Journal: Cell Death & Disease

Article Title: Activation of CD44 signaling in leader cells induced by tumor-associated macrophages drives collective detachment in luminal breast carcinomas

doi: 10.1038/s41419-022-04986-4

Figure Lengend Snippet: A Representative images of cell clusters in the vasculature of human primary breast tumor. The distributions of CD206 (brown), CD44 (brown), CK (brown), and CD31 (red, indicating vessels) in tissue serial sections from luminal-like BrCa ( n = 15) were determined by immunohistochemical staining. Nuclei are stained with hematoxylin. B Immunohistochemical staining of CD206, CD44, and CD31 was performed in serial tissue sections of human BrCa. Tumors with and without microemboli from a tissue microarray (85 human breast lesions) were analyzed. Using Image-Pro Plus software, the expression intensity of each molecule was evaluated by integral optical density and compared with and without microemboli. Data were analyzed with the Mann-Whitney test. * p < 0.05, *** p < 0.001. C The expression of CD44 and CD206 in the invasion front versus non-invasive front of serial sections from human BrCa tissues was analyzed. Data represents the mean ± SEM. Mann-Whitney test. D A positive association between high frequency of CD206 + -macrophage and CD44 high -cancer cells in BrCa.

Article Snippet: The mouse CD44 shRNA lentiviral particles (sc-35534-V) were purchased from Santa Cruz (Dallas, TX, USA).

Techniques: Immunohistochemical staining, Staining, Microarray, Software, Expressing, MANN-WHITNEY

Journal: Cell Death & Disease

Article Title: Activation of CD44 signaling in leader cells induced by tumor-associated macrophages drives collective detachment in luminal breast carcinomas

doi: 10.1038/s41419-022-04986-4

Figure Lengend Snippet: A Adaptive CD44 expression of luminal-like BrCa cells (MCF7 and T47D) upon THP-1-derived M2-like macrophages stimulation in a non-contact transwell system was assessed by immunoblotting analysis. GAPDH was used as control. The band intensities were analyzed by densitometry analysis. * p < 0.05. B Representative confocal images of CD44 expression in disseminated cell clusters on top-3D matrix membrane during collective detachment. DAPI was used to stain the nuclei. An acquired CD44 high state upon TAMs stimulation in primary BrCa cell clusters (purified from MMTV-PyMT tumor) was observed by immunofluorescence analysis. C Co-immunoprecipitation experiment from whole-cell extracts demonstrating the interaction between CD44 and Ezrin after co-cultured with THP-1-derived M2-like macrophages in a non-contact transwell system. The band intensities were analyzed by densitometry analysis. * p < 0.05. D The influence of CD44 knockdown on collective invasion of cell clusters in 3D matrix membrane. The sh-Control and sh-CD44 primary BrCa cell clusters, premixed with or without TAMs, were embedded in 3D system and recorded by time-lapse microscopy. TAMs were pre-stained with Vybrant CM-DiI (red). E Representative confocal images of localization of pEzrin, pMyosin, β-catenin and ZO-1 at the invasive and disseminating clusters. The sh-Control and sh-CD44 of primary BrCa cell clusters, premixed with or without TAMs, were cultured in top-3D basement. F Knockdown of CD44 inhibited TAMs-induced formation of mammosphere. The mammosphere-forming capacity of primary BrCa cells purified from MMTV-PyMT tumors was detected. Primary BrCa cells were directly sorted into wells of ultra-low attachment 24-well plate containing mammosphere growth medium, and then co-cultured with or without TAMs for 12 days in a non-contact transwell system. G Representative intravital images of subcutaneous xenografts derived from MCF7/sh-Control/GFP + and MCF7/sh-CD44/GFP + cells in vivo, which were injected with or without THP-1-derived M2-like macrophages, showing cohesive-invading MCF7/GFP + cells approaching around vascular vessel. Tomato lectin (DyLight649, Cat. L32472, Thermo Fisher) was injected via tail vein to label vascular structures. Scale bars, 100 μm. See Supplementary Video – . H Representative multiphoton confocal images of thick tumor tissues showed collective dissemination in subcutaneous xenografts. Tumor tissues were cut into 10- to 12-µm thick for paraffin sections, and the blood vessels were labeled by CD31 (Red channel) using immunohistochemical staining. Scale bars, 100 μm. See Supplementary Video – .

Article Snippet: The mouse CD44 shRNA lentiviral particles (sc-35534-V) were purchased from Santa Cruz (Dallas, TX, USA).

Techniques: Expressing, Derivative Assay, Western Blot, Control, Membrane, Staining, Purification, Immunofluorescence, Immunoprecipitation, Cell Culture, Knockdown, Time-lapse Microscopy, In Vivo, Injection, Labeling, Immunohistochemical staining

Journal: Cell Death & Disease

Article Title: Activation of CD44 signaling in leader cells induced by tumor-associated macrophages drives collective detachment in luminal breast carcinomas

doi: 10.1038/s41419-022-04986-4

Figure Lengend Snippet: A The mRNA levels of a panel of cytokines in TAMs versus PBMCs isolated from five primary breast tumors (MMTV-PyMT) were detected by qRT-PCR. B The intracellular CCL8 and IL-10 in TAMs versus PBMCs isolated from normal mouse or tumor-bearing mouse (MMTV-PyMT tumor) were determined by ELISA. n = 5/group. Bars correspond to mean ± SD. * p < 0.05, ** p < 0.01, ns, nonsignificant. C The secreted CCL8 and IL-10 in TAMs versus primary BrCa cells were determined by ELISA. Cells were isolated from five primary breast tumors (MMTV-PyMT) and cultured separately. Bars correspond to mean ± SD. ** p < 0.01, ns, nonsignificant. D Fluorescence in situ hybridization analysis of human BrCa tissue sections revealed that CCL8 mRNA is found in CD206 + TAMs but not in cancer cells. The expression of CD206 was detected using immunochemical staining. Inset representing a CD206 + macrophage-expressing CCL8 mRNA. Scale bars, 100 μm ( n = 3). E Blocking of CCL8 inhibited the collective migration induced by THP-1-derived M2-like macrophages. In vitro scratch assay of untreated MCF7 or treated with THP-1-derived M2-like macrophages, THP-1-derived M2-like macrophages plus normal IgG, or THP-1-derived M2-like macrophages plus CCL8 antibody for the indicated period of time. Red line, cell culture margins ( n = 4). F Blocking of CCL8 inhibited the re-distribution of invadopodia molecules in collective migration induced by THP-1-derived M2-like macrophages. Representative confocal images showed the re-distribution of Cortactin (red) and TKS5 (green) at the migrating front of clusters after CCL8 blocking for 3 days. MCF7 cells co-cultured with THP-1-derived M2-like macrophages in a non-contact transwell system, followed by treatment with normal IgG or CCL8 antibody. Scale bars, 20 μm. G Knocking down of CCL8 in THP-1-derived M2-like macrophages inhibited the increase of Vinculin at the migrating front of clusters. Wound healing assay was used to observe the changes of Vinculin expression at the migrating front. M2-like macrophages derived from THP-1 were transfected with siRNAs (si-Control or si-CCL8) and then co-cultured with MCF7 cells in a non-contact transwell system for 3 days. The expression of Vinculin was determined by immunofluorescence assay. Scale bars, 10 μm. H Blocking of CCL8 inhibited the onset of xenografts induced by THP-1-derived M2-like macrophages in vivo. MCF7 cells were orthotopically implanted into mammary pad of nu/nu mice, followed by intraperitoneal administration of a neutralizing antibody for CCL8. I HE staining of MCF7 xenografts in normal IgG or CCL8 antibody-treated mice. J CD44 (brown) expression of MCF7 xenografts in normal IgG- or CCL8 antibody-treated mice. K CD31 (red) expression of MCF7 xenografts in normal IgG- or CCL8 antibody-treated mice.

Article Snippet: The mouse CD44 shRNA lentiviral particles (sc-35534-V) were purchased from Santa Cruz (Dallas, TX, USA).

Techniques: Isolation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Cell Culture, Fluorescence, In Situ Hybridization, Expressing, Staining, Blocking Assay, Migration, Derivative Assay, In Vitro, Wound Healing Assay, Transfection, Control, Immunofluorescence, In Vivo

Journal: Cell Death & Disease

Article Title: Activation of CD44 signaling in leader cells induced by tumor-associated macrophages drives collective detachment in luminal breast carcinomas

doi: 10.1038/s41419-022-04986-4

Figure Lengend Snippet: A The influence of CCL8 on the expression of CD44 in MCF7 cells was evaluated by western blot. The band intensities were analyzed by densitometry analysis. * p < 0.05. B Blocking of CCL8 inhibited the acquisition of CD44 high state in MCF7 cells induced by M2-like macrophages, which was obtained from human monocytic cell line THP-1 stimulated by PMA/m-CSF/IL-4/IL-10/IL-13. The band intensities were analyzed by densitometry analysis. ** p < 0.01, ns: no significance. C Scheme of identifying differential CD44-interacting peptides upon THP-1-derived M2-like macrophages/CCL8 stimulation. The intersection showed the CD44-interacting proteins, which were analyzed by co-immunoprecipitation-based mass spectrometry assay. MCF7 and T47D cells were stimulated by THP-1-derived M2-like macrophages or CCL8 for 3 days, respectively. The proteins recruited to CD44 were precipitated by CD44 antibody, and then subject to mass spectrometry assay. D Functional associations of the regulatory networks of MDM2/p53-correlated genes from analysis of STRING data are presented. E The effects of CCL8 on the activation of MDM2 in MCF7 cells were evaluated by immunoblotting assay. The band intensities were analyzed by densitometry analysis. * p < 0.05. F Blocking of CCL8 inhibited the activation of MDM2 in MCF7 cells induced by THP1-derived M2-like macrophages. The band intensities were analyzed by densitometry analysis. * p < 0.05, ** p < 0.01, ns: no significance. G Repression of p53 expression by M2-like macrophages in human BrCa cells was detected by immunoblotting assay. The band intensities were analyzed by densitometry analysis. * p < 0.05. H Knocking down of MDM2 in primary BrCa cells inhibited the up-regulation of CD44 induced by TAMs. The band intensities were analyzed by densitometry analysis. *** p < 0.001, ns: no significance. I The influence of MDM2 inhibitor on collective dissemination of cell clusters induced by TAMs in 2D culture system. Before co-cultured with TAMs on 2D culture system, primary BrCa cell clusters were obtained from an ultra-low attachment culture system. TAMs were pre-stained with Vybrant CM-DiI (red).

Article Snippet: The mouse CD44 shRNA lentiviral particles (sc-35534-V) were purchased from Santa Cruz (Dallas, TX, USA).

Techniques: Expressing, Western Blot, Blocking Assay, Derivative Assay, Immunoprecipitation, Mass Spectrometry, Functional Assay, Activation Assay, Cell Culture, Staining

Journal: Cell Death & Disease

Article Title: Activation of CD44 signaling in leader cells induced by tumor-associated macrophages drives collective detachment in luminal breast carcinomas

doi: 10.1038/s41419-022-04986-4

Figure Lengend Snippet: A Activation of Ezrin and p38 after MCF7 cells co-cultured with THP-1-derived M2-like macrophages or CCL8 by a non-contact transwell system was determined by immunoblotting assay. The band intensities were analyzed by densitometry analysis. * p < 0.05, *** p < 0.001. B Knocking down of CD44 inhibited the activation of Ezrin and p38 induced by THP-1-derived M2-like macrophages or CCL8. The band intensities were analyzed by densitometry analysis. *** p < 0.001, ns: no significance. C Knocking down of Ezrin in primary BrCa cells inhibited collective dissemination induced by TAMs in 2D culture system. The primary BrCa cell clusters were transfected by si-Ezrin. TAMs were pre-stained with Vybrant CM-DiI (red). D The p38 inhibitor attenuated collective dissemination of primary BrCa cell clusters induced by TAMs. TAMs were prestained with Vybrant CM-DiI (red). E Knocking down of Ezrin inhibited phosphorylation of Cortactin and p38 triggered by CCL8. The band intensities were analyzed by densitometry analysis. *** p < 0.001, ns: no significance. F Knocking down of p38 repressed the phosphorylation of Cortactin induced by CCL8. The band intensities were analyzed by densitometry analysis. *** p < 0.001, ns: no significance. G Scheme summarizing the proposed mechanism by which MDM2-p53-p38 signaling pathway mediated the acquisition of CD44 high state, which leading to cohesive detachment.

Article Snippet: The mouse CD44 shRNA lentiviral particles (sc-35534-V) were purchased from Santa Cruz (Dallas, TX, USA).

Techniques: Activation Assay, Cell Culture, Derivative Assay, Western Blot, Transfection, Staining, Phospho-proteomics