{"id":440161,"date":"2024-01-01T19:00:00","date_gmt":"2024-01-02T00:00:00","guid":{"rendered":"https:\/\/platohealth.ai\/mcam-inhibits-macrophage-mediated-development-of-mammary-gland-through-non-canonical-wnt-signaling-nature-communications\/"},"modified":"2024-01-03T05:42:46","modified_gmt":"2024-01-03T10:42:46","slug":"mcam-inhibits-macrophage-mediated-development-of-mammary-gland-through-non-canonical-wnt-signaling-nature-communications","status":"publish","type":"post","link":"https:\/\/platohealth.ai\/mcam-inhibits-macrophage-mediated-development-of-mammary-gland-through-non-canonical-wnt-signaling-nature-communications\/","title":{"rendered":"Mcam inhibits macrophage-mediated development of mammary gland through non-canonical Wnt signaling – Nature Communications","gt_translate_keys":[{"key":"rendered","format":"text"}]},"content":{"rendered":"
To identify potential regulatory factors of MECs, we performed clone formation assay24<\/a><\/sup> and transplantation assay25<\/a>,26<\/a><\/sup> for screening using a pooled mouse GeCKO library containing 130 209 single guide RNAs (sgRNAs), targeting 20 611 protein-coding genes, and 1000 control sgRNAs (non-targeting sgRNAs) in the mouse genome27<\/a><\/sup>. The mammary progenitor-enriched population was isolated using standard protocols24<\/a><\/sup> and transduced with a lentivirus prepared by the GeCKO library. Multiplicity of infection (MOI) was tested and applied to ensure that each cell contained a single sgRNA. The non-transfected cells were screened out by puromycin. After 10-14 days, we selected large clones (diameter \u226b<\/span> 100 \u03bcm, enriched with mammary progenitor cells) for deep sequencing. The stably transfected cells were used for a 5-week transplantation assay, and the regenerated mammary tissues were collected for deep sequencing (Fig. 1a<\/a>). Thus, based on the above methods, we performed gene enrichment of representative sgRNAs from plasmids, stably transfected cells, large clones, and regenerated mammary tissues.<\/p>\n a<\/b> Schematic of screening strategy. Created with BioRender.com. b<\/b> tSNE plot showing expression pattern of Mcam in different lineages according to single-cell sequencing data29<\/a><\/sup>. c<\/b> qRT-PCR analysis of Mcam<\/i> mRNA expression in different subpopulations of MECs shown in Figs. Sa-3b. n\u2009=\u20093 biological replicates. d<\/b> Immunofluorescence (IF) images of Mcam co-stained with basal marker K14 and luminal marker K18. Nuclei were stained in blue by DAPI. Scale bar, 20 \u03bcm. e<\/b> IF images of Mcam staining at various developmental stages of mammary gland. 4\u2009W, 4-week old; I, involution. Scale bar, 20 \u03bcm. Representative images were taken from 3 mice for each stage. f<\/b> qRT-PCR analysis of Mcam<\/i> mRNA expression in MECs from WT and cKO mice by MMTV-Cre. n\u2009=\u20099 mice per genotype. g<\/b> IF staining showing knockout efficiency of Mcam in WT and cKO mice. Scale bar, 20 \u03bcm. h<\/b> Western blotting showing Mcam protein expression in WT and cKO mammary tissues. 3 mice for WT group and 4 mice for cKO group. i<\/b>, j<\/b> Representative images (i<\/b>) and their quantification (j<\/b>) of mammary duct extension of whole-mount staining in WT and cKO mice at 5 weeks of age. Arrow, length of epithelial extension. Scale bar, 5\u2009mm. n<\/i>\u2009=\u200910 mice in each group. k<\/b> Representative images of mammary tissues in 5-week-old WT and cKO mice by H&E staining. Scale bar, 20 \u03bcm. l<\/b> Quantification of mammary duct and TEB thickness in WT and cKO mice. n<\/i>\u2009=\u200950 ducts or n<\/i>\u2009=\u200930 TEBs in WT and cKO mice. Data are means\u2009\u00b1\u2009SEM. Two-sided Student\u2019s t<\/i> test was used to evaluate statistical significance. Source data are provided as a Source Data file.<\/p>\n<\/div>\n<\/div>\n For sequencing, we first compared the overall distributions of sgRNAs from all samples to demonstrate the dynamics of the sgRNA library (Fig. S1a<\/a>) and validate the sequencing quality. The detected number of sgRNAs in the plasmids was close to that in the original library, showing that most sgRNAs in the library were represented (Figs. S1b<\/a> and S1c<\/a>). The sgRNAs in the large clones and regenerated mammary tissues, retained less sgRNAs, reflecting that sgRNAs were enriched in these samples. The global patterns of sgRNA distribution in different samples were distinct, as evident by the strong shifts in respective cumulative distribution functions (Fig. S1d<\/a>). In addition, we compiled a list of genes (Supplementary Dataset 1<\/a>) showing enrichment of 1, 2, or 3 or more sgRNAs, along with the proportion of samples where these genes demonstrated enrichment (Fig. S1e<\/a>). Based on MAGeCK28<\/a><\/sup> (http:\/\/bitbucket.org\/liulab\/mageck-vispr<\/a>), we obtained a candidate gene list properly accounted for mammary gland morphogenesis and\/or MEC clonogenicity and regenerative capacity, and found Mcam<\/i> was one of the common genes in all conditions (Fig. S2a<\/a>). By further analysis, we found multiple sgRNAs targeting Mcam<\/i> are significantly enriched in more than half of the regenerated mammary tissue (Fig. S2b<\/a>) with two or more sgRNAs targeting Mcam<\/i> enriched in each sample (Fig. S2c<\/a>).<\/p>\n The candidates were selected according to the expression profile based on single-cell sequencing data of MECs29<\/a><\/sup>, and sgRNAs ranked in each sample. The single-cell sequencing data showed that Mcam<\/i> was highly expressed in basal cells (Fig. 1b<\/a>). To validate this result, we collected basal (Lin\u2212<\/sup>CD24+<\/sup>CD29+<\/sup>) and luminal (Lin\u2212<\/sup>CD24+<\/sup>CD29\u2212<\/sup>) cell populations using fluorescence-activated cell sorting (FACS) (Fig. S3<\/a>a, b), with quantitative real-time polymerase chain reaction (qRT-PCR) confirming the high expression of Mcam<\/i> in the basal cell population (Fig. 1c<\/a>). Immunofluorescence (IF) staining showed that Mcam was co-localized with K14 (basal cell marker) but not with K18 (luminal cell marker) (Fig. 1d<\/a>). Taken together, Mcam was identified as a potential key regulator of MECs.<\/p>\n To further investigate the role of Mcam in regulating MEC proliferation and mammary gland development in vivo, we firstly examined Mcam expression pattern in four stages of mammary gland development, and found Mcam highly expressed in puberty (4-5 weeks), pregnancy, and lactation (Fig. 1e<\/a>). Then we bred Mcam flox mice30<\/a><\/sup> with MMTV-Cre mice to generated Mcamfl\/fl<\/sup>\/MMTV-Cre mice (cKO mice)31<\/a><\/sup> and K14-Cre mice to generate Mcamfl\/wt<\/sup>\/K14-Cre mice (heterozygous cKO mice) with specific loss of Mcam in MECs or basal cells. Mcam<\/i> was specifically depleted in MECs or basal cells based on qRT-PCR (Figs. 1f<\/a> and S3c<\/a>), IF staining (Figs. 1g<\/a> and S3d<\/a>), and western blot analysis (Figs. 1h<\/a> and S3e<\/a>) of whole mammary tissues. We next explored the mammary duct phenotype. Loss of Mcam significantly lengthened mammary ducts at the puberty stage (5-weeks old), and therefore increased mammary duct occupation in the fat pad (Figs. 1<\/a>i, j<\/a> and S3<\/a>f, g<\/a>). In addition to ductal length, mammary duct and terminal end bud (TEB) thickness increased significantly (Figs. 1<\/a>k, l<\/a> and S3<\/a>h\u2013j<\/a>). The above effects were not observed in adult mice (8-weeks old) (Fig. S3k<\/a>), which could be due to rare expression level of Mcam at this period (Fig. 1e<\/a>).<\/p>\n These results showed that Mcam loss enhances mammary gland development, and this phenotype is likely due to increased proliferation and differentiation of the mammary epithelium. Based on IF staining with Ki67 (cell proliferation marker), both basal and luminal cells demonstrated a much higher percentage of Ki67-positive cells upon loss of Mcam (Figs. 2<\/a>a, b<\/a> and S4<\/a>a\u2013e<\/a>), which is also supported by the enrichment of DNA replication pathway in cKO mice based on RNA-seq data (Fig. S4f<\/a>). All cell differentiation markers, including Stat5<\/i>, Elf5<\/i>, Foxa1<\/i>, Esr1<\/i>, and Gata3<\/i>32<\/a><\/sup>, were increased at the mRNA level (Fig. S4g<\/a>). Immunohistochemistry (IHC) staining showed PR–<\/sup> and PR+<\/sup> cells occurred in cKO mice by MMTV-Cre, implying Mcam loss promoting cell differentiation (Fig. S4h<\/a>). The induction of both cell proliferation and differentiation suggests that loss of Mcam enhances mammary duct morphogenesis.<\/p>\n a<\/b> Immunostaining of K14 (red) and Ki67 (green) expression in mammary ducts and TEBs of WT and cKO mice by MMTV-Cre. Scale bar, 10 \u03bcm. b<\/b> Ki67+<\/sup> cells in basal and luminal cell populations of mammary ducts and TEBs in WT and cKO mice. n<\/i>\u2009=\u200910 sections in each group. c<\/b>, d<\/b> FACS analysis (c<\/b>) and statistical ratios of basal cell population (Lin\u2212<\/sup>CD24+<\/sup>CD29+<\/sup>) and luminal cell population (Lin\u2212<\/sup>CD24+<\/sup>CD29–<\/sup>) (d<\/b>) in WT and cKO mice. n<\/i>\u2009=\u20096 mice for each genotype. e<\/b> Representative images (left) and the reconstitution efficiency at limiting dilution (right) of whole-mount-stained mammary outgrowths derived from transplantation of isolating WT or cKO Lin–<\/sup> cells and harvested at 6 weeks after transplantation. Scale bar, 5\u2009mm. n<\/i>\u2009=\u20098 mice for each group. f<\/b> Representative images (left) and the reconstitution efficiency at limiting dilution (right) of whole-mount-stained mammary outgrowths derived from transplantation of WT or cKO basal cells and harvested at 6 weeks after transplantation. Scale bar, 5\u2009mm. n<\/i>\u2009=\u20095 in 200-cell of cKO group, n<\/i>\u2009=\u20096 mice in the other groups. g<\/b>\u2013i<\/b> Representative images (g<\/b>), clone numbers (h<\/b>, n<\/i>\u2009=\u20096 biological replicates), and clone diameters (i<\/b>, n<\/i>\u2009=\u200993 for WT and n<\/i>\u2009=\u2009125 for cKO by MMTV-Cre) of colonies formed by MECs derived from WT and cKO mice. Scale bar, 500 \u03bcm. Data are means\u2009\u00b1\u2009SEM. Two-sided Student\u2019s t<\/i> test was used to evaluate statistical significance. Source data are provided as a Source Data file.<\/p>\n<\/div>\n<\/div>\n To investigate whether the above phenotype is driven by MaSCs, we explored changes in the MaSC-enriched cell population (Lin\u2212<\/sup>CD24+<\/sup>CD29+<\/sup>) in cKO mice by MMTV-Cre. Compared to wild-type (WT) mice, the cKO mice exhibited a three-fold higher basal population (Figs. 2<\/a>c, d<\/a> and S5<\/a>a, b<\/a>), indicating that Mcam loss induces the basal cell compartment containing MaSCs. In addition, we examined the expression of stemness-related genes including Procr, K14, Lgr5<\/i>, and sSHIP<\/i> in basal cells derived from the mammary tissues of WT and cKO mice, and found these genes highly expressed in Mcam cKO mice (Fig. S5c<\/a>), supporting the potential regulatory roles of Mcam for MaSCs.<\/p>\n To determine whether Mcam negatively regulates MaSC function in vivo, we carried out limiting-dilution transplantation assays in non-obese diabetic\/severe combined immunodeficiency (NOD\/SCID) mice using isolated Lin–<\/sup> epithelial cells and basal population, primary MECs, respectively. All isolated Lin–<\/sup> epithelial cells and basal cells (regenerated mammary tissues were harvested at 6 weeks after transplantation), primary MECs (regenerated mammary tissues were harvested at 5 weeks after transplantation) from cKO mice displayed a significantly higher rate of successful engraftment and more extensive mammary outgrowth than the WT cells (Figs. 2<\/a>e, f<\/a> and S5d<\/a>). To illustrate whether Mcam directly regulates the MEC clonogenicity, we performed an in vitro clone assay using primary MECs (Figs. 2<\/a>g\u2013i<\/a> and S5<\/a>e\u2013g<\/a>). Mcam depletion significantly increased the numbers and diameters of colonies, representing MEC clonogenicity and proliferation ability. Moreover, Mcam<\/i> was knocked down using a lentivirus (Fig. S5h<\/a>) to explore its function in primary cultured MECs in vivo and in vitro. In the transplantation assay, knockdown of Mcam<\/i> significantly improved the regenerative capacity of MECs to reconstruct whole mammary tissue (Fig. S5i<\/a>). In the clone assay, both clone numbers and diameters increased (Fig. S5<\/a>j\u2013m<\/a>), suggesting loss of Mcam promotes MEC proliferation. These results provide strong evidences for the Mcam roles in inhibiting MEC proliferation and mammary reconstruction.<\/p>\n To identify the regulatory mechanism underlying the promotion function of MEC proliferation and mammary gland development in cKO mice by MMTV-Cre, RNA-sequencing (RNA-seq) was performed on mammary tissues from 5-week-old WT and cKO mice. First, we analyzed the differentially expressed genes (DEGs) identified between WT and cKO mammary tissues (total breast cells) (Fig. S6a<\/a>) and performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of upregulated DEGs in cKO mammary tissues. Results indicated strong activation of pathways associated with immune regulation (Fig. 3a<\/a>). Notably, the DEGs were significantly enriched in the GO categories \u201cimmune system process\u201d and \u201cimmune response\u201d (Supplementary Dataset 2<\/a>) and in the KEGG pathways \u201cactivation of innate immune response\u201d, \u201cregulation of immune effector process\u201d, and \u201cadaptive immune response\u201d (Fig. 3a<\/a>). Immune cells are key components of the mammary microenvironment33<\/a><\/sup>, therefore, we analyzed the abundance of various immune cells in WT and cKO mammary tissues using FACS.<\/p>\n a<\/b> GSEA of enriched immune response signatures using total mammary cells in cKO mice by MMTV-Cre compared with WT mice. NES, normalized enrichment score. b<\/b> Percentages of various immune cell populations in WT and cKO mice by FACS analysis. n\u2009=\u20098 mice for macrophages, neutrophils, and DCs, 3 mice for B cells and CD8+<\/sup> T cells, and 6 mice for CD4+<\/sup> T cells. c<\/b> FACS analysis of macrophage population (F4\/80+<\/sup>CD11b+<\/sup>) in WT and cKO mice. d<\/b>, e<\/b> IF images of CD206+<\/sup> (d<\/b>) and Cx3cr1+<\/sup> (e<\/b>) macrophages in WT and cKO mammary sections. Scale bar, 20 \u03bcm. f<\/b>, g<\/b> FACS analysis (f<\/b>) and their percentage quantification (g<\/b>) of macrophages in mammary tissues of WT and cKO mice. n<\/i>\u2009=\u20093 mice in each group. h<\/b>, i<\/b> Schematic of co-culture assay and representative images (h<\/b>) (The left panel was created with BioRender.com.), and quantification (i<\/b>) of migrating macrophages after WT and cKO CM treatment. Isolated macrophages by FACS in upper chamber were co-cultured with CM from WT and cKO primary MECs in lower chamber. CM, conditional medium. n<\/i>\u2009=\u20096 biological replicates. Scale bar, 200 \u03bcm. Data are means\u2009\u00b1\u2009SEM. Two-sided Student\u2019s t<\/i> test was used to evaluate statistical significance. Source data are provided as a Source Data file.<\/p>\n<\/div>\n<\/div>\n Data showed a significant increase in the abundances of macrophages and neutrophils in cKO mice, but not of dendritic cells (DC), B cells, or T cells (Figs. 3b<\/a> and S6b<\/a>). Furthermore, using macrophage markers (F4\/80 and CD11b), macrophages showed consistent and strong elevation in the Mcam cKO mice (Figs. 3c<\/a> and S6c<\/a>), implying that macrophages play important roles in Mcam cKO mice. In addition to stromal macrophages, tissue-resident ductal macrophages are also found in the mammary gland epithelium34<\/a><\/sup>. Thus, we explored changes in both types of macrophages in the mammary gland using CD206 (stromal macrophage marker)35<\/a><\/sup> and Cx3cr1 (tissue-resident macrophage marker)34<\/a><\/sup>. Data showed that both cell populations were significantly elevated (Figs. 3<\/a>d\u2013g<\/a> and S7<\/a>a\u2013d<\/a>). To further validate macrophages indeed were recruited by MECs, isolated primary macrophages and macrophage cell line RAW264.7 were co-cultured with conditional medium (CM) from either WT or cKO MECs by MMTV-Cre. Compared with WT-CM treatment, cKO-CM induced more macrophages migration (Fig. 3<\/a>h, i<\/a> and S7<\/a>e, f<\/a>). Thus, depletion of Mcam in the mammary tissue leads to an increase in macrophages.<\/p>\n As macrophages can maintain MaSC stemness36<\/a><\/sup>, we mixed macrophages with basal cells from WT or cKO mice by MMTV-Cre to implement a co-culture mammosphere assay for confirming Mcam roles on the clonogenicity promotion by macrophages. After co-culturing with isolated primary macrophages for 10-14 days, clone numbers and clone diameters increased significantly in the basal cells of cKO mice (Fig. 4<\/a>a\u2013c<\/a>). To confirm the mediating roles of macrophages in the above function, we performed rescue assays using clodronate liposome (CL) to specifically deplete macrophages in both WT and cKO mice37<\/a><\/sup>. After injection of CL, the percentages of macrophages in WT and cKO mice were effectively reduced, as shown by FACS analysis (Figs. 4d<\/a> and S8a<\/a>), as was the percentages in the spleen (Fig. S8<\/a>b, c<\/a>), a macrophage-enriched organ.<\/p>\n<\/a><\/div>\n
Loss of Mcam enhances mammary gland development<\/h3>\n
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Loss of Mcam promotes the regeneration of mammary gland<\/h3>\n
Knockout of Mcam promotes macrophage recruitment<\/h3>\n
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Depletion of macrophages blocks the elevating activity of MECs upon Mcam loss<\/h3>\n