Harnessing Induced Essentiality: Targeting Carbonic Anhydrase IX and Angiogenesis Reduces Lung Metastasis of Triple Negative Breast Cancer Xenografts

Abstract

Triple Negative Breast Cancer (TNBC) is aggressive, metastatic and drug-resistant, limiting the spectrum of effective therapeutic options for breast cancer patients. To date, anti-angiogenic agents have had limited success in the treatment of systemic breast cancer, possibly due to the exacerbation of tumor hypoxia and increased metastasis. Hypoxia drives increased expression of downstream effectors, including Carbonic Anhydrase IX (CAIX), a critical functional component of the pro-survival machinery required by hypoxic tumor cells. Here, we used the highly metastatic, CAIX-positive MDA-MB-231 LM2-4 orthotopic model of TNBC to investigate whether combinatorial targeting of CAIX and angiogenesis impacts tumor growth and metastasis in vivo to improve efficacy. The administration of a small molecule inhibitor of CAIX, SLC-0111, significantly reduced overall metastatic burden, whereas exposure to sunitinib increased hypoxia and CAIX expression in primary tumors, and failed to inhibit metastasis. The administration of SLC-0111 significantly decreased primary tumor vascular density and permeability, and reduced metastasis to the lung and liver. Furthermore, combining sunitinib and SLC-0111 significantly reduced both primary tumor growth and sunitinib-induced metastasis to the lung. Our findings suggest that targeting angiogenesis and hypoxia effectors in combination holds promise as a novel rational strategy for the effective treatment of patients with TNBC.

Keywords: angiogenesis; carbonic anhydrase IX; hypoxia; metastasis; resistance; sunitinib; triple negative breast cancer.

Figure 1

Pharmacologic inhibition of Carbonic Anhydrase IX (CAIX) activity and angiogenesis decrease tumor growth and metastasis in an in vivo model of Triple Negative Breast Cancer (TNBC). Mice with orthotopic MDA-MB-231 LM2-4Luc⁺ tumors were given SLC-0111 (50 mg/kg) and sunitinib (60 mg/kg), alone or in combination, daily by oral gavage. (a) Tumor growth curve. The schematic indicates the treatment schedule. For the combination, mice received sunitinib and SLC-0111 simultaneously from day 17 to day 27 post tumor cell inoculation, followed by SLC-0111 alone until endpoint. Data show the mean ± standard error of the mean (SEM). n = 8–10 animals/group. (b) Analysis of tumor volume at the tumor growth endpoint. Data show the mean ± SEM. n = 8–9 animals/group. ** p < 0.01, *** p < 0.001. (c) Analysis of tumor weight. Data show the mean ± SEM. n = 9 animals/group. * p < 0.05, ** p < 0.01. Representative images of primary tumors are shown below the graph. Scale bar = 1 cm. (d) Bioluminescence imaging (BLI) showing the change in metastatic burden with time. Representative images of animals from each group are shown. Heat map indicates increasing BLI intensity from low (blue) to high (red). (e) Quantification of BLI-derived metastatic burden at day 35 post tumor inoculation. Data show the mean ± SEM. n = 7–10 animals/group. * p < 0.05. (f) Images showing metastatic nodules harvested from animals in each group. Each image shows axillary and/or inguinal lymph node metastases (top) together with grossly resectable metastatic foci present within the abdominal cavity (bottom) of 1 representative animal/group. Scale bar = 1 cm. (g) Quantification of the weight of metastases harvested from animals described in panel f. Data show the mean ± SEM. n = 9–10 animals/group. Number of resectable metastatic foci/animal = 0–32. ** p < 0.01.

Figure 2

Sunitinib induces hypoxia and Carbonic Anhydrase IX (CAIX) expression in primary Triple Negative Breast Cancer (TNBC) tumors. (a) Images of MDA-MB-231 LM2-4Luc⁺ primary tumor tissue sections harvested at increasing tumor volumes from animals administered either vehicle or 60 mg/kg sunitinib and immunohistochemically stained for the indicated markers. Scale bars: upper panels, 1 mm; lower panels, 200 μm. (b to e) Image-based quantification of (b) CD31⁺ blood vessels, (c) CAIX expression, (d) pimonidazole and (e) proliferation. Data show the mean ± standard error of the mean (SEM). n = 3 animals/group with 10 images/animal. * p < 0.05, *** p < 0.001.

Figure 3

SLC-0111 reduces vessel density and vascular permeability in primary tumors, and decreases lung and liver metastases. (a) Representative 3D maximum projections of whole mount primary tumor tissue slices showing CD31⁺ blood vessels (green) and fluorescently labeled dextran (red). Merged images demonstrate the presence of both intravascular (yellow) and extravasated (arrowheads) dextran. Scale bar = 100 μm. (b) Quantification of the number of vessels in whole mount primary tumor slices. Data show the mean ± standard error of the mean (SEM). n = 13–14 images/group. ** p < 0.01, *** p < 0.001. (c) Quantification of vascular permeability as assessed by relative area of extravasated dextran in whole mount primary tumor slices. Data show the mean ± SEM. n = 13–16 images/group. * p < 0.05, ** p < 0.01. (d) Representative 3D maximum projections of whole mount primary tumor tissue slices showing levels of CAIX expression (green) and the number of CD31⁺ blood vessels (red). Lower panels, merge. Scale bar = 100 μm. (e) Quantification of Carbonic Anhydrase IX (CAIX) expression in whole mount primary tumor tissue slices in panel d. Data show the mean ± SEM. n = 11–15 images/group. * p < 0.05, *** p < 0.001. (f) Representative images of lung and liver tissues from animals with MDA-MB-231 LM2-4Luc⁺ orthotopic breast tumors and administered SLC-0111 and sunitinib, either alone or in combination. Visible metastatic nodules (arrows) are indicated. Scale bar = 1 cm. (g–i) Analysis of (g) lung weight (n = 9/group), (h) liver weight as a function of whole animal body weight (n = 9/group) and (i) number of metastatic nodules present on the liver surface (n = 7–8/group). For each graph, data show the mean ± SEM. * p < 0.05.

Figure 4

SLC-0111 inhibits sunitinib-induced Carbonic Anhydrase IX (CAIX) expression in liver metastases. (a) Representative 3D maximum projections of whole mount liver tissue slices from animals administered agents as indicated, showing rhodamine-labeled dextran (red) and vimentin-positive metastases (cyan). Scale bar = 100 μm. (b) Analysis of vimentin expression in the whole mount liver tissue sections described in panel a. Data show the mean ± standard error of the mean (SEM). n = 9–12 images/group: ** p < 0.01, *** p < 0.001. (c) Representative 3D maximum projections of whole mount liver tissue slices from animals administered agents as indicated, showing rhodamine-labeled dextran (red) and levels of expression of CAIX (green). Scale bar = 100 μm. (d) Analysis of CAIX expression in whole mount liver tissue sections described in panel c. Data show the mean ± SEM. n = 9–12 mages/group. *** p < 0.001.

Figure 5

Administration of SLC-0111 reduces lung metastases. (a) Representative 3D maximum projections of whole mount lung tissue slices from tumor-bearing mice, showing vimentin-positive metastases (cyan) and CD31⁺ blood vessels (red). Scale bar = 100 μm. (b) Analysis of vimentin positive metastases in whole mount lung tissue sections described in panel a. Data show the mean ± standard error of the mean (SEM). n = 8–10 images/group. * p < 0.05, *** p < 0.001. (c) Representative 3D maximum projections of whole mount lung tissue slices from tumor-bearing mice, showing Carbonic Anhydrase IX (CAIX)-positive metastases (green) and CD31⁺ blood vessels (red). Bar = 100 μm. (d) Analysis of CAIX positive metastases in the whole mount lung tissue sections described in panel c. Data show the mean ± SEM. n = 5–8 images/group: No statistically significant differences were observed among the groups.

Figure 6

Model for targeting angiogenesis and Carbonic Anhydrase IX (CAIX) to reduce metastasis of Triple Negative Breast Cancer (TNBC). Exposure of tumors to anti-angiogenic agents such as sunitinib leads to reduced tumor growth. However, major consequences can include enhanced hypoxia and increased vascular permeability. The exacerbation of hypoxia results in the upregulation of CAIX expression by tumor cells and the potentiation of metastasis. Inhibition of CAIX activity inhibits metastasis through several mechanisms, as shown by previous studies, but may also play a role in reducing vascular permeability and contributing to vascular normalization, potentially reducing the invasion of tumor cells to distant sites.