Fig. 8: Expression of a phosphomimetic Ser 301 lamin A/C mutant is sufficient to trigger CAF phenotype and induce tumor growth.

A Cancer cells expansion assays. HDFs infected with lentiviruses expressing the S301D mutant versus wild-type (WT) lamin A were co-cultured with SCC13 cells on a thin matrigel layer for 4 days. Immunofluorescence analysis with anti-Pan-keratin (red) and -Vimentin (blue) antibodies was used for cell type identification. Percentage of SCC positive area was quantified by Image J analysis of 5 random fields per well. Two-tailed unpaired t-test, n = 5 independent replicate wells. Mean ± SE. Scale: 100 µm. B Spheroid formation assays. HDFs expressing the S301D mutant versus WT lamin A/C were co-cultured with SCC13 cells in 50% Matrigel-coated eight well glass slide for 7 days. IF with anti-Pan-keratin (red) and -Vimentin (green) antibodies were used for cell type identification. Shown are representative images and quantification of diameter and number of spheroids, mean value as black bar. Two-tailed unpaired t-test, for diameter, n = 25 spheroids per condition; for spheroid numbers, n = 10 wells. Scale: 500 µm. C in vivo tumor formation assays. RFP-expressing SCC13 cells were co-injected with HDFs expressing S301D mutant versus WT lamin A/C intradermally into contralateral mouse back skin. Mice were sacrificed 14 days later. Shown are images of excised xenograft tumor nodules and quantification of excised tumor volumes calculated using the formula V = 3.14 × (W² × L)/6 V = volume, W = width, and L = length. Paired two-tailed t-test, n = 5 mice. Scale: 5 mm. D Representative H&E-stained images of the tumor lesions in (C). Scale bar: 500 µm. E IF analysis of RFP-expressing SCC cells and α-SMA positive fibroblasts respectively. Shown are representative images and quantification of RFP and α-SMA-positive area per lesion, mean value as black bar. Two-tailed unpaired t-test, n = 5 tumors. Scale: 100 µm. Source data for individual graphs are in the Source Data file.