Microphthalmia-associated transcription factor (MITF) is a key player in melanoma phenotype switching between proliferative and invasive states. We showed previously that MITF regulates phenotypic melanoma heterogeneity through changing extracellular matrix (ECM) composition and tumour microarchitecture. ROCK (Rho kinase) inhibition mimics the MITF-high phenotype by generating a loose spheroid architecture and a wider distribution of proliferating cells and thus a smaller G1-arrested core.
We hypothesise that MITF-upregulation or ROCK inhibition regulate immune cell infiltration through changed ECM structure.
We show that MITF-upregulation or ROCK inhibition affect immune cell infiltration in a cell line-dependent manner. The spatial distribution pattern of PBMCs infiltrating into spheroids matched the pattern of ECM fiber organization, suggesting that ECM structure, regulated by MITF expression and ROCK activity, plays a critical role in PBMC penetration.
Notably, PBMCs preferentially targeted proliferating cells. ROCK inhibition boosted PBMC activity regarding eliminating proliferating melanoma cells in the spheroid periphery. Natural Killer (NK) cells are likely responsible for the melanoma cell death, given the short duration of the experiment, which precludes T cell activation. Indeed, NK cell cytotoxicity in 2D culture resulted in an increase in cell death, indicating that NK cells were responsible for the melanoma cell elimination.
Given the melanoma cell death in PBMC-infiltrated groups primarily emerged in the peripheral zone, which was occupied by proliferating cells, we further sought to understand why NK cells specifically targeted the proliferating peripheral cell population. By investigating NK cell activation and inhibitory ligands in both melanoma subpopulations, we found that proliferative melanoma cells produced more MICA/B, a critical activation ligand for NK cells.
In summary, dormant cells are often responsible for melanoma relapse, which is a challenge for current clinical treatments. We propose that ROCKi will increase the total number of immune-sensitive proliferating cells and allow for a better innate immune response, suggesting a potential combination with immune checkpoint inhibitors.