primary human airway smooth muscle cells (asmcs)  (Lonza)

Journal: Nature Communications

Article Title: Collagen-rich airway smooth muscle cells are a metastatic niche for tumor colonization in the lung

doi: 10.1038/s41467-019-09878-4

Figure Lengend Snippet: Airway smooth muscle cell (ASMC) is a metastatic niche in the lung. a H&E staining of lung sections revealing typical metastatic lung foci within alveolar spaces that were formed from vector control T24 cancer cells. (*) denotes alveolar spaces. Scale bar:100 μm. b H&E staining of lung sections revealing morphologically distinct metastatic foci residing adjacent to ASMCs (outlined by arrows), which formed from CD167a-overexpressing T24 cancer cells. Scale bar:100 μm. c Quantification of unique lung metastases with proximal ASMCs localization (control, n = 7; CD167a, n = 7). d Immunofluorescent co-staining of α-SMA (an ASMCs marker, red), and CD167a (to label metastatic cancer cells, green), in a serial section to b . Scale bar:100 μm. e – i Serial sections demonstrating H&E staining, IF and IHC staining of various markers illustrating the colonization of CD167a-expressing cancer cells within COL3-rich ASMCs. Anti-CD167a antibody was used to outline metastatic cancer cells, anti-α-SMA and anti-collagen III antibodies were used to label ASMCs and secreted collagens, respectively. Scale bar:100 μm. j Serial transplantation scheme to establish a highly-metastatic model (T24-LungMET) that efficiently colonized into lung tissues, by serially transplanting parental T24 cells into tail vein five consecutive times, monitored by bioluminescence imaging. k , l Representative serial sections of metastatic foci stained with anti-CD167a to mark metastatic cancer cells and its relative localization to COL3 in T24-LungMET model. m Immunofluorescence co-staining images of CD167a (green) and α-SMA (red) showing the relative localization of CD167a+ metastatic cancer cells and their proximity to α-SMA+ ASMCs. Scale bar:100 μm. n Experimental scheme to establish a spontaneous human bladder metastatic PDX model. o , p Representative serial sections of metastatic foci stained with anti-CD167a to mark metastatic cancer cells and their proximity to COL3. q Immunofluorescence co-staining images of CD167a (green) and α-SMA (red) showing the relative localization of CD167a+metastatic cancer cells and their proximity to α-SMA+ASMCs. Scale bar:100 μm. r Western blot analysis comparing CD167a protein expression in parental T24 cells, T24-LungMET model, CD167a-overexpressing T24 model and the human bladder metastatic PDX model. s Quantification of lung metastases proximal to ASMCs, in the metastatic bladder carcinoma models evaluated in r . Statistical analysis: a two-tailed, unpaired student’s t-test. Error bar: mean ± SEM. ** p < 0.01, *** p < 0.005

Article Snippet: Normal human primary airway smooth muscle cell (ASMC) line was purchased from ATCC (ATCC® PCS-130-010TM) and cultured in vascular cell basal medium (ATCC® PCS-100-030TM) supplemented with vascular smooth muscle cell growth kit (ATCC® PCS-100-042TM).

Techniques: Staining, Plasmid Preparation, Control, Marker, Immunohistochemistry, Expressing, Transplantation Assay, Imaging, Immunofluorescence, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: Collagen-rich airway smooth muscle cells are a metastatic niche for tumor colonization in the lung

doi: 10.1038/s41467-019-09878-4

Figure Lengend Snippet: Effects of airway smooth muscle cells and collagen III in colony formation. a Schematic illustrating the overall scheme of conditioned media collection from airway smooth muscle cells (ASMCs) for the evaluation of its impact on colony formation in vitro, and subsequent molecular analysis. b Quantification and size measurement of colonies formed from control and CD167a-overexpressing T24 cancer cells, and c a human metastatic PDX model in a classical soft-agar assay, in the presence or absence of ASMC conditioned media over a 14-day period. Representative phase-contrast images depict the relative colony size from respective conditions. d Quantitative real-time qPCR analysis of COL3A1 mRNA in cultured human ASMCs. Human cDNA mix and cultured T24 cells were harvested as controls. GAPDH was used as reference and the relative expression was normalized against human cDNA mix. e Western blot analysis of COL3 expression in human ASMCs and T24 cancer cells. DNA-PKcs was used as the loading control. f Targeted mass spectrometry analysis to evaluate protein peptides specific toward COL3, that were secreted into the supernatant or conditioned media collected from cultured human ASMCs at 0 h and 16 h after incubation. The amount of COL3 was calculated based on MS1 signal area under curve using SKYLINE software based on top four strongly detected peptides. g Colony formation assay of control and CD167a-overexpressing T24 cancer cells upon COL3 treatment (50 μg ml −1 ). Number and size of colonies are shown on the left and right panel, respectively. h Colony formation assay of the human metastatic PDX model upon COL3 treatment (50 μg ml −1 ). Statistical analysis: a two-tailed, unpaired student’s t -test. Error bar: mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.005

Article Snippet: Normal human primary airway smooth muscle cell (ASMC) line was purchased from ATCC (ATCC® PCS-130-010TM) and cultured in vascular cell basal medium (ATCC® PCS-100-030TM) supplemented with vascular smooth muscle cell growth kit (ATCC® PCS-100-042TM).

Techniques: In Vitro, Control, Soft Agar Assay, Cell Culture, Expressing, Western Blot, Mass Spectrometry, Incubation, Targeted Proteomics, Software, Colony Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Collagen-rich airway smooth muscle cells are a metastatic niche for tumor colonization in the lung

doi: 10.1038/s41467-019-09878-4

Figure Lengend Snippet: CD167a-Stat3 axis in the COL3-rich airway smooth muscle cell metastatic niche in lung. a Western blot evaluating the relative protein expression of CD167a, Stat3 and phosphotyrosine Stat3 (Y705) in parental T24 cells and our newly-established metastatic model (T24-LungMET). b , c Effects of COL3, and ASMC-conditioned media and CD167a/DDR1 kinase inhibitor (DDR1-IN-1; 10μM) on Stat3 and phosphotyrosine Stat3 protein expression, in T24 and PDX models. d Coupling CD167a co-IP with mass spectrometry to discover CD167a-interacting partners. Selected CD167a associated gene protein products (GPs) and their corresponding iBAQ values are listed. e Coupling co-IP and western blot to evaluate CD167a-HSP90 interaction, via co-IP using anti-HSP90 antibody followed by western blotting of CD167a in control and CD167a-overexpressing T24 cancer cells. f Effects of CD167a kinase inhibitor (DDR1-IN-1, 10μM) and HSP90 inhibitor (Geldanamycin, 5 μM) on CD167a-HSP90 complex formation. Control cells and CD167a-overexpressing T24 cancer cells were treated with COL3 in the absence or presence of indicated inhibitors for 24 h. Cell lysates were collected for co-immunoprecipitation assay using anti-HSP90 antibody. g PDX cells were cultured in control (-ASMC) or ASMCs conditioned media (+ASMC) with or without DDR1-IN-1 inhibitor (10 μM) for 24 h. Cell lysates were assessed for CD167a-HSP90 interaction by co-immunoprecipitation assay followed by western blot analysis. h , i Western blot analysis of Stat3 tyrosine phosphorylation upon treatment with ASMC-conditioned medium in the presence or absence of HSP90 inhibitor (+HSP90i, 1, 5, and 10 μM) in T24 and PDX models. j , k Western blot analysis of COL3-induced Stat3 tyrosine phosphorylation in T24 and PDX models upon treatment with CD167a/DDR1 kinase specific inhibitor (DDR1-IN-1, 10 μM) and Geldanamycin (HSP90i, 5 μM)

Article Snippet: Normal human primary airway smooth muscle cell (ASMC) line was purchased from ATCC (ATCC® PCS-130-010TM) and cultured in vascular cell basal medium (ATCC® PCS-100-030TM) supplemented with vascular smooth muscle cell growth kit (ATCC® PCS-100-042TM).

Techniques: Western Blot, Expressing, Co-Immunoprecipitation Assay, Mass Spectrometry, Control, Cell Culture, Phospho-proteomics