A xenotransplantation mouse model to study physiology of the mammary gland from large mammals

Although highly conserved in structure and function, many (patho)physiological processes of the mammary gland vary drastically between mammals, with mechanisms regulating these differences not well understood. Large mammals display variable lactation strategies and mammary cancer incidence, however, research into these variations is often limited to in vitro analysis due to logistical limitations. Validating a model with functional mammary xenografts from cryopreserved tissue fragments would allow for in vivo comparative analysis of mammary glands from large and/or rare mammals and would improve our understanding of postnatal development, lactation, and premalignancy across mammals. To this end, we generated functional mammary xenografts using mammary tissue fragments containing mammary stroma and parenchyma isolated via an antibody-independent approach from healthy, nulliparous equine and canine donor tissues to study these species in vivo. Cryopreserved mammary tissue fragments were xenotransplanted into de-epithelialized fat pads of immunodeficient mice and resulting xenografts were structurally and functionally assessed. Preimplantation of mammary stromal fibroblasts was performed to promote ductal morphogenesis. Xenografts recapitulated mammary lobule architecture and contained donor-derived stromal components. Mammatropic hormone stimulation resulted in (i) upregulation of lactation-associated genes, (ii) altered proliferation index, and (iii) morphological changes, indicating functionality. Preimplantation of mammary stromal fibroblasts did not promote ductal morphogenesis. This model presents the opportunity to study novel mechanisms regulating unique lactation strategies and mammary cancer induction in vivo. Due to the universal applicability of this approach, this model serves as proof-of-concept for developing mammary xenografts for in vivo analysis of virtually any mammals, including large and rare mammals.


Introduction
The mammary gland is a defining feature of all mammals and is conserved in function and structure.It is a specialized, hormone-responsive organ that develops postnatally and undergoes cycles of growth, differentiation, lactation, and involution, and is comprised of a branching ductal network that terminates in functional secretory alveolar structures when fully developed [1][2][3].Despite its conserved nature, the mammary gland is highly variable across mammals regarding lactation strategy, disease incidence, and gross morphology, with glands from larger mammals displaying greater architectural complexity relative to more rudimentary glands in mice [4].These variations allow for comparative species approaches to identify novel regulatory factors and underlying molecular mechanisms of mammary gland (patho)physiology that are relevant to both human and veterinary mammary health [4][5][6][7].
While in vitro models, such as mammary organoids, mimic 3D mammary gland architecture [8,9], in vivo studies allow for studying mammary tissues within a vascularized supportive stromal environment as well as exposure to the complex hormonal milieu required for both normal and malignant mammary cell function [10,11].In vivo mammary studies, however, are difficult when assessing large and/or undomesticated mammals due to logistical challenges such as cost and/or limited availability.Thus, a manageable in vivo model is warranted.To this end, xenotransplantation models, i.e., immunodeficient laboratory mice containing functional tissues derived from other species, have notable value for in vivo tissue analysis, particularly since xenotransplanted tissues maintain a 3D architecture and are supported by host environmental factors.Historically, xenotransplantation models have served as valuable tools to understand cancer biology [12][13][14], assess stem/progenitor cell repopulation potential [15,16], and resolve critical developmental mechanisms [17].Furthermore, drug responses in xenotransplanted tissues, both healthy and malignant, often align with clinical responses [14,[18][19][20], indicating their value in modeling drug pharmacokinetics.Regarding the generation of mammary xenografts, one approach relies on the murine mammary fat pad de-epithelializing (clearing) method, in which the postnatally-developing endogenous mammary epithelium is excised during puberty, at about 3-4 weeks of age, followed by xenotransplantation of typically freshly obtained mammary tissues or sorted stem/ progenitor cells from other mammals into the residual stroma [21], as has been described for rat [22], cow [15,[23][24][25][26][27], and human [28][29][30].
Here, we report on a reproducible and functional xenotransplantation model that uses cryopreserved mammary tissue fragments from equine or canine donors.These mammals were selected based on (i) availability of tissues from research animals, (ii) their status as sentinel species, i.e., large mammals that share human environments [7], (iii) similarities to humans regarding mammary morphology [31,32], and (iv) variation in their natural mammary cancer incidence, with horses and dogs representing mammals with a low and high mammary cancer incidence, respectively [7,33].Importantly, the mammary tissue fragments used for xenotransplantation were isolated via an antibody-free mechanical and enzymatic digestion method, making this approach universally applicable to virtually any mammal.Moreover, the use of cryopreserved mammary tissue fragments for xenotransplantation allows for the processing and generation of large quantities of mammary tissue fragments for long term storage, which is critical for studies of rare and scarcely accessible wild mammals.
Our salient findings were that both equine and canine xenografts consisted of lobule-like mammary outgrowths, containing multiple acini, and recapitulated the cellular architecture of the donor mammary gland.Importantly, the xenografts responded to circulating murine mammotropic hormones, confirming tissue functionality.We propose this model using cryopreserved mammary gland tissue fragments to be appropriate for establishing mammary xenografts of virtually any mammal, especially those that are large and/or rare, and to represent the unique opportunity for physiology studies of the mammary gland of non-traditional, large mammals in vivo within the context of manageable laboratory mice.

Ethics statement
All experimental procedures were performed in accordance with relevant guidelines and regulations and were approved by the Institutional Animal Care and Use Committee (IACUC) at Cornell University (#2013-0022).Tissues obtained from equine (Equus caballus) and canine (beagles, Canis lupus familiaris) donors were recovered after euthanasia.All donors were euthanized for reasons unrelated to this study and all tissues were extracted post-mortem, therefore, IACUC approval was not required for the mammary gland tissue collections.

Generation and cryopreservation of mammary tissue fragments for xenotransplantation
Mammary gland tissues were recovered from clinically healthy, intact, virgin animals, that were euthanized for reasons unrelated to this study.Tissues were obtained from equine (Equus caballus) donors (various breeds) aged between 2 to 16 years, consisting of 5 cm 2 equine mammary gland tissue pieces bordering the mammary gland compartments, and canine (Canis lupus familiaris) donors (beagles) aged ~15 months, consisting of 2 cm 2 mammary gland tissue pieces from the mammary duct near the nipple.The large age range in equine tissue donors was due to the need to opportunistically obtain scare equine tissues for xenotransplantations, thus, tissues were extracted from animals of variable age.Notably, studies assessing multiple xenotransplanted tissue types have shown that donor age may impact xenograft function and behavior [34], however, it is well-acknowledged that mammary gland tissues from aged donors can still regenerate fully functional mammary glands [35].All tissues were extracted at Cornell College of Veterinary Medicine (CVM) or at the Cornell Animal Health Diagnostic Center (AHDC).Reproductive history was validated by caretaker/veterinarian documentation records.

Cleared fat pad surgeries and mating of immunodeficient mice
NOD-scid gamma (NSG) (NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ) mice were purchased from the Cornell Progressive Assessment of Therapeutics (PATh) patient-derived xenograft (PDX) Facility or the Jackson Laboratory (Bar Harbor, ME) and maintained on-site under sterile conditions.All mice were supplied with food (e.g., standard chow) and water ad libitum.Fat pad clearing surgeries were performed on 3-4-week-old female mice that were anesthetized with isoflurane (cat #: 200-129) (Dechra Pharmaceuticals, Northwich, UK) mixed with O 2 (3% vol/ vol in induction chamber, then maintained at 2-2.5% vol/vol on nose cone) and received a subcutaneous injection of ketoprofen (3 mg/kg) (cat #: 07-803-7389) (Patterson Veterinary Supply, Loveland, CO), followed by an additional abdominal subcutaneous injection of bupivacaine (5 mg/kg) (National Drug Code #: 0409-1163-18) to reduce post-procedural pain.Timelines for surgical procedures are detailed in S1 Fig. Timeline for baseline surgeries (without additional interventions, i.e., without induced pregnancy or fibroblast injections) is presented in S1a Fig.All surgical procedures were performed on a warming pad within a sterile biosafety cabinet.While under anesthesia, the developing mammary epithelia in the 4 th inguinal MFPs were removed to de-epithelialize (clear) the MFPs [21,24,29].Excised portions of the 4 th inguinal MFPs were mounted on glass slides and stained by acetocarmine (1.25 mg/ml carmine powder (cat #: C1022) (Sigma Aldrich) in 45% Glacial acetic acid (cat #: A38-212) (Thermo Fisher, Waltham, MA) (see below)) to visualize extracted murine mammary glands and confirm successful clearing.Immediately after clearing, a small incision was made in the remaining fat pad and a donor equine or canine mammary tissue fragment (~200-400 μm in diameter) containing mammary parenchyma and stroma was inserted.Incisions were subsequently sealed with surgical staples (cat #: INS750344) (Cellpoint Scientific, Inc., Gaithersburg, MD) and mice recovered on a warming pad until mobile.Following surgery, mice were given an oral antibiotic suspension of sulfamethoxazole (0.4mg/ml) and trimethoprim (0.08mg/ml) (cat #: 0121-0854-16) (PAI Pharma, Greenville, SC), dissolved in drinking water, for a minimum of 10 days.All mice received additional subcutaneous injections of ketoprofen (3 mg/kg) (Patterson Veterinary Supply) at 24 and 48 h after surgery to alleviate pain.Mice that underwent surgery were housed in cages of � 4 mice and were monitored daily for signs of distress and to ensure proper healing of the abdominal incisions.No signs of distress were observed during the xenograft engraftment period and no challenges were observed (e.g., infection, tumor formation).
For mating procedures, female mice bearing xenografts (6 weeks post-surgery) were paired and placed in mating cages with an adult male NSG mouse.Females were checked for vaginal copulation plugs daily to determine days post-coitus (dpc) and monitored for changes in external appearance to determine gestation progression [39].For experiments that assessed the mammotropic effects of circulating pregnancy hormones, pregnant mice were euthanized at 18 dpc (S1b Fig) .For mice that received primary mammary fibroblast injections, fibroblasts were injected immediately post-clearing, and mammary tissue fragment xenotransplantation surgeries occurred 2 weeks after MFP clearing and fibroblast injections, when mice were aged 5-6-weeks old (S1c Fig) .All mice were euthanized by CO 2 asphyxiation, with CO 2 flow maintained for 60 s after respiratory arrest, followed by cervical dislocation.Cleared MFPs (4 th inguinal MFPs) containing mammary xenografts were then removed and fixed for analysis.All xenografts were assessed ~9 weeks after xenotransplantation surgeries, unless otherwise noted.
To generate primary mammary fibroblast conditioned media (CM), early passage (� P5) equine or canine fibroblasts were seeded at 1.33 x 10 4 cells/cm 2 in T25 culture flasks (cat #: 07-000-226) (Thermo Fisher) and incubated overnight at 37˚C in 5% CO2 in fibroblast culture media.Fibroblast culture media was removed, then cell monolayers were washed twice in sterile 1x PBS (Corning Inc).Four ml of serum-free (sf) DMEM (Corning Inc.) was added to fibroblast cell monolayers and incubated for 24 h to generate fibroblast CM.CM was removed and centrifuged twice at 300 x g for 10 min, with supernatants moved to a sterile tube after each spin to remove cell debris before proceeding to BrdU proliferation analysis.
BrdU proliferation analysis was performed using the BrdU Cell Proliferation ELISA kit (colorimetric) (cat #: ab126556) (Abcam, Cambridge, UK), as per manufacturer's instructions.Briefly, biological replicates (n = 3) of equine and canine MDEC cultures were plated in triplicate at 5 x 10 3 cells/well in a 96-well cell culture plate (cat #: 3596) (Corning Inc.) and allowed to recover for 24 h.EpSC media was removed and fibroblast CM containing 1X BrdU reagent was added, after which cells were incubated for 24 h.Cells were then washed, fixed, and BrdU was labeled, as per manufacturer's instructions.BrdU incorporation was assessed via spectrophotometric analysis using a Tecan Infinite M200 Pro Microplate Reader (Tecan, Ma ¨nnedorf, Switzerland).Colorimetric reads were taken at 450 nm and background reads (550 nm reads, 450nm reads from "no BrdU" control wells) were subtracted prior to statistical analysis.

Xenotransplanted mammary tissue fragments from equine and canine donors engraft in murine mammary fat pads and establish mammary xenografts
Developing mammary glands were extracted from pubescent (3-4 weeks old) immunodeficient NSG mice to de-epithelialize (clear) the mammary fat pads (MFPs) [21,24,29], and mammary tissue fragments from equine or canine donors were bilaterally xenotransplanted   regionally-confined structures lack detectable ductal branching and resemble mammary xenografts derived from other mammalian donors such as human [29] and cow [15,24,27].Hematoxylin and eosin (H&E) staining showed that both equine and canine xenografts are comprised of multiple acinar-like structures containing hollow lumina that are surrounded by a layer of supportive stroma (Fig 2b).To confirm the correct species of origin and to assess the presence of secretory luminal cells in the mammary xenografts, immunohistochemistry (IHC) was performed to detect β-lactoglobulin (β-LG), a whey protein secreted in the milk of ruminants, horses, and dogs, but absent in humans and rodents [44,45].The presence of β-LG in luminal cells and within lumina verified that these structures were derived from equine or

Mammary xenografts proliferate and display parenchymal and stromal characteristics similar to donor equine and canine mammary glands
Equine and canine mammary xenografts were characterized by IHC using functional and structural markers (Fig 3).The proliferation marker Ki-67 (Ki-67) was used to assess proliferation of the engrafted mammary tissue fragments, indicating survival within the host MFP.Ki-67 + cells were observed in both equine and canine mammary xenografts with presence localized primarily to the inner luminal cell layer (Fig 3a).To determine if the mammary xenografts recapitulate the structural architecture and protein labeling patterns observed in donor glands, markers for myoepithelial cells (i.e., α-smooth muscle actin (α-SMA) and cytokeratin-14 .Presence of estrogen receptor-alpha (ERα) was also assessed, as mammary gland responsiveness to mammotropic hormones (e.g., estrogens) is a defining feature of mammary gland growth and functionality [46].As expected, we observed ERα presence in the nuclei of luminal cells of both equine and canine xenografts (Fig 3b), mirroring characteristics of the donor glands [31,32] (S2b(ii) Fig) and indicating that these mammary xenografts may be responsive to hormonal stimulation.
To visualize mammary xenografts in a three-dimensional space and assess vascularization, MFPs containing mammary xenografts were rendered optically transparent using a Clear Unobstructed Brain Imaging Cocktails (CUBIC)-based approach and imaged using light sheet microscopy [42].In concordance with observations of acetocarmine-and H&E-stained xenograft tissues using 2D imaging techniques (Fig 2a and 2b), 3D imaging also showed that equine and canine xenografts consist of multiple acini confined to the xenotransplantation site (Fig 4a, S1-S3 Videos).Importantly, autofluorescent imaging, which is sufficient to define anatomical boundaries within tissues [47,48], revealed blood vessels surrounding and penetrating equine and canine xenografts, indicating endothelial cell recruitment (Fig 4a, S1-S3 Videos).The extensive vasculature within the xenograft stroma of both species was corroborated by H&E staining (Fig 4b).Performing IHC analysis to evaluate vimentin presence, a filamentary protein present in both myoepithelial cells [49] and stromal fibroblasts [31,32], showed that the mammary parenchyma of each xenograft was surrounded by an extensive fibroblast-rich stroma (Fig 4c ), further demonstrating structural similarity to what is observed in donor mammary glands.Important to note is that: (i) the antibody used for vimentin labeling is non-reactive in mouse [50], as indicated by a lack of vimentin labeling in vimentin-rich murine tissues (i.e., skin and mammary gland [51][52][53][54]) following IHC (S4 Fig) , and (ii) positive labeling was localized solely to the xenotransplantation site and absent in other extracellular matrix (ECM)rich, fibrous regions of the MFP, suggesting that the observed stroma of equine and canine xenografts is largely derived from the stromal cells of the xenotransplanted tissue fragments.

Host pregnancy upregulates expression of milk-associated genes and alters mammary xenograft proliferation
Pregnancy promotes the production of circulating ovarian mammotropic hormones and growth factors that drive mammary development [55,56].It is well-established that many of these bioactive factors are species cross-reactive [29,[57][58][59].To validate hormone responsiveness, and thus, functionality of the mammary xenografts, host mice were mated and xenografts were assessed 18 days post-coitus (dpc) (S1b Fig), a period prior to parturition during which circulating levels of mammotropic ovarian hormones are high and the murine mammary gland has developed to facilitate lactation [29,60,61] [9,46] and β-lactoglobulin (LGB1) [9] were assessed by RT-qPCR analyses of MFPs containing xenografts using equine or canine-specific primers.Equine and canine xenografts recovered from pregnant hosts showed an upregulation in both CSN2 and LGB1, albeit this did not reach significance for the equine xenografts due to a high variation in gene expression in the nonpregnant group (Fig 5a).H&E imaging showed the presence of thickened eosinophilic deposits, suggestive of lipid synthesis and proteinaceous fluid production [29,56]   It is well-established that in addition to lactogenesis, pregnancy also drives increased cell number and glandular surface area [62].A characteristic of this process is increased proliferative index of epithelial cells as the gland develops to facilitate lactation [63][64][65].To that end, IHC labelling of Ki-67 in nonpregnant and pregnant equine and canine xenografts (Fig 6a(i)) showed a significant increase in the percentage of Ki-67 + mammary luminal cells was observed in canine, but not equine, xenografts recovered from pregnant mice (Fig 6a (ii)).To assess xenograft structural alterations, wholemounts containing equine and canine mammary xenografts from nonpregnant and pregnant mice were stained with acetocarmine, which showed a lobule-like, spherical morphology confined to the xenotransplantation site irrespective of donor species or host pregnancy status (Fig 6b).To determine if host pregnancy drives growth, xenograft surface area was assessed [28].A trend towards increased average xenograft surface area following pregnancy was observed, especially in the canine xenografts; however, sizes were highly variable within groups and thus, statistical significance was not reached (Fig 6c).Although the reasons for this remain elusive, the varied xenograft responses could be due to horses, dogs, and mice displaying in vivo differences in circulating hormone concentrations during pregnancy [66][67][68], or due to differences in mammary developmental stages between equine and canine xenografts in response to ovarian hormones, which would not be captured in these experiments as all xenografts were recovered and analyzed at the same time point; 18 dpc.Collectively, and despite some discrepancies between both mammals, these data

Preimplantation of mammary fibroblasts does not promote mammary ductal branching in equine and canine xenografts
While mammary lobule-like structures are commonly observed following equine and canine mammary cell xenotransplantation, indicated by xenografts consisting of clustered acini (Fig 2a and 2b, S1-S3 Videos), ductal branching into the MFP is typically absent [24,29].Previous reports using human mammary epithelial cells showed that pre-implantation of speciesmatched fibroblasts within the MFP were capable of driving xenograft ductal morphogenesis [29,38].This is based on the knowledge that soluble ligands produced by stromal fibroblasts, such as fibroblast growth factor (FGF), fibroblast-derived ECM components, such as collagen-I, and fibroblast-derived matrix remodeling proteins are essential for mammary developmental and ductal outgrowth [69][70][71].As seen in S1c Fig, we performed experiments by implanting cultured mammary gland-derived primary species-matched fibroblasts into cleared MFPs to generate a stromal environment that would be more conducive for ductal formation.First, we confirmed increased collagen deposition and successful engraftment of equine and canine fibroblasts in MFPs that were recovered 2 weeks following implantations by (i) staining with Picro-Sirius (Sirius) red and Masson's trichrome (MTC) and (ii) performing IHC for vimentin presence using the antibody clone that is non-reactive in mouse [50] (S4 Fig) .MFPs that received fibroblast implantations from both species were characterized by dense, collagen-rich populations of vimentin + stromal fibroblasts (Fig 7a), indicating that implanted fibroblasts successfully engrafted and deposited collagen.As expected, these dense regions were absent in MFPs that received the Matrigel vehicle injections (Fig 7a).Second, we xenotransplanted equine and canine mammary gland tissue fragments 2 weeks after the species-matched fibroblast implantations and analyzed the xenografts 9 weeks later (S1c Fig) .In contrast to previous reports with immortalized human fibroblast pre-implantations [29,38], we did not observe ductal growth in equine or canine xenografts in the presence of species-matched fibroblast engraftment prior to mammary xenotransplantations, as assessed by acetocarmine staining of wholemounts.Specifically, equine mammary xenografts within fibroblast pre-implanted MFPs showed a lobule-like morphology confined to the xenotransplantation site, which was similar to wholemounts that received vehicle injections (Fig 7b) or no pre-implantation procedure at all (Fig 2a).Despite a similar overall lobule-like morphology, it was noticed that some equine xenografts showed more blebbing formations after fibroblast pre-implantation (< 20% of xenografts) when compared to vehicle control (0% of xenografts in the vehicle group) (Fig 7b).The morphology of the canine xenografts was also lobule-like in both fibroblast preimplanted and vehicle groups, with interconnected outgrowth structures observed (Fig 7b).Both vehicle and fibroblast pre-implanted groups displayed similar proportions of blebbing formations (~35-50% for both groups), indicating that this morphology in canine xenografts may be a species-specific characteristic.When quantifying 2D surface areas, there were no statistically significant differences between treatment groups for both species, despite a trending increase in surface area in the fibroblast pre-implanted groups (Fig 7c).
Due to the trending increase in xenograft surface area, an in vitro experiment using a BrdU-ELISA assay was performed as a proxy to assess the capacity of paracrine signaling by primary mammary fibroblasts to drive mammary epithelial cell proliferation.Mammospherederived epithelial cells (MDEC), which are heterogenous cultures of primary mammary cells derived through the same approach used to generate mammary tissue fragments for xenotransplantation surgeries [43], were treated with species-matched primary fibroblast conditioned media (CM) and subjected to the BrdU-ELISA to determine changes in mammary cell proliferation (Fig 7d).Interestingly, MDEC cultures treated with primary mammary fibroblast CM showed decreased proliferation when compared to MDEC cultures treated with control base media (DMEM), suggesting that paracrine signaling from pre-implanted mammary fibroblasts is most likely not a major driver of mammary xenograft growth in vivo.

Discussion
Mammary gland function (i.e., lactation strategies), and disease incidence (i.e., mammary cancer) vary drastically across mammals [4,72].These variations allow for a unique comparative species approach to identify novel regulatory factors and underlying molecular mechanisms of mammary gland (patho)physiology that are relevant to both human and veterinary health.Such research, however, is challenging due to logistical constraints and limited model availability, especially for large and rare mammals, respectively.Thus, a more manageable in vivo model is warranted.Here, we describe a reproducible and functional xenotransplantation model that uses cryopreserved mammary tissue fragments from equine or canine donors.Characteristics such as bilayered architecture, vascularization, supportive fibrous stroma, milk production, and hormone responsiveness in mammary xenografts demonstrate the generation of functional mammary structures.Importantly, we used minimally processed cryopreserved tissue fragments containing both stroma and parenchyma, which overcomes the need for immediate access to fresh tissues and allows for long-term storage of mammary gland tissue fragments of virtually any mammal, especially those that are wild and/or rare.
While the overall features were similar for equine and canine donors, several differences were noted.Specifically, when demonstrating xenograft functionality by inducing pregnancy in host mice, we found an upregulation of lactation-associated genes (CSN2 and LGB1) and β-LG presence within xenografts for both donor mammals recovered from pregnant mice, whereas an increased proliferative index was observed in canine, but not equine xenografts.During host pregnancy, there was a mild increase in the presence of inspissated eosinophilic deposits that were evident solely in equine, but not canine, xenograft tissue sections.This discrepancy could be due to innately different responses to circulating hormone concentrations between these two donor species, particularly when considering species-specific differences in circulating pregnancy hormone levels and gestation [66][67][68]73], which may not be reflected adequately in the host mice at 18 dpc.Notably, the length of pregnancy varies across all three mammals, with ~20 day [74], ~62 day [75], and ~350 day [76] gestation periods for mice, dogs, and horses, respectively, indicating that pregnancy hormone-associated temporal signaling differs across mammals.Furthermore, circulating pregnancy hormone concentrations differ in the two donor species compared to mice.For example, the serum concentration of 17βestradiol (E2), a potent estrogen that promotes post-natal mammary growth, increases during pregnancy [77,78] and rises to ~35 pg/ml in dogs and ~50 pg/ml in horses at 2 weeks and 5 months gestation, respectively [79][80][81], but rises to a substantially greater concentration of 1000-1800 pg/ml in mice at 7 days gestation [68].Interestingly, administration of E2 in cows promotes mammary involution and decreases milk synthesis [82], indicating that high E2 levels within host mice may have potentially had an inhibitory effect on the pregnancy-associated phenotypes in the equine xenografts.Furthermore, prolactin, a hormone essential for mammary alveolar development [83], differs structurally in equine relative to multiple other mammals by the number of cysteine residues [84][85][86], indicating potential challenges with crossspecies signaling.To address these hormonal differences, ectopic hormone stimulation, administered either via injection [26] or subcutaneous implantations of slow-release hormone pellets [26,28,87], that more accurately reflects hormonal concentrations and timepoints observed during donor mammal pregnancies may be required to better accentuate xenograft growth and development [28].This would be particularly relevant for future studies intending to investigate variations in mammary lobule development and lactation.Despite these discrepancies across mammals, the findings demonstrate that both equine and canine mammary xenografts are hormonally responsive.
Mammary xenografts from both donor mammals displayed bilayered epithelial architectures that recapitulate the donor gland, however, these structures were confined to the xenotransplantation site and lacked extensive ductal branching.In an attempt to initiate mammary ductal formation, we pre-implanted species-matched primary mammary fibroblasts into the cleared MFPs 2 weeks prior to mammary tissue fragment xenotransplantation.Using this approach, we intended to introduce factors important for ductal morphogenesis, such as extracellular matrix (ECM) remodeling, which promote fibroblast-derived growth factor signaling [29,38,69].Unlike findings that observed immortalized fibroblast-driven mammary duct formation in human xenografts [38], our approach did not drive ductal morphogenesis in either equine or canine xenografts, with xenografts across treatment groups retaining a lobule-like morphology.Interestingly, an infrequent incidence of structural blebbing was observed in equine xenografts following fibroblast pre-implantation, as well as in both vehicle-treated and fibroblast pre-implanted canine xenograft groups.These observations may be a result of species-specific variations in donor fragments, variations in response to host-derived growth factors/stromal components, and/or responses to species-matched fibroblast or Matrigelderived ECM components/growth factors.Furthermore, it is possible that fibroblast preimplantation promotes the growth of the mammary xenografts, as indicated by overall increased 2D surface area in the fibroblast pre-implanted groups, however these analyses did not reach statistical significance.Since the pre-implanted primary mammary fibroblasts were observed in dispersed patches throughout the MFPs, these cells may not have been in direct contact with xenotransplanted mammary fragments, thus, we investigated whether paracrine signals from primary mammary fibroblast can drive mammary epithelial cell proliferation in a controlled in vitro setting using primary mammosphere-derived epithelial cell (MDEC) cultures.Interestingly, and in contrast to other reports assessing fibroblast CM stimulation in mammary epithelial cell cultures [88,89], treatment of MDEC cultures with primary mammary fibroblast CM resulted in decreased cell proliferation.However, studies where primary fibroblasts were co-cultured with mammary epithelial cells reported decreased mammary cell proliferation [70,90], indicating that mammary epithelial cell-fibroblast interactions are not always pro-proliferative, and thus, it is possible that direct signaling and/or ECM remodeling, but not paracrine signaling, by mammary fibroblasts, is the major driver for inducing xenograft ductal branching in this our model.To evaluate whether pre-implanted fibroblasts have some effects on the xenografts despite a lack of ductal morphogenesis, we could in future studies assess the expression of genes associated with ductal morphogenesis, such as transforming growth factor beta 1 (TGFβ-1) [91], or determine if cell polarity at apical regions of the mammary xenografts is altered, as previously reported [70,92].Also, further optimization of the fibroblast implantation process, potentially via fibroblast irradiation, fibroblast immortalization, or generation of transgenic fibroblast populations with expression profiles that promote fibroblast MFP colonization and/or ductal morphogenesis [29,38,69], may drive ductal formation in equine and/or canine xenografts.Following insights gained from mammary ductal morphogenesis experiments in human mammary organoids, one approach could be to encapsulate mammary fragments within collagen gels that reflect donor stroma collagen content prior to xenotransplantation [93,94].Furthermore, the addition of an inhibitor of the Rho-ROCKmyosin II signaling cascade has been shown to drastically increase organoid ductal branching, and thus, incorporating this inhibitor within an encapsulating collagen gel may be a promising approach to improve xenograft branching morphogenesis [93,94].Nevertheless, and despite a lack of extensive ductal morphogenesis, the xenotransplantation model at its current stage remains suitable to study lobule-related aspects of mammary health and disease in vivo.
Despite the fact that equine and canine mammary xenografts recapitulated stromal and parenchymal compartments, there are some limitations of this model.Firstly, xenograft viability was demonstrated up to only 9 weeks, thus, long-term viability of these structures must be considered and future studies assessing long-term xenograft survival and viability are warranted.One study demonstrated that human mammary xenografts in athymic nude mice were detectable at 25 weeks post-surgery, although they were present at a significantly lower frequency compared to 4 weeks post-surgery [28].Compared to NSG murine hosts, athymic nude mice are relatively immunologically competent, thus, NSG mice are considered more permissive to tissue engraftment [95] and may be more suited to long-term xenograft analyses compared to athymic nude hosts.Secondly, given that the xenografts lack drainage architecture, some xenografts exhibit a ductal ectasia-like phenotype [96], i.e., the accumulation of proteinaceous fluid within dilated lumina, which results in inflammation and fibrosis under normal immunologic conditions [97].To this end, effectively inducing xenograft duct formation may be particularly crucial to reduce proteinaceous buildup and alleviate this ectasia-like phenotype.Lastly, given that the NSG hosts are immunodeficient, experiments using this model do not account for the regulatory roles of the immune system in mammary health and disease [98][99][100].Despite these limitations, this model still allows for the in vivo assessment of stromal/epithelial cell responses to treatments (e.g., carcinogens), indicating value for cell-or compartment-specific analyses.
One of the reasons for selecting equine and canine mammary tissue fragments was based on their natural variation in mammary cancer incidences, despite both domesticated mammals sharing similar habitats with humans [4,7].Our group has published work describing molecular mechanisms that might potentially drive this difference in cancer incidence using in vitro primary cell cultures from these two, and other, mammals [33,36].With the successful generation of equine and canine xenografts, we are now able to confirm and extend this work in vivo.For example, and similar to induced carcinogenesis studies in rodent models, host mice containing equine and canine mammary xenografts could be treated with progestins (e.g., medroxyprogesterone acetate), followed by exposure to mammary carcinogens (e.g., 7,12-dimethylbenz(a)anthracene (DMBA) [101][102][103] or high levels of estrogens (e.g., 17-βestradiol) [5,13,96], and their xenografts could then be assessed for neoplastic features or other indications of early induced malignancy at the molecular level (S6 Fig) .Additionally, host mice containing mammary xenografts may also be adequate models for assessing variations in lactation strategy and developmental processes via hormone supplements or relevant pharmaceuticals (S6 Fig) .Given that xenografts have on average a surface area of ~1 mm 2 , they are of adequate size for single-cell RNA sequencing and/or spatial transcriptomics analysis [104] (S6 Fig) .Furthermore, current bioinformatics classification tools, such as Xenome [105] and XenoCell [106], providing the opportunity to discriminate host from xenograft sequences, thus allowing for accurate assessment of species-specific expression in vivo.
coordinating equine and canine mammary tissue extractions.We would also like to thank Rebecca Williams and the Cornell University Biotechnology Resource Center for assistance with light sheet imaging, and Sophie Nelissen for her assistance with histological data interpretation.
into the residual stroma (Fig 1a.Successful clearing of the MFP was confirmed by acetocarmine staining of the removed murine endogenous mammary ductal tree (Fig 1b).Mammary tissue fragments from both species ranged from ~200-400 μm in diameter and contained both parenchymal and stromal components, as shown by mammary ducts with characteristic mammary lumina surrounded by a fibrous supportive stroma (Fig 1c).Nine weeks after the xenotransplantation surgery (S1a Fig), MFPs containing xenografts were extracted for downstream analyses.Based on the presence or absence of mammary xenografts within formalinfixed paraffin-embedded (FFPE) serial sections and acetocarmine stained wholemounts, we report a ~50% engraftment success rate (Fig 1d).Mammary xenografts from both equine and canine mammary donors were observed in cleared MFPs (Fig 2).Wholemount staining showed that equine and canine xenografts resemble mammary lobules containing multiple clustered acini that are structurally distinct from the branching mammary ductal tree characteristic of the mouse mammary gland (Fig 2a).These

Fig 1 .
Fig 1. Overview of mammary fat pad clearing and xenotransplantation procedure.(a).Diagram depicting the mammary fat pad (MFP) clearing and xenotransplantation approach.(b).Acetocarmine-stained pubescent murine mammary gland extracted from the 4 th MFP of a 3-week-old NOD scid gamma (NSG) mouse.Dotted line indicates location of incision that separated the developing mammary gland from the remaining fat pad.Scale bar = 500 μm.(c).Representative mammary tissue fragments from equine and canine donors stained with H&E.Arrowheads indicate regions of fibrous supportive stroma and asterisks (*) indicate representative examples of mammary ductal structures.Scale bar = 100 μm.(d).Pie chart of percent (%) engraftment success rate of xenotransplantations, as determined by histological analysis.Counts determining engraftment success rate per experiment are presented in S3 Table. https://doi.org/10.1371/journal.pone.0298390.g001

Fig 3 .
Fig 3. Mammary cell proliferation and parenchymal characteristics.(a).IHC analysis of Ki-67 presence in equine and canine xenografts.No primary antibody control tissues (No 1˚ctrl) are presented.(b).IHC analysis of α-smooth muscle actin (α-SMA), cytokeratin-14 (CK14), cytokeratin-18 (CK18) and estrogen receptor-α (ERα) labeling in equine and canine mammary xenografts.Representative tissues treated with isotype controls are presented.Arrowheads indicate positive IHC labeling (red colorimetric indicator) Scale bar = 100 μm.https://doi.org/10.1371/journal.pone.0298390.g003 (S5 Fig).Gross anatomical changes in mated mice at 18 dpc are shown in S5a Fig, and representative developmental changes of the murine mammary gland within the 3 rd inguinal mammary fat pad are presented (S5b Fig) to indicate typical murine mammary development at this timepoint.Changes in expression of the milk protein-associated genes β-casein (CSN2) within the lumina of equine, but not canine, xenografts (Fig 5b), with slightly greater abundance of deposits observed in the equine xenografts from pregnant hosts.Despite a lack of dense thickened deposits, eosinophilic secretions were still present within the lumina of the canine xenografts (Fig 5b).IF imaging of β-LG in nonpregnant and pregnant equine (Fig 5c(i)) and canine (Fig 5d(i)) xenografts revealed a baseline level of β-LG presence