Abstract LB185: Lipid nanoparticle screening in 2D vs 3D cultures: Ovarian cancer cell lines exhibit different preference for optimal lipid formulation in monolayers vs spheroids

Abstract Ovarian cancer is a complex disease with various subtypes and diverse molecular mechanisms of pathogenesis. Chemoresistance is a common challenge in ovarian tumors, where inherent resistance or adaptive changes in the tumor microenvironment may antagonize the effects of chemotherapy through various mechanisms such as promoting metastasis or inhibition of immune cell activation. Lipid nanoparticle (LNP) mediated co-administration of RNA with chemotherapy drugs presents an opportunity to modify protein expression within the tumor microenvironment and enhance chemosensitivity. siRNA-mediated knockdown of chemo resistance genes or intratumoral administration of mRNAs encoding immunostimulatory proteins are two such examples. LNP efficacy is contingent upon effective uptake into the target tissue and subsequent cargo release from endosomes. These two processes vary significantly depending on the nature of the cargo, LNP lipid composition and target tissue; the ability to screen different lipid formulations and cargo types against tumors in vitro may be a powerful tool in development of new therapies. In vitro cell-based screening methods are valuable tools for assessing cytotoxicity of candidate drugs, however the tumor microenvironment differs starkly from conventional two-dimensional culture. Heterogeneity of substrate availability, oxygen tension and pH are all defining characteristics of tumors and these factors may also contribute to altered responses to lipid nanoparticle formulations. In this study, we compared LNP-mediated cargo uptake & expression in three different ovarian cancer cell lines cultured in 2D monolayers or 3D spheroids to investigate whether efficacy in 2D culture is recapitulated in the spheroid model. SK-OV-3, PA-1 & CaOV-3 cells were grown in monolayers and 3D spheroids and transfected with four different LNP formulations containing the ionizable lipids SM-102, ALC-0315, C12-200 or DLin-MC3-DMA. Empty pre-formed loadable LNPs were loaded with mCherry mRNA, GFP DNA or Cy5-labelled 2’3’cGAMP and expression/uptake in each model was determined via fluorescence imaging. BODIPY-cholesterol labelled versions of each LNP exhibited similar dose-dependent uptake into monolayers & spheroids independent of ionizable lipid formulation. In contrast cargo expression varied substantially across conditions in cell, cargo and culture model-dependent manners. SM-102 exhibited superior efficacy at mRNA expression in monolayer cultures across all three cell types, while C12-200 generally led to the highest DNA expression in monolayer cultures. Notably the optimal LNP formulation for cargo expression in spheroids frequently differed from 2d culture: SK-OV-3 spheroids exhibited optimal mCherry mRNA expression with C12-200 while GFP DNA expression was highest with SM-102. These results highlight the importance of 3D models for lipid nanoparticle formulation testing and demonstrate the utility of preformed loadable LNP screens in early formulation development. Citation Format: David Taylor, Valerie Forsyth, Caleb Riddering, Aileen R. Lipid nanoparticle screening in 2D vs 3D cultures: Ovarian cancer cell lines exhibit different preference for optimal lipid formulation in monolayers vs spheroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB185.