微孔阵列中抗原感知调控下 CAR-T 细胞在 3D 乳腺癌球体核心内的浸润与细胞毒杀伤作用

The success of chimeric antigen receptor (CAR) T cells in blood cancers has intensified efforts to develop CAR T therapies for solid cancers. In the solid tumor microenvironment, CAR T cell trafficking and suppression of cytotoxic killing represent limiting factors for therapeutic efficacy.

Here, we present a microwell platform to study CAR T cell interactions with 3D breast tumor spheroids and determine predictors of anti-tumor CAR T cell function. To precisely control antigen sensing, we utilized a switchable adaptor CAR system that covalently attaches to co-administered antibody adaptors and mediates antigen recognition.

Following the addition of an anti-HER2 adaptor antibody, primary human CAR T cells exhibited higher infiltration, clustering, and secretion of effector cytokines. By tracking CAR T cell killing in individual spheroids, we showed the suppressive effects of spheroid size and identified the initial CAR T cell to spheroid area ratio as a predictor of cytotoxicity.

We demonstrate that larger spheroids exhibit higher hypoxia levels and are infiltrated by CAR T cells with a suppressed activation state, characterized by reduced expression of IFN- , TNF- , and granzyme B. Spatiotemporal analysis revealed lower CAR T cell numbers and cytotoxicity in the spheroid core compared to the periphery.

Finally, increasing CAR T cell seeding density resulted in higher CAR T cell infiltration and cancer cell elimination in the spheroid core.

Our findings provide new quantitative insight into CAR T cell function within 3D cancer spheroids. Given its miniaturized nature and live imaging capabilities, our microfabricated system holds promise for screening cellular immunotherapies.