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Multiple primary lung cancers (MPLCs) harbour various genetic profiles among the tumours, even from individuals with same non-intrinsic risk factors. Paired mutational analyses were performed to obtain a census of mutational events in MPLC and assess their relationship with non-intrinsic risk factors. Thirty-eight surgical specimens from 17 patients diagnosed as MPLC were used. Extracted DNAs were sequenced for somatic mutations in 409 cancer-associated genes from a comprehensive cancer panel. We statistically analysed the correlation between each driver mutation frequency and non-intrinsic risk factors using Fisher's exact test, and whether genetic mutations occurred concomitantly or randomly in MPLC using an exact test. Comprehensive genetic analyses suggested different mutation profiles in tumours within the same individuals, with some exceptions. EGFR, KRAS, TP53, or PARP1 mutations were concomitantly detected in some MPLC cases. EGFR mutations were significantly more frequent in never or light smokers and females. Concomitant EGFR or KRAS mutations in MPLCs were significantly more frequent than expected by chance (P = .0023 and .0049, respectively) suggesting a more prominent role of non-intrinsic risk factors in EGFR and KRAS mutations than other mutations, which occurred more randomly. Concomitant EGFR or KRAS mutations were particularly prominent in never or light smokers and males.

Cancer-associated fibroblasts (CAFs) play critical roles in the tumor progression. However, it remains unclear how cancer cells migrate in the three-dimensional (3D) matrix of cancer tissues and how CAFs support the cancer invasion. Here we propose a novel mechanism of fibroblast-dependent cancer cell invasion in the 3D collagen matrix. Human cancer cell lines from the pancreas (Panc-1), lung (A549) and some other organs actively adhered to normal fibroblasts and primary lung CAFs in cultures. To show its significance in tumor invasion, we designed a new invasion assay in which homogeneous microspheroids consisting of cancer cells and fibroblasts were embedded into collagen gel. Time-lapse experiments showed that cancer cells adhered to and quickly migrated on the long protrusions of fibroblasts in the 3D collagen matrix. Fibroblast-free cancer cells poorly invaded the matrix. Experiments with function-blocking antibodies, siRNAs, and immunocytochemistry demonstrated that cancer cells adhered to fibroblasts through integrin α5β1-mediated binding to fibronectin on the surface of fibroblasts. Immunochemical analyses of the co-cultures and lung cancers suggested that cancer cells could acquire the migratory force by the fibronectin/integrin signaling. Our results also revealed that the fibroblast-bound fibronectin was a preferential substrate for cancer cells to migrate in the collagen matrix.