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.

Alarm anti-proteases are secreted locally in response to inflammation and have been shown to be elevated in cancers. Secretory leukocyte protease inhibitor (SLPI), an alarm anti-protease, is amplified in ovarian carcinoma and is induced and binds to and protects progranulin (prgn) in inflammation. We reported prgn is a survival protein in ovarian cancer and now hypothesize that SLPI/prgn would promote proliferation and survival. Neutralizing anti-SLPI antibody treatment of HEY-A8 and OVCAR3 ovarian cancer cells decreased cell number (P < 0.001), induced apoptosis and reduced prgn quantity. This was confirmed using SLPI small interfering RNA. Prgn and SLPI were co-immunoprecipitated and co-localized by confocal microscopy. Prgn is a substrate of the serine protease elastase and SLPI is an inhibitor of elastase. Elastase reduced prgn expression, inhibited proliferation in a dose-dependent manner (P

The alarm anti-protease secretory leukocyte protease inhibitor (SLPI) is frequently overexpressed in ovarian cancer cells and has been proposed for inclusion in biomarker panels but function remains unclear. We hypothesized that SLPI overexpression promotes ovarian cancer growth and survival. Low SLPI-expressing Hey-A8 ovarian cancer cells were engineered to produce functional (WT) or protease inhibitor-null (PI-) mutant SLPI; lack of PI activity was confirmed by enzymatic assay. WT/SLPI and PI-mutants stimulated significant proliferation and survival of Hey-A8 ovarian cancer cells under basal culture conditions (P £ 0.02), in soft agar colony number and size (P £ 0.05), and in anoikis resistance (P £ 0.005). SLPI protected the ovarian cancer survival factor, progranulin (PRGN), and HEY-A8 cells from degradation and apoptosis due to neutrophil elastase. PI-/SLPI cells had greater protective activity than WT/SLPI cells. HEY-A8 murine xenografts revealed enhanced solid tumor formation, dissemination, and invasion in WT/SLPI and PI-/SLPI mutants. (1) Modest improvements in survival of ovarian cancer patients have been achieved over the last decade, driving a need for a better understanding of the mechanisms underlying ovarian cancer formation and progression. Secretory leukocyte protease inhibitor (SLPI) has been reported as one of the most commonly amplified genes in epithelial ovarian cancer, (2)(3)(4)(5) an observation that is explained in part by genomic amplification of the SLPI locus, 20q12-13. (6) Significantly elevated serum concentrations of SLPI have been found in ovarian cancer patients compared to healthy women or women with benign ovarian cysts, leading to the inclusion of SLPI in some experimental ovarian cancer biomarker panels. (7) Secretory leukocyte protease inhibitor is a secreted pleiotropic protein with tissue-protective, regenerative, and anti-inflammatory properties. (8) It protects normal mucosal tissues against detrimental proteolytic actions of inflammatory serine proteases, such as neutrophil elastase, through direct inhibition of these enzymes. (9,10) Evidence also points toward direct roles for SLPI in tumor development and progression. (2,11,12) Some of these studies are conflicting depending upon the model used, suggesting context dependence for SLPI function. SLPI was reported to both stimulate and counteract tumor formation, invasion and/or metastasis of cancer cells. (10,(12)(13)(14)(15) The role of the anti-protease properties of SLPI in cancer remains under investigation.Progranulin (PRGN) is a potent growth and survival factor for both normal and cancer cells. (16)(17)(18)(19)(20)(21) Mutations in PRGN are associated with frontotemporal dementia and other neuromuscular degenerative syndromes, (22,23) consistent with our reported prosurvival role. We have shown that anti-sense silencing of PRGN in Hey-A8 ovarian cancer cells reduced in vitro proliferation rates and soft agar colonization. (16) Treatment of ovarian cancer cell lines with neutralizing anti-PRGN antibodies induced a...