(P 66) ARHGEF4 EXPRESSION IN SOFT TISSUE SARCOMA CORRELATES WITH LOCAL GROWTH PATTERN: WHOLE TRANSCRIPTOME SEQUENCING ON SPATIALLY SEPARATED REGIONS, PATIENT-DERIVED MODELS, AND ORTHOTOPIC IN VIVO ANALYSIS

Objective: Soft tissue sarcoma (STS) is known to respect barriers to tumor spread, while its infiltrative and expanding growth patterns have not been studied at the molecular level. We hypothesized the presence of genes up- or down- regulated during local growth of STS. We explored the discovery of candidate genes involved in the growth pattern of STS and sought their correlation with clinical outcomes including local recurrence and metastasis. In addition, we investigated the role of ARHGEF4, which is mainly located in the cytoplasm and involved in the regulation of the actin cytoskeleton.

Methods: We collected tumor cells from central and peripheral areas separately and performed whole transcriptome sequencing (WTS) in 3 undifferentiated pleomorphic sarcoma (UPS) and one pleomorphic liposarcoma (P-LPS) tissues. Among the 58 selected candidate genes, ARHGEF4 showed higher expression in the peripheral than in the central areas of the tumors by real-time PCR. To investigate the role of ARHGEF4 in cell invasion, migration, and cell viability, Boyden chamber assay, wound healing assay, and MTT assay were performed using patient-derived STS cell lines after knockdown of ARHGEF4 by short interfering RNA (siRNA). We also performed immunohistochemistry for ARHGEF4 expression in biopsy specimens from 66 patients with UPS and myxofibrosarcoma (MFS). Finally, we developed a patient-derived STS organoid system using sarcoshpere and established an orthotopic mouse model by injecting luciferase-expressing STS organoid cells into Balb/c nude mice.

Results: Knockdown of ARHGEF4 by siRNA resulted in decreased cell invasion, migration, and cell viability in patient-derived STS cell lines. ARHGEF4 expression correlated with an infiltrative and expanding histological growth pattern (p=0.023). Increased ARHGEF4 expression was associated with a trend towards decreased local recurrence-free survival (p=0.046). Immunofluorescence staining with anti-ARHGEF4 antibodies showed significant reduction in Alexa Fluor 488 (green) signal in ARHGEF4 siRNA-treated cells compared to control siRNA. For in vivo validation, serial IVIS imaging on days 5, 7, 9 and 12 showed a significant suppression of tumor growth in ARHGEF4 siRNA group compared to controls.

Conclusion: The in vitro and in vivo results suggest that ARHGEF4 could serve as a biomarker and a therapeutic target of local growth patterns in STS.