301 Novel microfluidic-based approach to study CAR-T cell trafficking into solid tumours and cytotoxicity evaluation

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Background Chimeric Antigen Receptor T (CAR-T) cell therapy has demonstrated remarkable success in treating haematological malignancies, yet its efficacy in solid tumours remains limited due to the challenging tumour microenvironment. 1 This reduced effectiveness is largely attributed to physical infiltration barriers, as well as to the nutrient-deprived and hypoxic conditions within the tumour niche.2 While biochemical factors have been extensively studied, the role of mechanical cues remains underexplored. To overcome this limitation, it is essential to investigate these mechanical mechanisms in physiologically relevant models to gain deeper insights into CAR-T cell functionality in solid tumours.Methods To replicate the biomechanical characteristics of solid tumours, we employed an innovative approach using microfluidic devices designed to mimic the tumour microenvironment. 3 These platforms were fabricated from polydimethylsiloxane (PDMS), a biocompatible silicone selected for its transparency, flexibility, and gas permeability.4 CAR-T cells were seeded onto the microdevices and visualised via time-lapse microscopy for later processing and analysis using ImageJ and MATLAB software. CAR-T cell efficacy was further evaluated using transwell assays that simulated the spatial constraints and mechanical stress conditions typical of solid tumour tissues. These experiments were analysed via resazurin assay using a microplate reader in the corresponding emission range.Results We found that CAR-T cells present significant decrease in migration velocity and number of migrating cells under hypoglycaemic and hypoxic conditions - prevalent features of the tumour microenvironment. Furthermore, transwell migration experiments demonstrated that these immunotherapy cells show altered efficacy after undergoing physical stimuli. Therefore, these biomechanical constraints play a decisive role in immunotherapy against solid tumours.Conclusions Our results highlight that CAR-T cell migration, strongly related to correct infiltration in solid neoplasms, is remarkably influenced by mechanical cues under nutrient-deprived and hypoxic conditions. This methodology holds significant potential for future applications, including improved characterisation of CAR-T cell migration and the ability to predict infiltration efficiency in patient-specific contexts. Additionally, we present a simple yet effective way to examine how the biophysical environment alters CAR-T cells effectiveness, thereby contributing to the development of more effective immunotherapies for solid tumours.This work was supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (ICoMICS grant agreement No 101018587).Acknowledgements This work was supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (ICoMICS grant agreement No 101018587). Jack Zhang-Zhou was granted by the Ministerio de Educacion y Formacion Profesional (grant FPU21/06003).References Newick K, O’Brien S, Moon E, Albelda SM. CAR T cell therapy for solid tumors. Annu. Rev. Med. 2017;68:139–152.Onozuka H, Tsuchihara K, Esumi H. Hypoglycemic/hypoxic condition in vitro mimicking the tumor microenvironment markedly reduced the efficacy of anticancer drugs. Cancer Sci. 2011;102:975-982.Zhang-Zhou J, Movilla Meno N, Oñate Salafranca C, Gomez-Benito MJ, Guerrero PE, Pardo Jimeno J, Garcia-Aznar JM. CAR-T cells are more affected than T lymphocytes by mechanical constraints: a microfluidic-based approach. Life Sci. 2025;363:123335.Shin Y, Han S, Jeon JS, Yamamoto K, Zervantonakis IK, Sudo R, Kamm RD, Chung S. Microfluidic assay for simultaneous culture of multiple cell types on surfaces or within hydrogels. 2012;7:1247-1259.Ethics Approval Human peripheral blood mononuclear cells (PBMCs) were collected and obtained from healthy donors and patients who provided informed consent, in accordance with protocols approved by the Ethical Committee of Clinical Research of Aragon, number: C.I.PI11/006.

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