Aim
Gastric cancer (GC) remains the third leading cause of cancer‑related death worldwide. Unfortunately, only a subset of GC patients, characterised by tumours with high high microsatellite instability (MSI), responds to immune checkpoint (ICI) therapy. In the context of GC, the mechanisms of how MSI improves ICI therapy responses remain poorly understood. Here we are undertaking a proof of principle study to demonstrate in novel GC mouse models that loss of the mismatch repair protein MLH1 confers MSI phenotype and impairs tumour growth via altered anti-tumour immune responses.
Methods
To study the functional and mechanistic effects of loss of MLH1 protein, we established MLH1-deficient (Kras-, Pi3kca-, Tp53-mutant) murine tumour organoids via CRISPR/Cas9 technology. These organoids were subcutaneously allografted into immunocompetent C57BL/6 mice to study tumour progression, immune surveillance, and responses to immunotherapy in vivo.
Results
We successfully generated MLH1-deficient (Kras-, Pi3kca-, Tp53-mutant) murine tumour organoids. MSI testing confirmed that MLH1-proficient parental organoids are MSI low, whereas MLH1-deficient organoids are MSI high. Low passage MLH1-deficient organoid tumours grew similarly to MLH1-proficient tumours. However, after culturing of organoids in vitro for a prolonged time prior to injection to allow accumulation of mutations, subcutaneous allograft MLH1-deficient tumours were considerably smaller compared to MLH1-proficient tumours. In addition, MLH1‑deficient tumours showed a significantly higher number of CD8+ T cells. However, treatment with the ICI anti-PD-1 only marginally reduced tumour mass further. We are currently investigating the underlying mechanism for the impaired growth of MLH1‑deficient organoid allograft tumours via tumour mutational burden analysis and neoantigen testing.
Conclusion
Taken together, we provide evidence that loss of MLH1 leads to high MSI in gastric tumours, reduces tumour growth after prolonged in vitro culture but does not increase anti-PD-1 therapy. Our findings encourage further studies to investigate the mechanisms of impaired tumour growth after MLH1 loss in GC and may provide insights leading to improve ICI therapy responses for GC patients.