Adhesion and Beyond: CD44 in Ovarian Cancer Spheroids

Epithelial ovarian cancer is a leading cause of death from gynecological malignancies. Challenges in effectively treating patients with metastatic disease and preventing chemo-resistance can be attributed to the insufficient understanding of the biology and the mechanisms involved in ovarian cancer metastasis. Epithelial ovarian carcinoma (EOC) metastasis is characterized by the shedding of malignant cells from the surface of the primary tumor and their implantation onto the peritoneal surface lining the abdominal cavity in addition to more distant sites. This shedding of malignant cells from the primary site results as either single cells or free-floating multicellular aggregates, known as spheroids, in the ascites. Although single cells and spheroids may potentially seed metastases, considerable evidence now suggests that the aggregation of cells is important for anchorage-independent cell survival and growth, and spheroid formation may represent an important intermediate survival mechanism to facilitate EOC dissemination. This project, thus, aimed to characterize CD44s in EOC spheroids and its functional significance in epithelial ovarian carcinoma metastasis. Recent studies have uncovered human transmembrane cell adhesion molecule CD44 standard (CD44s) isoform expression correlating with high grade and advance-stage ovarian carcinoma. Moreover, it has also been suggested that CD44s may be an important mediator of ovarian cancer cell implantation in the peritoneal cavity enhancing the metastatic potential of ovarian cancer cells. Therefore, we investigated the functional significance of CD44 standard by means of in vitro culture experiments with ovarian cancer spheroids. After characterizing 8 EOC cell lines, a spectrum of CD44 expression was observed and we chose two cell lines with high CD44s (ES-2 and SKOV-3) and two cell lines low in CD44s (OVCAR-4 and OVSAHO) for further testing. Interestingly, the high CD44s-expressing cell lines formed larger spheroids that adhered significantly faster to a monolayer of primary mesothelial cells. Silencing of CD44 using CRISPR/Cas9 in these 2 cell lines reduced spheroid formation suggesting that differential expression of CD44s plays a role in cell-cell adhesion. When we i.p. injected ES-2 CD44-/- cells into athymic nude mice, decreased ascites production and mesentery tumor burden were observed as well as increased overall survival. However, CD44 knockout also significantly increased metastasis to the lung suggesting a suppressive role for CD44 in EOC distant metastasis, as well. This data implies that CD44 enhances the metastatic potential in ovarian cancer spheroids and regulates organ-specific dissemination of ovarian cancer cells, favoring peritoneal dissemination. However, high CD44 expression prevents metastatic dissemination to distant sites, including lungs, thereby limiting its use as a therapeutic target for disease management.