New metastatic model of human small-cell lung cancer by orthotopic transplantation in mice

Abstract

Small-cell lung cancer (SCLC) is an aggressive cancer with high metastatic ability and novel strategies against the metastasis are urgently needed to improve SCLC treatment. However, the mechanism of metastasis of SCLC remains largely to be elucidated. For further studies of SCLC metastasis, we developed a new orthotopic transplantation model in mice. We established a GFP-labeled subline from the human SCLC cell line DMS273 and transplanted them orthotopically into the lung of nude mice with Matrigel. The GFP-labeled cells showed significant metastatic activity and formed metastatic foci in distant tissues such as bone, kidney, and brain, as observed in SCLC patients. From a bone metastasis focus of the mouse, we isolated another subline, termed G3H, with enhanced metastatic potential and higher hepatocyte growth factor (HGF) expression than the parental line. Further studies indicated that the HGF/MET signaling pathway was involved in in vitro motility and invasion activities of the G3H cells and treatments with MET inhibitors decreased formation of distant metastases in our orthotopic model using G3H cells. These data indicated that our model mimics the clinical aspect of SCLC such as metastatic tropism and autocrine of HGF/MET signaling. Compared with other orthotopic SCLC models, our model has a superior ability to form distant metastases. Therefore, our model will provide a valuable tool for the study of SCLC metastasis.

Keywords: Hepatocyte growth factor; MET; metastasis; orthotopic transplantation; small-cell lung cancer.

Fig 1

Green fluorescent protein-labelled metastatic sublines of human small-cell lung carcinoma (SCLC) cell line DMS273 and effect of cisplatin treatment. (a) Establishment of GFP-labelled metastatic sublines of DMS273. (b) Cell morphology of DMS273-GFP and G3H cells. Bar = 50 μm. (c) In vitro growth rate of the DMS273 sublines as determined by MTT assay. The growth rate was calculated as the ratio of the absorbance from cultured cells compared with that of day 0. Values are expressed as means of triplicate experiments ± SD. (d) In vitro sensitivity of the DMS273 sublines and MKN45 cell line against cisplatin. Cells were treated in triplicate with the indicated concentrations of cisplatin for 72 h. The growth rate was calculated as the ratio of MTT absorbance from cisplatin-treated cells compared with that of untreated cells. Results are expressed as means ± SD from three independent experiments. (e, f) Effect of cisplatin treatment on orthotopic and metastatic tumor formation in our model using G3H cells. The mice were killed and assessed on day 25 or 26. (e) Orthotopic tumor formation. Results are expressed as means ± SD (n = 5–6). (f) Distant metastatic tumor formation. The dotted lines show the means of the number of metastasis-positive organs of each group. Percentages show distant metastases incidence. *P < 0.05, Fisher's exact test.

Fig 2

Orthotopic and metastatic tumor formation of G3H, a GFP-labelled subline of human small-cell lung carcinoma cell line DMS273, in nude mice. Intrapulmonary injected cells formed tumor at the injected site of lung and metastatic colonies in various distant organs. (a–j) Representative GFP fluorescent images of orthotopic and metastatic tumor formation of G3H cells in lung (a, b), femur (c), vertebra (d), brain (e, f), liver (g), mesentery lymph node (h), adrenal gland (i), and kidney (j). (k–v) Representative histological appearance of orthotopic tumors (k, l) and metastatic (Meta) tumors in bone (m, n), brain (o, p), adrenal gland (q, r), kidney (s, t), and a blood vessel of mesentery (u, v). (k, m, o, q, s, u) H&E staining; (l, n, p, r, t, v) GFP immunostaining. Magnification, ×40 (k, l), ×100 (m–r), ×200 (s–v).

Fig 3

Hepatocyte growth factor (HGF)/MET signal in the metastatic sublines of small-cell lung carcinoma cell line DMS273. (a) Images of RayBio C-Series Human Cytokine Antibody Array 5 that were incubated with conditioned media (CM) or control media. The HGF, granulocyte/macrophage colony-stimulating factor (GM-CSF), insulin-like growth factor-binding protein-2 (IGFBP-2), interleukin-8 (IL-8), and monocyte chemotactic protein-1 (MCP-1) signals are marked with circles. (b) Quantitative analysis of HGF mRNA. Total RNA (1 μg) extracted from subconfluent cells were subjected to real-time RT-PCR. Results are expressed as means of three independent experiments ± SD. (c) ELISA of HGF protein in CM. Cells were incubated with DMEM containing 10% FBS for 3 days and the CM was harvested. Results are expressed as means of three independent experiments ± SD. (d) Western blot analysis for MET. Whole cell lysate (20 μg) was separated on a 7.5% SDS-PAGE and membranes were blotted with anti-phosphorylated MET (pY1234/1235) (top panel), anti-MET (second panel), or α-tubulin (bottom panel, loading control). (e, f) Effect of anti-HGF neutralizing antibodies on MET phosphorylation induced by CM of G3H. G3H (e) or PC-3 (f) cells were starved in DMEM containing 0.5% FBS overnight and pre-incubated with anti-HGF neutralizing antibody (10 μg/mL) for 2 h. Cells were then incubated with CM of G3H or recombinant HGF (50 ng/mL) for 15 min. Cells were lysed immediately and whole cell lysate (20 μg) was analyzed by Western blotting.

Fig 4

In vitro effect of PHA665752, a small molecule inhibitor of MET, on DMS273 small-cell lung cancer sublines. (a) Effect of PHA665752 on MET phosphorylation. G3H cells were starved in DMEM containing 0.5% FBS overnight, and then pre-incubated with PHA665752 (0.2 μM) for 2 h. Cells were then incubated with recombinant hepatocyte growth factor (HGF; 50 ng/mL) for 15 min. Cells were lysed immediately and whole cell lysate (20 μg) was analyzed by Western blotting. (b) In vitro sensitivity of the DMS273 sublines and MKN45 cell line against PHA665752. Cells were treated with the indicated concentrations of PHA665752 for 72 h. Assays were carried out in triplicate. The growth rate was calculated as the ratio of the MTT absorbance of PHA665752-treated cells compared with that of untreated cells. Results are expressed as means of three independent experiments ± SD. (c) Effect of PHA665752 and anti-HGF neutralizing antibodies on in vitro motility and invasion activities of G3H cells. In vitro motility and invasion assays using conditioned media of G3H as chemoattractant were carried out. Relative migrated or invaded cells were calculated as the ratio of numbers of migrated or invaded cells of the treated groups compared with the untreated group. Results are expressed as means of three independent experiments ± SD. *P < 0.01, **P < 0.005, Student's t-test, compared with untreated cells.

Fig 5

Effect of MET inhibitors on orthotopic and metastatic tumor formation in our model of human small-cell lung cancer using G3H cells. (a–d) Immunostaining of hepatocyte growth factor (HGF)/MET in orthotopic tumors of our model using G3H cells. (a) H&E staining, (b) GFP immunostaining, (c) HGF immunostaining, (d) MET immunostaining, and (e) phosphorylation of MET (pY1230/Y1234/Y1235) immunostaining. (f, g) Effect of PHA665752 (PHA) treatment on orthotopic and metastatic tumor formation in our model using G3H cells. The mice were killed and assessed on day 27 or 28. (f) Orthotopic tumor formation. Results are expressed as means ± SD (n = 8 or 9). (g) Distant metastatic tumor formation. The dotted lines show the means of the number of metastasis-positive tissues of each group. Percentages show distant metastases incidence. *P < 0.05, Fisher's exact test. (h, i) Effect of ARQ-197 (ARQ) treatment on orthotopic and metastatic tumor formation in our model using G3H cells. The mice were killed and assessed on day 26. (h) Orthotopic tumor formation. Results are expressed as the means ± SD (n = 6). (i) Distant metastatic tumor formation. The dotted lines show the means of the number of metastasis-positive organs of each group. Percentages show distant metastases incidence. *P < 0.05, Fisher's exact test.