The antifibrotic adipose‐derived stromal cell: Grafted fat enriched with CD74+ adipose‐derived stromal cells reduces chronic radiation‐induced skin fibrosis

Abstract Fat grafting can reduce radiation‐induced fibrosis. Improved outcomes are found when fat grafts are enriched with adipose‐derived stromal cells (ASCs), implicating ASCs as key drivers of soft tissue regeneration. We have identified a subpopulation of ASCs positive for CD74 with enhanced antifibrotic effects. Compared to CD74− and unsorted (US) ASCs, CD74+ ASCs have increased expression of hepatocyte growth factor, fibroblast growth factor 2, and transforming growth factor β3 (TGF‐β3) and decreased levels of TGF‐β1. Dermal fibroblasts incubated with conditioned media from CD74+ ASCs produced less collagen upon stimulation, compared to fibroblasts incubated with media from CD74− or US ASCs. Upon transplantation, fat grafts enriched with CD74+ ASCs reduced the stiffness, dermal thickness, and collagen content of overlying skin, and decreased the relative proportions of more fibrotic dermal fibroblasts. Improvements in several extracellular matrix components were also appreciated on immunofluorescent staining. Together these findings indicate CD74+ ASCs have antifibrotic qualities and may play an important role in future strategies to address fibrotic remodeling following radiation‐induced fibrosis.

damaged by RT. [11][12][13][14][15] Fat grafting restores a more "normal" skin architecture by decreasing dermal collagen content and overall dermal thickness, creating greater alignment of collagen fiber networks, 9,16,17 and increasing skin perfusion. 9,17 The beneficial effects of grafted fat on tissue texture, color, and elasticity cannot be explained through tissue expansion alone, 18,19 and the adipose-derived stromal cells (ASCs) within the stromal vascular fraction (SVF) of lipoaspirate are thought to orchestrate tissue regeneration, primarily via the secretion of growth factors with potent adipogenic, angiogenic, and antifibrotic effects. 13,[20][21][22][23][24][25] Recent work has highlighted the existence of multiple distinct subpopulations of stem and progenitor cells contained within the SVF.
For example, ASCs with low expression of the surface marker endoglin (CD105) 26 or those expressing the surface receptor Thy-1 (CD90) 27 possess enhanced osteogenic capacities. Similarly, bone morphogenetic protein receptor-1A marks ASCs with enhanced capacity for adipogenesis, 28 while endosialin (CD248) characterizes ASCs with angiogenic potential. 29 Although ASCs have been described to have antifibrotic effects within transplanted tissue, a specific ASC subpopulation characterized by an ability to reduce soft tissue fibrosis has yet to be described. Recent work has highlighted CD74 as a surface marker present on cells with antifibrotic qualities in a number of tissue types. CD74 −/− mice show increased liver fibrosis when treated with carbon tetrachloride (CCl 4 ) in vivo, 30 and also develop spontaneous lung injury by 6 months of age. 31 Given these findings, we hypothesized the existence of a subpopulation of CD74+ ASCs with enhanced antifibrotic actions able to restore irradiated soft tissue defects and modify both critical cell subpopulations and molecular signals involved in radiation fibrosis.

| Human SVF isolation
Human lipoaspirate samples were obtained from healthy female patients (n = 5) with informed consent under a protocol approved by Stanford Institutional Review Board (IRB #2188). Fat was harvested from the abdomen, flank, and/or thigh under local or general anesthesia using the Coleman technique. The SVF of adipose tissue was isolated as previously described. 17,32,33 In brief, the lipoaspirate was first washed with 1X sterile phosphate-buffered saline (PBS, #10010023, Thermo Fisher Scientific, Waltham, Massachusetts), and allowed to sit for 30 minutes at 4 C for separation into layers of blood/debris, fat, and lipid. The layer of fat was retrieved by aspiration and digested using collagenase (Collagenase from Clostridium histolyticum, ile-filtered, density: 1.077 g/mL, #10771, Sigma Aldrich) by pipette, and then centrifuged at 1500 rpm (27 C, 30 minutes, no deceleration), to remove the red blood cells and cellular debris. The SVF cell layer (buffy coat) was then retrieved, washed in FACS buffer, and centrifuged (450g, 5 minutes, 4 C) to leave the purified SVF cell pellet.

| Gene expression
CD74+, CD74−, and US ASCs were FACS sorted directly into TRIzol lysing solution (#15596026, ThermoFisher) and immediately frozen in Significance statement CD74+ adipose-derived stromal cells have antifibrotic qualities both in vitro and in vivo and may play an important role in future strategies to address fibrotic remodeling following radiation-induced fibrosis. dry ice and kept at −80 C until processing. RNA was harvested using RNeasy Mini Kit (#74104, Qiagen, Hilden, Germany). Reverse transcription was performed using TaqMan Reverse Transcription Reagents (#4304134, Invitrogen, ThermoFisher) and an ABI Prism 7900HT Sequence Detection System (#4317596, ThermoFisher) was used to perform quantitative real-time polymerase chain reaction to evaluate expression levels for genes known to be associated with antifibrotic activity-hepatocyte growth factor (HGF), fibroblast growth factor 2 (FGF2), and transforming growth factor β3 (TGF-β3)and the pro-fibrotic growth factor TGF-β1. All experiments were run in triplicate and data were standardized to glyceraldehyde 3-phosphate dehydrogenase expression for statistical analysis. Significant differences in gene expression levels between the CD74+, CD74−, and US ASCs were determined using the relative threshold cycle method. 34 To obtain ΔCT values, averaged CT values of the reference transcripts were subtracted from CT values of the candidate transcripts. ΔCT values of each gene in the analysis were compared to determine statistically significant differences. Cell suspensions were pelleted (200 g, 5 minutes, 4 C), resuspended in 500 μL of FACS buffer, carefully pipetted onto 1 mL of Histopaque, and centrifuged. The "buffy coat" of dermal cells was retrieved by pipette, washed, repelleted, and resuspended in 500 μL of fibroblast media (10% FBS, 1% antibiotic-antimycotic [Gibco, #15240062], 1% GlutMax in DMEM) and expanded in gelatin (0.1%)-coated wells at low oxygen conditions (2% O 2 and 7.5% CO 2 ) until confluence in 60 mm wells. All cells were kept below passage 3. The media was then aspirated, washed, and dermal fibroblasts were then incubated with media from the cultured CD74+, CD74−, and US ASCs. An amount of 10 ng/60 mm well of transforming growth factor β1 (TGF-β1, R&D Systems) was added and protein production was assessed following 24 hours. Incubation with an appropriate horseradish peroxidase (HRP)-linked secondary antibody and enhanced chemiluminescence were used for protein detection (#34075 SuperSignal West Dura Extended Duration Substrate, ThermoFisher).

| Mice scalp irradiation and fat grafting
Mice received irradiation to the scalp, using methodology previously described. 9,17 A total of 30 Gy was delivered, fractionated into six 5 Gy doses on alternate days over a total of 12 days. Lead shielding was used to ensure only the scalp was irradiated. A 4-week recovery period followed irradiation to allow for the chronic fibrotic effects of radiation to develop ( Figure 1A). ASC-enriched grafts were prepared by mixing

| Skin mechanical strength testing of irradiated skin
Eight weeks postgrafting, the mice were sacrificed and the full-thickness scalp skin overlying the grafted fat was harvested for mechanical strength testing (MST), histological assessment of skin structure, and FACS-sorting of fibroblast subpopulations. MST was performed using methodology previously described. 7 In brief, the skin tissue was attached to custom grips of a microtester (model 5848, Instron, Norwood, Massachusetts) equipped with a 100 N load cell using doublesided tape to provide a gauge length of 1 cm. The tissue specimen was stretched until a break in the skin was detected, observed as a decrease in stress despite increasing strain. Change in length divided by gauge length was used to calculate true strain. True stress was determined by dividing force by the original tissue cross-sectional area. Ultimate tensile strength corresponds to the greatest true stress achieved prior to breakage.

| Histological staining of irradiated skin and fat explants
The skin and explanted fat explants were immediately fixed in 4% paraformaldehyde for 16 hours at 4 C. Samples were then washed with PBS, dehydrated in gradients of alcohols, and embedded in paraffin blocks. Blocks were sectioned into 8-μm slices and fat was stained with H&E (#H-3502, Vector Laboratories, Burlingame, California) for assessment of integrity, and skin was stained with H&E to assess dermal thickness and with Masson's Trichrome (#HT15-1KT, Sigma Aldrich) and Picrosirius Red to determine collagen content. Slides were imaged using a Leica DM5000 B Light microscope (Leica Microsystems, Buffalo Grove, Illinois) using a ×10 objective. Dermal thickness was defined as the distance from the epidermis to the hypodermis, and measurements were made on 10 stained samples from each specimen using image J software (https://imagej.nih.gov/ij/, NIH, Bethesda, Maryland). Collagen content was determined using ImageJ based upon pixel-positive area per high power field using the same intensity threshold for all images. Five measurements were made per sample, and the mean of the total 10 measurements per sample was recorded as the value for that sample. Images of H&E-stained fat explants were imaged using a

| FACS analysis of fibroblast subpopulations within the irradiated skin
To explore the effect of fat grafts on the fibroblast subpopulations within the overlying skin, mouse skin was digested and prepared for

| CD74 marks an antifibrotic subset of ASCs
Flow cytometry of fresh human lipoaspirate (n = 5) confirmed existence of a subpopulation of CD74+ ASCs that comprised almost 5% of the SVF ( Figure 1B). Compared to CD74− ASCs and US ASCs, the CD74+ ASCs had significantly increased expression of HGF, FGF2, and TGF-β3 (all *P < .05), growth factors with potent antifibrotic actions ( Figure 1C). [40][41][42] Conversely, TGF-β1 expression was found to be lower in CD74+ ASCs compared to CD74− and US cells (Figure 1C). These findings were confirmed by Western blot which revealed greater TGF-β3 protein and decreased TGF-β1 protein levels with CD74+ ASCs ( Figure 1D). Primary cultures of human dermal fibroblasts incubated in conditioned media from CD74+ ASC exhibited decreased production of procollagen type 1 as well as collagen type 1 upon stimulation with TGF-β1 ( Figure 1E). Though less marked, Collagen type 3 production was also found to be decreased with CD74+ ASC conditioned media ( Figure 1D). These data are consistent with potential antifibrotic activity by CD74+ ASCs which may be mediated by paracrine signaling.

| Fat grafts enriched with CD74+ ASCs reduced fibrosis in overlying skin
To explore whether fat grafts enriched with CD74+ ASCs had an antifibrotic influence on surrounding irradiated skin and soft tissue, the skin overlying the fat grafts was harvested for biomechanical testing and histological assessment 8-weeks postgrafting. Calculation of Young's modulus indicated that the skin overlying fat grafts enriched with CD74+ ASCs was less stiff (*P < .05) ( Figure 3A,B), had reduced dermal thickness (****P < .0001) ( Figure 3C,D), and had significantly less collagen (***P < .001) ( Figure 3C,E) than the skin of mice receiving fat grafts enriched with CD74− and US ASCs or fat alone.

| Fat grafts enriched with CD74+ ASCs promote regeneration of elastic fibers
Radiation is known to alter several different fibers within the extracellular matrix. 36 To explore whether fat grafting had a beneficial effect on these components in addition to the collagen content and dermal thickness, we stained for elastin, fibrillin, and versican. While we observed no significant change in elastin fibers between any of the groups, there were notable differences in fibrillin and versican fibers.

| DISCUSSION
Significant soft tissue fibrosis following RT can distort skin form, impair skin function, and negatively impact patient quality-of-life. [1][2][3][4][5][6] Despite these negative consequences, RT remains an important anticancer treatment, and is used to cure or palliate over 50% of cancer patients. 51,52 With increasing numbers of patients surviving cancer and increasing risks of patients experiencing the long-term effects of RT, it is of paramount importance to prevent or reverse the pathological fibrotic process. 22,53 Fat grafting can improve the quality, hyperpigmentation, and thickness of irradiated skin. 8,[11][12][13][14][15] ASCs within the grafted fat are thought to drive tissue regeneration, and recent work has identified numerous subpopulations of ASCs with distinct properties. [26][27][28][29] Here, we describe an additional subpopulation of ASCs, positive for the surface marker CD74, with enhanced antifibrotic qualities.
CD74 is a nonpolymorphic type II transmembrane glycoprotein which functions as a major histocompatibility complex class II chaperone and has a high affinity for the macrophage inhibitory factor (MIF) receptor. CD74 is thought to be anti-inflammatory via its interaction with MIF; in hematopoietic stem cells MIF binds CD74, and this instigates intracellular signaling culminating in phosphorylation of AMPactivated protein kinase (AMPK). AMPK, in turn, inhibits platelet derived growth factor induced migration and proliferation of hepatic stellate cells. 54 This pathway may also mediate the antifibrotic activity of cells known to express the CD74 surface marker in other tissues, such as adipose tissue.
In our study, we observed ASCs positive for the surface marker CD74 to have antifibrotic qualities. Though this may derive, in part, from an anti-inflammatory function, we also found CD74+ ASCs to express antifibrotic genes and media from cultured CD74+ ASCs reduced collagen production, particularly collagen type 1 which has been shown to be significantly increased relative to collagen type III in fibrosis/scar, 55 in stimulated human dermal fibroblasts in vitro. Fat grafts enriched with CD74+ ASCs had greater improved histological quality, underwent less resorption, and had an antifibrotic influence on surrounding irradiated soft tissue. Specifically, CD74+ ASCenriched fat grafts decreased stiffness, thickness, and collagen content of overlying skin, and decreased the proportions of fibrotic fibroblast subpopulations. Furthermore, this was also associated with enhanced staining for fibrillin which may play a role in modulation of TGF-β1 activity. Fibrillin may interact with latent TGF-β1 50 and murine knockouts of fibrillin-1 have been found to have greater interstitial fibrosis secondary to increased TGF-β1 activation. 56,57 Finally, versican, a chondroitin sulfate proteoglycan known to promote fibrogenic cellular functions, 58 was noted to be lower in irradiated soft tissue grafted with CD74+ ASC-enriched fat compared to CD74− ASC or US ASC-enriched fat.
The transforming growth factor beta (TGF-β) superfamily are critical regulators of tissue repair and fibrosis and three isoforms of TGF-β (TGF-β1, 2, and 3) are known to possess distinct roles. 59 Interestingly, all TGF-β isoforms act through the same receptors, suggesting antagonizing functions. 59 Differential activation of downstream small mothers against decapentaplegic (SMAD) signaling intermediates may also contribute to their contrasting functions. In particular, recent studies have shown Smad7 to suppress fibrosis in multiple organs, and further exploration of this may be warranted with respect to our observations with CD74+ ASCs. 60,61 In our study, we found that CD74+ ASCs express greater levels of TGF-β3, the isoform with the most antifibrotic activity, 62 and had decreased levels of TGF-β1 transcripts, which is known to mediate fibrosis. 63 Supporting our findings, recent reports have shown that ASCs can inhibit fibroblast proliferation by decreasing TGF-β1 expression, and promote collagen remodeling by increasing TGF-β3. 64,65 Furthermore, connective tissue growth factor has been implicated as a cofactor with TGF-β1 in mediating fibrosis, 66,67 and it may be of interest to evaluate the impact CD74+ ASCs may play in production of this growth factor in subsequent studies.
Identification and isolation of ASC subpopulations with antifibrotic potential can both expand the current understanding of adipose tissue biology and expedite the application of specific ASC subpopulations for therapeutic benefit. While enrichment of fat grafts with ASCs (cell-assisted lipotransfer or CAL) can improve retention rates, enhance the quality of grafted fat, and further attenuate radiation-induced dermal thicknening 9,68-72 compared to fat alone, we demonstrate here that these effects are more pronounced when grafted fat is enriched with CD74+ ASCs, relative to CD74− or US ASCs. Thus CD74+ ASCs may have a potentially important role in the treatment of radiation-induced soft tissue fibrosis. While the CD74+ subpopulation comprised a small fraction of the SVF and may require substantial in vitro expansion prior to for grafting in the clinical setting, one option may be to expand or enhance the activity of the CD74+ within lipoaspirate in vivo using targeted molecules. And aside from this consideration, our findings of a potential role CD74+ ASCs may play in improving fibrosis also help to begin explaining the regenerative effects of fat grafting already observed clinically. 8

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available on request from the corresponding author.