Figure 4 from Metastasis-Associated Wound Repair Promotes Reciprocal Lung Epithelium Activation and Breast Cancer Metastatic Outgrowth

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AT2 cell activation by breast cancer (BC) cells. A, Model indicating the cellular interactions being interrogated: BC activation of AT2 cells. B, Schematic of no-contact co-culture experimental design used to investigate the effects of TNBC cells on lung AT2 cells. C, AT2 cell viability was measured by crystal violet assay following co-culture with TNBC cells: 5 days co-culture for A549 cells and 7 days for iAT2 cells. Relative cell numbers were normalized to the mean confluence of AT2 cells cultured alone (−). Mean ± SEM (unpaired t tests with Welch correction); *, P ≤ 0.05; **, P < 0.01; ****, P < 0.0001. D, AT2 cell morphology was examined by H&E staining following co-culture with TNBC cells for 3 days. Control cells were cultured alone (−). Shown are representative images of cell size and shape; scale bars, 10 μm, inset zoom 4x for A549 cells and 6x for iAT2 cells. E, iAT2 lamellar bodies were imaged using lysotracker staining following co-culture with TNBC cells for 3 days. Control cells were cultured alone (−). Shown are representative images of lamellar body size and shape; scale bars, 10 μm, inset zoom 3x. F, iAT2 lamellar body levels were measured by quantifying lysotracker staining in iAT2 cells co-cultured with TNBC cells for 3 days. Lamellar body fluorescence was normalized to cell number and the mean fluorescence of iAT2 cells cultured alone (−). Mean ± SEM (unpaired t tests with Welch correction); *, P ≤ 0.05. G, Bulk RNA-seq was performed on RNA collected from iAT2 cells co-cultured with SUM159PT and MDA453 cells for 5 days, whereas control iAT2 cells were cultured alone. Function biological pathways in AT2 cells were determined using DEG lists from mouse AT2 cells in metastatic lungs and human iAT2 cells co-culture with TNBC cells. The enriched pathways from these datasets were then compared to identify overlapping biological pathways. ECM, extracellular matrix.

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