Abstract 6766: Assessing the impact of immunotherapies and cell therapies in 3D heterospheroid models mimicking tumor microenvironments
Authors: Madsen Natasha Helleberg , Nielsen Boye Schnack , Holmstrøm Kim , Olofsson Gitte , thor Straten Per , Skov Søren , Rodriguez-Pardo Carlos , Ormhøj Maria , Larsen Jesper , Gad Monika 
Published: March 2024

Abstract
Immunotherapies and cell therapies show substantial potential in the treatment of cancer. However, their efficacy within the complex microenvironment of solid tumors remains a critical area of investigation. In this study, we employ physiologically relevant 3D multicellular heterospheroid models to quantitatively assess the impact of these therapies on immune and tumor cells residing in tumor-like microenvironments. The spheroids are composed of cancer cell lines, fibroblasts and various primary immune cell types matched to the specific target of immunotherapy.
We have previously reported that interactions between cancer cells and fibroblasts promote a tumor-like environment, recruiting and polarizing monocytes into M2 macrophages. Treatment of spheroids with immunomodulating compounds successfully reprogrammed the macrophages into M1-like macrophages with increased pro-inflammatory cytokine secretion.
To mimic tumor-specific T cell responses observed in cancer patients, we here educated T cells to recognize target cell antigens through a 14-day co-culture of PBMCs and PANC-1 tumor cells. Subsequent re-challenge with PANC-1 heterospheroids revealed 70% specific killing by educated T cells, whereas T cells educated with a breast cancer cell line did not exhibit this targeted cytotoxicity. FACS analysis demonstrated increased expression of activation markers and immune checkpoint molecules such as PD-1, LAG-3 and TIGIT on educated T cells. Furthermore, we observed infiltration of educated CD4+ and CD8+ T cells into heterospheroids by immunohistochemistry, indicating the replication of a tumor-specific response within solid tumors. We are currently investigating the enhancement of educated T cell killing using various immune checkpoint inhibitors including antibodies targeting PD-1, TIGIT and LAG-3. Another means of eliciting tumor-specific responses is by employing CAR T cells. Antigen-specific CAR-T cells or untransduced CAR-T cells derived from the same donor were added to HT-29 and HCT-116 heterospheroids at various target-to-effector ratios. By using cancer cells genetically engineered to express a luciferase enzyme, we assessed the specific killing of cancer cells within the heterospheroids. Analysis showed a clear dose-dependent increase in luminescence, indicating greater antigen-specific CAR-T cell killing potential (60-80% specific lysis) compared to the non-specific killing observed with untransduced CAR-T cells (&lt;5% specific lysis). Additionally, preliminary data demonstrate the killing of heterospheroids by γδ T cells and that this killing is enhanced by immune checkpoint inhibitors.
In conclusion, we have developed 3D heterospheroids from various cancer types that faithfully mimic tumor-like microenvironments, enabling us to evaluate the effects of immunotherapies and cell therapies in physiologically relevant models.
Citation Format: Natasha Helleberg Madsen, Boye Schnack Nielsen, Kim Holmstrøm, Gitte Olofsson, Per thor Straten, Søren Skov, Carlos Rodriguez-Pardo, Maria Ormhøj, Jesper Larsen, Monika Gad. Assessing the impact of immunotherapies and cell therapies in 3D heterospheroid models mimicking tumor microenvironments [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 6766.