Identification and characterization of ovarian cancer-initiating cells from primary human tumors - PubMed (original) (raw)

Identification and characterization of ovarian cancer-initiating cells from primary human tumors

Shu Zhang et al. Cancer Res. 2008.

Abstract

The objective of this study was to identify and characterize a self-renewing subpopulation of human ovarian tumor cells (ovarian cancer-initiating cells, OCICs) fully capable of serial propagation of their original tumor phenotype in animals. Ovarian serous adenocarcinomas were disaggregated and subjected to growth conditions selective for self-renewing, nonadherent spheroids previously shown to derive from tissue stem cells. To affirm the existence of OCICs, xenoengraftment of as few as 100 dissociated spheroid cells allowed full recapitulation of the original tumor (grade 2/grade 3 serous adenocarcinoma), whereas >10(5) unselected cells remained nontumorigenic. Stemness properties of OCICs (under stem cell-selective conditions) were further established by cell proliferation assays and reverse transcription-PCR, demonstrating enhanced chemoresistance to the ovarian cancer chemotherapeutics cisplatin or paclitaxel and up-regulation of stem cell markers (Bmi-1, stem cell factor, Notch-1, Nanog, nestin, ABCG2, and Oct-4) compared with parental tumor cells or OCICs under differentiating conditions. To identify an OCIC cell surface phenotype, spheroid immunostaining showed significant up-regulation of the hyaluronate receptor CD44 and stem cell factor receptor CD117 (c-kit), a tyrosine kinase oncoprotein. Similar to sphere-forming OCICs, injection of only 100 CD44(+)CD117(+) cells could also serially propagate their original tumors, whereas 10(5) CD44(-)CD117(-) cells remained nontumorigenic. Based on these findings, we assert that epithelial ovarian cancers derive from a subpopulation of CD44(+)CD117(+) cells, thus representing a possible therapeutic target for this devastating disease.

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Figures

Figure 1

Figure 1

A subpopulation of human ovarian tumor cells form self-renewing, anchorage-independent spheroids under stem cell—selective conditions and are capable of epithelial differentiation. A, cell suspensions form small, nonadherent clusters 1 wk after plating (top left). Magnification, 100×. After ∼10 passages, a minor (1%) fraction of spheres persist as larger, symmetric, prototypical spheroids (top right). Magnification, 100×. Typical spheroids contained ∼100 viable cells and could be serially passaged for >6 mo (bottom left). Magnification, ×320. Under differentiating conditions for 11 to 14 d, dissociated sphere-forming cells adhere to plates and form symmetric holoclones (bottom right). Magnification, 100×. B, immunofluorescence of undifferentiated (top) or differentiated (bottom) spheroids or single cells under differentiating conditions, using antibodies against the stem cell markers Oct-4 (left), Nanog (center), and nestin (right). Nuclei were stained with DAPI. Magnification, 20×. C, under differentiating conditions, sphere-forming cells express the epithelial markers CK-7 and ovarian CA-125, as shown by fluorescence microscopy. Nuclei were stained with DAPI. D, as shown by RT-PCR, sphere-forming cells (OCICs), under stem cell—selective conditions, overexpress several stem cell marker genes compared with parental bulk tumor population cells (OC) and OCICs under differentiating conditions (Different.). Lanes 1 to 3 correspond to tumor T1 gene expression under the three conditions: lane 1, stem cell–selective (OCICs); lane 2, bulk tumor (OC); lane 3, differentiating (Different.). Lanes 4 to 6 similarly denote tumor T2 sphere-forming cell gene expression under the same conditions (β-actin used as a control).

Figure 2

Figure 2

OCICs, under stem cell conditions, are highly resistant to conventional chemotherapies. A, sphere-forming OCICs from tumor T1, under stem cell—selective (black columns) or differentiating (white columns) conditions, were treated with cisplatin (CDDP;30 μmol/L; left), paclitaxel (Ptx;2 μmol/L; center), or cisplatin (30 μmol/L) and paclitaxel (2 μmol/L) for 3 h (CDDP + Ptx; right); cell survival was determined by MTT assays. B, same treatments as in A for OCICs derived from tumor T2, with white columns denoting differentiating conditions and black columns indicating stem cell conditions; *, P < 0.05; **, P < 0.01.

Figure 3

Figure 3

Robust in vivo propagation of human ovarian tumors (with reproducible histologic phenotypes) in nude mice by sphere-forming OCICs. A, xenograft tumor formed after injection of sphere-forming OCICs derived from patient tumors T1 and T2. Injection of ∼100 OCICs per mouse from T1 (left top)or T2(right top) dissociated spheroids generated tumors with 2/2 efficiency. I.p. injection of T2 OCICs gave rise to bloody ascites (left bottom) and peritoneal metastasic lesions (right bottom); black arrows denote metastases on the colon. B, representative H&E staining sections of T2 primary tumor (top left; magnification, 100×) and subcutaneous graft tumor from T2-derived spheroids (top right; magnification, 200×). Both tumors were classified as advanced grade (2/3) serous adenocarcinomas. Expression of the epithelial tumor marker CA125 in human xenograft tumor derived from T2 spheroids, as determined by immunohistochemistry; specific peroxidase staining is indicated by the brown color, and nuclei (blue) were counterstained with hematoxylin magnification at 100× (bottom left). H&E staining of intraperitoneal tumor derived from T2 spheroids (bottom right; magnification, 200×). C, representative double staining for CD44 and CD117 in T1 spheroids by immunofluorescence; similar results were obtained for tumors T2—T5. Immunofluorescence staining of anti-CD44 monoclonal antibodies (PE-conjugated secondary antibody, red) in ovarian tumor spheroid (top left); immunofluorescence staining of anti-CD117 monoclonal antibodies (FITC-conjugated secondary antibody, green) in ovarian tumor sphere cells (top right); CD44+ sphere cells colocalize with CD117+ cells (orange overlay, bottom left) or overlaid and additionally stained with DAPI (blue; bottom right). Magnification, 200×.

Figure 4

Figure 4

Tumor-derived spheroids stably coexpress CD44 and CD117, and those markers can be used to isolate highly malignant progenitors from whole tumors that reproduce their original phenotype. A, isolation of CD44+CD117+ cells by FACS. Scatter plots represent typical examples of patterns of CD44+CD117+ expression in a panel of human ovarian tumors T1—T3 (top) or spheroids generated from tumors T4 and T5 (bottom left and center). FACS experiments were repeated in duplicate, and the purity of CD44+CD117+ population was >99%, as revealed by postresorting FACS analysis (bottom right). B, H&E staining of the T3 xenograft tumors (left) generated from CD44+CD117+ cells is histologically identical to the corresponding T3 patient primary tumor (right). Both tumors were classified as poorly differentiated (G3) serous adenocarcinoma; magnification, 100×. C, after 30 d in culture, CD44+CD117+-generated spheroids from the T3 primary tumor retained CD44+CD117+ (orange overlay) expression. Nuclei were stained with DAPI (blue). Magnification, 200×.

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