ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Endocrine Abstracts (2019) 63 OC5.2 | DOI: 10.1530/endoabs.63.OC5.2

Pheochromocytoma aggressiveness induced by tumor microenvironment depends on the SDH subunit involved

Serena Martinelli1, Maria Riverso1, Tommaso Mello1, Giuseppina De Filpo1, Letizia Canu1, Mario Maggi1, Massimo Mannelli1 & Elena Rapizzi2

1Department of Experimental and Clinical Biomedical Science, University of Florence, Florence, Italy; 2Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy.

Pheochromocytoma/paraganglioma (Pheo/PGL) are rare neuroendocrine tumors generally benign. About 30–40% of Pheo/PGL are due to germ-line mutations in one of the susceptibility genes, including those encoding the succinate dehydrogenase subunits A-D (SDHA-D). Up to 80% of patients affected by SDHB mutated Pheo/PGL develop metastatic disease with no successful cure at present. In this study, we evaluated the different effect of tumor microenvironment on tumor cell migration/invasion by co-culturing tumor cell spheroids with primary cancer-activated fibroblasts (CAFs). To this end, we used three dimension (3D) cultures of a mouse pheochromocytoma cell line (MTT) silenced or not (wild type = Wt) for the catalytic SDHB or the anchoring SDHD subunit. We measured matrigel invasion of spheroid cells by the computation of the migratory areas. Intriguingly, we observed that SDHD silenced spheroids had an intermediate migration pattern compared to the highest migration capability of SDHB and the lowest one of the Wt spheroids. Interestingly, by confocal microscopy, we found that all the conditioned spheroids (Wt, SDHB and SDHD silenced ones) developed long filamentous formations, but only SDHB silenced cells invaded the surrounding space moving collectively probably using those filamentous as binaries. Wt and SDHD silenced spheroids tended to move individually. Ongoing experiments are aimed to understand if these long outgrowths are neuronal structures (dendrites or axon) or not. To characterize the molecules involved in promoting tumor cell migration, conditioned medium of CAFs was divided into two fractions depending on molecular weights of its components by using cut-off filters. The upper fraction contains molecules with a molecular weight higher than 3000 Da, while the lower fraction is constituted by small molecules (MW < 3000 Da). When the spheroids were conditioned with only the upper or only the lower fraction of conditioned medium we did not observe a significant increase in migration compared with unconditioned spheroids, as suggesting a synergistic role of both fractions in inducing cell migration. This synergy is particularly evident in SDHB silenced spheroids. These results suggest that the interplay between tumor microenvironment and SDHB silenced spheroids is distinctive compare to the ones of Wt or SDHD silenced spheroids. The characterization of the specific factors causing migration may open new approaches for medical therapy.

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