1 Project Summary 2 3 Autologous fat grafting is the transfer of fat from one anatomical region to another and is commonly used in 4 breast reconstruction after mastectomy. Accurate prediction of graft retention remains an enduring clinical 5 problem, as resorption is reported to range between 10% to 50% in the literature. While there are several devices 6 available for fat grafting, no randomized, controlled trials have been completed to compare outcomes between 7 them, including volume retention and factors that would affect volume retention. Given the lack of evidence- 8 based guidelines, the decision of how best to process graftable fat remains up to physician preference. Moreover, 9 much work fails to assess the microscopic alterations to the grafted tissue resulting from each processing 10 technique, the study of which might reveal important predictive factors of complications or graft take. In response, 11 we are seeking to compare outcomes between three fat grafting devices which process the lipoaspirate in different 12 ways before it is injected into the graft site. We will utilize fat samples collected from patients undergoing fat grafting 13 who have been enrolled in an ongoing randomized, controlled trial to receive fat processed by techniques that 14 actively wash and filter the fat, known as “active closed wash and filtration” (ACWF), passively filter the fat 15 through a collecting system with minimal processing, known as a “passive low pressure closed system” (LPCS), 16 or actively wash and filter the fat with the addition of surfactant, known as “active wash and filtration plus 17 surfactant” (AWFPS). Originally, the ACWF and LPCS devices were compared to standard decantation; however 18 interim analysis showed superior volume retention with the ACWF and LPCS devices. Standard decantation has been 19 replaced with AWFPS to identify the optimal fat grafting device to use for patients. We will take the lipoaspirate that has 20 been harvested and processed via ACWF, LPCS, or AWPS, and inject a predefined quantity into the dorsa of 21 immunocompromised mice. We will assess the volume and projection of the grafted material in the mice at 1, 2, 22 and 3 months, as the literature states that reabsorption happens within the first 3 months and then the graft 23 becomes stable. We will also characterize the cellular environment around and within each graft after 3 months, 24 including any immune or inflammatory response as well as the viability and potential hormonal activity of the 25 grafted fat. Further, we will compare the retention rates and complication profiles observed in our mouse models 26 with those of their patient counterparts in the companion trial to provide further confirmation of any preliminary 27 conclusions drawn. Lastly, we will complete this analysis with a small subset of humanized mice as well in order 28 to compare outcomes and the ability to measure volume retention in immunocompromised versus humanized 29 mice. Measurement of volume retention of human lipoaspirate has not yet been documented in humanized mice, 30 and including a pilot study would allow us to assess immunogenicity effects on volume retention of lipoaspirate 31 and whether future studies of this nature could be completed in humanized mice. Immunogenicity effects will be 32 assessed by measuring angiogenesis in the grafted fat after 3 months and comparing between mouse species.

Project Narrative This project holds the potential to establish best-practice guidelines for surgeons seeking to safely and reliably use a patient's own fatty tissue to improve outcomes in breast reconstruction. While many methods of using patient-derived fat for this purpose exist, there is a lack of consensus in the surgical community regarding which method is superior, and this study would provide new and important data characterizing not only the clinical outcomes of three of the most common fat processing methods used, but also highly impactful, time-dependent information on the cellular-level differences in the fat after it is processed by each method. Finally, this study will evaluate differences in the levels of estrogen-producing proteins in the processed fat that is ultimately placed in close proximity to what was, in many cases, estrogen-responsive cancer, any differences between which have yet to be studied and hold the potential for direct influence over clinical practice should they prove substantial.