Chemotherapeutic drug-containing microspheres for tumor suppressing adipose regeneration Wakako Tsuji1,2, Jacquelene M Bliley1, Kacey G Marra1, J Peter Rubin1 1. Adipose Stem Cell Center, Plastic Surgery Department, University of Pittsburgh, PA, USA 2. Department of Breast Surgery, Shiga Medical Center for Adults, Shiga, Japan Background Results One mg of doxorubicin, paclitaxel, and 4-OH tamoxifen double-walled MS contained 4.4±0.9µg, 9.2±0.8µg, and 5.4±0.4µg of each drug, respectively. Fat grafting to the oncologic breast is still in question because fat component may promote breast cancer cells (BCCs). Previously, we reported that IC50 of doxorubicin is higher on adipose stem cells (ASCs) than BCCs (Cancer Research, December 2013, 73, 24 supplement). Thus, a fat graft incorporating chemotherapeutic drugs at concentration to inhibit BCC growth while also allowing for tissue preservation would be ideal after breast cancer surgery. The aim of this study was to manufacture doxorubicin, paclitaxel, and 4-OH tamoxifen–containing microspheres that suppress BCCs growth while also maintaining normal cellular growth of adipose tissue components, including adipocytes and adipose-derived stromal cells (ASCs). Drug release kinetics assay result showed that doxorubicin was released from double-walled MS for more than one year. Materials and Methods Microscopic observation of BT-474 72 hours after co-incubation with graded dose of doxorubicin MS. Scale bar = 200 µm Microspheres (MS) encapsulating doxorubicin-HCl (Enzo Lifesciences Inc., Farmingdale, NY) were manufactured with standard water-oil-water emulsion. Paclitaxel (TSZ CHEM) and 4OH tamoxifen (Sigma-Aldrich, St. Louis, MO) containing MS were manufactured similarly. Double-walled MS were encapsulated with two layers of polymer (inner poly(lactic-co-glycolic acid) (PLGA) core with outer PLA layer). Drug loading and release kinetics were determined from chemotherapeutic MS. The amount of doxorubicin released into the PBS was detected by measuring the fluorescence of the supernatant at 470 (excitation wavelength) - 585 nm (emission wavelength) using a plate reader. Doxorubicin MS 10mg Doxorubicin MS 3mg Doxorubicin MS 0.3mg Doxorubicin MS 1mg PBS Doxorubicin MS Empty MS Doxorubicin MS 0.1mg Doxorubicin MS 0.03mg Doxorubicin 0 µM Doxorubicin 100 µM Double-walled MS Using 24-well transwell basket system (pore size was 0.4 µm, Falcon, NY) , drug-loaded MS and 1×104 BCCs were co-incubated in 1 mL of regular media for 72 hours. BT-474, MCF-7 and MDA-MB-231were purchased by ATCC (Manassas, VA). We had seven experimental groups: 10 mg, 3 mg, 1 mg, 0.3 mg, 0.1 mg, 0.03 mg, and 0 mg of MS in 1mL of regular media. Empty MS was used for the negative control group. All the experiments were done with triplicate. * * * 1×104 BCCs MS 1 mL of media Cell viability assay results with doxorubicin MS. Ten mg of doxorubicin MS was enough to kill 1×104 MCF-7, BT-474, and MDA-MB-231. Three mg of doxorubicin MS was also toxic to 1×104 BT-474. A toxic effect of doxorubicin, paclitaxel, and 4OH-tamoxifen was found on BCCs with a dose of 10, 1, and 10 mg, respectively. These doses were also found to allow for normal ASC proliferation. *P<0.05 vs negative control group Empty MS 3mg/well * Discussion Dox MS 1mg/well Dox MS 10mg/well Dox MS 3mg/well Considering IC50 of doxorubicin, paclitaxel, and 4-OH tamoxifen is higher on ASCs than BCCs, certain drug levels should exist to kill BCCs not ASCs. Toxic doses of drug containing MS on BCCs were determined in this study. This technology is promising for tumor suppressing adipose regeneration. Future work consists of completing studies to confirm these dose effects in vivo. Acknowledgements Dox MS 0.3mg/well Dox MS 0.03mg/well Dox MS 0.1mg/well 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. We appreciate all the members of Adipose Stem Cell Center. Scale bar = 200 µm Bioactivity of chemotherapeutic-loaded MS and drug in media alone was determined by assessing death of BCCs after 72 hours of co-incubation in culture. After microscope observation, CyQUANT® cell proliferation assay kit (Invitrogen, Carlsbad, CA) was used to assess cell viability