# Therapeutic efficacy of external beam radiotherapy combined with anti-PD-L1 inhibition in a preclinical syngeneic head and neck cancer model

## TL;DR

This study explores how combining radiation therapy with an anti-PD-L1 drug improves survival in a mouse model of head and neck cancer.

## Contribution

The study reveals how radiation schedules affect immune cell dynamics and optimizes combination therapy with anti-PD-L1 in preclinical models.

## Key findings

- MTCQ1 cells showed the strongest response to radiation therapy in vitro.
- Combining 2 Gy x 6 radiation with anti-PD-L1 treatment increased survival in mice.
- Radiation therapy altered CD8a T cell infiltration in tumors.

## Abstract

•The preclinical therapeutic impact of radiotherapy with anti-PD-L1 in head and neck squamous cell carcinoma was studied.•In vitro, MTCQ1 cells demonstrated significant radiosensitive responses compared to MOCL1 and MOCL2 cells.•Anti-PD-L1 monotherapy significantly reduced tumor volume, only when treatment was initiated at lower tumor volume.•Significant changes in CD8a T cells dynamics were observed post 2 Gy x 6 radiotherapy.•Combining 2 Gy x 6 radiotherapy with sequential anti-PD-L1 treatment led to extended survival by reducing tumor ulceration.

The preclinical therapeutic impact of radiotherapy with anti-PD-L1 in head and neck squamous cell carcinoma was studied.

In vitro, MTCQ1 cells demonstrated significant radiosensitive responses compared to MOCL1 and MOCL2 cells.

Anti-PD-L1 monotherapy significantly reduced tumor volume, only when treatment was initiated at lower tumor volume.

Significant changes in CD8a T cells dynamics were observed post 2 Gy x 6 radiotherapy.

Combining 2 Gy x 6 radiotherapy with sequential anti-PD-L1 treatment led to extended survival by reducing tumor ulceration.

Treating high-grade head and neck squamous cell carcinoma (HNSCC) has recently combined immunotherapy with conventional therapies. However, optimizing the scheduling of anti-PD-L1 with external beam radiation therapy (EBRT) requires further research to improve efficacies.

In vitro, MTCQ1, MOCL1, and MOCL2 murine HNSCC cell responses to 2 Gy x 6 X-ray EBRT were assessed in metabolic, clonogenic and γH2AX assays. Ex vivo, syngeneic tumors in C57BL/6 mice were stained for haematoxylin and eosin (H&E) and immune cell infiltration. Combination therapeutic in vivo studies using MTCQ1 tumors treated with 2 Gy x 6 or 8 Gy x 1 EBRT with sequential and/or concurrent dosing with anti-PD-L1 were also performed.

MTCQ1 cells exhibited the most marked responses to EBRT in vitro. H&E analysis revealed highest cellular density and most disperse extracellular matrix in MTCQ1 tumors with infiltration of CD8a+ T cells in tumor centres and macrophages predominantly peripheral. The 2 Gy x 6 EBRT regimen slowed tumor progression; average tumor volumes were 129.2 ± 49.0 mm3 on day 10, compared to 234.1 ± 130.7 mm3 in the CT-only control (P = 0.039). However, there was also a reduced CD8a+ T cell infiltration on day 3 post complete treatment, with 0.19 ± 0.17 % CD8a+ T cell area compared to 0.91 ± 0.31 % in the CT-only control. Combining EBRT with anti-PD-L1 (delivered either concurrently or sequentially), resulted in greater median survival compared to the CT-only control (33 and 32 days versus 28 days, respectively). Similarly, statistically insignificant improved survival with 8 Gy x 1 EBRT regimen combined with concurrent anti-PD-L1 was observed.

These results reveal spatial distribution of immune cells in the tumor microenvironment and underscore the role of EBRT regimens in modulating immune cell dynamics. This highlights the importance of optimising radiation protocols to inform combination therapy designs in preclinical models.