Understanding Chemoresistance in High-Grade Serous Carcinoma

<p dir="ltr">High-grade serous carcinoma (HGSC), accounting for approximately 90% of all epithelial ovarian cancer cases, is the most lethal gynaecological malignancy due to the lack of early diagnosis and acquired drug resistance. HGSC primarily spreads through the peritoneal cavity partly via cancer spheroids, which derive from cellular aggregates of tumor cells that have detached from the primary sites and became suspended the in peritoneal fluid (ascites); these cancer spheroids are known to also play important roles in HGSC chemoresistance, but the molecular mechanisms are not well understood. High expression of podocalyxin (PODXL), a large transmembrane protein, has been associated with a significant decrease in disease-free survival in HGSC patients, however, the underlying reasons remain unclear. Recent studies by our laboratory and others have discovered an important intrinsic function of PODXL in safeguarding barrier functions in epithelial and endothelial cells. It was thus hypothesized that the barrier protecting function of PODXL may play an important role in enabling HGSC cells to form robust and compact cancer spheroids that are able to evade chemotherapy and immune surveillance. Therefore, removing/lowering PODXL may sensitize these spheroids to chemotherapy drugs and immune cells. This thesis aimed to provide experimental evidence to support or refute these hypotheses. </p><p dir="ltr">The first aim of this thesis, Chapter 2, investigated the functional importance of PODXL in promoting chemoresistance of HGSC spheroids. The study first confirmed PODXL expression in HGSC patient tissues (n=17) and various ovarian cancer cell lines (a total of 28 different lines). Next, Kuramochi cells, a HGSC cell line expressing the highest level of PODXL among HGSC cancer cell lines, were used as a model and spheroid characteristics were investigated by knockout (KO) of PODXL via CRISPR/Cas9 gene editing; key features observed here were then examined in primary cancer cells derived from ascites of HGSC patients. No or low PODXL expression impacted spheroid characteristics by significantly reducing their compactness while increasing fragility; importantly these spheroids were more sensitive to the chemotherapy drug carboplatin, resulting in fewer live and proliferating cells following exposure to carboplatin, whereas spheroids with high PODXL showed greater survival against carboplatin. These data thus provided in vitro evidence that PODXL promotes the formation of compact and chemoresistant HGSC spheroids. </p><p dir="ltr">The second aim of this thesis, Chapter 3, investigated whether PODXL may also influence HGSC spheroid susceptibility to NK cell infiltration and cytotoxicity, using control/PODXL-KO Kuramochi cells as well as ascites-derived primary HGSC cells. HGSC spheroids were co-cultured with primary NK cells isolated from human peripheral blood mononuclear cells and the impact on the spheroids were assessed over time. Following co-culture with NK cells, spheroids expressing no or lower levels of PODXL endured more NK cell infiltration and cytotoxicity, while spheroids with higher PODXL were more resistant to NK cell infiltration and showed higher proliferation. These data suggest that PODXL may also play an important role in aiding immune evasion of HGSC spheroids, at least partly by conferring resistance to NK cell infiltration and the related cytotoxicity. </p><p dir="ltr">The third aim of this thesis, Chapter 4, explored clinically applicable approaches that may down-regulate PODXL to sensitize HGSC spheroids to carboplatin and NK cell infiltration. Progesterone, a steroid hormone widely used as a medication in women’s health, was recently reported to down-regulate PODXL in endometrial epithelial cells. This study thus utilized the Kuramochi cell line (wildtype only) as a HGSC model and examined whether progesterone could lower PODXL to make Kuramochi spheroids more vulnerable to chemotherapy drugs and infiltration by NK cells. Progesterone was found to be able to significantly reduce PODXL in Kuramochi cells, and Kuramochi spheroids that were pre-treated with progesterone were more sensitive to carboplatin. Furthermore, progesterone-treated spheroids also exhibited higher NK cell infiltration and worse cytotoxicity than control spheroids. These in vitro data thus advocate the potential utility of progesterone as an adjunct treatment to improve clinical outcomes of HGSC expressing high levels of PODXL. </p><p dir="ltr">Lastly, the fourth aim, Chapter 5, initiated studies towards understanding the molecular mechanisms underpinning the above observed role of PODXL in HGSC by RNAseq analysis of control and PODXL-KO Kuramochi cells. Preliminary analysis of the RNAseq data indicated several differently expressed genes that are involved in cell-cell junction, cell proliferation and drug resistance which may partly explain the spheroid characteristics observed throughout the studies of this thesis, but further research is needed to complete this line of enquiry. </p><p dir="ltr">In summary, this thesis provides new evidence that high PODXL expression in HGSC correlates with the formation of compact and hardy cancer spheroids, and that removal/lowering of PODXL can increase HGSC spheroid sensitivity to carboplatin and NK cells. Furthermore, using Kuramochi cell line as a model, the study indicates that progesterone may lower PODXL levels in HGSC to render cancer spheroids more vulnerable to carboplatin and NK cells, suggesting the potential benefit of progesterone as an adjunct medication to improve clinical outcomes of some HGSC patients.</p>