Microphthalmia-associated transcription factor (MITF) is a key player in phenotype switching in melanoma. We showed previously that MITF regulates phenotypic melanoma heterogeneity through changing extracellular matrix (ECM) composition and tumour microarchitecture. ROCK (Rho kinase) inhibiton phenocopies the MITF-high phenotype by generating a “spongy” spheroid architecture and an increase in proliferative cells. We hypothesise that MITF and ROCK inhibition regulate immune cell infiltration through changed ECM structure in our 3D melanoma spheroid model.
Our results show that MITF and ROCK inhibition affect immune cell infiltration, though this depends on the cell line and additional factors, such as chemoattractants and ECM structure. We observed distinct patterns of peripheral blood mononuclear cell (PBMC) distribution related to MITF-high and MITF-low conditions, suggesting that ECM structure, regulated by MITF expression and ROCK activity, plays a role in PBMC penetration.
Notably, PBMCs preferentially targeted proliferating cells transiting through G1 phase. High MITF levels correlated with increased melanoma cell death mediated by PBMCs. Moreover, ROCK inhibition in MITF-low spheroids (i.e. mimicking an MITF-high phenotype) significantly boosted PBMC activity regarding eliminating melanoma cells. Natural Killer (NK) cells are likely responsible for this G1 cell elimination, given the short duration of the experiment, which precludes spontaneous T cell activation. This finding aligns with 2D tissue culture results showing NK cells are cytotoxic to melanoma cells, leading to increased melanoma cell death in G1 phase.
In summary, we report for the first time that PBMCs may prefer proliferative cells in melanoma, and ROCK inhibition promotes the cytotoxicity against proliferating tumour cells from innate immunity. This is a major challenge for current clinical treatments, as dormant cells are often responsible for melanoma relapse. Moreover, this finding is significant beyond melanoma, as phenotypic heterogeneity occurs in a range of solid tumours, including prostate, breast, ovarian and lung cancer.