**4. pgMEC characteristics**

30 s to 1 min in ice cold acetone/methanol (1:1) or for several minutes in 4% paraformaldehyde. Fixation was followed by permeabilization (not necessary when using acetone-methanol fixation or in case of membrane-bound markers) with 0.3% Triton X-100 for 10 min. After washing with phosphate buffered saline (PBS), cells were blocked with 5–10% fetal serum (it is recommended to use fetal serum from species in which secondary antibodies were produced) and 1–3% bovine serum albumin (BSA) for 60 min. Incubation with primary antibodies was performed overnight at 4°C. Next day, cells were washed with PBS several times and incubated with fluorescently labeled secondary antibodies at room temperature for 1 h. After washing with PBS, cell nuclei can be counterstained with 4′,6-diamidino-2-phenylindole (DAPI), washed, and visualized under microscope. In case of paraffinized tissue, sections were deparaffinized using xylene and rehydrated in decreasing concentrations of ethanol. Rehydrated tissue slices were washed in PBS, followed by performing heat-induced antigen retrieval in a microwave oven, using 10 mM sodium-citrate buffer (pH 6). Afterward, the same protocol was used for immunofluorescent staining as described previously for pgMECs. The more detailed protocols and the antibodies used were described in our previ-

When performing mammosphere formation assay, a single-cell suspension of the mammary cells was grown in DMEM/F12 medium, supplemented with EGF (20 ng/mL), bFGF (human, 20 ng/mL), heparin (4 μg/mL), cholera toxin (10 ng/mL), hydrocortisone (0.5 μg/mL), insulin (0.5 μg/mL), and B27 supplement (2%), and grown in 6-well ultralow-attachment plates with or without extracellular membrane matrix or in hanging drops, according to the described

Growth medium was aspirated and the pgMECs were fixed in 4% paraformaldehyde for 15 min. Oil Red O (0.5 g) was dissolved in 50 ml of isopropanol and diluted with water (3:2) and then left for 10 min, and the solution filtered through a 20-μm filter. Cells were briefly washed with isopropanol (60%) and incubated with solution of Oil Red O for 15 min at room temperature. The cells were then rinsed with isopropanol and washed under tap water. The formation of lipid droplets (red stain) was observed under bright

Mammary gland development occurs in stages; proliferation and differentiation of mammary cells are dependent on sexual development and reproduction, which are under control of endocrine system [12]. The gland is primarily composed of mesenchymal and epithelial tissue and the latter is subjected to significant remodeling during lactation cycles. The tissue remodeling involves proliferation and differentiation of the epithelial cells forming functional

**3. Mammary tissue–derived primary cells as an** *in vitro* **model**

ous publications [8–10].

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protocol [11].

*2.2.4. Oil Red O staining*

field microscope.

*2.2.3. Mammosphere formation assay*

## **4.1. Morphology and growth**

The derived primary cell culture consisted of a heterogeneous population of mostly epithelial and mesenchymal (fibroblast-like) cells. Epithelial cells grew in round-shaped densely packed islands of cells with multiple nucleoli and exhibited typical cobblestone morphology. Cells randomly spreading around these islands were larger, spindle-shaped cells, morphologically resembling fibroblasts (**Figure 2**).

Cell proliferation was slow for the first week after seeding dissociated cells in plastic dishes. After the first passage, the cells started to proliferate much faster and overgrew the surface

The method of choice for characterization of different cell types in a cell culture is staining cells with tissue/cell type–specific antibodies, which reveal presence and localization of markers in the cells. The analysis of whole-transcriptome mRNA expression and review of previous studies, regarding distinctive mammary-specific markers in different species, represented a rationale for selection of antibodies, potentially useful for characterization of major cell types in goat mammary tissue and the derived cell cultures. Antibody-based characterization is a challenge in ruminants (especially goats) as most of the commercially available antibodies are targeted against human or rodent antigens, while their reactivity in ruminants is generally unknown and has to be determined empirically. To determine the presence of mammary-specific protein markers, immunofluorescent staining with different antibodies was performed. Based on our results, we suggest cytokeratins (KRT) 14 and 18, as well as vimentin (VIM) as suitable markers for basic characterization of primary mammary cell cultures (**Figure 4**). Namely, cells of mesenchymal origin (e.g. fibroblasts) express VIM (**Figure 4A** and **B**), KRT 14 is distinctive of myoepithelial (**Figure 4E** and **F**), whereas KRT18 of luminal epithelial cells (**Figure 4C** and **D**). Based on these three markers, it is possible to distinguish epithelial cells from mesenchymal cells and distinguish between basal/myoepithelial and

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**Figure 4.** Basic characterization of the pgMECs. Fixed pgMECs under bright field (A, C, and E) and fluorescent illumination (B, D, and F) under 40× magnification (A–D; scale bars = 20 μm) and 20× magnification (E and F; scale bars = 50 μm) (photo: J. Ogorevc). Fluorescently labeled secondary antibodies were used to visualize expression and localization of the markers and a DAPI counterstain was used to visualize the nuclei. (A and B) The cells immunostained with primary antibodies against VIM. Spindle-shaped fibroblasts stained for VIM. (C and D) Double staining for KRT14 and KRT18. Luminal epithelial cells stained for KRT18 (D), whereas myoepithelial for KRT14 (D). Interestingly, when grown at low confluency, cells tended to organize as in alveoli, myoepithelial cells encircling luminal cells. (E and F) Staining for KRT14. Two islands of epithelial cells visible; myoepithelial cells stained for KRT14 (upper right corner),

whereas no staining with KRT14 is visible in luminal epithelial cells (left).

luminal cells.

**Figure 2.** Primary culture 5 days after seeding under bright field microscope (photo: J. Ogorevc). (A) Heterogeneous cell types visible in the primary culture (40× magnification; scale bar = 200 μm). (B) Islands of epithelial cells surrounded by mesenchymal cells (fibroblasts). Cell debris can be observed in primary culture prior to first passaging (100× magnification; scale bar = 100 μm). (C) Densely packed island of epithelial cells (200× magnification; scale bar = 50 μm).

every several days. No changes in proliferation, morphology, or growth patterns were noticed for over five passages (**Figure 3**). When cells were kept at full confluency (without passaging) for extended period of time, they started to show signs of senescence.

#### **4.2. Expression of specific markers**

Different cell types express cell type–specific genes, which can be considered characterization markers. A draft of such markers was used to characterize the derived pgMECs and to distinguish different cell types in a heterogeneous primary cell culture.

Transcription profile generated by NGS showed that markers typical of basal/myoepithelial and luminal epithelial cells were highly expressed. The most expressed were different keratins, desmoplakin, and actins. The expression of markers varies based on the number of different cell types/lineages present in the culture and culture conditions, which may favor proliferation of a specific cell type and may promote differentiation (e.g. epithelialto-mesenchymal transitions).

**Figure 3.** Mammary cell lines after passaging (photo: J. Ogorevc). (A) Epithelial and mesenchymal cells under 40× magnification (scale bar = 200 μm). (B) Island of epithelial cells (right) and mesenchymal cells (left) (200× magnification; scale bar = 50 μm). (C) Enriched culture of epithelial cells after differential trypsinization and removal of fibroblasts (200× magnification; scale bar = 100 μm).

The method of choice for characterization of different cell types in a cell culture is staining cells with tissue/cell type–specific antibodies, which reveal presence and localization of markers in the cells. The analysis of whole-transcriptome mRNA expression and review of previous studies, regarding distinctive mammary-specific markers in different species, represented a rationale for selection of antibodies, potentially useful for characterization of major cell types in goat mammary tissue and the derived cell cultures. Antibody-based characterization is a challenge in ruminants (especially goats) as most of the commercially available antibodies are targeted against human or rodent antigens, while their reactivity in ruminants is generally unknown and has to be determined empirically. To determine the presence of mammary-specific protein markers, immunofluorescent staining with different antibodies was performed.

Based on our results, we suggest cytokeratins (KRT) 14 and 18, as well as vimentin (VIM) as suitable markers for basic characterization of primary mammary cell cultures (**Figure 4**). Namely, cells of mesenchymal origin (e.g. fibroblasts) express VIM (**Figure 4A** and **B**), KRT 14 is distinctive of myoepithelial (**Figure 4E** and **F**), whereas KRT18 of luminal epithelial cells (**Figure 4C** and **D**). Based on these three markers, it is possible to distinguish epithelial cells from mesenchymal cells and distinguish between basal/myoepithelial and luminal cells.

every several days. No changes in proliferation, morphology, or growth patterns were noticed for over five passages (**Figure 3**). When cells were kept at full confluency (without passaging)

**Figure 2.** Primary culture 5 days after seeding under bright field microscope (photo: J. Ogorevc). (A) Heterogeneous cell types visible in the primary culture (40× magnification; scale bar = 200 μm). (B) Islands of epithelial cells surrounded by mesenchymal cells (fibroblasts). Cell debris can be observed in primary culture prior to first passaging (100× magnification; scale bar = 100 μm). (C) Densely packed island of epithelial cells (200× magnification; scale

Different cell types express cell type–specific genes, which can be considered characterization markers. A draft of such markers was used to characterize the derived pgMECs and to distin-

Transcription profile generated by NGS showed that markers typical of basal/myoepithelial and luminal epithelial cells were highly expressed. The most expressed were different keratins, desmoplakin, and actins. The expression of markers varies based on the number of different cell types/lineages present in the culture and culture conditions, which may favor proliferation of a specific cell type and may promote differentiation (e.g. epithelial-

**Figure 3.** Mammary cell lines after passaging (photo: J. Ogorevc). (A) Epithelial and mesenchymal cells under 40× magnification (scale bar = 200 μm). (B) Island of epithelial cells (right) and mesenchymal cells (left) (200× magnification; scale bar = 50 μm). (C) Enriched culture of epithelial cells after differential trypsinization and removal of fibroblasts (200×

for extended period of time, they started to show signs of senescence.

guish different cell types in a heterogeneous primary cell culture.

**4.2. Expression of specific markers**

bar = 50 μm).

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to-mesenchymal transitions).

magnification; scale bar = 100 μm).

**Figure 4.** Basic characterization of the pgMECs. Fixed pgMECs under bright field (A, C, and E) and fluorescent illumination (B, D, and F) under 40× magnification (A–D; scale bars = 20 μm) and 20× magnification (E and F; scale bars = 50 μm) (photo: J. Ogorevc). Fluorescently labeled secondary antibodies were used to visualize expression and localization of the markers and a DAPI counterstain was used to visualize the nuclei. (A and B) The cells immunostained with primary antibodies against VIM. Spindle-shaped fibroblasts stained for VIM. (C and D) Double staining for KRT14 and KRT18. Luminal epithelial cells stained for KRT18 (D), whereas myoepithelial for KRT14 (D). Interestingly, when grown at low confluency, cells tended to organize as in alveoli, myoepithelial cells encircling luminal cells. (E and F) Staining for KRT14. Two islands of epithelial cells visible; myoepithelial cells stained for KRT14 (upper right corner), whereas no staining with KRT14 is visible in luminal epithelial cells (left).

Additional markers useful to distinguish epithelial cells from other cell types and to determine epithelial subtypes are different keratins (e.g. 5, 19), epithelial cell adhesion molecule (EPCAM), estrogen receptor 1 (ESR1), tumor protein p63 (TP63), integrin subunit beta 1 (ITGB1/CD29), integrin subunit alpha 6 (ITGA6/CD49f), progesterone receptor (PGR), alpha smooth muscle actin (ACTA2), caseins (e.g. CSN2), and mucin 1 (MUC1).

Additionally, paraffin-embedded sections of goat mammary tissue were stained to compare the expression of the markers between the pgMECs and the mammary tissue (**Figure 5**). Most of the markers showed reactivity in both—the cell cultures and the tissue, whereas some of the markers showed reactivity only in pgMECs (ESR1, CD49F, and KRT5) or only in the tissue (TP63). Tissue sections undergo chemical and physical treatment, which might result in changed conformation and antigen masking. On the other hand, pgMECs adapt to *in vitro* environment, which may alter cell metabolism and expression of markers. Therefore, discrepancies in immunostaining results are possible between the tissue and pgMECs. For example, EpCAM marker was localized in cytoplasmic compartment of pgMECs (**Figure 5A** and **B**) and was also found in epithelial compartments of goat mammary tissue (**Figure 5C** and **D**). In case of MUC1, weak signal was observed in pgMECs (**Figure 5E** and **F**) and strong signal, showing distinctive localization of MUC1 only to apical plasma membranes of secretory (luminal) epithelial cells, was detected in the mammary tissue (**Figure 5G** and **H**).

#### **4.3. 3D organization—mammosphere formation**

Under conditions that do not allow adherence to the surface, differentiated epithelial cells undergo anoikis. Growth under nonadherent, serum-free conditions is a characteristic of mammary stem/progenitor cells, which in such conditions form spherical structures called mammospheres. Spherical structures formed by human mammary epithelial cells contain enriched population of cells capable to differentiate into luminal or myoepithelial cells (bipotent progenitors) [19]. The molecular and cellular processes in mammospheres are similar as those in developing alveoli of the mammary gland [20]. Hierarchically, mammary cells range from terminally differentiated cells to undifferentiated progenitors and stem cells, the latter two being likely targets for malignant transformations in cancer [21]. It was shown that an entire mammary gland can be reconstituted from a single mammary stem cell [22]. Existence of mammary stem/progenitor cells in goat was first demonstrated by [10].

Under nonadherent conditions, irregularly shaped floating masses (organoids) were formed after several days. Aggregates that arose in ultralow-attachment plates (**Figure 6A**) in medium supplemented with basement membrane matrix were rounder and larger in shape as those grown in medium without basement membrane matrix. Immunostaining of fixed mammospheres revealed that luminal (KRT18—positive) and basal/myoepithelial (KRT14—positive) cells were the main constituents of the mammospheres [8]. Additionally, mammospheres were grown using hanging drop method. Hanging drop method is used as one of the *in vitro* tests for determining the pluripotent character of putative stem cells. The spherical structures appeared after several days of growth in hanging drops. They were fewer in number, but larger and more round in shape (**Figure 6B**), compared to mammospheres grown in ultralowattachment plates. The mammospheres were fixed to glass slides and stained with DAPI and

**Figure 5.** Immunofluorescence of pgMECs and goat mammary tissue under bright field and fluorescent illumination, stained against EpCAM (A–D) and MUC1 (E–H) (20× magnification; scale bars = 50 μm) (photo: J. Ogorevc). Fluorescently labeled secondary antibodies (green) were used and a DAPI counterstain was used to visualize nuclei (blue). The mammary cell culture (A and B) and epithelial cells of the tissue (C and D) stained against epithelial cell–specific marker EpCAM. pgMECs showed weak staining against MUC1 (E and F), whereas strong signal, localized to apical membranes

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of alveolar structures, was observed in the tissue (G and H).

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Additional markers useful to distinguish epithelial cells from other cell types and to determine epithelial subtypes are different keratins (e.g. 5, 19), epithelial cell adhesion molecule (EPCAM), estrogen receptor 1 (ESR1), tumor protein p63 (TP63), integrin subunit beta 1 (ITGB1/CD29), integrin subunit alpha 6 (ITGA6/CD49f), progesterone receptor (PGR), alpha

Additionally, paraffin-embedded sections of goat mammary tissue were stained to compare the expression of the markers between the pgMECs and the mammary tissue (**Figure 5**). Most of the markers showed reactivity in both—the cell cultures and the tissue, whereas some of the markers showed reactivity only in pgMECs (ESR1, CD49F, and KRT5) or only in the tissue (TP63). Tissue sections undergo chemical and physical treatment, which might result in changed conformation and antigen masking. On the other hand, pgMECs adapt to *in vitro* environment, which may alter cell metabolism and expression of markers. Therefore, discrepancies in immunostaining results are possible between the tissue and pgMECs. For example, EpCAM marker was localized in cytoplasmic compartment of pgMECs (**Figure 5A** and **B**) and was also found in epithelial compartments of goat mammary tissue (**Figure 5C** and **D**). In case of MUC1, weak signal was observed in pgMECs (**Figure 5E** and **F**) and strong signal, showing distinctive localization of MUC1 only to apical plasma membranes of secretory (luminal)

Under conditions that do not allow adherence to the surface, differentiated epithelial cells undergo anoikis. Growth under nonadherent, serum-free conditions is a characteristic of mammary stem/progenitor cells, which in such conditions form spherical structures called mammospheres. Spherical structures formed by human mammary epithelial cells contain enriched population of cells capable to differentiate into luminal or myoepithelial cells (bipotent progenitors) [19]. The molecular and cellular processes in mammospheres are similar as those in developing alveoli of the mammary gland [20]. Hierarchically, mammary cells range from terminally differentiated cells to undifferentiated progenitors and stem cells, the latter two being likely targets for malignant transformations in cancer [21]. It was shown that an entire mammary gland can be reconstituted from a single mammary stem cell [22]. Existence

Under nonadherent conditions, irregularly shaped floating masses (organoids) were formed after several days. Aggregates that arose in ultralow-attachment plates (**Figure 6A**) in medium supplemented with basement membrane matrix were rounder and larger in shape as those grown in medium without basement membrane matrix. Immunostaining of fixed mammospheres revealed that luminal (KRT18—positive) and basal/myoepithelial (KRT14—positive) cells were the main constituents of the mammospheres [8]. Additionally, mammospheres were grown using hanging drop method. Hanging drop method is used as one of the *in vitro* tests for determining the pluripotent character of putative stem cells. The spherical structures appeared after several days of growth in hanging drops. They were fewer in number, but larger and more round in shape (**Figure 6B**), compared to mammospheres grown in ultralowattachment plates. The mammospheres were fixed to glass slides and stained with DAPI and

smooth muscle actin (ACTA2), caseins (e.g. CSN2), and mucin 1 (MUC1).

epithelial cells, was detected in the mammary tissue (**Figure 5G** and **H**).

of mammary stem/progenitor cells in goat was first demonstrated by [10].

**4.3. 3D organization—mammosphere formation**

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**Figure 5.** Immunofluorescence of pgMECs and goat mammary tissue under bright field and fluorescent illumination, stained against EpCAM (A–D) and MUC1 (E–H) (20× magnification; scale bars = 50 μm) (photo: J. Ogorevc). Fluorescently labeled secondary antibodies (green) were used and a DAPI counterstain was used to visualize nuclei (blue). The mammary cell culture (A and B) and epithelial cells of the tissue (C and D) stained against epithelial cell–specific marker EpCAM. pgMECs showed weak staining against MUC1 (E and F), whereas strong signal, localized to apical membranes of alveolar structures, was observed in the tissue (G and H).

**5.1. Mastitis model**

Because of the economic importance for dairy industry and possible health and milk quality risks for consumers, there is a great interest to understand and enhance natural immunity of the mammary gland. Mammary epithelial cells are capable of innate immune response during intramammary infections and represent important barrier against invading pathogens.

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In small ruminants, coagulase-negative staphylococci account for most of the mastitis cases, followed by Streptococci, *Staphylococcus aureus*, and other bacteria [23]. Additionally, contagious agalactia caused by *Mycoplasma agalactiae* (*Ma*) is a common cause of intramammary infections (contagious agalactia), especially in Mediterranean regions [24]. Mammary cell lines are often used to study immune response to mastitis, instead of *in vivo* infections. In our study, next-generation sequencing (NGS) was used to assess whole-transcriptomic response

The results show that the infection induced an innate immune response in the infected cells. The pgMECs were capable of recognizing and responding to the pathogen infection (**Figure 7**). The pgMECs responded by induced expression of cytokines (interleukins and chemokines) and other chemotactic agents, activation of complement system, apoptosis pathways, and induction of genes coding for antimicrobial effector molecules (e.g. defensins, lysozyme, and nitric oxide synthase) (**Figure 7A**). The changes in expression were moderate, with no phenotypically visible changes in cell morphology, which corresponds to subclinical course of contagious agalactia *in vivo*. The pathway enrichment analysis showed that the most affected pathways were associated with immune response, steroid and fatty acid metabolism, apoptosis signaling, transcription regulation, and cell cycle regulation. We speculate that physiologically, the *in vivo* immune contribution of the pgMECs is important for recruitment of

**Figure 7.** Transcriptomic studies on *Mycoplasma agalactiae*–infected pgMECs (modified from Ref. [25]). (A) Induction of immune-associated genes interleukin 8 (IL8), chemokine (C-X-C motif) ligand 5 (CXCL5), Toll-like receptor 2 (TLR2), and S100 calcium-binding protein 9 (S100A9). (B) Possible immune response mechanisms in pgMEC, suggested based

on differential expression of genes and analysis of genetic networks and metabolic pathways.

of *Ma*-challenged pgMECs 3, 12, and 24 h postinfection [25].

**Figure 6.** Formation of spherical structures in the pgMECs after 7 days of growth under nonadherent conditions (photo: J. Ogorevc). (A) Spherical structures in low-attachment plates (20× magnification; scale bar = 50 μm). (B) Organoids (mammospheres) grown in hanging drops (4× magnification; scale bar = 200 μm). (C and D) DAPI-stained fixed mammospheres, grown in hanging drops under bright field (C) and fluorescent illumination (D) (100× magnification; scale bar = 100 μm). Number of cells in the organoid structures can be estimated based on the number of stained nuclei (blue).

antibodies raised against KRT14 and 18. They consisted of several hundred cells (**Figure 6D**), which were KRT14- or KRT18-positive. Hanging drop is an efficient method to grow mammospheres from primary mammary cultures. The cells avoiding anoikis and forming organoids probably represent mammary progenitors.
