Abstract 4162: Lentivirally delivered shRNA knockdown of CXCL12 is effective at preventing radiation fibrosis in normal tissues

Abstract

Abstract Late Adverse Effects (LAEs) following adjuvant radiotherapy (RT) are common sequelae in free flaps used for breast reconstruction and result in ongoing healthcare burdens during the phase of cancer survivorship. Intra-arterial delivery of gene therapy to an isolated vascular region permits localized, targeted genetic modulation of reconstructed tissue, avoiding potential off-target effects in microscopic residual disease. We studied the immunological component of LAE development in a validated accelerated rodent model recapitulating autologous flap reconstruction, across 90 days after RT (50 Gy/3). CXCL12 was significantly up-regulated from 2-21 days after RT, peaking at 7 days (p<0.001), and corresponded to an increase in CXCR4 expression (p=0.047). Multiplexed confocal imaging co-localised CXCL12 and γH2AX in fibroblast stromal cells after RT, confirming that CXCL12 was a flap-originating signal suitable for our therapy modality. This was associated with increased macrophage ingress and fibroblast activation as characterized by immunohistochemical (p=0.002) and flow cytometric end-points (p=0.035). In vitro work corroborated the relevance of the CXCL12 axis in the development of LAEs, demonstrating significant increase in CXCL12's transcriptome expression (p=0.013) and protein secretion (p=0.03) in primary rat fibroblasts stimulated with RT, high expression of CXCL12's receptors, CXCR4 and CXCR7 on M1 and M2 rat macrophages, and increased fibroblast activation, assayed with αSMA expression, with co-stimulation of CXCL12 and TGFβ. We hypothesized that LAEs in animals with flap-targeted knockdown of CXCL12 would be reduced. Flaps were infected with vascularly-delivered, lentiviral particles encoding shRNA against CXCL12 (LVshCXCL12), scrambled controls (LVSCR) alongside sham (phosphate-buffered saline (PBS)) and un-irradiated controls. Flaps infected with LVshCXCL12 exhibited reversal of LAE-mediated flap contracture and were not significantly different from un-irradiated flaps at 90 days post-RT, but were significantly greater in area compared to sham flaps or those infected with LVSCR (p<0.0001). Radiation Therapy Oncology Group (RTOG) LAE scores were also significantly improved compared with LvSCR (p<0.0001) and PBS groups (p=0.0011). Endpoint assays demonstrated significant vascular preservation (p=0.049) and reduced tissue collagen deposition (p=0.019). In conclusion, targeted knockdown of CXCL12 is effective at preventing the development of LAEs in flap tissue exposed to RT and is a promising strategy for clinical translation. Citation Format: James T. Paget, Martin McLaughlin, Joan N. Kyula, David Mansfield, Henry G. Smith, Victoria Roulstone, Paul A. Harris, Alan A. Melcher, Kevin J. Harrington, Aadil A. Khan. Lentivirally delivered shRNA knockdown of CXCL12 is effective at preventing radiation fibrosis in normal tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4162.