Abstract 4566: 3D cancer cell-fibroblast heterospheroids forming tumor-like microenvironments are valuable tools to study immune cell infiltration and as pre-clinical drug testing models

Abstract

Abstract 3D cancer cell cultures have enabled new opportunities for replacing compound testing in experimental animals and provide a basis for high content work-flows for drug testing. Here we present data obtained from 3D multicellular tumor spheroids (MCTS) composed of cancer cell lines, fibroblasts and macrophages. We cultured HT29 and SW480 CRC cell lines (as well as others) with fibroblasts and obtained heterospheroids that could be evaluated in cell viability and apoptosis assays, by flow cytometry and high content imaging (HCI). In addition, mature MCTS were embedded in paraffin and used for traditional biomarker characterization using immunohistochemistry and in situ hybridization. The cancer cell x fibroblast interactions promoted a tumor-like microenvironment that recruited monocytes isolated from primary donors. FACS analysis indicated that the infiltrating monocytes polarized into CD163-positive M2 macrophages in HT29 MCTS, and in pancreatic MIA PaCa-2 MCTS, using Pexidartinib and anti-CSF1R mAb, we found that monocyte infiltration was strongly CSF1R dependent. Preliminary data suggest that also infiltration of T cells into the heterospheroids is possible, thus making the models useful for the study of immune cell infiltration. Whole transcriptomic analysis of mono-cultures of SW480 cancer cells and fibroblast spheroids and co-cultures (MCTS) identified traditional cell-type-related transcripts as well as a group of de novo expressed genes induced by co-culturing, such as serpin peptidase inhibitor clade A1 (serpinA1) and inhibin-β A (INHBA). The NGS analysis as well as immunohistochemical staining also showed that SW480 expressed high levels of P-glycoprotein (P-Gp, MDR1) in contrast to the fibroblasts. On the other hand, the HT29 MCTS were negative for P-Gp. Histological analysis and the apoptosis assay showed that the fibroblasts were more sensitive to 5FU and cisplatin treatment than the cancer cells. We are currently testing if the P-Gp inhibitor, PSC-833, can re-sensitize the cancer cells to 5FU and cisplatin. In conclusion, we have developed 3D multicellular heterospheroids mimicking tumor-like microspheres that can be used for drug testing of traditional drug compounds, biologics and even immunotherapeutic agents. Citation Format: Boye Schnack Nielsen, Natasha H. Madsen, Jesper Larsen, Hjalte M. Larsen, Monika Gad, Kim Holmstrom. 3D cancer cell-fibroblast heterospheroids forming tumor-like microenvironments are valuable tools to study immune cell infiltration and as pre-clinical drug testing models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4566.