We evaluated the effects of SDHB silencing in a three dimension (3D) culture using tumor spheroids of a mouse pheochromocytoma cell line silenced or not (wild type = wt) for the catalytic SDHB subunit. Moreover, by co-culturing SDHB silenced or wt spheroids with primary cancer-activated fibroblasts (CAFs), we investigated the role played by the microenvironment on spheroid growth and migration/invasion processes.
Spheroid growth was rapid and similar in both wt and SDHB silenced spheroids. When spheroids were conditioned by CAFs medium, both wt and SDHB silenced spheroid diameter was significantly smaller, but the cloud of cells surrounding SDHB silenced spheroids was wider. Indeed, when spheroids were co-cultured with fibroblasts, SDHB silenced cells showed a significant increase in matrigel invasion as demonstrated by the computation of the migratory areas (p<0.001). Moreover, cells detaching from the SDHB silenced spheroids moved collectively, unlike the cells of wt spheroids that moved individually. Additionally, SDHB silenced spheroids developed long filamentous formations along which clusters of cells migrated far away from the spheroid, while these structures were not present in wt spheroids. Looking for environmental factors responsible for these effects, we added lactate, that we found to be largely secreted by CAFs, to the culture medium and we found that lactate plays a specific role in promoting migration only of SDHB silenced cells.
In this work, we demonstrated that SDHB silencing per se increases tumor cell migration/invasion and that microenvironment, as represented by CAFs, plays a pivotal role in enhancing collective migration/invasion in Pheo SDHB silenced tumor cells, suggesting their role in increasing the tumor metastasizing potential.