Abstract 6784: Cancer cell heterospheroids with stromal component used as drug testing and immune cell infiltration models
Authors: Nielsen Boye Schnack , Madsen Natasha H. , Larsen Jesper , Gad Monika , Holmstrøm Kim 
Published: March 2024

3D cancer cell cultures have enabled new opportunities for replacing compound testing in experimental animals. However, driven by increased understanding of the need for more complex cancer cell models that consider drug resistance and penetration, hypoxia, and immuno-oncology applications, we have developed multicellular tumor heterospheroids composed of cancer cell lines, fibroblasts and immune cells. We have studied heterospheroids of cancer cell lines, HT29, MCF7, PANC1 and SW480 co-cultured with fibroblasts and 1) explored the architectural organization, 2) performed whole transcriptome analysis (WTA) of mono and co-cultures and 3) tested their capacity of immune cell infiltration. The architectural organization was studied in embedded heterospheroids. HT29 and MCF7 cells developed spheroids with the cancer cells surrounding the fibroblasts, whereas PANC1 cells mingled with the fibroblasts and SW480 cells were surrounded by fibroblasts. The fibroblasts expressed FAP-α mRNA as determined by ISH as well as collagen-1 as determined by immunohistochemistry. The hypoxia marker, Ca-IX, and the apoptosis marker, cleaved-Caspase-3, were dominating in the central core of the spheroids but the Ca-IX and Caspase-3 expression was not coordinated. In addition, WTA performed on day-7 heterospheroids, showed upregulated LOX, indicating that hypoxia-related markers known from genuine cancers are upregulated also in heterospheroids. WTA of day-7 mono-cell cultured and hetero-cultured spheroids, showed abundant ECM-, EMT-, and fibrosis-related expression in heterospheroids, thus reflecting a representative tumor-like microenvironment in heterospheroids compared to mono-culture spheroids. In particular, PANC1 spheroids showed a strong EMT profile with abundant FOXC2, TGF-β and TWIST expression. Genes related to Akt/mTOR and EGF pathways were consistently expressed in the tested heterospheroids. Evaluation of the cytotoxicity of anti-cancer compounds was performed using both cell viability and apoptosis assays. Exposure of the NCI drug library composed of 166 cytotoxic compounds to the heterospheroids, interestingly, revealed a complex output with limited correlation between cytotoxic and apoptotic effects. A subset of drug candidates is currently being further examined. PBMC-derived immune cells infiltrate heterospheroids and their infiltration is strongly enhanced after activation. The T cells' ability to infiltrate heterospheroids of different cancer types may serve as models of cold and hot tumors. We conclude that the cancer cell lines determine the architectural structure of heterospheroids, that ECM development is a prevalent part of the heterospheroids, and that the different heterospheroids possess varying ability of immune cell infiltration.

Citation Format: Boye Schnack Nielsen, Natasha H. Madsen, Jesper Larsen, Monika Gad, Kim Holmstrøm. Cancer cell heterospheroids with stromal component used as drug testing and immune cell infiltration models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6784.