Combining losartan with radiotherapy increases tumor control and inhibits lung metastases from a HER2/neu-positive orthotopic breast cancer model

Abstract

Background: Patients with metastatic HER2/neu-positive (HER2/neu +) breast cancer (BC) often experience treatment resistance, disease recurrences and metastases. Thus, new approaches for improving the treatment of HER2/neu + BC to prevent metastatic dissemination are urgently needed. Our previous studies have shown that losartan, an angiotensin receptor blocker, increases tumor perfusion and decreases hypoxia in a number of tumor models. Hypoxia reduces the efficacy of radiation and increases metastases. We therefore hypothesized that by modifying tumor stroma and increasing oxygenation, losartan will improve the outcome of radiotherapy and inhibit disease progression in a highly metastatic HER2/neu + murine BC model.

Methods: We established a metastatic HER2/neu + murine BC line (MCa-M3C) and used it to generate mammary fat pad isografts in syngeneic female FVB/N mice. Starting on day 3 after orthotopic tumor implantation, we administered a 7-day losartan treatment (40 mg/kg BW, gavage daily); or a 7-day losartan treatment followed by 20 Gy single dose local irradiation (S-IR) on day 10 (tumor size ~ 100 mm3), or 20 Gy local fractionated (5 × 4 Gy daily) irradiation (F-IR) on days 10-14. We analyzed tumor-growth delay (TGD), development of spontaneous lung metastases, animal survival, tumor vascular density, and tumor hypoxia.

Results: Treatments with S-IR, F-IR, Losartan + S-IR, or Losartan + F-IR resulted in a significantly increased TGD (8-16 days) in MCa-M3C tumors versus controls. However, the combination of Losartan + S-IR and Losartan + F-IR further enhanced tumor response to radiation alone by increasing TGD an additional 5 to 8 days for both single and fractionated dose irradiation (P < 0.01), decreasing lung metastasis (Losartan + IR vs. Control, P < 0.025), and increasing animal survival (Losartan + IR vs. Control, P = 0.0303). In addition, losartan treatment significantly increased tumor vascularity (P = 0.0314) and decreased pimonidazole positive (hypoxic) area (P = 0.0002).

Conclusions: Combining losartan with local irradiation significantly enhanced tumor response, at least in part via reduced tumor hypoxia presumably due to increased tumor perfusion. Our findings suggest that combining losartan with radiotherapy is a potential new treatment strategy for local control and inhibiting metastasis in HER2 + BC.

Keywords: Angiotensin receptor blocker; Losartan; Metastatic breast cancer; Mouse model; Radiotherapy.

Conflict of interest statement

RKJ received honorarium from Amgen and consultant fees from Chugai, Elpis, Ophthotech, Merck, SPARC, and SynDevRx; owns equity in Accurius, Enlight, SPARC, and SynDevRx, and serves on the Boards of Trustees of Tekla Healthcare Investors, Tekla Life Sciences Investors, Tekla Healthcare Opportunities Fund and Tekla World Healthcare Fund. RKJ and DF received a research grant from Boehringer Ingelheim. DF received consultant fees from Merck. DGD received consultant fees from Bayer, Simcere, Surface Oncology and BMS and research grants from Bayer, Exelixis and BMS. No funding or reagents from these companies were used in this study.

Figures

Fig. 1

MCa-M3C cell response to treatment, morphological features, and HER2/neu expression. Colony formation assay for a MCa-M3C cell response to irradiation, and b MCa-M3C cell response to losartan. c The absence of an effect of losartan alone on MCa-M3C cell proliferation at doses up to 20 µmol/L. d Morphological features of MCa-M3C cells in vitro. The large number of polygonal and a few spindle-shaped cells are seen under the phase contrast microscopy. Bar = 100 µm. e HER2/neu expression in MCa-M3C cells in vitro by Western blot

Fig. 2

MCa-M3C tumor histological features and lung metastases. a Spontaneous lung metastases from MCa-M3C tumor implanted in FVB/N mouse MFP. b Photomicrograph of the section of MCa-M3C MFP tumor isograft showing adenocarcinoma histological features, with a glandular-epithelial growth patterns (*); abundant tumor stroma (S); and tumor cell mitotic figures (arrows). H&E stain; Bar = 100 µm. c Photomicrograph of a section of MCa-M3C metastasis in lung (M), showing tumor cells and the growth pattern similar to that in the primary MFP tumor. H&E stain; Bar = 100 µm. d  Masson’s Trichrome staining of MCa-M3C MFP tumor isograft reveals a high collagen density (S, positive light blue stain) Bar = 100 µm

Fig. 3

MCa-M3C orthotopic tumor response to combined losartan and single-dose or fractionated dose radiotherapy. a MCa-M3C orthotopic tumor growth and response curves of control, losartan alone, 20 Gy local single-dose irradiation alone (S-IR), 20 Gy five fractionated dose irradiation alone (F-IR), combined losartan with single-dose irradiation (Los + S-IR), and losartan with fractionated dose irradiation (Los-F-IR). Los indicates the start losartan treatment; RT indicates single-dose irradiation; and the FRT + arrows indicate the schedules of 5 fraction irradiation treatment. b Mean tumor growth time (days) for tumors to reach a mean volume of 500 mm3. Results show that S-IR, F-IR, Los + S-IR, or Los + F-IR treatment significantly delays tumors growth for 8 to 16 days compared to control, or losartan alone treatment (Unpaired t test, all P < 0.0001; 95% confidence interval between 5.292 to 10.04 and 10.35 to 16.05). In addition, the combination of Los + S-IR, or Los-F-IR treatment significantly enhanced tumor response compared to radiation alone (S-IR or F-IR) by increasing TGD an additional 5 days (Los + S-IR vs S-IR, or Los + F-IR vs F-IR; Unpaired t test, both P < 0.025; 95% confidence interval between 1.405 to 8.150 and 1.457 to 8.343). Losartan alone did not show an anti-tumor effect (Losartan vs Control, P > 0.05). Data are presented as Mean ± SEM; P < 0.05 is considered statistically significant (N = 9–10/group)

Fig. 4

Combining losartan with radiotherapy increased MCa-M3C tumor host survival; and losartan treatment increased tumor vessel fraction. a Kaplan–Meier survival curve shows that combining losartan with 20 Gy IR (Los + IR) significantly increased host survival (Los + IR vs. Control P = 0.0303). b Tumor vessels imaging measurement of MCa-M3C tumors growth in MFP windows by MPLSM at day 0 and day 3 post-treatment. Result showed losartan treatment significantly increased tumor vessel fraction (Area of blood vessels/field area, Unpaired t test, P = 0.0314)

Fig. 5

Representative immunohistochemistry of CD31 staining and pimonidazole immunofluorescence (IF) in treated and control MCa-M3C tumor tissues. a CD31 and pimonidazole IF staining in the control tumor sections, b CD31 and pimonidazole IF staining in losartan-treated tumors. CD31 positive vessels are shown in red and pimonidazole positive hypoxic tissue staining in green. Bar = 50 µm

Fig. 6

Losartan treatment increased tumor vascular density and significantly decreased tumor hypoxia in MCa-M3C orthotopic tumors. a Increases in CD31 positive vascular area after losartan treatment, which is not statistically significant (Losartan vs. Control, P = 0.2147). b Losartan treatment significantly deceases hypoxia measured as pimonidazole positive area (Losartan vs. Control, Unpaired t test, P = 0.0002) in MCa-M3C tumor tissues (N = 5–10 areas/group)