In Vitro Effects of Adipose-Derived Stem Cells on Breast Cancer Cells Harvested From the Same Patient.

R ESEARCH P APER In Vitro Effects of Adipose-Derived Stem Cells on Breast Cancer Cells Harvested From the Same Patient Heath J. Charvet, MD,* Hakan Orbay, MD PhD,† Lindsey Harrison, BS,† Kamaljit Devi, BS,† and David E. Sahar, MD† Introduction: Fat grafting for breast cancer (BrCa) reconstruction and breast augmentation has become increasingly more popular. A major area of debate and controversy is the effect of adipose-derived stem cells (ASCs) on remnant or undetected BrCa cells. We investigate the in vitro response of BrCa to ASCs in a coculture model with regards to cell migration. Methods: The study was approved by the institutional review board. BrCa and adi- pose tissue specimens either from subcutaneous breast tissue or abdominal lipoaspirate were obtained from the same patient. BrCa cells and ASCs were harvested with either explant culture and/or enzymatic digestion. Tissues were grown in cell culture flasks until adequate cell libraries were established. Adipose-derived stem cells from adipose specimens were characterized with flow cytometry. Immunofluorescence (IF) stain- ing of the initial cell population harvested from the BrCa specimens confirmed the presence of CD24, an epithelial marker of BrCa. A homogenous CD 24+/CD 90− BrCa cell population was obtained with flowcytometric cell sorting. The in vitro mi- gration of BrCa cells was examined in coculture with and without ASCs. Results: Adipose-derived stem cells harvested from the adipose specimens were positive for mesenchymal stem cell markers CD 105, CD 90, CD 73, and CD 44 and negative for lymphocyte cell marker CD 34 and leukocyte marker CD 45. The percentage of the CD 24+/CD 90− BrCa cells in the initial cell population harvested from BrCa specimens was 0.61%. The BrCa cells morphologically had large nuclei and small cytoplasm in clusters under the light microscope, sug- gesting a cancer cell phenotype. CD 24 expression on the surface of BrCa cells was confirmed with IF staining. The number of BrCa cells migrated in ASCs co- culture was approximately 10 times higher than the number of BrCa cells mi- grated in BrCa cell only cultures. Conclusions: Adipose-derived stem cells significantly increase the migration ca- pacity of BrCa cells in vitro in cocultures. This should be taken into consideration when performing fat grafting to the breast especially in patients with a history of BrCa or strong family history of BrCa. Key Words: fat grafting, breast cancer recurrence (Ann Plast Surg 2016;76: S241–S245) B reast cancer (BrCa) is the most common cancer in women and an increasing number of women are seeking postmastectomy breast re- construction since the passage of the Women's Health and Cancer Rights Act in 1998. 1 In conjunction with the increased number of cases, autologous fat grafting to the breast has become increasingly popular for postmastectomy reconstruction as well as cosmetic augmentation. 2 However, debate and controversy between surgeons and scientist over the safety of autologous fat grafting to the breast with regards to BrCa re- currence has been a hot topic in the plastic surgery community, especially after the discovery of adipose-derived stem cells (ASCs) in fat tissue. 3–5 The approach of plastic surgeons to fat grafting also changed considerably in response to ongoing debate and new scientific findings. Received June 30, 2015, and accepted for publication, after revision February 8, 2016. From the *Department of Surgery, and †Division of Plastic Surgery, Department of Surgery, University of California Davis Medical Center, Sacramento, CA. Conflicts of interest and sources of funding: none declared. Reprints: Heath Charvet, MD, Surgical Bioengineering Laboratory, University of California Davis Medical Center, 4625 2nd Avenue, Room 3004 Sacramento, CA 95817. E-mail: heath.charvet@ucdmc.ucdavis.edu. Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved. ISSN: 0148-7043/16/7605–S241 DOI: 10.1097/SAP.0000000000000802 Annals of Plastic Surgery • Volume 76, Supplement 3, May 2016 In 1987 the American Society of Plastic Surgeons (ASPS) banned au- tologous fat grafting to the breasts with concerns over the ability to detect and differentiate BrCa from fat necrosis. 6 In 2007, with ad- vancements in BrCa screening technology, the ASPS revisited autol- ogous breast fat grafting agreeing the procedure can be useful and safe, but results remained unpredictable. 7 In 2009, the ASPS lifted the ban and set forth the Fat Graft Task Force to monitor the procedure warning surgeons to proceed with “caution when considering fat grafting procedure in patients at high-risk for BrCa.” 8 As of today, there is still no consensus on the safety of breast fat grating in terms of BrCa recurrence. The discrepancy between clinical and basic science studies with regards to BrCa recurrence after breast fat grafting is rampant in the literature. 9 Although clinical studies have failed to show an increase in BrCa recurrence after fat grafting to the breasts, many basic science studies have shown potentially dangerous effects of ASCs on BrCa cells. On one hand, advocates of clinical fat grafting to the breast argue that the procedure is technically safe with low complication rates, can be performed in an outpatient setting with minimal donor site morbidity, and offers autologous tissue transfer without microsurgical expertise or resources. They further argue that the majority of basic science studies use purchased banked BrCa cell lines, which are more hardy and resilient than what is often clinically encountered. These studies are also limited by using adipose tissue and BrCa specimens harvested from different patients. On the other hand, scientists opposed to clinical fat grafting argue that the clinical studies are limited by few prospective trials, short overall follow-up time frames, and small study populations. 10–17 Our aim in this study is to investigate the in vitro response of BrCa cells to ASCs from the same patient in a coculture model with regards to cell migration in a more clinically applicable model, specifically eliminat- ing the use of banked resilient cancer cell lines and the use of adipose and BrCa specimens from different patients. We strive to answer the question: Is ASC enriched fat grafting to breast safe following oncologic surgery of the breast with potential remnant BrCa and is this procedure safe for aug- mentation in young woman with a strong family history of BrCa with po- tentially undetected or dormant cancer cells in the breast? METHODS The study was approved by the institutional review board (ap- proval 254494). Adipose tissue, in the form of lipoaspirate or subcuta- neous tissue, and primary BrCa specimens, were obtained from the same patient, a 67-year-old white woman. Adipose-derived stem cells and BrCa cells were harvested from human tissues as follows. Harvesting of ASCs To harvest ASCs, adipose tissue was digested enzymatically using 0.15% (w/v) type I collagenase (Calbiochem, San Diego, CA) at 37°C with vigorous shaking. The effect of the enzyme was neutral- ized by addition of an equal volume of cell growth medium (Dulbecco Modified Eagle Medium [DMEM; Gibco, Grand Island, NY] contain- ing 10% (v/v) fetal bovine serum [FBS; Corning, Manassas, VA], and 1% antibiotic-antimycotic solution [Sigma, St Louis, MO]). The mix- ture was centrifuged at 1500 rpm for 5 minutes. The resultant cell pellet was seeded the onto 75-mm 2 tissue culture flasks (Nunclon, Denmark) after cell counting using trypan blue. The cells in culture flasks were www.annalsplasticsurgery.com Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved. S241