Poster S12 - Abnormal Nuclear Structure and Metabolism of Ovarian Cancer Polyploid Giant Cancer Cells

Introduction: Dysregulated lipid metabolism is a hallmark of epithelial ovarian cancer (EOC) (Chaudhry, 2022). This deadly gynecological malignancy is associated with widespread and rapid metastasis of cells from the primary EOC tumor into the abdominal cavity, which is rich in bioactive lipids and fat cells. Invasive cells adapt to metabolic and treatment stress by activating lipid transport pathways that allow for the uptake and processing of exogenous lipids to form cytosolic lipid droplets. These lipid droplets provide a rich energy source for nutrient deprived cells and sequester cytotoxic lipids and drugs to limit their toxicity (Chaudhry, 2022). Polyploid giant cancer cells (PGCCs) represent a novel and understudied subpopulation of drug-resistant cancer cells that are often found in late stage tumors (Richards, 2021; Zhang, 2023). PGCCs have increased nutrient demands, in part to their larger size and more invasive phenotype, and undergo metabolic reprogramming in response to stress, which may allow them to use lipid metabolism for energy production. Previous work has shown that some cancer cells can use lipophagy, where cells take up lipids from their environment, store them in lipid droplets, and later catabolize them for energy production, to increase their chances of survival under metabolic stress (Kostecka, 2021). The goal of our study is to investigate PGCC metabolism. We hypothesize that PGCCs’ ability to use lipid metabolism and lipophagy for energy production is directly linked to their highly resistant phenotype.

Materials and

Methods: Cell culture and PTX treatment: We cultured high grade serous ovarian cancer Kuramochi cell line in RPMI 1640 supplemented with 10% FBS and 1% penicillin-streptomycin. Cells were maintained at 37°C and 5% carbon dioxide in ambient oxygen (normoxia) or 2% oxygen (hypoxia). To enrich for PGCCs, we treated cells for 72 hours with 500nM paclitaxel (PTX) before returning to normal growth media.
Epifluorescent imaging: Cells were fixed and stained with Hoechst and imaged with a Nikon Eclipse Ti inverted fluorescence microscope at 10x magnification.
Metabolic terturbation and lipid content measurement: Kuramochi PGCCs were treated with or without lipid mixture (1:100) in media for 24 hours, then treated overnight with serum free (SF) media or EBSS to induce autophagy. Cells were then stained with BODIPY™ 493/503 for 45 min, and fluorescence was measured using a Thermo Fisher Varioskan Lux plate reader.
Metabolic perturbation and immunofluorescence staining: Kuramochi PGCCs were seeded on glass coverslips. They were treated with lipid mix (1:100), then fixed and stained for LAMP1, TOMM20, Hoechst 33342 and BODIPY™ 493/503. Coverslips were imaged with Olympus Yokogawa Spinning Disk Confocal Microscope at 60X magnification.
Epifluorescent Image Analysis: Nuclear physical properties were analyzed using FIJI software (Schindelin, 2012). PGCCs were characterized as having at least 2.5x the mean nuclear size of naive cells (Xuan, 2020).

Results, Conclusions, and Discussions: In this study, we use PTX treatment to enrich for resistant PGCCs and investigate differences in PGCC formation and senescence escape in ambient oxygen (normoxia) and reduced oxygen (hypoxia) conditions. We also observe lipid droplet accumulation in high lipid and low nutrient environments.
In hypoxia and normoxia, cells reach similar peak PGCC percentages. Within our study timespan, hypoxia group recovers faster, based on decreased PGCC percentage and increased cell density after the peak (Fig. 1A), likely attributed to PGCCs’ ability to escape senescence and undergo amitotic budding to form proliferative daughter cells. Cells in normoxia show a similar trend, but starts recovering later than in hypoxia condition.
PGCCs form through various mechanisms, distinguishable by the resulting nuclear structures. Endocycle formation often produces multilobular nuclei with grape-like cluster morphology; endomitosis often results in mononucleated cells with enlarged nuclear morphology; fusion often produces PGCCs containing separate nuclei of different sizes (Zhou, 2022). These structures are all observed in Kuramochi PGCCs, indicating they likely form through a combination of these mechanisms (Fig. 1B-i-iii). We also visually observe daughter cell colonies near PGCCs at later time points, indicating amitotic budding has occurred (Fig. 1B-iv).
We treated PGCCs with lipid mix to simulate a lipid-rich microenvironment, and measured increased cytosolic lipid content, suggesting that exogenous lipids were uptaken by cells and stored. When cells were cultured in nutrient-deficient media (EBSS), lipid content decreased, suggesting that nutrient deprivation leads to catabolism of cytosolic lipids (Fig. 1C). Visually, lipid mix-supplemented PGCCs store more lipids as droplets compared to control cells, whereas regular media induced very few lipid droplets in either population (Fig. 1D).
These studies investigate the abnormal nuclear structure and lipid metabolism of ovarian cancer PGCCs. We plan to further probe lipophagy mechanistically, by treating cells with metabolic inhibitors and analyzing changes in their metabolic and autophagic network. We will also inspect the cytoskeletal structures of PGCCs in an attempt to connect their physical and metabolic properties. Understanding these unique properties of PGCCs can potentially contribute to the development of new treatment strategies for targeting chemoresistant cells important in tumor recurrence after therapy.

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