T cell exhaustion presents a major challenge for the efficacy of both immune checkpoint inhibitors (ICBs) and chimeric antigen receptor T (CAR-T) cell immunotherapies. To address this issue, we generate hypofunctional CAR-T cells that imitate the exhaustion state. By screening a Food and Drug Administration (FDA)-approved small molecule library using this model, we identify miltefosine as a potent molecule that restores the impaired function of CAR-T cells in a PD-1/PD-L1-independent manner. Impressively, in the terminally exhausted state where PD-1 antibody treatment is ineffective, miltefosine still enhances CAR-T cell activity. Single-cell sequencing analysis reveals that miltefosine treatment significantly increases the population of effector cells. Mechanistically, miltefosine improves impaired glycolysis and oxidative phosphorylation in hypofunctional CAR-T cells. In both allogeneic and syngeneic tumor models, miltefosine effectively enhances the solid tumor clearance ability of CAR-T cells and T cells, demonstrating its potential as an effective immunotherapeutic drug.
Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.

There is a compelling need for approaches to predict the efficacy of immunotherapy drugs. Tumor-on-chip technology exploits microfluidics to generate 3D cell co-cultures embedded in hydrogels that recapitulate simplified tumor ecosystems. Here, we present the development and validation of lung tumor-on-chip platforms to quickly and precisely measure ex vivo the effects of immune checkpoint inhibitors on T cell-mediated cancer cell death by exploiting the power of live imaging and advanced image analysis algorithms. The integration of autologous immunosuppressive FAP+ cancer-associated fibroblasts impaired the response to anti-PD-1, indicating that tumors-on-chips are capable of recapitulating stroma-dependent mechanisms of immunotherapy resistance. For a small cohort of non-small cell lung cancer patients, we generated personalized tumors-on-chips with their autologous primary cells isolated from fresh tumor samples, and we measured the responses to anti-PD-1 treatment. These results support the power of tumor-on-chip technology in immuno-oncology research and open a path to future clinical validations.
Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.

ABSTRACT There is a compelling need for new approaches to predict efficacy of immunotherapy drugs. Tumor-on-chip technology exploits microfluidics to generate 3D cell co-cultures embedded in hydrogels that recapitulate immune and stromal characteristics of a simplified tumor ecosystem. Here, we present the development and validation of lung-tumor-on-chip platforms to quickly and precisely measure ex vivo the effects of immune check-point inhibitors on T-cell-mediated cancer cell death, by exploiting the power of live imaging and advanced image analysis algorithms. These tumor-on-chips were generated with patient-derived autologous primary cells isolated from fresh lung cancer samples, opening the path for applications in personalized medicine. Moreover, cancer-associated fibroblasts were shown to impair the response to anti-PD-1, indicating that tumor-on-chips are capable of recapitulating stroma-dependent mechanisms of immunotherapy resistance. This interdisciplinary combination of microfluidic devices, clinically-relevant cell models, and advanced computational methods, can innovatively improve both the fundamental understanding and clinical efficacy of immuno-oncology drugs.