mcf7 luminal human breast cancer cell lines  (ATCC)

Journal: bioRxiv

Article Title: Immunosuppressive myeloid cells induce mesenchymal-like breast cancer stem cells by a mechanism involving membrane-bound TGF-β1

doi: 10.1101/2025.02.21.639264

Figure Lengend Snippet: (A) CellTrace Violet (CTV) dilution of unstimulated and CD3/CD28 stimulated T cells after 5-day co-culture with monocytes or HuMoSC at the indicated ratios (T cell:HuMoSC). (B) Tumorsphere formation assay of MCF7 cells alone or in co-culture with Human Monocyte-derived Suppressive Cells (HuMoSC) at the indicated ratio or monocytes at a 1:5 (MCF7:monocyte) ratio. (C) Live videomicroscopy representative images of ZsGreen-expressing MCF7 cells (green) alone or in co-culture with CellTrace Far Red-stained HuMoSC or monocytes (red) in tumorsphere forming conditions (MCF7:HuMoSC or monocyte ratio of 1:5). (D) Tumorsphere formation live videomicroscopy quantification of ZsGreen intensity over time of MCF7 cells alone (green) or in co-culture with HuMoSC (red) or monocyte (grey) (MCF7:HuMoSC or monocyte ratio of 1:5). Dashed line: 16h timepoint. (E-K) All analyses were performed on FACS-sorted MCF7 pre-cultured alone or with HuMoSC for 72 hours in adherent, classical 2D culture conditions (MCF7:HuMoSC or monocyte ratio of 1:5). (E) Normalized tumorsphere-forming efficiency of MCF7 cells pre-cultured alone or with HuMoSC or monocytes prior to seeding in tumorsphere forming conditions. (F) In vitro Extreme Limiting Dilution Assay (ELDA) of the tumorsphere formation ability of MCF7 cells. The number of wells presenting tumorspheres and total number of wells assayed in each condition is indicated (n). TFF, Tumorsphere Forming Frequency. (G and H) Representative FACS plots (left) and normalized proportion (right) of CD44 + CD24 - MCF7 cells (G) and ALDH + MCF7 cells (H). (I) GSEA plot showing the Normalized Enrichment Score (NES) and associated p-value for the EMT Hallmark gene set in the variance-ranked genes for MCF7 cells cultured 24h with HuMoSC compared to MCF7 cells cultured alone. FDR: False Discovery Rate. (J) Bubble plot showing the upregulation of EMT-related Kegg signaling pathways in MCF7 cultured for 24 hours with HuMoSC compared to MCF7 cultured alone. Pathways of interest are highlighted in red. (K) Monocyte-to-HuMoSC immunosuppression potential upon T-cell coculture (left, red) and MCF7 tumorsphere formation assay after co-culture with HuMoSC (right, blue) at each day of their polarization from monocytes (Day 0) to HuMoSC (Day 7). (L) CellTrace Violet dilution of T cells unstimulated (Unstim), stimulated with anti-CD3/CD28 (Stim) or stimulated and co-cultured with CD33 low or CD33 high myeloid cells isolated from pleural effusion of breast cancer patients. (M) Tumorsphere formation assay of MCF7 cells alone or in co-culture with CD33 low or CD33 high isolated from pleural effusion of breast cancer patients. Data are means ± S.E.M. ∗p < 0.05, ∗∗p < 0.01, $$$/∗∗∗p < 0.001, $$$$/∗∗∗∗p < 0.0001. ns, not significant. Kruskal-Wallis test with Dunn’s multiple comparisons post-test (B, D, E, K, M); Tumorsphere Forming Frequency (TFF) and associated p-value was calculated using ELDA software (F); Student’s unpaired t-test (G, H).

Article Snippet: MCF7 human Luminal A breast cancer cell line was purchased from the ATCC.

Techniques: Co-Culture Assay, Tube Formation Assay, Derivative Assay, Expressing, Staining, Cell Culture, In Vitro, Limiting Dilution Assay, Protein-Protein interactions, Isolation, Software

Journal: bioRxiv

Article Title: Immunosuppressive myeloid cells induce mesenchymal-like breast cancer stem cells by a mechanism involving membrane-bound TGF-β1

doi: 10.1101/2025.02.21.639264

Figure Lengend Snippet: (A) scRNAseq workflow of the three different conditions. See also Figure S2A. (B) Two-dimensional UMAP of scRNA-seq data of HuMoSC (n = 8643 cells) and MCF7 (n = 7259 cells) after integration of the three experimental conditions “2D-culture”, “Tumorsphere conditions” and “Tumorsphere co-cultures”. (C) Split two-dimensional UMAP of MCF7 cells based the three experimental conditions. (D) Bar plot of cell proportion of each MCF7 cluster in each condition. (E) Density Plot representing the expression of CSC and EMT genes in MCF7 cell clusters. (F) Feature Plot representation of core signature associated with the 55 genes from Mesenchymal Cell Differentiation gene set. (G) Relative differences in cell proportions for each MCF7 cluster between the “Tumorsphere conditions” versus “Tumorsphere co-culture” conditions.

Article Snippet: MCF7 human Luminal A breast cancer cell line was purchased from the ATCC.

Techniques: Expressing, Cell Differentiation, Co-Culture Assay

Journal: bioRxiv

Article Title: Immunosuppressive myeloid cells induce mesenchymal-like breast cancer stem cells by a mechanism involving membrane-bound TGF-β1

doi: 10.1101/2025.02.21.639264

Figure Lengend Snippet: (A) Split two-dimensional UMAP of HuMoSC cells based the three experimental conditions. (B) Bar plot of cell proportion of each HuMoSC cluster in each condition. (C) Relative differences in cell proportions for each HuMoSC clusters between “Tumorsphere conditions” and “Tumorsphere co-culture” conditions. (D) Boxplot showing the predicted ordering by cytoTRACE as assessment of cells differentiation potency. (E) Feature Plots representation of core signature associated with “MDSC” gene sets extracted from PangloDB. (F) Top 5 most significant differentially expressed genes across all HuMoSC clusters. (G) Violin plot expression of CD52 gene across HuMoSC clusters. (H) CD52 flow cytometry analysis of HuMoSC. See also Figure S3A. (I-J) CellTrace Violet dilution of T cells (I) and MCF7 tumorsphere formation assay (J) after co-culture with total, CD52 - and CD52 + HuMoSC at a 1:5 ratio (MCF7:HuMoSC). (K) Tumorsphere formation assay of MCF7 cells alone or in co-culture with myeloid cells isolated from pleural effusion of breast cancer patients and differentially expressing CD33 (high or low) and CD52. See also Figure S3B. (J and K) Statistical significance was assessed using a Kruskal-Wallis test with Dunn’s multiple comparisons test. ****p<0.0001; ns, not significant. Data are represented as mean values ± S.E.M.

Article Snippet: MCF7 human Luminal A breast cancer cell line was purchased from the ATCC.

Techniques: Co-Culture Assay, Expressing, Flow Cytometry, Tube Formation Assay, Isolation

Journal: bioRxiv

Article Title: Immunosuppressive myeloid cells induce mesenchymal-like breast cancer stem cells by a mechanism involving membrane-bound TGF-β1

doi: 10.1101/2025.02.21.639264

Figure Lengend Snippet: (A) Immunofluorescence images of live ZsGreen-expressing MCF7 tumorspheres alone or in co-culture with HuMoSC (1:5 ratio) after staining with anti-CD14 (red) anti-Epcam (green) antibodies. Nuclei stained with Hoechst (blue). All images are representative of at least three replicates. White arrows points towards tumorspheres-associated HuMoSC. (B) Tumorsphere formation assay of MCF7 cells cultured alone, with CD52 + HuMoSC or with conditioned medium (CM) from HuMoSC (CM HuMoSC ) or from co-culture of MCF7 and HuMoSC (CM co-culture ). In some conditions, MCF7 were physically separated from HuMoSC cells by a transwell. Statistical significance was assessed using a Kruskal-Wallis test with Dunn’s multiple comparisons test. ***p<0.001; ns, not significant. Data are represented as mean values ± S.E.M. (C) CellChat inferred outgoing communication patterns of the different cell clusters (left panel) and associated signaling pathways (right panel). (D) Surfaceome workflow for the identification and quantification of total cell surface proteins. (E) Talkien-predicted protein interaction network of HuMoSC and MCF7 surfaceome. (F-G) Kernel density plot showing log2 intensity of TGFB1 in HuMoSC surfaceome (F) and TGF-BR1 (G) in MCF7 surfaceome. CD45 (F) and EPCAM (G) were used as reference for protein intensities. Dashed line: calculated standard deviation (SD) of the three replicates for the corresponding protein. (H) Chord diagram showing predicted TGFb signaling between clusters. Red arrows highlight the signaling specificity of CD52 + HuMoSC towards MCF7_1 and MCF7_2 clusters.

Article Snippet: MCF7 human Luminal A breast cancer cell line was purchased from the ATCC.

Techniques: Immunofluorescence, Expressing, Co-Culture Assay, Staining, Tube Formation Assay, Cell Culture, Protein-Protein interactions, Standard Deviation

Journal: bioRxiv

Article Title: Immunosuppressive myeloid cells induce mesenchymal-like breast cancer stem cells by a mechanism involving membrane-bound TGF-β1

doi: 10.1101/2025.02.21.639264

Figure Lengend Snippet: (A and B) Secreted TGF-β1 quantification by ELISA assay on supernatants from D0 to D7 HuMoSC (A) and supernatant from MCF7 cells alone or in co-culture with HuMoSC, with or without treatment with Ly2109761 (B). Secretion was normalized to D0-monocyte secretion (A). “MCF7 medium” condition correspond to basal TGF-β detected in the medium (B). (C) Heatmap comparing all significant surfaceome changes in HuMoSC compared to monocyte. (D) TGF-β western blot using Monocyte or HuMoSC total protein fractions before streptavidin pulldown (Whole Cell Lysate) or cell surface protein fractions of biotinylated proteins only (Cell Surface Fraction). α-Tubuline was used as a control for whole cell lysate loading. (E) Tumorsphere formation assay of MCF7 cells alone or after co-culture with CD52 + HuMoSC and treated or not with 5 μM TGF-βRI/II inhibitor Ly2109761 or 1 μg/mL αTGF-β neutralizing antibody. (F) Tumorsphere formation assay of MCF7 cells alone after in co-culture with CD33 high CD52 + myeloid cells isolated from pleural effusion of breast cancer patients and differentially expressing CD33 (high or low) and CD52 and treated or not with 5 μM TGF-βRI/II inhibitor Ly2109761 or 1 μg/mL αTGF-β neutralizing antibody. Data are means ± S.E.M. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. ns, not significant. Kruskal-Wallis test with Dunn’s multiple comparisons post-test (A-B, E-F). Data are means of n = 3 independent biological replicates. (C) Log2 FC (HuMoSC/Monocyte) above 2 or below -2 (4-fold) were considered as significantly upregulated (red) or downregulated (blue), respectively. P-values were determined by unpaired two-tailed Student’s t-tests; P < 0.05.

Article Snippet: MCF7 human Luminal A breast cancer cell line was purchased from the ATCC.

Techniques: Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Western Blot, Control, Tube Formation Assay, Isolation, Expressing, Two Tailed Test