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Therapeutic Flap Targets Residual Cancer Cells In Therapeutic Breast Reconstruction
Christopher R. Davis, BSc, MB ChB, MRCS, Peter A. Than, MD, Sacha Khong, PhD, Melanie Rodrigues, PhD, Michael W. Findlay, MD, PhD, Daniel Navarrete, Shadi Ghali, Adriaan O. Grobbelaar, Geoffrey C. Gurtner, MD, FACS.
Stanford University, Stanford University, CA, USA.
Purpose: One million new cases of breast cancer are diagnosed each year worldwide. With appropriate care, five-year survival is currently 91%. However, local-regional recurrence causes five-year survival to decrease to 42-49%. Breast reconstruction rates are increasing, but currently, reconstruction offers no direct survival advantage. We sought to develop a novel means to control local recurrence by converting the autologous tissue used for reconstruction into a therapeutic flap releasing anti-cancer proteins directly to the tumor bed.
Methods: Under appropriate institutional ethics approval, female Fischer rats underwent free-flap surgery equivalent to abdominally-based autologous flaps used in human breast reconstruction, along with the introduction of a luciferase positive breast cancer cell line (1x106 MAD-B106 cells) into the wound bed to simulate residual microscopic disease. We selected interferon gamma (IFNγ) as our model anti-cancer agent based on in vitro experiments where we quantified:
(1) Anti-cancer properties of IFNγ against a breast adenocarcinoma cell line (MAD-B106-Luc)
(2) Transfection efficiency of adeno-associated viral (AAV) vector encoding in adipose tissue akin to the free flap for breast reconstruction
(3) Concentration of IFNγ released by transfected rat adipose cells by enzyme-linked immunosorbent assay (ELISA)
In vivo rodent therapeutic flap surgery was performed by raising free fasciocutanous flaps based on the superficial inferior epigastric (SIE) vessels (SIE flap). After dividing the pedicle, the flap was detached and perfused for one hour with AAV encoding either IFNγ or green fluorescent protein (GFP), before saline irrigation to clear any residual AAV. The therapeutic flap was then inset into the wound bed and re-anastomosed under the operating microscope with transfection confirmed by fluorescence microscopy and flow cytometry. In vivo cancer burden was monitored non-invasively by IVIS imaging of the luciferase positive cancer cell line.
Results: IFNγ demonstrated potent anti-cancer properties against MAD-B106-Luc breast adenocarcinoma cells. Cancer cell populations were reduced to when exposed to IFNγ and macrophages versus macrophages alone (<5% versus 100% survival respectively). AAV-GFP and AAV- IFNγ constructs successfully transfected the SIE flaps, as demonstrated by GFP presence via fluorescence microscopy and flow cytometry showing positive GFP cell presence. IFNγ release occured in a dose-dependent relationship when measured by ELISA, showing sustained therapeutic protein release for weeks (14ng, 86ng, 148ng IFNγ released at week 3 by 1ul AAV-IFNγ for 1 hour, 1ul AAV-IFNγ for 48 hours, 10ul AAV-IFNγ for 1 hour respectively). The AAV-IFNγ therapeutic flap had lower local recurrence and disease progression compared to the AAV-GFP non-therapeutic flap control, supported by IVIS data.
Conclusion: Therapeutic breast reconstruction using AAV-IFNγ to deliver a gene of interest is a feasible technique to convert autologous tissue into a therapeutic flap releasing therapeutic proteins. Future breast reconstruction using a therapeutic flap may permit personalized and tailored local chemotherapy release to target residual disease and reduce the risk of recurrent disease.
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