Ovarian Cancer Spheroid Cells with Stem Cell-Like Properties Contribute to Tumor Generation, Metastasis and Chemotherapy Resistance through Hypoxia-Resistant Metabolism

Abstract

Cells with sphere forming capacity, spheroid cells, are present in the malignant ascites of patients with epithelial ovarian cancer (EOC) and represent a significant impediment to efficacious treatment due to their putative role in progression, metastasis and chemotherapy resistance. The exact mechanisms that underlie EOC metastasis and drug resistance are not clear. Understanding the biology of sphere forming cells may contribute to the identification of novel therapeutic opportunities for metastatic EOC. Here we generated spheroid cells from human ovarian cancer cell lines and primary ovarian cancer. Xenoengraftment of as few as 2000 dissociated spheroid cells into immune-deficient mice allowed full recapitulation of the original tumor, whereas >105 parent tumor cells remained non-tumorigenic. The spheroid cells were found to be enriched for cells with cancer stem cell-like characteristics such as upregulation of stem cell genes, self-renewal, high proliferative and differentiation potential, and high aldehyde dehydrogenase (ALDH) activity. Furthermore, spheroid cells were more aggressive in growth, migration, invasion, scratch recovery, clonogenic survival, anchorage-independent growth, and more resistant to chemotherapy in vitro. 13C-glucose metabolic studies revealed that spheroid cells route glucose predominantly to anaerobic glycolysis and pentose cycle to the detriment of re-routing glucose for anabolic purposes. These metabolic properties of sphere forming cells appear to confer increased resistance to apoptosis and contribute to more aggressive tumor growth. Collectively, we demonstrated that spheroid cells with cancer stem cell-like characteristics contributed to tumor generation, progression and chemotherapy resistance. This study provides insight into the relationship between tumor dissemination and metabolic attributes of human cancer stem cells and has clinical implications for cancer therapy.

Ovarian cancer stem cells express ROR1, which can be targeted for anti–cancer-stem-cell therapy

“…S2E). Consistent with previous reports (17,22), the cells within such spheroids were enriched for cells that had high levels of ALDH1 expression (Fig. S2F).…”

“…Furthermore, ovarian CSCs may express various surface antigens, such as CD44, CD117, or CD133 (7,8,(17)(18)(19). Functionally, human ovarian CSCs apparently can form nonadherent cellular spheres, called "spheroids," which in turn are enriched for ovarian cancer cells that have high ALDH1 activity, are relatively resistant to chemotherapeutic drugs, and have enhanced potential for seeding metastatic sites or engrafting immune-deficient mice (5,17,18,(20)(21)(22).…”

<em>In vitro</em> Enrichment of Ovarian Cancer Tumor-initiating Cells

“…Count viable cells using trypan blue assay. NOTE: Although this study compared tumorigenicity of TICs versus adherent cells for the first generation only, in vivo serial passaging of TICs grown under similar conditions has been completed by others 11,20 .…”

“…Several markers have been proposed to identify ovarian TICs with enhanced tumorigenicity including CD133, ALDH1A1, CD117, CD44, and MyD88, although the exact contribution of each marker is unclear and may be cell type specific [11][12][13][14][15][16] . While a universal marker or set of markers has not been unequivocally established for ovarian TICs, different groups have isolated ovarian TICs more commonly by selecting for CD44+, CD133+ and/or cells with high aldehyde dehydrogenase (ALDH) activity 13,[17][18][19][20][21] . CD44 is a transmembrane glycoprotein that acts as a receptor for hyaluronic acid and regulates several processes important for tumor progression, including adhesion, proliferation, migration, angiogenesis and differentiation 22 .…”

Hepatocyte selection medium-enriched hepatocellular carcinoma cells are positive for α-fetoprotein and CD44

“…In future studies, cells obtained from culture in HSM require propagation and investigation for cancer stem cell characteristics including analysis of cell-surface markers and spheroid formation assay (27,28).…”