Poster T1 - Targeting Metabolism in Paclitaxel Driven Chemoresistance in High Grade Serous Ovarian Carcinoma

Introduction: High grade serous ovarian carcinoma (HGSOC) is the most predominant and lethal subtype of epithelial ovarian cancer (EOC). It accounts for 70% of EOC incidence, 70-80% of EOC death, and a 5-year survival rate less than 30%. HGSOC patients present with a diverse set of non-specific symptoms making it difficult to diagnose accurately and early. Once symptoms are seen there is a 75-80% chance of HGSOC already at an advanced stage (Lisio, 2019; Shepard, 2022). For those with HGSOC, the gold standard of treatment is through surgical debulking and chemotherapy (Pokhriyal, 2019). Paclitaxel (PTX) is one of the most used chemotherapeutics in HGSOC patients. PTX is highly effective in killing rapidly dividing cancer cells by triggering the activation of the mitotic checkpoint leading to cell cycle arrest and apoptosis in cancer cells (Weaver, 2014). However, resistance to chemotherapeutic agents is common in HGSOC. Early-stage patients have a 25% chance of developing chemoresistance, which rises to 80% in late-stage patients. The lack of an anatomical barrier between the pelvis and abdomen aids in the ease of metastatic implementation and invasion, as well as a higher chance of chemoresistance development (Pokhriyal, 2019; Kaur, 2019). A subpopulation of cells in HGSOC, polyploidal giant cancer cells (PGCCs), are large, multi-nucleated cells that form under therapeutic stress, give rise to a chemoresistant subpopulation, contribute to tumor heterogeneity, and create genomic instability (Bowers, 2022; Richards, 2021). Thus, it is important to understand why this chemoresistance develops in order to improve HGSOC chemotherapy and progression-free survival.

Materials and

Methods: Cell Culture: The HGSOC cell line OVCAR-8 was cultured in RPMI-1640 supplemented with 10% fetal bovine serum and 1% penicillin streptomycin, and maintained at 37 degrees C and 5% carbon dioxide. PTX-resistant (TR) cells were derived from the OVCAR-8 parent cell line after 3-4 months of weekly treatments with 50 nM PTX for 72 hours allowing intermediate recovery periods in between.

Viability assays: Treatment resistance was confirmed through MTT assays with varying concentrations of PTX on the PTX-sensitive (TS) and TR cell lines. Absorbance was measured at 590 nm using Varioskan Lux plate reader.

Immunostaining: All cells were fixed before immunostaining. Cytoskeletal structure was investigated through staining for actin, tubulin and vimentin. Adhesion was examined through staining for β1 integrin and vinculin. Nuclei were stained with Hoescht-33342.

Imaging: All images used in the data presented in this abstract were acquired through confocal microscopy on the Olympus Yokogawa Spinning Disk Live Cell Microscopy system at 100X magnification (150.1 um x 150.1 um, 0.065 um/pixel).

Data Analysis and Processing: Nuclear area and cytoskeletal density measurements were computed using FIJI (Schindelin, 2012). The FIJI plugin, StarDist (Schmidt, 2018), was adapted to measure nuclear area and form factor, while a custom FIJI macro was used to examine the cytoskeletal density values across the image. Graphs were made in GraphPad Prism. Statistical analysis was carried using a 2-way analysis of variance, considering p< 0.05 to be statistically significant (****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05).

Results, Conclusions, and Discussions:

Results: TS and TR cells displayed IC50 values 1.86 and 54.06 nM, respectively – an approximate 30-fold difference (Figure 1a). Similar nuclear and microtubule structure are seen in the DMSO control, yet PTX-treatment induces microtubule disruption and nuclear deformation seen through fractured and multi-nucleated nuclei in TS cells. The structure of TR cells remains unaffected with PTX-treatment compared to the DMSO control (Figure 1b-d). No statistically significant differences were found in actin and vimentin cytoskeletal density between TS and TR cells. TS cells demonstrate higher β1 integrin expression than TR cells. Upon PTX-treatment, β1 integrin levels in TS cells drop below the level seen in TR cells, while β1 integrin levels in TR cells are unaffected (Figure 1e). Collectively, our data suggests TR cells possess a more stable cell and nuclear structure than TS cells, which likely contributes to their resistant phenotype.

Conclusions: Our current findings demonstrate we have an in vitro model able to recapitulate PTX-treatment resistance based on viability assay data and immunofluorescent staining of cellular nuclei and microtubule structure. PTX-treatment in TS cells induces nuclear multinucleation and a decrease in β1 integrin expression suggesting PTX treatment is altering cellular adhesion in TS cells, but not in TR cells. Preliminary metabolomic results (data not shown) and visual assessment of TR proliferation rates suggest TR cells display lower metabolic function than TS cells, which may highlight a mechanism of PTX resistance.

Discussion: Future viability assays will be performed to determine if TR cells are also resistant to platinum and/or combined treatment. The presence of PGCCs only in TS cells will need to be further explored to see if PTX-treatment causes PGCC development or selects for naive PGCCs in the untreated population. TS and TR proliferation rates will be calculated, as well as metabolic activity, to validate our hypothesis that TR cells proliferate slowly to avoid cellular death upon PTX-treatment. However, after PTX-treatment, TR cells proliferate more rapidly than TS cells, which slowly recover. Unchanged levels of β1 integrin and the pause in cellular proliferation support this theory and demonstrate a high metastatic potential for TR cells.

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