PO-121 Lentivirally-delivered ShRNA knockdown of CXCL12 prevents fibrosis in a rodent model of radiation late adverse effects

Abstract

IntroductionLate Adverse Effects (LAEs) following adjuvant radiotherapy (RT) are common sequelae in free flaps used for breast reconstruction. Intra-arterial delivery of gene therapy to an isolated vascular region permits localised, targeted genetic modulation of reconstructed tissue, avoiding potential off-target effects in microscopic residual disease. We hypothesised that targeted knockdown of CXCL12 would reduce LAEs.Material and methodsLAE development was studied across 8 time points to 90 days after 50 Gy/3 in 24 male Fischer 344 rats in a validated LAE flap model. RT and control human skin and subcuticular tissue was obtained for validation from the PRADA study.Efficacy of ShCXCL12 against LAEs was assessed in 72 animals. 1 × 108 lentiviral particles encoding ShRNA against CXCL12 (ShCXCL12), RNA overexpressing CXCL12 (OeCXCL12), or scrambled control (SCR), or sham infection with PBS were delivered intra-arterially to the flap tissue 30 days before RT. Acute and Late toxicities were assessed using Radiation Therapy Oncology Group (RTOG) scores with tissue endpoints at 7, 21 and 90 days after RT.Results and discussionsCXCL12 and CXCR4 were up-regulated after RT, peaking at 7 days (p=0.001 and p=0.047 respectively). Confocal imaging co-localised CXCL12 and γH2AX in fibroblast stromal cells after RT, confirming that CXCL12 was flap-originating and suitable for our therapy. This was associated with increased macrophage ingress and fibroblast activation characterised by immunohistochemical and flow cytometric end-points (p=0.035). We corroborated these findings in human RT tissue, demonstrating increased CXCL12 (p=0.035) and CD68 (p=0.027) expression compared to paired controls.ShCXCL12 animals had reduced acute toxicities compared to LVSCR or PBS groups. OeCXCL12 animals had significantly worse toxicity. ShCXCL12 animals exhibited reversal of flap contracture and retained significantly greater flap area overall. RTOG late toxicity scores were significantly improved (p<0.0001).At day 7 and day 21 ShCXCL12 tissue contained fewer macrophages. This was not seen by day 90. Fibroblasts did not differ at day 7, but were reduced by day 21 and at day 90. Endpoint assays confirmed amelioration of the LAE phenotype with reduced tissue collagen deposition and reduced Acta2 expression.ConclusionCXCL12 over-expression post-RT signals an innate immune response mediated primarily by macrophages. Targeted ShCXCL12 can prevent the development of LAEs. Further work aims to understand the mechanism of immune-fibroblast cross-talk.