Abstract A122: Deciphering macrophage targeting cancer immunotherapies using a novel in vitro assay modelling the tumor microenvironment
Authors: Turner Darryl , Dobie Ross , Rzepecka Justyna , Gooding Hayley , Bingham Matilda 
Published: December 2023

Background: Macrophages are one of the major infiltrating immune cells in the tumor microenvironment and have been proposed as a primary driver of epithelial to mesenchymal transition (EMT) through interactions with tumour cells. These interactions play a key role in tumor progression, metastasis and fibrosis, processes which are common to all types of cancer. Understandably therefore, targeting macrophages is an attractive approach for treating wide range of tumors including those resistant to checkpoint inhibitors. Evaluation of cancer therapeutics is hampered by a lack of suitable in vitro models which closely recapitulate the complexity of tumor microenvironment. We addressed this challenge by developing a model system with potential to faithfully represent the key molecular interactions occurring between tumor associated macrophages and cancer cells, driving the latter to become invasive and metastatic. In order to demonstrate suitability of the assay, galunisertib, a TGFbRI/ALK5 inhibitor was used to block one of pathways that has been evaluated clinically in cancer patients.

Methods:  Human macrophages were differentiated using key growth factors prior to polarisation with various cytokines to generate different putative macrophage phenotypes as evidenced by differences in cytokine secretion, gene transcription and protein expression. Epithelial cells were stimulated with macrophage conditioned medium in a scratch wound assay (Incucyte) and analysed by immunocytochemistry.

Results: Macrophages derived supernatants were shown to drive EMT, both in migratory capacity and EMT associated protein expression. Our results demonstrate that whilst galunisertib could potently inhibit TGF-b driven EMT it was only partly capable of reversing the effects associated with addition of macrophage conditioned media.

Conclusions: The data show that macrophages can directly drive the EMT process in vitro via multiple mechanisms. One such mechanism is through the secretion of TGF-b. Indeed, blockade of TGF-b signalling appears to be a tangible approach in cancer immunotherapy, but to date this approach has delivered mixed results for clinical efficacy as a monotherapy. By way of example, development of galunisertib has recently been halted. As shown herein, macrophages effects on cancer migration and metastasis span beyond TGF-b only effects. We offer a potential explanation for why single therapies such as galunisertib alone are ineffective in cancer treatment. The described model provides a powerful tool to researchers to better understand and replicate in vitro the complex interactions occurring in the tumor microenvironment. Thus, enabling the identification of molecular targets and combination therapies with potential to block cancer cell migration and metastasis and to evaluate candidate drug efficacy.

Citation Format: Darryl Turner, Ross Dobie, Justyna Rzepecka, Hayley Gooding, Matilda Bingham. Deciphering macrophage targeting cancer immunotherapies using a novel in vitro assay modelling the tumor microenvironment [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A122.