In the present study, we demonstrated the following: (1) co-inoculation of mice with lung cancer cells and lung fibroblasts results in higher susceptibility to tumorigenesis and higher tumor progression in those mice, (2) lung fibroblast-conditioned medium contributes to lung cancer cell survival through, at least in part, the production of HGF, (3) inhibition of HGF/Met signaling can suppress cancer cell survival and tumor progression, and (4) lung cancer cells stimulate HGF production in lung fibroblasts, as summarized in Fig. 7.

Fig. 7 Schema of the interaction between lung cancer cells and lung fibroblasts. Lung fibroblasts enhance lung cancer cell survival and tumorigenesis via the production of HGF. Lung cancer cells stimulate HGF production in lung fibroblasts. Full size image

In the present study, all three tested lung fibroblast-conditioned media led to a prolonged survival of lung cancer cells. Consistent with this, in a 3D co-culture model, the co-culture of MRC5 fibroblasts and cancer cell lines increased the survival of several types of cancer cells [1]. In lung cancer, however, only 2 of the 7 cell lines tested exhibited increased survival when co-cultured with MRC5 cells for 5 days, and no increased survival was observed in A549 cells [1]. In the present study, however, MRC5-conditioned medium clearly prolonged the survival of A549 cells. One possible reason for this difference is that the culture period was different, which may be important because in the present study, the effect of fibroblast-conditioned medium was more obvious after one week (Fig. 2).

The co-inoculation of mice with lung cancer cells and lung fibroblasts increased the incidence of tumorigenesis and promoted tumor progression. The fibroblasts surrounded by cancer cells tested in the current study are akin to CAFs. A two-week period seems to be sufficient for the effect of fibroblasts on the stimulation of cancer progression to be observed. Interestingly, it has been reported that the subcutaneous co-inoculation of SCID mice with A549 cells and CAFs significantly enhanced tumor growth compared with co-inoculation with normal fibroblasts [19]. Although we attempted to detect co-inoculated fibroblasts within the tumor, no significant difference was found in the stroma between the conditions with or without co-inoculation with fibroblasts even at 2 weeks after co-inoculation. This might be due to the difference in cell proliferation rates between cancer cells and fibroblasts; that is, cancer cells grow very rapidly so that fibroblasts are soon outnumbered. Another possibility is that lung fibroblasts might be induced by lung cancer cells to undergo apoptosis.

The present study suggests that the strategy of lung cancer treatment should include the regulation of either fibroblasts or some of the factors produced by fibroblasts, such as HGF. In this regard, the effects of the inhibition of HGF/Met signaling on lung cancer progression have been reported. PHA-665752 has been shown to reduce the tumorigenicity of lung cancer cell lines (NCI-H69 and NCI-H441) in mouse xenografts [10]. In the current study, PHA-665752 also significantly suppressed tumor progression after the co-injection of EBC1 and HFL1 cells. One mechanism for the inhibition of tumor progression might be the occurrence of cell cycle arrest, as evidenced by a decrease in mitosis. PHA-665752 also reportedly inhibits the formation of vascular structures [10].

In the treatment of NSCLC cases that harbor epidermal growth factor receptor (EGFR) mutations, EGFR-tyrosine kinase inhibitors (TKIs), such as gefitinib, erlotinib, and afatinib, can block the survival signals that are mediated by this driver oncogene and can induce marked tumor regression [14]. However, tumors eventually acquire resistance to EGFR-TKIs and regrow in almost all cases. The mechanisms of EGFR-TKI resistance include alterations of the EGFR gene (e.g., T790M mutation) and activation of bypass signaling pathways (e.g., Met gene amplification and HGF overexpression). Indeed, in one study, exogenous HGF induced resistance to an anti-EGFR antibody in lung cancer cells (via the Met/Gab1/Akt signaling pathway) [11]. In addition, resistance to an anti-EGFR antibody was also induced in tumor cells by the co-culture of cancer cells and HGF-producing lung fibroblasts [11]. Moreover, the usefulness of the inhibition of the HGF/Met pathway for the treatment of EGFR-TKI-resistant lung cancer cells has been reported [9, 12, 13]. The Met inhibitor SU11274 exerted a pro-apoptotic effect on EGFR-TKI-resistant H1975 cells and induced tumor regression in H1975 xenografts [9]. The combination of SU11274 and erlotinib treatment induced complete tumor regression in a xenograft model [9]. E7050, a dual inhibitor of Met and vascular endothelial growth factor receptor two kinase, and EGFR-TKIs suppressed tumor progression in erlotinib-resistant cancer cell lines and in HGF-induced EGFR-TKI-resistant lung cancer cell lines that also have a mutation in EGFR [12, 13].

Two phase III clinical trials using a Met inhibitor tivantinib (ARQ 197) and erlotinib have been conducted [20, 21]. The combination of erlotinib plus tivantinib increased the progression-free survival of patients with previously treated advanced non-squamous NSCLC [21]. In a subgroup of patients with high Met expression, tivantinib also improved the overall survival. However, another trial targeting Asian patients with EGFR-WT non-squamous NSCLC was prematurely terminated due to the increased incidence of interstitial lung disease in patient group treated with erlotinib and tivantinib, although preliminary data revealed that the PFS was longer in that group [20]. Met amplification is an excellent predictor of sensitivity to Met inhibitors such as PHA-665752 and crizotinib [22, 23]. Impaired Met degradation mediated by Met exon 14 mutations has also been documented in NSCLC [24,25,26]. Met exon 14 skipping was identified in 2.7% of patients with NSCLC, and a robust response to crizotinib was observed regardless of the Met amplification status [26]. Ethnicity and Met activation as a predictive marker (e.g., Met expression, Met amplification or Met exon 14 skipping) should be considered prior to the clinical application of Met inhibitors.

Another important finding of the current study is that HGF production in fibroblasts was stimulated by lung cancer cells, which seemed to exploit the fibroblasts for their own growth. Unfortunately, we could not identify the factor that stimulates HGF production in fibroblasts. Although transforming growth factor-β is known to change the characteristics of lung fibroblasts and is produced by lung cancer cells, its exogenous addition did not stimulate HGF production by fibroblasts (data not shown). The identification of the specific factor that is released from lung cancer cells that stimulates HGF production by fibroblasts might also be useful for the development of a novel strategy for lung cancer treatment.