Journal: bioRxiv
Article Title: TGF-β induces an atypical EMT to evade immune mechanosurveillance in lung adenocarcinoma dormant metastasis
doi: 10.1101/2024.10.15.618357
Figure Lengend Snippet: a , 3D reconstructed images of H2087-LCC and M802T4-LCC cells disseminated in brain parenchyma at indicated time point after intracardiac inoculation into athymic mice and B6(Cg)-Tyrc-2J/J (B6-albino) mice, respectively. Cancer cells were visualized by GFP IF (green) and brain capillaries by CD31 IF (magenta). Scale bar: 10 μm. b , Quantification of elongated, spheroidal, and microclustered cancer cells (fewer than 10 cells) in A. n=33 and 18 for H2087-LCC cells on day7 and day 49 respectively. n=253 and 135 for M802T4-LCC cells on day 7 and day 49 respectively. Chi-square test. c , Representative images of H2087-LCC and M802T4-LCC cells expressing a TGF-β reporter in the lungs of athymic mice and B6129SF1/J mice, respectively, 7 weeks after intravenous injection. Cancer cells were visualized by GFP IF (green), the TGF-β mCherry reporter activity in red, Ki67 proliferation marker in white, and DAPI in blue. Scale bar: 20 μm. d , Quantification of TGF-β mCherry reporter and Ki67 expression in H2087-LCC (n=129) and M802T4-LCC (n=63) cells in the lungs of athymic and B6129SF1/J mice, respectively, 7 weeks after intravenous injection. e and f , Representative IF images of H2087-LCC in the lungs of athymic mice 7 weeks after intravenous injection. GFP + cancer cells (green), TGF-β mCherry reporter (red), E-cadherin or FN1 (white), and DAPI (blue). g , Quantification of TGF-β mCherry reporter and E-cadherin (n=87) or FN1 (n=53) expression in disseminated H2087-LCC cells in the lungs of athymic mice. h and i , Representative IF images of M802T4-LCC in the lungs of B6129SF1/J mice 7 weeks after intravenous injection. GFP + cancer cells (green), TGF-β mCherry reporter (red), E-cadherin (white), and DAPI (blue). Scale bar: 20 μm. j , Quantification of TGF-β mCherry reporter and E-cadherin (n=71) or FN1 (n=51) expression in M802T4-LCC in the lungs of B6129SF1/J mice 7 weeks after intravenous injection. k-m , Schematic representation of the experimental design, created with Biorender.com (k), and tracking of wild-type and Tgfbr2 KO M802T4-LCC cells intravenously inoculated in B6129SF1/J mice followed by antibody-mediated depletion of NK, CD4+ and CD8+ T cells from day 3 (l) or day 35 (m) after inoculation. n=6 or 7 mice per group.
Article Snippet: To validate the depletion of each immune population, peripheral blood was collected and stained with violetFluor 450 Anti-Mouse CD45 (30-F11) (Tonbo Biosciences, Cat# 75-0451-U100), FITC Anti-Mouse CD4 (GK1.5) (Tonbo Biosciences, Cat# 35-0041-U100), PE-Cy7 Anti-Mouse CD8a (53-6.7) (Tonbo Biosciences, Cat#60-0081-U100), APC Anti-Mouse NK1.1 (CD161) (PK136) (Tonbo Biosciences, Cat# 20-5941-U100), followed by flow cytometric analysis on an LSRFortessa (BD Biosciences) instrument.
Techniques: Expressing, Injection, Activity Assay, Marker
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