BIBF 1120 (nintedanib), a triple angiokinase inhibitor, induces hypoxia but not EMT and blocks progression of preclinical models of lung and pancreatic cancer

Abstract

Signaling from other angiokinases may underlie resistance to VEGF-directed therapy. We evaluated the antitumor and biologic effects of BIBF 1120 (nintedanib), a tyrosine kinase inhibitor that targets VEGF receptor, platelet-derived growth factor receptor, and fibroblast growth factor receptor in preclinical models of lung and pancreatic cancer, including models resistant to VEGF-targeted treatments. In vitro, BIBF 1120 did not show antiproliferative effects, nor did it sensitize tumor cells to chemotherapy. However, in vivo BIBF 1120 inhibited primary tumor growth in all models as a single agent and in combination with standard chemotherapy. Analysis of tumor tissue posttreatment revealed that BIBF 1120 reduced proliferation (phospho-histone 3) and elevated apoptosis (cleaved caspase-3) to a greater extent than chemotherapy alone. Furthermore, BIBF 1120 showed potent antiangiogenic effects, including decreases in microvessel density (CD31), pericyte coverage (NG2), vessel permeability, and perfusion, while increasing hypoxia. Despite the induction of hypoxia, markers of epithelial-to-mesenchymal transition (EMT) were not elevated in BIBF 1120-treated tumors. In summary, BIBF 1120 showed potent antitumor and antiangiogenic activity in preclinical models of lung and pancreatic cancer where it induced hypoxia but not EMT. The absence of EMT induction, which has been implicated in resistance to antiangiogenic therapies, is noteworthy. Together, these results warrant further clinical studies of BIBF 1120.

Figure 1. BIBF 1120 inhibits primary tumor growth in subcutaneous lung and orthotopic pancreatic cancer xenografts

A) Chemical structures of BIBF 1120 (first panel) and chemotherapeutics Gemcitabine and Cisplatin (second and third panels). B) The indicted NSCLC cells were injected subcutaneously into mice. Treatment by with vehicle (Control), BIBF 1120 (BIBF, 50 mg/kg, daily), CHEMO (gemcitabine [25 mg/kg 2x/week] + Cisplatin [1 mg/kg 1x/week]) was initiated when average tumor volume reached 100–150 mm³ (n=10/group). Tumor growth curves displaying mean tumor volume +/− SEM and C) mean final tumor weights +/− SEM are shown. D) The indicated pancreatic cancer cells were injected orthotopically into SCID mice. Treatment was initiated 7–10 days post tumor cell injection (TCI). Animals were treated as above with the exception of chemotherapy; gemcitabine was given at 12.5 mg/kg 3x/week. Mean final pancreas weight +/− SEM is shown (n=10/group). *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001 by Dunn’s post-test.

Figure 2. BIBF 1120 decreases gross metastatic burden in orthotopic pancreatic xenografts

A) At sacrifice, the number of gross metastatic events was recorded. Gross metastatic burden was decreased significantly in BIBF 1120-treated animals compared to control groups in each model. B) Representative image of a HPAF-II tumor presentation at the time of sacrifice. Dashed lines, primary tumor borders; Arrows, metastatic foci. Bar graphs indicate mean + SEM. ***, p<0.001; ****, p<0.0001 by student’s t-test / Dunn’s post-test.

Figure 3. BIBF 1120 shows potent anti-angiogenic effects, decreases perfusion and induces hypoxia in lung cancer xenografts

Vascular parameters of NSCLC tumors were evaluated by immunohistochemistry and perfusion studies. A) Representative images of microvessel density (CD31) and pericyte coverage (NG2) in Calu-6 xenografts at 200x magnification (inset: 400x), with DAPI labeling nuclei. B) Representative images of perfusion of high molecular weight (FITC, MW: 2 million) and low molecular weight (Rhodamine, MW 10000) dextrans in A549 xenografts. C) Representative images of hypoxia as assessed by pimonidazole staining in H1993 xenografts. Scale bar, 100 μm. Quantification of microvessel density (D), pericyte coverage (E) and dextran perfusion data and hypoxia (F). Vessel patency reflects signal from high molecular weight dextran and perfusion reflects signal from low molecular weight dextran. Bar graphs indicate means + SEM. A minimum of 5 images were acquired per group. Results were given as mean percentage of thresholded area or absolute vessel counts per 200x field. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001 by Dunn’s post-test.

Figure 4. BIBF 1120 shows potent anti-angiogenic effects, induces hypoxia and decreases drug delivery in pancreatic cancer xenografts

Vascular parameters of pancreatic tumors were evaluated by immunohistochemistry and perfusion studies. A) Representative images of microvessel density (CD31) and pericyte coverage (NG2) in Mia PaCa-2 xenografts at 200x magnification (inset: 400x), with DAPI labeling nuclei. Scale bar, 100 μm. B) Representative images of CD31 and pimonidazole reactivity in MIA PaCa-2 xenografts. Scale bar, 100 μm. C) Representative images of doxorubicin perfusion in AsPC-1 xenografts after acute or chronic exposure to BIBF 1120. Quantification of microvessel density (D), pericyte coverage (E), hypoxia (F), and doxorubicin (perfusion) fluorescence (G) is shown. Bar graphs indicate means + SEM. A minimum of 5 images were acquired per group. Results are mean percentage of thresholded area or absolute vessel counts per field. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001 by Dunn’s post-test.

Figure 5. BIBF 1120 does not drive an invasive phenotype in lung cancer

EMT markers from lung tumors were evaluated by immunohistochemistry. A) Representative images of mature myofibroblasts (α-SMA) at 200x magnification with DAPI labeling nuclei. B) Representative images of E-cadherin and vimentin at 200x magnification. C) Quantification of fibroblasts, E-cadherin and vimentin. Bar graphs indicate mean +/− SEM. A minimum of 5 images were acquired per group. Results were given as mean percentage of thresholded area per field. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001 by Dunn’s post-test.

Figure 6. BIBF 1120 does not drive an invasive phenotype in pancreatic cancer

EMT markers from pancreatic tumors were evaluated by immunohistochemistry. A) Representative images of zeb1 at 200x magnification with DAPI labeling nuclei. B) Representative images of mature myofibroblasts (α-SMA) at 200x magnification with DAPI labeling nuclei. C) Representative images of E-cadherin and vimentin at 200x magnification. Scale bar, 100 μm. D) Quantification of zeb1, fibroblasts, E-cadherin and vimentin. Bar graphs indicate means +/− SEM. A minimum of 5 images were acquired per group. Results were given as mean percentage of thresholded area per field. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001 by Dunn’s post-test.