DG. (2008) PC3 Human prostate carcinoma cell holoclones contain self-renewing tumor-initiating cells. Cancer Res 68, 1820–1825.PubMedCrossRefGoogle Scholar
  33. 33.
    Miki J, Furusato B, Li H, et al. (2007) Identification of putative stem cell markers, CD133 and CXCR4, in hTERT-immortalized primary nonmalignant and malignant tumor-derived human prostate epithelial cell lines and in prostate cancer specimens. Cancer Res 67, 3153–3161.PubMedCrossRefGoogle Scholar
  34. 34.
    Gu G, Yuan J, Wills M, Kasper S. (2007) Prostate cancer cells with stem cell characteristics reconstitute the original human tumor in vivo. Cancer Res 67, 4807–4815.PubMedCrossRefGoogle Scholar
  35. 35.
    Goodell MA, McKinney-Freeman S, Camargo FD. (2005) Isolation and characterization of side population cells. Methods Mol Biol 290, 343–352.PubMedGoogle Scholar
  36. 36.
    Bhatia B, Tang S, Yang P, et al. (2005) Cell-autonomous induction of functional tumor suppressor 15-lipoxygenase 2 (15-LOX2) contributes to replicative senescence of human prostate progenitor cells. Oncogene 24, 3583–3595.PubMedCrossRefGoogle Scholar
  37. 37.
    Cunha GR, Lung B. (1978) The possible influence of temporal factors in androgenic responsiveness of urogenital tissue recombinants from wild-type and androgen-insensitive (Tfm) mice. J Exp Zool 205, 181–193.PubMedCrossRefGoogle Scholar
  38. 38.
    Xin L, Lukacs RU, Lawson DA, Cheng D, Witte ON. (2007) Self-renewal and multilineage differentiation in vitro from murine prostate stem cells. Stem Cells 25, 2760–2769.PubMedCrossRefGoogle Scholar
  39. 39.
    Wang Y, Revelo MP, Sudilovsky D, et al. (2005) Development and characterization of efficient xenograft models for benign and malignant human prostate tissue. Prostate 64, 149–159.PubMedCrossRefGoogle Scholar
  40. 40.
    Wang Y, Xue H, Cutz JC, et al. (2005) An orthotopic metastatic prostate cancer model in SCID mice via grafting of a transplantable human prostate tumor line. Lab Invest 85, 1392–1404.PubMedCrossRefGoogle Scholar
  41. 41.
    Cunha GR, Hayward SW, Wang YZ, Ricke WA. (2003) Role of the stromal microenvironment in carcinogenesis of the prostate. Int J Cancer 107, 1–10.PubMedCrossRefGoogle Scholar
  42. 42.
    Cher ML, Towler DA, Rafii S, et al. (2006) Cancer interaction with the bone microenvironment: a workshop of the National Institutes of Health Tumor Microenvironment Study Section. Am J Pathol 168, 1405–1412.PubMedCrossRefGoogle Scholar
  43. 43.
    Olumi AF, Grossfeld GD, Hayward SW, et al. (1999) Carcinoma-associated fibroblasts direct tumor progression of initiated human prostatic epithelium. Cancer Res 59, 5002–5011.PubMedGoogle Scholar
  44. 44.
    Studeny M, Marini FC, Dembinski JL, et al. (2004) Mesenchymal stem cells: potential precursors for tumor stroma and targeted-delivery vehicles for anticancer agents. J Natl Cancer Inst 96, 1593–1603.PubMedCrossRefGoogle Scholar
  45. 45.
    Karnoub AE, Dash AB, Vo AP, et al. (2007). Mesenchymal stem cells within tumour stroma promote breast cancer metastasis. Nature 449, 557–563.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Hangwen Li
    • 1
  • Ming Jiang
    • 2
  • Sofia Honorio
    • 1
  • Lubna Patrawala
    • 1
  • Collene R. Jeter
    • 1
  • Tammy Calhoun-Davis
    • 1
  • Simon W. Hayward
    • 2
  • Dean G. Tang
    • 1
  1. 1.Department of CarcinogenesisUniversity of Texas M.D. Anderson Cancer CenterSmithvilleUSA
  2. 2.Department of Urologic SurgeryVanderbilt University Medical CenterNashvilleUSA

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