Phenotypic modifications in ovarian cancer stem cells following Paclitaxel treatment

Abstract

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. Despite initial responsiveness, 80% of EOC patients recur and present with chemoresistant and a more aggressive disease. This suggests an underlying biology that results in a modified recurrent disease, which is distinct from the primary tumor. Unfortunately, the management of recurrent EOC is similar to primary disease and does not parallel the molecular changes that may have occurred during the process of rebuilding the tumor. We describe the characterization of unique in vitro and in vivo ovarian cancer models to study the process of recurrence. The in vitro model consists of GFP+/CD44+/MyD88+ EOC stem cells and mCherry+/CD44−/MyD88− EOC cells. The in vivo model consists of mCherry+/CD44+/MyD88+ EOC cells injected intraperitoneally. Animals received four doses of Paclitaxel and response to treatment was monitored by in vivo imaging. Phenotype of primary and recurrent disease was characterized by quantitative polymerase chain reaction (qPCR) and Western blot analysis. Using the in vivo and in vitro models, we confirmed that chemotherapy enriched for CD44+/MyD88+ EOC stem cells. However, we observed that the surviving CD44+/MyD88+ EOC stem cells acquire a more aggressive phenotype characterized by chemoresistance and migratory potential. Our results highlight the mechanisms that may explain the phenotypic heterogeneity of recurrent EOC and emphasize the significant plasticity of ovarian cancer stem cells. The significance of our findings is the possibility of developing new venues to target the surviving CD44+/MyD88+ EOC stem cells as part of maintenance therapy and therefore preventing recurrence and metastasis, which are the main causes of mortality in patients with ovarian cancer.

TRX-E-002-1 Induces c-Jun–Dependent Apoptosis in Ovarian Cancer Stem Cells and Prevents Recurrence In Vivo

“…Proliferation was calculated as described previously (28,34,35) using metrics from either confluence or fluorescent count from nuclear-restricted RFP or GFP. Cell death was quantified similarly using CellTox Green Cytotoxicity Assay (Promega).…”

“…Intraperitoneal tumors were established in athymic nude mice using 4 Â 10 6 mCherry þ OCSC1-F2 ovarian cancer cells as described previously (35). Injection of cancer cells is designated as day 0, and treatment commenced between days 3 to 5.…”

“…TRX-E-002-1 prevents OCSC recurrence in vitro One of the main characteristics of OCSCs is their ability to overcome the cytotoxic effect of chemotherapeutic drugs even after 24 hours of treatment in vitro (35). To determine whether the cytotoxic effect of TRX-E-002-1 is sustained even after its removal, we treated OCSC2 with equivalent doses of TRX-E-002-1 or cisplatin for 24 hours and then removed the treatment allowing the culture to recover in growth media for another 48 hours.…”

“…This allows the detection of intraperitoneal tumors and real-time measurement of response during the course of treatment (35)(36)(37). This model recapitulates the clinical profile typically seen in patients with ovarian cancer and allows us to answer three distinct questions: (i) in vivo activity of TRX-E-002-1 against primary disease; (ii) ability of TRX-E-002-1 given in combination with chemotherapy to prevent/delay recurrence; and (iii) ability of TRX-E-002-1 given as maintenance after chemotherapy to prevent/delay recurrence.…”

“…As such, carcinomatosis is always observed and more importantly, although animals initially respond to paclitaxel, with some exhibiting complete response (i.e., no visible mCherry fluorescence detected by imaging), tumors eventually recur once paclitaxel treatment is terminated. Upon recurrence, tumors acquire resistance and become unresponsive to a second round of paclitaxel regimen (46), as observed in ovarian cancer patients. Therefore, the model allows us to assess the value of novel compounds in preventing recurrence when given as maintenance treatment.…”