Investigating Ras homolog gene family member C (RhoC) and Ki67 expression following external beam radiation therapy show increased RhoC expression in relapsing prostate cancer xenografts

Ras homolog gene family member C (RhoC) is a GTPase involved in cell migration, implicated in epithelial-mesenchymal transition and treatment resistance and metastasis of cancer. For example, RhoC has been shown to be involved in resistance to radiation in cervical carcinoma. Here, the effect of X-ray irradiation on RhoC expression in prostate cancer (PCa) xenografts was investigated in both xenografts in regression and relapse. Male BALB/cAnNRj-Foxn1^nu/nu mice were inoculated with 4–6 million LNCaP-FGC cells and established xenografts were irradiated with X-rays (200 kV, 1 Gymin^-1), 5, 10 or 15 Gy using a Gulmay Medical X-ray system. Expression of RhoC and Ki67, a known proliferation marker, was investigated in xenografts, given 15 Gy, 7 days (midst response as measured by size) or 3 weeks (relapse) post irradiation. Staining was quantified using the Halo software (v2.3.2089.34) with the Indica Labs – cytonuclear v1.6 algorithm. RhoC and Ki67 staining was divided into weak, medium, and strong staining and the percentage of cells stained, single and dual staining, was quantified. The HALO software was further used to classify the tissue in each section so that analysis of RhoC and Ki67 expression in cancer cells, stroma and necrotic areas could be done separately. The results showed that RhoC expression in cancer and stroma cells was significantly higher in relapsed xenografts than in those in regression. This was not seen for Ki67 staining, where the percentage of stained cells were the same in regressing and relapsing tumors. RhoC could be a useful biomarker to confirm relapse following external beam radiation therapy.