(604a) Engineering Ovarian Cancer Spheroids and Analyses of Drug Resistance and Migration

Ovarian cancer is the most lethal gynecological disorder, with a fatality more than 70% within 5 years of diagnosis¹. High mortality rates are due to peritoneal metastasis facilitated by disseminated tumor spheroids within the peritoneal cavity². Spheroids within patient ascites vary in size from 50 to 750 mm as well as shape; loose aggregates or compact spheroids³. Moreover, epithelial-to-mesenchymal (EMT) markers are known to play role in spheroid formation and functionalities of spheroids like migration and invasion. We formed ovarian cancer spheroids by using ultra-low attachment technique with OVCAR-3 and OVCAR-8 cells with different initial cell numbers; 50-100-200-500 and 5000 cells. We characterized the surface area, circularity and solidity of the spheroids over 6 days. Then we analyzed the morphology of the spheroids as well as expression of EMT markers, E-cadherin and Vimentin by imaging and western blotting. OVCAR-3 cells formed loose aggregates and OVCAR-8 cells formed compact spheroids. We analyzed their morphology by using water dipping objective (Figure 1A), which provides a higher penetration depth than conventional confocal imaging. We analyzed solidity at day 6 (Figure 1B). Next, we analyzed the expression of E-cadherin and Vimentin by using western blotting (Figure 1C). OVCAR-3 spheroids were found positive for E-cadherin and negative for vimentin expression, on the other hand, OVCAR-8 spheroids were negative for E-cadherin and positive for Vimentin expression. We, then checked the cytotoxicity of clinically used Taxol and Cisplatin and found that anti-cancer drug resistance was independent from spheroid size. Lastly, OVCAR-8 spheroids were found to be nearly 15-folds more migratory than OVCAR-3 loose aggregates (Figure 1D). Our results provide an insight into the effects of spheroid size variation on anti-cancer drug resistance, and spheroid morphology on migratory profiles for in vitro studies⁴. Our next step is moving to larger spheroids (>500 mm) to understand the hypoxia formation and changes in the stem cell markers. We found that CD44, a cancer stem cell marker, is upregulated as spheroids become larger and increase in hypoxic region also increases the expression of CD44 in ovarian cancer spheroids.

References

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2. Al Habyan, S., Kalos, C., Szymborski, J. & McCaffrey, L. Multicellular detachment generates metastatic spheroids during intra-abdominal dissemination in epithelial ovarian cancer. Oncogene (2018) doi:10.1038/s41388-018-0317-x.
3. Casey, R. C. et al. Beta 1-integrins regulate the formation and adhesion of ovarian carcinoma multicellular spheroids. Am. J. Pathol. 159, 2071–2080 (2001).
4. Gunay, G. et al. The effects of size and shape of the ovarian cancer spheroids on the drug resistance and migration. Gynecologic Oncology S0090825820338579 (2020) doi:10.1016/j.ygyno.2020.09.002.