Combined targeting of TGF-β1 and integrin β3 impairs lymph node metastasis in a mouse model of non-small-cell lung cancer

Abstract

Background: Transforming Growth Factor beta (TGF-β) acts as a tumor suppressor early in carcinogenesis but turns into tumor promoter in later disease stages. In fact, TGF-β is a known inducer of integrin expression by tumor cells which contributes to cancer metastatic spread and TGF-β inhibition has been shown to attenuate metastasis in mouse models. However, carcinoma cells often become refractory to TGF-β-mediated growth inhibition. Therefore identifying patients that may benefit from anti-TGF-β therapy requires careful selection.

Methods: We performed in vitro analysis of the effects of exposure to TGF-β in NSCLC cell chemotaxis and adhesion to lymphatic endothelial cells. We also studied in an orthotopic model of NSCLC the incidence of metastases to the lymph nodes after inhibition of TGF-β signaling, β3 integrin expression or both.

Results: We offer evidences of increased β3-integrin dependent NSCLC adhesion to lymphatic endothelium after TGF-β exposure. In vivo experiments show that targeting of TGF-β and β3 integrin significantly reduces the incidence of lymph node metastasis. Even more, blockade of β3 integrin expression in tumors that did not respond to TGF-β inhibition severely impaired the ability of the tumor to metastasize towards the lymph nodes.

Conclusion: These findings suggest that lung cancer tumors refractory to TGF-β monotherapy can be effectively treated using dual therapy that combines the inhibition of tumor cell adhesion to lymphatic vessels with stromal TGF-β inhibition.

Figure 1

TGF-β exposure enhances NSCLC cell adhesion and transmigration across lymphatic endothelial cells. (A) Adhesion of H157 cells to LEC monolayers after treatment for 5 days with TGF-β (2 ng/ml), and in the presence or absence of the TGF-βRI inhibitor SB 431542 (**p < 0.001, Student’s t-test). (B) Time-lapse microphotographs of the movement of H157 NSCLC cells, treated as in A, across LEC monolayers (63× water objective). Dots indicate the initial position of the cell and arrows indicate the same cell in motion. (C) Quantification of the number of H157 cells in transit over LEC monolayers, expressed as a percentage of the total number of cells counted in a single XY plane. (D) Quantification of NSCLC cell transmigration across monolayers of primary human LECs in the presence or absence of TGF-β. Data are presented as fold-change with respect to untreated control H157 cells (**p < 0.001, Student’s t-test).

Figure 2

TGF-β treatment induces integrin expression, FAK phosphorylation, β3 integrin-dependent adhesion and transmigration of H157 NSCLC cells across LEC monolayers. (A) mRNA expression of several integrins in H157 cells following treatment with TGF-β and its inhibitors (fold-change with respect to untreated cells) and confirmation by real-time PCR of the differential expression of β3 and β5 integrins after exposure to TGF-β. (B) FAK phosphorylation after TGF-β treatment of β3 integrin-deficient (shRNAβ3) and β3 integrin-competent H157 NSCLC cells. (C) Adhesion of TGF-β-treated H157 cells to LEC monolayers in the presence or absence of the FAK inhibitor, PF-573228 (**p < 0.001, Student’s t-test). (D) Quantification of H157 cell transmigration across LEC monolayers in the presence of the TGF-βRI inhibitor SB431542, the FAK inhibitor PF-573228, a blocking mAb against β3 integrin (Anti-β3) and that of β3 integrin-deficient H157 clones (β3). Samples were pretreated with or without TGF-β as described in the Materials and methods (**p < 0,001 compared against non-treated cells, ^## p < 0,001 compared against TGFβ-treated cells, Mann–Whitney U-test).

Figure 3

Integrin β3 silencing in H157 cells significantly diminishes cell movement across LEC monolayers. (A) Microphotographs showing the adhesion of TGF-β-pretreated H157 cells (integrin-competent and integrin-deficient) to LEC monolayers (63× water objective). (B) Average distance covered and speed of TGF-β-pretreated H157 cells (integrin-competent and integrin-deficient): ** p = 0.001, one-way ANOVA with Dunnett’s multiple comparison test. (C) Representation of cell directionality for one representative cell from each group. A minimum of 30 cells were analyzed per group.

Figure 4

H157 cell transmigration across endothelial monolayers is inhibited by blocking antibodies against the β3 integrin ligands L1CAM and CD31. Quantification of the number of TGF-β-pretreated β3 integrin-competent (A) and β3 integrin-deficient (B) H157 cells transmigrating across LEC monolayers in the presence of blocking antibodies against the L1CAM RGD binding region (L1.35.9 mAb), the L1CAM homotypic binding region (L1 9.3 mAb) and CD31: **p = 0.001, one-way ANOVA with Dunnett’s multiple comparison test.

Figure 5

Integrin β3 silencing and inhibition of stromal TGF-β improves survival and attenuates tumor cell growth in an orthotopic mouse model of lung carcinoma. (A) Kaplan-Meier survival curves for mice injected with β3 integrin-deficient or β3 integrin-competent cells that were pretreated with or without TGF-β. Mice were injected intraperitoneally with 200 μg of the TGF-β inhibitor peptide P144 as described in Materials and methods. (B) Tumor size determined from microphotographs obtained from tumor sections and expressed in cm² (maximum diameter). Image analyses were performed using fiji software. Data were analyzed using a one-way ANOVA followed by a Dunnett’s multiple comparison test (** p < 0.001: H157 TGF-β PBS vs H157 control PBS; ^§§p = 0.001: shRNA β3 TGF-β PBS vs H157 control PBS; ^##p = 0.004: H157 TGF-β PBS vs H157 TGF-β P144). (C) Micro-CT analyses of mouse lungs before sacrifice. A representative image from each group is shown.

Figure 6

Combined targeting of TGF-β and integrin β3 attenuates lymph node metastasis of H157 NSCLC cells in the lungs of immunodeficient mice. (A) Percentage of affected lymph nodes (axillary and brachial) in the different experimental groups analyzed. Statistical analyses were performed by means of Mann–Whitney U- test. Significant values are depicted with double asterisks and correspond to p values of 0.009 for both comparisons (B) Representative microphotograph of GFP tumor detection in the histological samples obtained from lungs (upper row) and lymph nodes (lower row). (C) Immunohistochemical detection of integrin β3 in tumors obtained from mice.