Interrogating and targeting mechanisms by which cancer-associated fibroblasts limit radiotherapy responses in bladder cancer.

Background: Radiotherapy is an important bladder-sparing curative treatment for patients with muscle-invasive bladder cancer but survival outcomes are poor, only 50% of patients are alive five years after radiotherapy or surgery.

The tumour microenvironment (TME) modulates the effectiveness of radiotherapy and the radiation-induced immune response is increasingly recognised to be clinically important, yet the function of cancer-associated fibroblasts (CAFs) in radiotherapy response is poorly understood.

CAF infiltration varies widely between bladder tumours; my unpublished transcriptomic analysis of diagnostic bladder tumour biopsies indicates that increased CAF infiltration associates with poor radiotherapy outcomes.

This builds on my published insights showing poor radiotherapy responses in CAF-rich rectal tumours, plus my observations in murine melanoma models that radiation can rapidly reprogram podoplanin (PDPN)+ CAFs to sequester effector immune cells out at the tumour periphery.

Overall Aim: Identify the mechanisms by which CAFs, particularly PDPN+ CAFs, limit radiotherapy cure rates for bladder cancer and establish CAF-related therapeutic targets to combine with radiation to improve survival.

Specific Aims: (1): Identify tumour cell/CAF phenotype and transcriptomic signalling, plus related immune cell neighbourhoods, which associate with radiotherapy outcomes in human bladder tumours. (2): Interrogate CAF/tumour cell dialogue, and related immune cell neighbourhoods, to identify mechanisms of radioresistance in pre-clinical murine models and 3D co-culture systems. (3): Improve radiotherapy responses through CAF modulation in combination with radiotherapy in pre-clinical models.

Methods: (1): I will perform an integrative analysis of biological datasets I have generated from two unique cohorts (n=454) of patient bladder cancer diagnostic tissue (these datasets encompass bulk transcriptomics, multiplex immunofluorescence (mIF) of four CAF subtypes plus immune cells, and extracellular matrix features).

These data will be linked to survival after radiotherapy and will inform deeper interrogation using spatial transcriptomics and Phenocycler highly mIF in a subset (n=40) of tumours from patients with very good versus very poor survival. (2): Mechanistic insights to findings in (1) will be generated using syngeneic immunocompetent bladder cancer murine models with different immune phenotypes and in vitro spheroid and organotypic CAF/tumour cell co-culture systems.

These will be interrogated pre- and post-radiation to identify radioresistance signals. (3): The efficacy of CAF-related targets that confer radioresistance will be tested with radiotherapy in the above pre-clinical models.

How the results of this research will be used: New CAF targets showing synergistic combination with radiation will be incorporated into early phase clinical trials to improve radiotherapy cure rates in bladder cancer, and potentially other solid tumours.