Mcam stabilizes luminal progenitor breast cancer phenotypes via Ck2 control and Src/Akt/Stat3 attenuation

2.1. Cell lines and Cell culture:

Met-1 and NDL-1 cell lines were provided by Dr. Alexander Borowsky.^32,33 Authenticated 4T1 (RRID: CVCL_0125), E0771 (RRID: CVCL_GR23), MCF10A (RRID: CVCL_0598), MDA-MB-231 (RRID: CVCL_0062) and MDA-MB-468 (RRID: CVCL_0419) cell lines were obtained from ATCC. 4T1, E0771, NDL-1, Met-1, MDA-MB-231, and MDA-MB-468 cells were cultured in Dulbecco’s modified Eagle’s media (DMEM) with ciprofloxacin 10 μg/ml and 10% fetal calf serum. MCF10A cells were cultured in Dulbecco’s modified Eagle’s medium/F12 with 5% horse serum, 10 μg/ml insulin, 20 ng/ml epidermal growth factor, 100 ng/ml cholera toxin, 0.5 μg/ml hydrocortisone, and 10 μg/ml ciprofloxacin. Py230 cell line was kindly provided by L. Ellies ⁹. Identity of gifted lines is authenticated herein through molecular assays (e.g., scRNASeq,) that match profiles reported for these lines. Passage numbers were tracked and minimized, and experiments were repeated 2–8 times including repetition with early passage frozen aliquots. Py230 cells were cultured in “Py230 Media” composed of F12-Kaighn’s Modified Media with 5% fetal calf serum, ciprofloxacin 10μg/ml, amphotericin B 2.5μg/ml, and Mito+ serum extender (Corning #355006). Py230 organoids were cultured in Py230 media with 4% Matrigel. HCI-011 organoids were kindly provided by Dr. Alana Welm and cultured according to their published protocol.³⁴ They were grown in PDxO base medium (Advanced DMEM/F12 with 5% FBS, 10 mM HEPES, 1× Glutamax, 1 μg ml⁻¹ hydrocortisone, 50 μg ml⁻¹ gentamicin and 10 ng ml⁻¹ hEGF) with 10μM Y-27632, 100 ng ml⁻¹ FGF2 and 1 mM NAC. All cells were grown in sterile humidified tissue culture incubators at 37 °C with 5% CO₂ and ambient (~ 17–18%) O₂. Cells were transduced with lentiviral concentrates in the presence of 7 μg/ml polybrene for up to 16 h with transduction efficiency (not shown) suggesting MOI <<1. CX-4945/Silmitasertib (Selleck Chem #S2248), (E/Z)-GO289 (MedChem Express, #HY-115519), Ag490 (Selleck Chem #S1143), Stattic/S7947 (S7947, Sigma Aldrich #19983–44-9), and tamoxifen citrate (Selleck Chem #S1972) were resuspended in DMSO according to manufacturers’ recommendations and diluted to final concentrations in media immediately prior to being added to cells.

2.2. Lentiviral vectors:

Viral vector plasmids were obtained from OriGene Technologies, Inc. (Rockville, MD), including the mouse Mcam shRNA lentiviral plasmid (Cat # TL514377), a scrambled shControl, and a custom shRNA-resistant (via silent-mutation) mouse Mcam construct subcloned into a lentiviral gene expression vector (pLenti-C-mCFP-P2A-BSD), (# PS100107). Completed vectors were sequenced confirmed.

2.3. siRNA construct:

siRNA constructs were obtained from OriGene Technologies, Inc. (Rockville, MD). This includes the three unique 27mer mouse Mcam siRNA duplexes (SR418656A-C) and a Trilencer-27 Universal Scrambled Negative Control siRNA Duplex (SR30005). Cells were transfected utilizing Lipofectamine 3000 Transfection Reagent (Invitrogen, #L3000008) according to manufacturer’s instructions.

2.4. Western Blot:

Cell were lysed in RIPA buffer (1X PBS (137mM NaCl; 2.7mM KCl; 4.3mM Na2PO4; 1.47 mM KH2PO4) with 1% Nonidet P-40 Substitute (Sigma #74385), 0.5% Sodium deoxycholate, 0.1% SDS) with the Halt Protease & Phosphatase inhibitor cocktail (Thermo Fisher Scientific, #78440), quantified with the RC-DC Protein Quantification Assay (BioRad) and equivalent concentrations (10–16ug) loaded per sample on precast NuPAGE 4–12% Bis-Tris gradient gels (Invitrogen). Separated proteins were transferred to nitrocellulose and probed overnight with primary antibodies under agitation. Secondary antibodies were incubated with blots for 1hr. Intervening washes used TBST pH7.5 (10mM Tris, 15mM NaCl, 0.05%Tween 20), Blots were imaged on an Odyssey CLx Imager (LI-COR).

2.5. Immunoprecipitation:

HEK293A cells were transfected with ps14-Mock ³⁵, an rTA expression construct to later induce CK2 expression, utilizing Lipofectamine 3000 (Invitrogen; L3000008). 16 hours later cells were sorted for GFP+ cells and plated. After 32 hours cells were transfected with an equimolar mix of CK2α/β plasmid (Addgene; #27093) and the relevant MCAM tail variant. Following another 16-hour incubation cells were treated with DOX. After 24 hours cells were washed with PBS+EDTA, collected, resuspended in IP Lysis buffer (Pierce; 87787) with the Halt Protease & Phosphatase inhibitor cocktail (Thermo Fisher Scientific, #78440) and allowed to rotate at 4°C for 1 hour. 10ug of rabbit anti-FLAG antibody (Invitrogen; #740001) was added and allowed to incubate overnight at 4°C. The following day, magnetic Protein A/G beads (Pierce; 88802) were added according to the manufacturer’s instructions and samples were rotated at 4°C for 1 hour prior to magnetic separation, washing the A/G beads, and sample preparation for western blot analysis. Westerns were probed for HA-Tag (Invitrogen; #26183) and Myc (Invitrogen; #MA1–980) for CK2 α and β respectively.

2.6. Immunohistochemistry:

Freshly dissected tissues were fixed overnight at 4°C in 10% neutral buffered formalin (Sigma; HT501128). Fixed tissues were transferred to 70% ethanol for storage at 4 °C. Processing for histology followed standard protocols with paraffin embedding, sectioning at 5 μm thickness, baking for 1 h at 55 °C, and deparaffinizing with CitriSolv (Decon Labs, 89426–268) and rehydration through graded alcohol/water washes. Antigen retrieval was achieved by boiling samples 15min in citrate buffer (10 mM citric acid, 0.05% Tween 20, pH 6.0) prior to overnight primary antibody staining. Slides were mounted in Fluormount-G (Electron Microscopy Sciences). Slides were imaged on a Leica SP8 White light Laser Confocal microscope.

2.7. Antibodies:

2.8. scRNA Sequencing:

All protocols used to generate scRNA-seq data on 10x Genomics Chromium Controller platform including library prep, instrument and sequencing setting can be found on: https://www.10xgenomics.com/support/single-cell-gene-expression/documentation. The Chromium Next GEM Single Cell 3’ Kit v3.1 (PN-1000268) was used to barcode individual cells with 16nt 10x Barcode, to tag cell specific transcript molecules with 12 nt Unique Molecular Identifier (UMI) and to capture poly (A) mRNA with 30 nt ploy(dT) sequence according to the manufactures. The following protocol based on 10x Genomics user guide (CG000315) was performed by High-Throughput Genomics Shared Resource at Huntsman Cancer Institute, University of Utah. Briefly, Py230 single cell suspension was isolated by trypsinization and resuspended in phosphate buffered saline with 0.04% bovine serum albumin. The cell suspension was filtered through 40-micron cell strainer. Viability and cell count were assessed on Countess 2 (Invitrogen, Carlsbad, CA). Equilibrium to targeted cell recovery of 6,000 cells along with 10x Gel Beads and reverse transcription reagents were loaded to Chromium Single Cell Chip G (PN-1000120) to form Gel-Bead-In Emulsions (GEMs), the nano-droplets. Within individual GEMs, barcoded cDNA generated from captured mRNA was synthesized by reverse transcription at the setting of 53°C for 45 min followed by 85°C for 5 min. Subsequent fragmentation, end repair and A-tailing, adaptor ligation and sample indexing with dual index (PN-1000215) were performed in bulk according to the user guide. The resulting barcoded libraries were qualified using Agilent D1000 ScreenTape on Agilent Technology 2200 TapeStation system and quantified by quantification PCR using KAPA Biosystems Library Quantification Kit for Illumine Platforms (KK4842). Multiple libraries were then normalized, pooled and sequenced on NovaSeq 6000 with 150×150 paired end mode.

2.9. Bulk RNA-sequencing:

RNA was collected from snap frozen biological replicates of 10e6 Py230 shControl/shMcam cell-derived tumors that were collected 12-weeks after inoculation. RNA was isolated via QIAzol-chloroform extraction followed by column-based purification and On-Column DNase Digestion (Qiagen #79254). The aqueous phase was brought to a final concentration of 50% ethanol, and RNA was purified using the RNeasy Lipid Tissue Mini Kit according to the manufacturer’s instructions (Qiagen #74804). Library preparation was performed using the Illumina TruSeq Stranded mRNA Library Prep with UDI (Illumina; poly(A) selection). Sequencing was performed using the NovaSeq 6000 (50 × 50 bp paired-end sequencing; 25 million reads per sample).

2.10. Bioinformatic Analysis:

All downstream analysis of sequencing data was completed using Loupe Browser (6.0.0) (RRID:SCR_018555), Enrichr (RRID:SCR_001575), Appyters (RRID:SCR_021245), GraphPad Prism (RRID:SCR_002798) and RStudio (4.1.2) (RRID:SCR_000432). For differential gene expression analysis, we utilized the default clustering method of Seurat with the Louvain algorithm to iteratively group cells together ³⁶. Reanalysis of primary mouse mammary data from Giraddi et al. used the published diffusion map coordinates and the markers Krt14, Krt8 and Wfdc18, to designate 4 adult groups/cell-types, basal, luminal differentiated, luminal progenitor, and alveolar ¹. We used the Seurat default differential expressed gene (DEG) test [FindMarkers()] (non-parametric Wilcoxon rank sum test). The top 100 DEGs were then used for downstream comparative analysis. From the Py230 dataset, DEG lists were generated by comparing the shCon luminal to shMcam luminal clusters and similarly for the alveolar clusters identified through marker gene comparisons. We used GSEA software (RRID:SCR_005724), and Molecular Signature Database (MSigDB; RRID:SCR_016863) to compare gene sets with 100,000 permutations in GSEA Pre-ranked. GSEA data was plotted with GraphPad Prism (RRID:SCR_002798). Kaplan Meier plots were generated using TCGA data visualized utilizing KMplot and the auto-select cutoff values for Basal (3.45), Luminal A (3.35), Luminal B (3.29) and Her2 (2.97). ³⁷. Heatmaps and MCAM CNV analysis utilized the UCSC Xena Browser ³⁸ (RRID:SCR_018938).

2.11. Tumor studies:

10-week-old nude female mice were from Charles River Laboratories (Wilmington, MA). 1,000 or 1,000,000 MMTV-PyMT Py230 cells were resuspended in complete Matrigel (Corning) and orthotopically injected into the #4 mammary glands of mice. At endpoint, mice were euthanized according to AVMA guidelines and tissues were harvested for processing. The maximum mammary tumor size permitted is 2cm diameter, which was not exceeded in these studies.

2.12. Statistics:

Replicates and experimental repetition are indicated in figures and figure legends. For statistical analysis, One-Way ANOVA with Tukey Kramer Multiple Comparisons, Repeated Measures One-Way ANOVA (Geisser-Greenhouse correction) w/ Tukey multiple comparison tests, and Two-Way ANOVA with Sidak’s multiple comparisons were used. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Gene mapping and differential gene expression analysis utilized established default methods embedded in the Cell Ranger, CLoupe browser and Seurat computational packages.

3.1. Mcam maintains epithelial phenotypes in luminal mammary cancer cells.

Although experimental OE of MCAM has been reported to drive EMT and basal phenotypes in breast cancer cells ^21,24, we found that diverse murine/human breast cancer cell lines and patient derived organoid models expressed MCAM including luminal and LP-like models (Fig. 1A–C).^9,39 To test the requirements for Mcam in Luminal breast cancer cell state control and tumorigenicity, we focused our initial attention on the murine Py230 cell line, given its experimental tractability, LP-like phenotype, robust Mcam expression, and reported subtype plasticity.^9,39 We knocked Mcam down by transient transfection with a panel of Mcam-directed siRNAs (Figs. S1A–C) or by stable transduction with lentiviral vectors (LV) bearing an Mcam-directed shRNA construct (Figs. 1D–H, Fig. S1D–G). Although transfection efficiency and KD were modest in transient transfections, a subset of cells in siMcam-transfected cultures exhibited qualitative differences in adhesion and cellular morphology with an elongated spindeloid-like phenotype (Figs. S1A–C). Although mesenchymal phenotypes were unexpected in KD cells, we obtained corroborating results using sorted stable LV-transduced Mcam KD Py230 cells compared to cells bearing scrambled control vectors (Figs. 1E–H). Three independently derived stable Mcam KD clones showed the same cellular and molecular phenotypes observed in transient KD cells including elongated cellular morphology and augmented Paxillin levels (Figs. 1D–H, S1D–E). Py230 shMcam cells also exhibited alterations in focal adhesion signaling including elevated activation of focal adhesion kinase and alterations in Src family kinases including Fyn and Lyn (Fig. 1H).²⁸ These changes were correlated with enhanced migration in scratch wound assays but no significant alteration to proliferation or survival relative to scrambled shRNA controls (Fig. 1G, S1F). However, MCAM KD did alter growth kinetics of Py230 in 3D organoid culture as well as the 3D growth of the patient derived organoid model, HCI-011 (Fig. S1G).³⁴ These data indicate that in contrast to studies where Mcam OE drives mesenchymal phenotypes in epithelial cells, MCAM can also sustain the luminal phenotypes in breast cancer cells.

3.2. Mcam stabilizes a luminal progenitor cell state in Py230 cells.

Changes in cell morphology and morphological heterogeneity were also evident in confluent Mcam KD Py230 cultures relative to controls, suggesting alterations in cellular differentiation states or their relative distributions (Fig. 2A). Examination of the lineage related keratins, Krt8 and Krt14, revealed a marked reduction in the frequency of uncommitted Krt8/14 co-expressing cells upon Mcam KD (Fig. 2B). To further characterize Mcam-associated cell state changes, we sequenced transcriptomes from several thousand individual control and Mcam KD Py230 cells (Fig. 2C). UMAP graphical representation of the data revealed three major transcriptional cell states with differential representation between control and Mcam KD cells (Fig. 2C, S2A,B). Surveying the levels of previously described markers of distinct mammary cell states ², we observed that transcript levels for Epcam and Sca1 delineate the major transcriptional states identified in the UMAPs as Epcam+Sca1^High, Epcam+Sca1^Low and Epcam-Sca1^High. We further determined that the Epcam− cells are Ncam1+ whereas Epcam+ cells are Ncam− (Fig. 2C). Flow cytometric analysis (FACS) of surface protein expression mirrored scRNA-seq data and confirmed population skewing upon Mcam KD (Fig. 2D, S2C,D). FACS and scRNA-seq of additional independent clones demonstrated consistent loss of the Epcam+Sca1^High cell state in Mcam KD cells, although some variance in the final population frequencies was observed (Figs. S2B–D). Upon Mcam OE with a silent mutation-bearing shRNA-resistant vector in our Py230 Mcam KD cells, we restored predominance of the Epcam+Sca1^High profile while Epcam+Sca1^low and Epcam-Ncam+ cell populations were restored to near baseline proportions (Figs. S2E–H). We also determined that all three populations were able to interconvert and re-establish parental distributions within 3 weeks of cell sorting by FACS, although kinetics for redistribution from the Epcam-Ncam+ of each genotype were slower than other populations (Fig. 2E). Critically, while sorted Epcam+Sca1^Low cells of both genotypes rapidly (within one week) generated Epcam+Sca1^High cells, the Epcam+Sca1^High cell type was depleted in Mcam KD cells within two weeks (Fig. 2E, arrows). Thus, Mcam KD alters cell state distributions in Py230 by destabilizing the Epcam+Sca1^High phenotype.

3.3. Mcam loss triggers a Stat3 driven alveolar/basal lineage switch.

Epcam and Sca1 have previously been proposed to distinguish alveolar progenitors (AP; Epcam^highSca1^low) from hormone sensing luminal progenitors (HSP/LP; Epcam^highSca1^high).² We took a comparative transcriptomics approach to determine whether Py230 transcriptomes mimic these progenitor states more broadly. We determined that Py230 cells shared expression similarity with previously published expression profiles for HSP/LP transcriptional states, while the majority of Mcam KD Py230 cells resembled AP and basal cell states of the normal gland (Figs. 3A–C, S3A–C).^1,40 Additionally, although we noted upregulation of genes previously ascribed to neural crest fates in Mcam KD Py230 cells in vivo, we determined that several of these genes are also expressed in normal mammary alveolar and basal cell types and do not necessarily indicate loss of mammary epithelial specification (Fig. 3C, S3A).^1,40,41

To identify underlying regulatory programs contributing to MCAM mediated cell state control, we examined enriched transcription factor target categories among genes that were differentially expressed between control and Mcam KD Py230 cells using EnrichR.⁴² In addition to Elf5, a classic alveolar lineage specifier (Fig. 3C, S3B)⁴³, genes upregulated in Mcam KD cells were significantly enriched for target genes of the STAT family of transcription factors, including alveolar markers Tfap2c, Igfbp5, and Wfdc18 (Figs. 3C,D).^{17,18,44–46} We therefore examined Stat3 and Stat5a as potential mediators of Py230 cell state change consequent to Mcam KD. Activated Stat3 and Stat5A were upregulated in Mcam KD Py230, providing a potential mechanistic basis for the alveolar cell state switch adopted by Mcam KD cells and for the OE of Stat target genes described above (Fig. 3E, S3D–F). While increased Stat5a could be explained by its overexpression, total Stat3 protein levels were only modestly upregulated though it was strongly overactivated in Mcam KD cells relative to MCAM expressing controls (Fig. 3E, S3D,E).

3.4. Mcam governs Ck2 substrate utilization attenuating Stat3 activity in Py230 cells.

To determine whether hyper-activation of Stat3 was necessary for the HSP/LP → AP switch, we treated Py230 cells with Stattic, a selective STAT3 inhibitor. Additionally, we looked at the effects of inhibitors of upstream regulators of STAT signaling including Ag490, an inhibitor of JAK2 and EGFR, and the CK2 inhibitors CX-4935 and GO289, (Figs. 4A–E, 4G, S4A–D).^44,47,48 Levels of phosphorylated Stat3 (pStat3) in Py230 Mcam KD cells were decreased to near baseline levels with Stattic or by inhibition of either upstream kinase (Fig. 4A). Cell state skewing associated with Mcam KD was dramatically reversed by CX-4945, and to a lesser degree by Stattic, whereas effects of Ag490 were not significant with respect to restoration of a predominant Epcam+Sca1^High LP-like cell state (Figs. 4B–E, S4A,B,E–H). Cytotoxicity was negligible for all three reagents. The role of Ck2 in Mcam KD phenotypes was intriguing as CK2 has not previously been implicated as a functional downstream target of MCAM, although CK2 has previously been implicated in breast tumorigenesis and cell state control of mammary cells.^49,50 In previous studies, reduction of CK2 levels in the colorectal cancer cell line, LoVo, led to reduced E-cadherin and other signs of EMT.⁵¹ However, in our study, Ck2 inhibition did not significantly alter EMT features and Ck2 levels were unaffected by Mcam KD (Figs. S4D,H). Rather, we noted Mcam KD was associated with augmented Ck2 activity toward Stat3 and phosphorylation of the PI3K negative regulator Pten at Ck2 specific sites (Ser380/Thr382/Thr383) that promote its degradation and consequent Akt hyperactivation (Fig. 4F, S4I).^51,52 Ck2 inhibition reversed these effects and restored expression of luminal markers while depleting alveolar markers (Fig. 4G, S4D). These data indicate that Mcam controls cell state in Py230 cells in a manner dependent on Ck2 and Stat3.

Using an antibody that detects phosphorylated Ck2 (pCK2) sites across diverse proteins, we observed altered Ck2 phosphorylation target patterns as a function of MCAM status in a variety of lines, including Py230, another PyMT derived line, Met1, and the human non-transformed epithelial cells MCF10A (Fig. 4H, S4J,K). CX-4945 treatment confirmed Ck2 specificity of the majority of detected proteins (Fig. S4L). Further, Flag ‘pull downs’ of Flag-tagged MCAM rescue constructs co-precipitated tagged CK2α/β proteins when co-expressed in HEK293A cells, indicating Mcam regulation of Ck2 may be direct and dependent on the terminal 72 amino acids of MCAMs’ cytoplasmic (Fig. 4I). Mutation of a putative Ck2 phosphorylation site target residue (S629A) did not compromise binding (Fig. 4I).³¹

3.5. MCAM loss hallmarks aggressive luminal tumors.

Considering the MCAM expression we observed in luminal tumor cell lines and its activity in HSP/LP/AP cell state transitions in Mcam KD Py230 cells, we re-examined MCAM’s relationship to tumor aggressiveness and subtype using The Cancer Genome Atlas (TCGA) database. While we confirmed that elevated MCAM levels correlated with poorer prognosis in Basal-like and Her2-enriched subtypes, the inverse was true in a subset of luminal cell types when examining patients treated with chemotherapy (Fig. 5A). Chemotherapy treated luminal cancers tend to reflect more aggressive tumors than those treated with endocrine therapy as an adjuvant monotherapy. Examining MCAM expression across the various breast cancer subtypes, we found that MCAM was generally reduced across all breast cancer subtypes relative to normal samples, yet more highly expressed in Basal-like, and Her2-enriched tumors than in Luminal A/B tumors as previously reported (Fig. 5B, S5A).²² It should be noted that to some extent MCAM expression levels in TCGA may also reflect different levels of stromal involvement since stromal cells can express high levels of MCAM.

The Luminal B intrinsic subtype is distinguishable from Luminal A tumors by its poorer prognosis, more frequent endocrine resistance, and greater proliferation.^3,6 However, underlying mechanisms that give rise to these distinctions remain unclear. We noted that reduced MCAM gene copy number was surprisingly frequent in breast cancer, especially in luminal tumors (Fig. 5C, S5A). In the Luminal A subtype MCAM loss was associated with a trend toward increased expression of proliferation associated PAM50 genes and therefore a trend toward a more Luminal B like phenotype (Fig. 5C, S5B).^7,38,53 This likely reflects concomitant amplification of Cyclin D1 at 11q, in many cases.⁵³ Interestingly, among patient derived models (PDMs) that formed estrogen independent outgrowths, two out of four instances showed concomitant reduction in MCAM, one of which (HCI-040) showed evidence of the same 11q perturbations observed in TCGA (Fig. S5C).³⁴ We also noted a correlation between MCAM copy number reduction and ‘triple positive’ (TPBC) status in archival TCGA data (Fig. 5C). Clinically, despite their amplified ERBB2, such tumors are usually treated as HR+ luminal tumors (Fig. 1C, S5D).

Frontline treatments for luminal, HR+ breast cancers include a variety of estrogen blocking therapies such as the selective estrogen receptor modulator, tamoxifen. When grown in media containing estrogens, Py230 cells express estrogen receptor and become estrogen sensitive.⁹ In light of this, our observation that Stat3 inhibition reverts Mcam KD Py230 cells to an HSP/LP-related transcriptional state, and prior reports that inhibition of Stat3 can increase the sensitivity to tamoxifen treatment, we tested the sensitivity of control and Mcam KD Py230 cells to tamoxifen using an in vitro proliferation assay (Figs. 5D,E). We found control cells are more sensitive to tamoxifen than Mcam KD cells, and that sensitivity can be restored in Mcam KD Py230 cells by treatment with inhibitors of Stat3 or Ck2 (Figs. 5D,E). Together, these data further indicate that Mcam maintains a hormone-sensing cell state through modulation of Ck2 and downstream Stat3 signaling.

3.6. MCAM promotes tumorigenicity through cell state control.

Finally, we examined whether cell state and signaling changes observed in Mcam KD cells in vitro alter tumorigenic potential of the cells or whether they alter the neural crest associated, Basal-like, Sox10-positive phenotype previously associated with Py230 cells in vivo (Fig. 5C).³⁹ Surprisingly, given the apparent increase in mesenchymal traits observed when Mcam is KD in these cells in 2D culture, they were less tumorigenic than control cells in the graft setting when low cell numbers (1,000) were injected (Fig. 6A). When 1,000,000 cells were orthotopically transplanted into the mammary fat pads, we saw formation of both control and Mcam KD-derived tumors, but the tumors that form were fundamentally different in terms of size, histopathological appearance, and expression of select cell type markers (Fig. 6B). We found that Py230 cells exhibit expression of Sox10 in vivo though they lack such expression in 2D culture, consistent with a prior report (Fig. 6B).³⁹ Sox10 expression in vivo marked large invasive peripheral regions of Py230 tumors consistent with the previously proposed neural crest like differentiation state. We determined cells in such regions co-express Krt14 suggesting they are likely basal-like or luminal progenitor-like regions (Figs. 5C, 6B). However, these large uniform Sox10+Krt14+ positive regions were absent in Mcam KD tumors and the many fewer Sox10+ cells that were found in Mcam KD tumors were usually either Krt8/14 negative or were found in organized Krt8+ epithelial structures resembling the Sox10+,HR− luminal cells previously described in the normal mouse mammary epithelium (Fig. 6B).⁴¹ Gene expression profiling of control and Mcam KD tumors confirmed cell state skewing by Mcam KD that was consistent with a block to the luminal progenitor related basal-like phenotype (Fig. 6C).