normal human lung fibroblasts  (ATCC)

Journal: bioRxiv

Article Title: Tenascin-C in the early lung cancer tumor microenvironment promotes progression through integrin αvβ1 and FAK

doi: 10.1101/2024.09.17.613509

Figure Lengend Snippet: a Representative KPT LUAD, 10 weeks, with TnC ISH (red) and immunohistochemistry co-detection of tdTomato or Vimentin (DAB-brown). 9 ROIs, from 8 tumors in n =3 mice, female. b Quantification of TnC particles in (a). Two-tailed T-test with Welch’s correction. c, d Representative immunohistochemistry for TNC and fibroblast markers in serial sections of transitioning LUAD, n =3 female and 3 male KPT mice at each time point. e, f TNC mRNA in human LUAD, as a function of tumor purity and stromal content, TCGA Pan Cancer Atlas. Counts are normalized Log2 fold change. g Representative western blot and quantification of TNC in LUAD tumor cell lines and fibroblasts, n =4. Human LUAD cell lines H1299, A549, H23. Mouse LUAD cell lines 1783, 3658, 4043, 7865. MEF, mouse embryonic fibroblast. NHLF, normal human lung fibroblast, immortalized. CAF, lung cancer-associated fibroblasts. Error bars SD. h H&E and immunohistochemistry images from human stage I LUAD. Off-tumor is tumor >1 mm from tumor boundary. Representative of n =15 cases of stage I or II LUAD. Arrow shows TNC overlapping with fibroblast regions and arrowhead TNC overlapping with tumor cells bordering fibroblasts. i Representative ISH-immunohistochemistry codetection of TNC (red) and fibroblasts (TE7 antibody, DAB-brown), n =7 cases of stage I and II LUAD. j Distribution of TNC particles/cell in off-tumor and LUAD ROIs in (i), manual counting. K-S test. n= cells. k Distribution of TNC expression in tumor, stroma and immune regions for cases in (i), automated detection described in ). l Representative immunohistochemistry for TNC and tumor cell markers tdTomato and NKX2-1 in serial sections of late KPT tumors. m TNC isoforms in LUAD. RNAseq by Expectation Maximization (RSEM) expected counts from RNAseq of primary LUAD patient samples, pre-treatment . n TNC isoforms expressed in U251 cells. RSEM, calculated from RNAseq of U251 cells , and plotted as a % total TNC .

Article Snippet: Normal human lung fibroblasts (NHLF) and cancer-associated lung fibroblasts (CAF) were cultured in Lonza Fibroblast Media and MSCGro media, respectively and after two passages, immortalized by infection with retrovirus expressing pBabe-hTERT-p53 DD generated in Phoenix-AMPHO cells, then cultured in DMEM, 5% FBS.

Techniques: Immunohistochemistry, Two Tailed Test, Western Blot, Expressing

Journal: bioRxiv

Article Title: A Nerve-Fibroblast Axis in Mammalian Lung Fibrosis

doi: 10.1101/2024.09.09.611003

Figure Lengend Snippet: (A-D) Immunofluorescence imaging displayed BrdU (green) and nuclear staining with propidium iodide (PI) (red) in MRC5 human lung fibroblasts. A dose- dependent increase in BrdU incorporation was observed in MRC5 cells stimulated with increasing concentrations of noradrenaline (NA), peaking at 25 µM (D, P = 0.0159). This response was reversed upon co-incubation with terazosin (E, P = 0.0441). (F) MTT assays demonstrated a significant reduction in the number of viable cells in MRC5 human lung fibroblasts and IPF fibroblasts when stimulated with NA and treated with terazosin ( P = 0.0493 and P = 0.0156, respectively). This effect was not observed in normal human lung fibroblasts, indicating that IPF fibroblasts are poised to receive and respond to noradrenergic signals via an α1-AR dependent mechanism. (G-I) Immunofluorescence imaging demonstrated expression of ADRA1D (red) and α-SMA (green), with nuclear staining by DAPI (blue) in human precision-cut lung slices. Following exposure to a fibrotic cocktail, a marked increase in α-SMA (ACTA2) expression was observed in stromal cells adjacent to alveoli, with some cells showing co- expression of ADRA1D (white arrows, G, H). This expression pattern was consistent with trichrome staining and matched ACTA2 expression quantified by PCR analysis (J, P = 0.0236). The introduction of terazosin to the culture media reversed these fibrotic effects (I, J, P = 0.0106). Images were captured at 20x magnification. Data are presented as mean ± SEM or median ± IQR, with statistical analysis performed using Student’s t-test for normally distributed data and Mann-Whitney or Kruskal-Wallis tests with Dunn’s multiple comparisons for non- normally distributed data. * P < 0.05, ** P < 0.01. ADRA1D, α1-adrenoreceptor subtype D; α-SMA, alpha-smooth muscle actin; BrdU, bromodeoxyuridine; DAPI, 4′,6-diamidino-2-phenylindole; FC, fibrotic cocktail; IPF, idiopathic pulmonary fibrosis; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide; NA, noradrenaline; NHLF, normal human lung fibroblast; PCR, polymerase chain reaction; PI, propidium iodide; TZ, terazosin.

Article Snippet: MRC5 fibroblasts, normal human lung fibroblasts, and IPF fibroblasts were procured from ATCC (Manassas, VA), Lonza (Allendale, NJ), and Asterand Bioscience (Detroit, MI), respectively.

Techniques: Immunofluorescence, Imaging, Staining, BrdU Incorporation Assay, Incubation, Expressing, MANN-WHITNEY, Polymerase Chain Reaction