1. Effect of Doxorubicin and Paclitaxel on Adipose-Derived Stem Cells: Can we incorporate chemotherapy into our reconstructive strategies? Materials and MethodsAbstract Materials and Methods Results Conclusion Acknowledgements Cells: Human adipose tissues were obtained from non-diabetic female patients between 35 and 60 years of age with informed consent (n=3). All patients had a body mass index less than 30. ASCs were isolated by collagenase enzymatic digestion and expanded in vitro until passage 3. BT-474 and MDA-MB-231 breast cancer cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA). Breast surgery often results in physical deformities that significantly diminish a patient’s quality of life. A natural and aesthetically pleasing breast reconstruction can be achieved through autologous fat grafting and the success of this technique has largely been attributed to the regenerative properties of adipose-derived stem cells (ASCs) within fat grafts. (Kølle et al., Lancet, 2013) However, in the setting of resected breast cancer, the growth stimulating, angiogenic, and immunomodulatory effects of ASCs pose a risk of increasing local recurrence rates. (Petit et al., Annals of Oncology, 2013) Although the safety of autologous fat grafting in the breast cancer population has yet to be determined, potential recurrence risk may be minimized through incorporating tumor-suppressing drugs in the graft. This study aimed to determine if doxorubicin and paclitaxel could be used to inhibit breast cancer cells while maintaining the viability and functionality of ASCs in vitro. We have identified a concentration of doxorubicin and paclitaxel that kills breast cancer cells but does not affect ASC bioactivity. Therefore, tumor suppressing fat grafts can be a feasible and promising reconstructive strategy for breast cancer patients. Cytotoxicity assay: ASCs and BT-474 cells were seeded at a density of 10×10 3 cells/well. MDA-MB-231 cells were seeded at a density of 8×10 3 cells/well. Doxorubicin-HCl (EnzoLife Sciences, Inc., Farmingdale, NY) was added to ASCs to breast cancer cells in appropriate maintenance media at concentrations of 0, 10, 30, 100, 300, 1000, 3000, or 100000 nM (n=4). Paclitaxel (TSZ CHEM) was added at the concentrations of 0, 0.1, 0.3, 1, 3, 10, 30, 100, and 300 nM (n=4). Proliferation and viability were assessed with commercially available CyQUANT® cell proliferation assay kit (Invitrogen™, Carlsbad, CA) and Colorimetric (MTT) kit (Millipore, Billerica, MA) according to the manufacturers’ instructions, respectively. Plates were read using a plate reader, TECAN infinite M200 PRO (TECAN US, Inc., Morrisville, NC) plate reader. IC 50 of the drugs were calculated with GraphPad Prism software (GraphPad Software Inc., La Jolla, CA). Adipogenic differentiation assay: Adipogenic differentiation capacity was assessed with commercially available AdipoRed™ adipogenesis assay reagent (Lonza Inc., Walkersville, MD). ASCs were seeded at the density of 10×10 3 cells/well on 96-well plate on day 0. Doxorubicin was added at concentrations of 0, 10, 30, 100, 300, 1000, 3000, or 100000 nM (n=4) on day 1. Similarly, paclitaxel was added at concentrations of 0, 0.1, 0.3, 1, 3, 10, 30, 100, and 300 nM (n=4) on day 1. On day 2, media was changed to 50% of adipogenic differentiation media (ZenBio Inc., Chapel Hill, NC) and then incubated for 2 weeks. On day 17, plates were read with fluorescence at 470 and 575 nm. Imaging: Images were taken using a Nikon Eclipse TS100 (Nikon Instruments Inc., NY) microscope. Summary In vitro cytotoxicity studies demonstrated greater doxorubicin and paclitaxel sensitivity in BT-474 and MDA-MB-231 breast cancer cell lines compared to human ASCs. Adipogenic differentiation ability was retained in the presence of doxorubicin up to 1000 nM and paclitaxel up to 300 nM concentrations. Paclitaxel may be a superior candidate for tumor suppressing fat grafts. Wakako Tsuji, Christopher W Chung, Meghan M McLaughlin, Jolene E Valentin, Kacey G Marra, J Peter Rubin Adipose Stem Cell Center, Plastic Surgery Department, University of Pittsburgh, PA, USA MTT Assay CyQuant Assay Doxorubicin Paclitaxel AdipoRed Assay Scale bar = 200µm BT-474 ASCs This study was supported by AFIRM Soft Tissue Regeneration Using Cell Based Therapies, Department of Defense. We thank the Center of Biologic Imaging in University of Pittsburgh for imaging. Vehicle 0.1 nM 0.3 nM 1 nM 10 nM 30 nM 100 nM Fluorescent Microscopy - Paclitaxel Fluorescent Microscopy- Doxorubicin 1K nM 300 nM 100 nM Vehicle 10 nM30 nM 3K nM10K nM 100K nM MDA-MB-231 IC50 with BT-474 = 169.5 nM IC50 with MDA-MB-231 = 293.8 nM IC50 with ASCs = 9187 nM IC50 with MDA-MB-231 = 15.65 nM IC50 with BT-474 = 3.467 nM Stromal Vascular Fraction (SVF) Collagenase digestion and centrifugation Aspirated adipose tissue Adipose- derived stem cells (ASCs) Seeding of SVF on a plastic flask with overnight incubation 3 nM 300 nM Scale bar = 200µm