*Article* **An In Vitro Approach for Investigating the Safety of Lipotransfer after Breast-Conserving Therapy**

**Theresa Promny 1,\* , Chiara-Sophia Kutz <sup>1</sup> , Tina Jost <sup>2</sup> , Luitpold V. Distel <sup>2</sup> , Sheetal Kadam <sup>1</sup> , Rafael Schmid <sup>1</sup> , Andreas Arkudas <sup>1</sup> , Raymund E. Horch <sup>1</sup> and Annika Kengelbach-Weigand <sup>1</sup>**


**Abstract:** The application of lipotransfer after breast-conserving therapy (BCT) and irradiation in breast cancer patients is an already widespread procedure for reconstructing volume deficits of the diseased breast. Nevertheless, the safety of lipotransfer has still not been clarified yet due to contradictory data. The goal of this in vitro study was to further elucidate the potential effects of lipotransfer on the irradiated remaining breast tissue. The mammary epithelial cell line MCF-10A was co-cultured with the fibroblast cell line MRC-5 and irradiated with 2 and 5 Gy. Afterwards, cells were treated with conditioned medium (CM) from adipose-derived stem cells (ADSC), and the effects on the cellular functions of MCF-10A cells and on gene expression at the mRNA level in MCF-10A and MRC-5 cells were analyzed. Treatment with ADSC CM stimulated transmigration and invasion and decreased the surviving fraction of MCF-10A cells. Further, the expression of cytokines, extracellular, and mesenchymal markers was enhanced in mammary epithelial cells. Only an effect of ADSC CM on irradiated fibroblasts could be observed. The present data suggest epithelial–mesenchymal transition-like changes in the epithelial mammary breast cell line. Thus, the benefits of lipotransfer after BCT should be critically weighed against its possible risks for the affected patients.

**Keywords:** adipose-derived stem cells; ADSC; MCF-10A; mammary epithelial cells; irradiation; fibroblasts; epithelial-mesenchymal transition; mesenchymal markers

### **1. Introduction**

Multimodal treatment of breast cancer consists of surgical treatment as well as systemic therapies and adjuvant radiation, depending on the underlying breast cancer subtype and breast cancer stage. Thereby, surgical therapy has become more conservative over the last decades and breast-conserving therapy (BCT), usually followed by radiation therapy, has evolved into a widespread alternative to mastectomy for patients with early breast cancer [1,2]. However, BCT might provide disfiguring results, so patients and surgeons aim to reconstruct the original anatomical contours of the breast [3–5]. This can be achieved by lipofilling or lipotransfer, a procedure in which fat is harvested via liposuction from typically used donor-sites (e.g., the abdomen, flank, thighs) and subsequently transplanted into the breast. Besides its many advantages including a natural appearance and texture, low donor-site morbidity, and easy availability, autologous fat transplants often show a low rate of graft survival [6]. The fat grafts include a minor fraction of adipose-derived stem cells (ADSC). To ameliorate graft viability, supplementation of the fat grafts with ADSC has been proposed [7,8]. ADSC have immunomodulatory and paracrine characteristics and secrete several cytokines, chemokines, and growth factors, e.g., vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF-2), and insulin-like growth factor 1 (IGF-1) [9–11]. These capacities are associated with regenerative effects and might contribute to higher fat engraftment, especially in irradiated tissues [12]. On the other hand, they might

**Citation:** Promny, T.; Kutz, C.-S.; Jost, T.; Distel, L.V.; Kadam, S.; Schmid, R.; Arkudas, A.; Horch, R.E.; Kengelbach-Weigand, A. An In Vitro Approach for Investigating the Safety of Lipotransfer after Breast-Conserving Therapy. *J. Pers. Med.* **2022**, *12*, 1284. https://doi.org/ 10.3390/jpm12081284

Academic Editor: Raimund Winter

Received: 14 June 2022 Accepted: 30 July 2022 Published: 5 August 2022

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confer an oncological potential on the grafted fat. The issue of the oncological safety of lipotransfer, particularly to a site where malignancy was treated, has been discussed widely and controversially in the literature. Whereas predominantly clinical studies claim a safe use of lipotransfer [13–18], pre-clinical studies suggested an ADSC-induced promotion of breast cancer growth and tumor angiogenesis [9,19–22]. Among other factors, the role of vascularization in any recipient tissue for cell ingrowth or proliferation is certainly relevant [23–25]. Further, previous studies showed that ADSC can also influence healthy breast tissue and induce an epithelial-to-mesenchymal transition in mammary epithelial cells [26]. This observation should not be neglected, as many patients undergo lipotransfer after BCT with the remaining tissue consisting partially of mammary epithelial cells. In most of the cases, this tissue has also been irradiated. Radiotherapy has an outstanding role in the treatment of breast cancer to eliminate (residual) cancer cells. However, it has also an effect on the surrounding tissue. There is evidence that the microenvironment contributes to the initiation, proliferation, and metastasis of a tumor [27–29]. Thereby, fibroblasts, the predominant cellular component of the tumor microenvironment, play a decisive role. Previous investigations showed that stromal fibroblasts are involved in the control of the growth and morphogenesis of normal and tumorigenic breast epithelial cells [30,31]. Additionally, they can be activated by endogenous stimuli, such as cytokines and growth factors secreted by tumor cells, or by external stimuli, such as radiation therapy [32–34]. Those activated "cancer-associated fibroblasts" (CAF) are considered fundamental actors in tumor progression and were shown to promote tumor growth, invasion, and metastasis [35]. Further, they have been attributed a role in stroma-mediated radiotherapy resistance [36]. Thus, tumor development, growth, recurrence, and resistance to therapies is dependent on various factors. When investigating the oncological safety of lipotransfer after BCT, the interplay of different cell types and external stimuli should be considered.

The present experimental in vitro study aims to further elucidate the effect of lipotransfer on breast tissue. Therefore, we investigated the influence of ADSC on mammary epithelial cells after exposure to irradiation while taking into account an important element of the microenvironment, the fibroblasts, by using a co-cultivation model. Thereby, functional analysis was performed, and alterations in gene expression profiles were investigated.

#### **2. Materials and Methods**
