# Microphysiological Solid Tumor Models in Hydrogel Beads for CAR T Cell Immunotherapy Evaluation

## TL;DR

This paper introduces a 3D tumor model using hydrogel beads to study how CAR T cell therapy works against micrometastases, which are hard to detect and treat.

## Contribution

The novel contribution is a 3D micrometastasis model that mimics the tumor microenvironment to evaluate CAR T cell therapy efficacy in vitro.

## Key findings

- A hydrogel bead model with prostate cancer and fibrosarcoma cells mimics micrometastases and supports long-term culturing.
- CAR T cells showed successful infiltration and synergistic efficacy when targeting both FAP and PSCA antigens.
- The model's morphology aligns with clinical and murine metastatic samples, validating its relevance.

## Abstract

Micrometastases are challenging to resect surgically and to detect with in vivo imaging. Immunotherapy is highly anticipated to revolutionize their treatment, but its overall efficacy still remains limited for solid tumors. Here, a 3D micrometastases model is developed to mimic key microenvironmental cues, enabling in vitro evaluation of chimeric antigen receptor (CAR) T cell immunotherapy. Prostate cancer that preferentially metastasizes to, e.g., liver or bone marrow, is utilized as a model. Hydrogel beads with an elastic modulus matching those of soft organs are used to support long‐term culturing, immunostaining, and monitoring of the spheroids. As a biochemical cue, the impact of fibroblast activation protein (FAP), an emerging target in the tumor microenvironment, is investigated on prostate cancer spheroids and on the efficacy of CAR T cell therapy. The multi‐spheroid model consists of prostate stem cell antigen (PSCA)‐expressing prostate cancer cells and FAP‐producing fibrosarcoma cells in varying ratios. The morphological features of the model are compared to clinical histopathology and metastatic murine model samples. Finally, CAR T cell trials demonstrate successful chemoattraction and infiltration through the hydrogel matrix, with a dual‐targeting approach against FAP and PSCA antigens showing synergistic efficacy. This research provides invaluable insights for engineering 3D tumor models and modeling therapies targeting small metastatic or residual tumors, suggesting that co‐targeting may be a more effective strategy to unlock the tumor microenvironment's suppression.

The complex physical and biochemical barriers of the tumor microenvironment are major triggers of immune exclusion. Here, a 3D micrometastasis model is developed to mimic key microenvironmental cues, enabling in vitro evaluation of chimeric antigen receptor (CAR) T cell immunotherapy.