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. 2016 Jul 19;7(29):45702-45714.
doi: 10.18632/oncotarget.10157.

Fibrotic microenvironment promotes the metastatic seeding of tumor cells via activating the fibronectin 1/secreted phosphoprotein 1-integrin signaling

Chong Zhang  1 , Mengzhi Wu  1 , Lizhen Zhang  1 , Li-Ru Shang  1 , Jian-Hong Fang  1 , Shi-Mei Zhuang  1
Affiliations

Fibrotic microenvironment promotes the metastatic seeding of tumor cells via activating the fibronectin 1/secreted phosphoprotein 1-integrin signaling

Chong Zhang et al. Oncotarget. .

Abstract

The seeding of tumor cells is a critical step in the process of metastasis, but whether and how the microenvironment of target organs affects metastatic seeding remain largely unknown. Based on cell and mouse models, we found that the metastatic seeding and outgrowth of tumor cells were significantly enhanced in fibrotic lungs. The conditioned medium from both fibrotic lungs and the fibrotic lung-derived fibroblasts (CM-FLF) had a strong activity to chemoattract tumor cells and to inhibit the apoptosis of tumor cells. Subsequent investigations revealed that the levels of fibronectin 1 (FN1) and secreted phosphoprotein 1 (SPP1) were significantly increased in fibrotic lungs. Silencing of FN1 in the fibrotic lung-derived fibroblasts dramatically decreased the chemoattracting activity of CM-FLF, while silencing of FN1 or SPP1 in fibroblasts attenuated the anti-apoptosis activity of CM-FLF. Moreover, the CM-FLF-induced apoptosis resistance or chemotaxis of tumor cells was attenuated when ITGAV, the common receptor of FN1 and SPP1, was silenced by RNA interference or blocked by GRGDS treatment in tumor cells. Consistently, ITGAV silencing or GRGDS treatment significantly inhibited the seeding and outgrowth of tumor cells in fibrotic lungs in vivo. Collectively, we suggest that fibrotic microenvironment may enhance the metastatic seeding of tumor cells in the lung by chemoattracting tumor cells and inhibiting their apoptosis via activating the FN1/SPP1-ITGAV signaling. These findings give a novel insight into the regulatory mechanisms of cancer metastasis and provide a potential target for anti-metastasis therapy.

Keywords: FN1; ITGAV; SPP1; fibrotic microenvironment; metastatic seeding.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1. Fibrotic microenvironment promotes the seeding and outgrowth of tumor cells in the lungs
(A, B) Fibrotic microenvironment promoted the outgrowth of tumor cells in the lungs. Hepa1-6-GFP (A, 2 × 106) and 4T1-luc (B, 2 × 105) cells were injected into the tail vein of saline or bleomycin-treated C57BL/6 and BALB/c mice, respectively. Metastasis burden was analyzed by HE staining 21 days post-injection (A, Hepa1-6-GFP) or monitored by in vivo bioluminescence imaging nine days after inoculation (B, 4T1-luc). Scale bar, 100 μm in (A). (C, D) Fibrotic microenvironment enhanced the seeding of tumor cells in the lungs. Hepa1-6-GFP (C, 1 × 106) and 4T1-luc (D, 1 × 106) cells were injected into the tail vein of saline or bleomycin-treated C57BL/6 and BALB/c mice, respectively. Ten hours later, murine lungs were subjected to the realtime quantitative PCR (qPCR) for the mRNA levels of GFP (C) or the in vivo bioluminescence imaging (D). The number of mice in each group is indicated on the top of cartogram. *P < 0.05; **P < 0.01.
Figure 2
Figure 2. The conditioned medium from fibrotic lungs has chemoattracting and anti-apoptosis activity
(A, B) The conditioned medium from fibrotic lungs enhanced the chemotaxis of tumor cells. Serum-free CM-NL or CM-FL was added to the lower chamber of transwell, while Hepa1-6 (A) and 4T1 (B) cells were added to the upper chamber. Total number of migrated cells from five random fields (100×) was calculated for each sample. Scale bar, 100 μm. (C, D) CM-FL inhibited the apoptosis of tumor cells. Hepa1-6 (C) and 4T1 (D) cells were cultured with serum-free CM-NL or CM-FL for 48 hours. Apoptosis was analyzed by 4′-6′-diamidino-2-phenylindole (DAPI) staining and at least 500 cells were counted for each sample. Scale bar, 50 μm. (E, F) CM-FL decreased the level of active-caspase-3 in tumor cells. Hepa1-6 (E) and 4T1 (F) were cultured in serum-free CM-NL or CM-FL for 22 hours, followed by detection for pro- and active-caspase-3 using immunoblotting. β-actin, internal control. Data are derived from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 3
Figure 3. The conditioned medium of fibrotic lung-derived fibroblasts possesses chemoattracting and anti-apoptosis activity
(A, B) The conditioned medium of fibrotic lung-derived fibroblasts had a strong chemoattracting activity. Ctrl (control) medium containing 1% FBS or CM-FLF containing 1% FBS was added to the lower chamber of transwell, while Hepa1-6 (A) and 4T1 (B) cells were added to the upper chamber. Scale bar, 100 μm. (C, D) CM-FLF inhibited the apoptosis of tumor cells. Hepa1-6 (C) and 4T1 (D) cells were grown in serum-free Ctrl medium or CM-FLF for 36 hours, followed by DAPI staining. Scale bar, 50 μm. (E, F) CM-FLF decreased the level of active-caspase-3 in tumor cells. Hepa1-6 (E) and 4T1 (F) were cultured in serum-free Ctrl medium or CM-FLF for 22 hours, followed by detection for pro- and active-caspase-3 using immunoblotting. β-actin, internal control. Data are derived from three independent experiments. *P < 0.05; **P < 0.01.
Figure 4
Figure 4. Silencing of FN1 or SPP1 in fibrotic lung-derived fibroblasts attenuates the chemoattracting and anti-apoptosis activity of CM-FLF
(A, B) FN1 but not SPP1 silencing in fibrotic lung-derived fibroblasts attenuated the ability of CM-FLF to chemoattract tumor cells. The fibrotic lung-derived fibroblasts were transfected with the indicated siRNAs for 48 hours, followed by refreshment with 1% FBS-supplemented RPMI 1640. The conditioned medium was harvested 24 hours later, and then applied to the lower chamber of transwell. (C, D) FN1 and SPP1 silencing in fibrotic lung-derived fibroblasts abrogated the anti-apoptosis activity of CM-FLF. The fibrotic lung-derived fibroblasts were transfected with the indicated siRNAs for 48 hours, followed by refreshment with serum-free RPMI 1640. The conditioned medium was harvested 24 hours later, and applied to incubation with Hepa1-6 (C) and 4T1 (D) for 36 hours before DAPI staining. iMAX, treatment with transfection reagent RNAiMAX. NC, transfection with negative control duplex for siRNAs. Data are derived from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.
Figure 5
Figure 5. Blocking the ITGAV pathway in tumor cells attenuates the chemoattracting and anti-apoptosis activity of CM-FLF
(A, B) GRGDS treatment reduced the chemotaxis of tumor cells towards CM-FLF. Hepa1-6 (A) or 4T1 (B) cells were resuspended in the medium containing 25 ug/ml GRGES (control) or GRGDS and added to the upper chamber of transwell, while CM-FLF containing 1% FBS was added to the lower chamber. (C) GRGDS treatment blocked the anti-apoptosis effect of CM-FLF on tumor cells. Hepa1-6 or 4T1 cells were cultured in the serum-free CM-FLF containing 25 ug/ml GRGES or GRGDS for 36 hours before DAPI staining. (D, E) Silencing of ITGAV decreased the chemotaxis of tumor cells towards CM-FLF. Hepa1-6 (D) or 4T1 (E) cells were transfected with the indicated siRNAs for 36 hours, then added to the upper chamber of transwell, while CM-FLF containing 1% FBS was added to the lower chamber. (F) Silencing of ITGAV blocked the anti-apoptosis effect of CM-FLF on tumor cells. Hepa1-6 or 4T1 cells were transfected with the indicated siRNAs for 24 hours, then replaced with serum-free CM-FLF for 36 hours before DAPI staining. iMAX, treatment with transfection reagent RNAiMAX. NC, transfection with negative control duplex for siRNAs. Scale bar, 100 μm. Data are derived from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 6
Figure 6. Blocking the ITGAV pathway in tumor cells inhibits the seeding and outgrowth of tumor cells in murine fibrotic lungs
(A) GRGDS treatment inhibited the seeding of tumor cells in fibrotic lungs. Fourteen days after bleomycin treatment, GRGES or GRGDS (3 mg in 100 ul) was injected into the tail vein of bleomycin-treated mice, followed by injection of Hepa1-6-GFP (1 × 106) or 4T1-luc (2 × 105) cells. Ten hours after injection, the amount of seeding tumor cells in murine lungs was quantified by qPCR analysis on the mRNA level of GFP or luciferase. (B) Silencing of ITGAV inhibited the seeding of tumor cells in fibrotic lungs. Hepa1-6-GFP (1 × 106) and 4T1-luc (2 × 105) cells that were transfected with NC or siItgav-2 duplex for 36 hours were injected into the tail vein of bleomycin-treated mice. Ten hours post-injection, the amount of seeding tumor cells in murine lungs was quantified by qPCR analysis. (C, D) GRGDS treatment inhibited the outgrowth of tumor cells in fibrotic lungs. GRGES or GRGDS (3 mg in 100 ul) was injected into the tail vein of bleomycin-treated mice, followed by injection of Hepa1-6-GFP (C, 2 × 106) and 4T1-luc (D, 2 × 105) cells. (E, F) Silencing of ITGAV attenuated the outgrowth of tumor cells in fibrotic lungs. Hepa1-6-GFP (E, 2 × 106) and 4T1-luc (F, 2 × 105) cells that were transfected with NC or siItgav-2 duplex for 36 hours were injected into the tail vein of bleomycin-treated mice. For (C–F), HE staining was used to analyze the metastasis of Hepa1-6-GFP cells 21 days post-injection, and in vivo bioluminescence imaging was employed to monitor the outgrowth of 4T1-luc cells nine days after inoculation. The number of mice in each group is indicated on the top of cartogram. NC, transfection with negative control duplex for siRNAs. *P < 0.05; **P < 0.01.
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