# Protease‐Activated Plasmonic Nanosensors for Predictive Ultrasound‐Guided Photoacoustic Imaging of Tumor Responses to Adoptive T Cell Therapy

## TL;DR

This paper introduces a new imaging tool using nanosensors to track T cell activity in tumors during cancer treatment.

## Contribution

A protease-activated plasmonic nanosensor for noninvasive photoacoustic imaging of T cell therapy responses is developed.

## Key findings

- The nanosensor detects granzyme B with high specificity and amplifies photoacoustic signals upon cleavage.
- In mouse models, the nanosensor identifies T cell activity in tumors before changes in tumor size are visible.
- Systemic delivery of the nanosensor correlates with antigen-specific T cell-mediated cytotoxicity.

## Abstract

Adoptive T cell therapy (ACT) is a promising strategy for cancer treatment that harnesses a patient's own T lymphocytes to enhance antitumor immunity. A major challenge in assessing therapeutic responses following ACT is the lack of robust, noninvasive tools to monitor cytotoxic T cell activity within tumors with anatomical context. Here, a protease‐activated plasmonic nanosensor is reported for noninvasive photoacoustic (PA) imaging of ACT responses. The nanosensor comprises gold nanospheres functionalized with peptide substrates of granzyme B (GzmB), a key effector protease secreted by cytotoxic T cells. Upon peptide cleavage by GzmB, nanosensor aggregation is induced, leading to plasmon coupling and enhanced optical absorption with ≈90% efficiency in the near‐infrared optical window. This aggregation significantly amplifies PA signals, enabling sensitive detection of GzmB. The nanosensor exhibits high specificity for GzmB over other proteases and correlates optical and PA responses with antigen‐specific T cell‐mediated cytotoxicity in vitro. In murine ACT models, systemic nanosensor administration enables detection of tumor‐infiltrating cytotoxic T cell activity, producing elevated PA signals in antigen‐positive tumors compared to antigen‐negative controls before any measurable differences in tumor volume. This study presents a noninvasive approach for assessing ACT efficacy via GzmB‐activated plasmonic nanosensors combined with ultrasound‐guided PA imaging.

Protease‐activated plasmonic nanosensors are demonstrated to noninvasively monitor antitumor T cell activity in tumors with anatomical information upon adoptive T cell transfer via ultrasound‐guided photoacoustic imaging. These nanosensors are actuated by granzyme B, a protease secreted by activated cytotoxic T cells during tumor cell killing, which triggers interparticle plasmon coupling and thus amplifies optical and photoacoustic responses. Upon systemic nanosensor delivery during adoptive T cell therapy, the nanosensors enable detection of tumor‐infiltrating cytotoxic T cell activity, with enhanced photoacoustic signals correlating with therapeutic responses.