Recent observations indicate that, in several types of human cancer, only a phenotypic subset of cancer cells within each tumor is capable of initiating tumor growth. This functional subset of cancer cells is operationally defined as the “cancer stem cell” (CSC) subset. Here we developed a CSC model for the study of human colorectal cancer (CRC). Solid CRC tissues, either primary tissues collected from surgical specimens or xenografts established in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice, were disaggregated into single-cell suspensions and analyzed by flow cytometry. Surface markers that displayed intratumor heterogeneous expression among epithelial cancer cells were selected for cell sorting and tumorigenicity experiments. Individual phenotypic cancer cell subsets were purified, and their tumor-initiating properties were investigated by injection in NOD/SCID mice. Our observations indicate that, in six of six human CRC tested, the ability to engraft in vivo in immunodeficient mice was restricted to a minority subpopulation of epithelial cell adhesion molecule (EpCAM) ^high /CD44 ⁺ epithelial cells. Tumors originated from EpCAM ^high /CD44 ⁺ cells maintained a differentiated phenotype and reproduced the full morphologic and phenotypic heterogeneity of their parental lesions. Analysis of the surface molecule repertoire of EpCAM ^high /CD44 ⁺ cells led to the identification of CD166 as an additional differentially expressed marker, useful for CSC isolation in three of three CRC tested. These results validate the stem cell working model in human CRC and provide a highly robust surface marker profile for CRC stem cell isolation.

Although the concept that cancers originate from stem cells (SC) is becoming scientifically accepted, mechanisms by which SC contribute to tumor initiation and progression are largely unknown. For colorectal cancer (CRC), investigation of this problem has been hindered by a paucity of specific markers for identification and isolation of SC from normal and malignant colon. Accordingly, aldehyde dehydrogenase 1 (ALDH1) was investigated as a possible marker for identifying colonic SC and for tracking them during cancer progression. Immunostaining showed that ALDH1⁺ cells are sparse and limited to the normal crypt bottom, where SCs reside. During progression from normal epithelium to mutant (APC) epithelium to adenoma, ALDH1⁺ cells increased in number and became distributed farther up the crypt. CD133⁺ and CD44⁺ cells, which are more numerous and broadly distributed in normal crypts, showed similar changes during tumorigenesis. Flow cytometric isolation of cancer cells based on enzymatic activity of ALDH (Aldefluor assay) and implantation of these cells in nonobese diabetic–severe combined immunodeficient mice (a) generated xenograft tumors (Aldefluor⁻ cells did not), (b) generated them after implanting as few as 25 cells, and (c) generated them dose dependently. Further isolation of cancer cells using a second marker (CD44⁺ or CD133⁺ serially) only modestly increased enrichment based on tumor-initiating ability. Thus, ALDH1 seems to be a specific marker for identifying, isolating, and tracking human colonic SC during CRC development. These findings also support our original hypothesis, derived previously from mathematical modeling of crypt dynamics, that progressive colonic SC overpopulation occurs during colon tumorigenesis and drives CRC development. [Cancer Res 2009;69(8):3382–9]

Th17 cells play an active role in autoimmune diseases. However, the nature of Th17 cells is poorly understood in cancer patients. We studied Th17 cells, the associated mechanisms, and clinical significance in 201 ovarian cancer patients. Tumor-infiltrating Th17 cells exhibit a polyfunctional effector T-cell phenotype, are positively associated with effector cells, and are negatively associated with tumor-infiltrating regulatory T cells. Tumor-associated macrophages promote Th17 cells through interleukin-1β (IL-1β), whereas tumor-infiltrating regulatory T cells inhibit Th17 cells through an adenosinergic pathway. Furthermore, through synergistic action between IL-17 and interferon-γ, Th17 cells stimulate CXCL9 and CXCL10 production to recruit effector T cells to the tumor microenvironment. The levels of CXCL9 and CXCL10 are associated with tumor-infiltrating effector T cells. The levels of tumor-infiltrating Th17 cells and the levels of ascites IL-17 are reduced in more advanced diseases and positively predict patient outcome. Altogether, Th17 cells may contribute to protective human tumor immunity through inducing Th1-type chemokines and recruiting effector cells to the tumor microenvironment. Inhibition of Th17 cells represents a novel immune evasion mechanism. This study thus provides scientific and clinical rationale for developing novel immune-boosting strategies based on promoting the Th17 cell population in cancer patients.

Foxp3⁺CD4⁺ regulatory T (Treg) cells inhibit immune responses and temper inflammation. IL-17⁺CD4⁺ T (Th17) cells mediate inflammation of autoimmune diseases. A small population of IL-17⁺Foxp3⁺CD4⁺ T cells has been observed in peripheral blood in healthy human beings. However, the biology of IL-17⁺Foxp3⁺CD4⁺ T cells remains poorly understood in humans. We investigated their phenotype, cytokine profile, generation, and pathological relevance in patients with ulcerative colitis. We observed that high levels of IL-17⁺Foxp3⁺CD4⁺ T cells were selectively accumulated in the colitic microenvironment and associated colon carcinoma. The phenotype and cytokine profile of IL-17⁺Foxp3⁺CD4⁺ T cells was overlapping with Th17 and Treg cells. Myeloid APCs, IL-2, and TGF-β are essential for their induction from memory CCR6⁺ T cells or Treg cells. IL-17⁺Foxp3⁺CD4⁺ T cells functionally suppressed T cell activation and stimulated inflammatory cytokine production in the colitic tissues. Our data indicate that IL-17⁺Foxp3⁺ cells may be “inflammatory” Treg cells in the pathological microenvironments. These cells may contribute to the pathogenesis of ulcerative colitis through inducing inflammatory cytokines and inhibiting local T cell immunity, and in turn may mechanistically link human chronic inflammation to tumor development. Our data therefore challenge commonly held beliefs of the anti-inflammatory role of Treg cells and suggest a more complex Treg cell biology, at least in the context of human chronic inflammation and associated carcinoma.