Lipid Metabolic Modulation to Increase Car-T Cell Attack Efficacy on Ovarian Cancer

Introduction: Ovarian cancer is one of the most lethal gynecological cancers in the world and has one of the highest recurrence rates. Recent advances and development in Chimeric Antigen Receptor T- cell (CAR-T) therapy holds much promise in cancer treatment, but its efficacy in solid tumors like ovarian cancer is hindered by challenges such as antigen escape and CAR-T cell exhaustion. This study aims to explore the metabolic changes during ovarian cancer and CAR-T cell interactions and identify the therapeutic target to increase the CAR-T cell mediated cancer cell killing efficacy. Here we showcase the application of the recently developed label-free high-content stimulated Raman imaging to uncover dynamic metabolic changes in ovarian cancer cells during CAR-T cell attack. Furthermore, we show employment of cholesterol biomarker-guided use of Avasimibe treatment for metabolic modulation, enhancing CAR-T cell cytotoxic efficacy.

Materials and

Methods: We employ high-content Stimulated Raman Scattering (SRS) imaging to analyze cells, which is hyperspectral SRS (hSRS) imaging followed by spectral unmixing. We use hSRS imaging and acquire hyperspectral stacks of SKOV3 (Ovarian Cancer) cells with and without the presence of HER2+ CAR-T cells. We then perform spectral unmixing using LASSO (Least Absolute Shrinkage and Selection Operator) algorithm to decompose the stacks into chemical maps of proteins, lipids, cholesterol, nucleic acids and analyse these maps for any statistical significance. Based on these analyses, then we use Avasimibe to treat the SKOV3 and CAR-T cells before coculturing them together to investigate any changes in the CAR-T mediated killing of the SKOV3 cells including cytotoxicity analysis, cytokines secretion panels, metabolism changes using fluorescence plate reader, flow cytometry and our SRS imaging system.

Results, Conclusions, and Discussions: Results – We generated proteins, lipids, cholesterol, and nucleic acids chemical maps of hSRS images acquired of the SKOV3 cells with and without HER2+ CAR-T cells. We performed individual cell analysis of the cholesterol and lipids signals relative to the protein signal to identify upregulation of free cholesterol in the SKOV3 cells when under CAR-T cell attack demonstrating that the cell is under stress. This cholesterol upregulation is also accompanied with lipid upregulation which is marker of cell response for migration. Cholesterol esterification inhibition in other cancers leads to free cholesterol accumulation leading to Endoplasmic Reticulum stress and apoptosis. So, using Avasimibe treatment of SKOV3 cells we notice that these cells become more susceptible to CAR-T killing. We also see that Avasimibe treatment of both the SKOV3 and CAR-T cells before coculture shows an increase in the SKOV3 killing. We however see very minimal difference in the activation of CAR-T cells under the Avasimibe treatment.
Conclusions – We successfully used high-content SRS imaging to demonstrate the cholesterol and lipid regulation in SKOV3 cells under CAR-T attack. Using the cholesterol upregulation as a therapeutic target, we employed Avasimibe treatment of SKOV3 cells and CAR-T cells to improve the CAR-T cell mediated killing efficacy. This study demonstrates the promise of our high-content SRS imaging tool and its application in immunometabolism studies and shows that drug interventions can be used to improve CAR-T cell killing efficacy in solid tumors.
Discussions – Using High-content SRS imaging, we demonstrated the cholesterol upregulation in the SKOV3 cells cocultured with CAR-T cells and used Avasimibe to inhibit cholesteryl esterification causing ER stress in the SKOV3 cells making them susceptible to CAR-T killing. Confirming this, we further plan to implement this study in-vivo to study the impact of Avasimibe treatment followed by CAR-T therapy on tumors growth. High-content SRS imaging is excellent to study immunometabolism especially label-free at single cell level. We can explore multiple chemical maps to study changes in the metabolism to identify therapeutic targets in the CAR-T therapy. These biomarker-based drug interventions then can be employed to increase the CAR-T therapy impact in solid tumors.

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