The HGF-cMET axis is recapitulated in the Calu-1 FB co-culture model and induces single cell invasion.

<p>(A) and (B) Proteome analysis of cell lysates and supernatants derived either from mono-cultures (Calu-1, NCI-H1437 (H1437), HDF) or from a 2D co-culture (Calu-1+HDF, H1437+HDF). All samples were taken from 24 h cultures as triplicates. For detection of ERK1(T202/Y204), ERK2(T185/Y187) and CREB(S133) the Phospho-MAPK Array (R&D) was used, for AXL(Y779) and MET(Y1234/Y1235) the Phospho-Receptor Tyrosine Kinase Array (R&D), lysis buffer served as control. For HGF the RayBio Cytokine Antibody Array was used, McCoy’s medium plus 10% FCS served as control. (C) MET Western blot analyses utilizing whole cell lysates from mono-cultures (Calu-1, HDF) and from corresponding 2D co-cultures (Calu-1+HDF). Calu-1 and HDF mono-culture lysates were artificially mixed (Calu-1+HDF Mix) to serve as control. (D) p-MET Western blot analyses of whole cell lysates from Calu-1 mono-cultures prior starved in OPTI-MEM for 6 h. Cells were harvested after 15 min of incubation in supernatants (SN) from Calu-1 or HDFs. OPTI-MEM and McCoy‘s 10% FCS medium served as a control. (E) Addition of either recombinant human HGF (rhHGF; 10 ng/ml; left panel) or rhHGF plus anti-hHGF antibody (1 μg/ml; right panel) in a 3D assay (n = 3). The number of invasive single cells (ISC) is depicted on the right for 24 h and 48 h of incubation (n = 3) (Student’s t-test: *p<0.05) (F) Calu-1 spheroids cultured with HDFs (left panel) or with 10 ng/ml recombinant human HGF (rhHGF, right panel). The upper panel shows treatment with 1 μM MET inhibitor crizotinib, whereas the lower panel serves as control (n = 3). Number of ISC is shown on the right. Statistical analysis was performed on the means of ISC by unpaired comparison with Calu-1+HDF (DMSO) or Calu-1+rhHGF (DMSO) samples using Student’s t-test (**p<0.01, ***p<0.001). Scale bar = 100 μM. (G) Calu-1 cells were starved in OPTI-MEM for 6 h and then incubated with HDF conditioned medium (HDF SN) alone or together with 1 μM of crizotinib (HDF SN+crizotinib 1μM). Cells were harvested after 15 min of incubation. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. p-MET = Phospho-MET (Y1234/Y1235).