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PNAS 109 (45): 3119-3127

Copyright © 2012 by the National Academy of Sciences.

Combined targeting of HER2 and VEGFR2 for effective treatment of HER2-amplified breast cancer brain metastases

David P. Kodacka,1, Euiheon Chunga,b,1, Hiroshi Yamashitaa,1, Joao Incioa, Annique M. M. J. Duyvermana, Youngchul Songc, Christian T. Farrard, Yuhui Huanga, Eleanor Agera, Walid Kamouna, Shom Goela, Matija Snuderla,e, Alisha Lussieza, Lotte Hiddingha, Sidra Mahmooda, Bakhos A. Tannousf, April F. Eichlerg, Dai Fukumuraa,2, Jeffrey A. Engelmanc,2, and Rakesh K. Jaina,2

aEdwin L. Steele Laboratory for Tumor Biology, Department of Radiation Oncology, cDepartment of Medicine, dMartinos Center for Biomedical Imaging, eDepartment of Pathology, fDepartment of Neurology, and gStephen E. and Catherine Pappas Center for Neuro-Oncology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114; and bDepartment of Medical System Engineering and School of Mechatronics, Gwangju Institute of Science and Technology, Gwangju 500-712, South Korea


Figure 01
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  		Fig. 1.. Imaging of the breast cancer brain metastasis model and the effect of anti-HER2 therapies on tumor growth. (A) Imaging of an established BT474-Gluc tumor after direct injection into the brain parenchyma. The cranial window was used for intravital microscopy. (Left) Representative bioluminescence image of the tumor symbolizes its approximate size at treatment initiation. (Scale bar: 5 mm.) Intravital multiphoton microscopy images illustrate the brain metastatic lesion boundary (Center) and the abnormality of the tumor vasculature (Right). (Scale bars: 100 μm.) Tumor cells expressing CFP are green, and blood vessels (red) are contrast-enhanced by i.v. injection of tetramethylrhodamine dextran (2,000,000 molecular weight). (B) Effect of trastuzumab (red, 5 mg/kg twice a week) or lapatinib (green, 100 mg/kg daily) on the growth of BT474-Gluc breast cancer cells when growing in the mammary fat pad (Left) or brain parenchyma (Right). Tumor growth curves for each treatment group are shown. Data are expressed as the mean ± SEM. (Left) BT474-Gluc mammary fat pad tumors were allowed to reach ~75 mm3 in volume before treatment initiation; the y axis is the fold-change of tumor volume (n = 6–8 mice). (Right) BT474-Gluc brain metastatic tumors were allowed to reach a blood Gluc activity of roughly 10 RLU/s, corresponding to a volume of ~10 mm3, before treatment initiation; the y axis is the fold-change of blood Gluc activity (n = 8–13 mice). (Treatment was initiated for BT474-Gluc brain metastatic tumors at ~10 mm3 in all subsequent experiments. Data are expressed as the mean ± SEM in all subsequent figures.)

 

Figure 02
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  		Fig. 2.. Effects of anti-HER2 and anti-VEGFR2 therapies, and their combination on breast cancer growth in the brain parenchyma and mouse survival. Trastuzumab and lapatinib were dosed as previously mentioned, and DC101 was dosed at 40 mg/kg via i.p. injection twice a week. Tumor size was monitored twice a week via blood Gluc activity (Left), and animal survival was ascertained (Right). (A) Tumor growth plot (Left) and Kaplan–Meier survival plot (Right) of brain metastatic tumor-bearing mice treated with control (black), trastuzumab (red), DC101 (blue), or trastuzumab and DC101 (magenta) (n = 8–10 mice; except in the case of trastuzumab treatment, where n = 5 mice). **P < 0.01. (B) Tumor growth plot (Left) and Kaplan–Meier survival plot (Right) of brain metastatic tumor-bearing mice treated with control (black), lapatinib (green), DC101 (blue), or lapatinib and DC101 (orange) (n = 8–10 mice). ***P < 0.001. (Final tumor growth points occur when at least 3 mice are still alive.)

 

Figure 03
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  		Fig. 3.. Imaging of metastatic breast cancer growth in the brain parenchyma and response to the combination of anti-HER2 and anti-VEGFR2 therapy. (A) MRI analysis of control or trastuzumab and DC101 treatment on BT474-Gluc metastatic tumors in the brain. MRI tumor volume was measured on days 0, 5, and 14 of treatment. (Left) Representative MRI of control- and combination-treated tumors is shown. (Right) Tumor volume, calculated using MRI, is plotted for each individual mouse (# corresponds to the tumors illustrated by the MRI scans in A). (B) Bioluminescence imaging analysis of control or trastuzumab and DC101 treatment on metastatic BT474-Gluc tumors in the brain. (Left) Images of control- and combination-treated tumors 14 d after treatment initiation are shown. (Right) Average bioluminescence signal for the three control- and three combination-treated tumors is depicted. *P < 0.05.

 

Figure 04
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  		Fig. 4.. Effects of the indicated treatments on the necrotic fraction of the tumor. The percentage of necrotic area in the total tumor area for each BT474-Gluc tumor was determined 15 d after treatment initiation using an in-house MATLAB program. **P < 0.01; *P < 0.05. DC101 vs. control, trastuzumab, or lapatinib (P < 0.001). Representative H&E-stained sections used for quantification of the necrotic area are shown in Fig. S4.

 

Figure 05
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  		Fig. 5.. Effects of the indicated treatments on blood vessels. CD31-positive blood vessel area (A) and lectin-positive perfused blood vessel area (B) in the viable regions of BT474-Gluc tumors 15 d after treatment initiation. Tissues were analyzed by an in-house MATLAB program. *P < 0.05; **P < 0.01. DC101 vs. control, trastuzumab, or lapatinib (P < 0.001). (C) Representative images of CD31-positive endothelial cells in the viable tumor tissue. (Scale bar: 100 μm.) (Representative images of lectin-positive blood vessels in the viable tumor tissue are shown in Fig. S5A).

 

Figure 06
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  		Fig. 6.. Effects of dual HER2 targeting with and without anti-VEGFR2 therapy on BT474-Gluc brain metastatic tumors. (A) Tumor growth plot (Left) and Kaplan–Meier survival plot (Right) of tumor-bearing mice treated with control (black), trastuzumab (red), lapatinib (green), or trastuzumab and lapatinib (brown) (n = 8–20 mice). ***P < 0.001. (B) Tumor growth plot (Left) and Kaplan–Meier survival plot (Right) of tumor-bearing mice treated with control (black); trastuzumab and DC101 (magenta); lapatinib and DC101 (orange); trastuzumab and lapatinib (brown); and the triple combination of trastuzumab, lapatinib, and DC101 (cyan) (n = 6–9 mice). ***P < 0.001. (Final tumor growth points occur when at least three mice are still alive.)

 

Figure 01
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  		Fig. P1.. Effect of various combinations of anti-HER2 and anti-VEGFR2 treatments on the growth of established breast cancer brain metastases. Breast cancers were allowed to grow to roughly 10 mm3 in size in the brain parenchyma before the initiation of treatment, which consisted of either a dual or triple combination of anti-HER2 agents (trastuzumab and lapatinib) and an anti-VEGFR2 antibody (DC101). (A) MRI scans of control and the combination treatment of trastuzumab and DC101 at days 0, 5, and 14. Arrows indicate tumor location. (B) Bioluminescence images of control- and trastuzumab plus DC101-treated mice at day 14. ph, photons; sr, steradian. (C) Tumor growth plot (Upper, mean ± SEM) and Kaplan–Meier survival plot (Lower) of tumor-bearing mice treated with control (black); trastuzumab plus DC101 (magenta); lapatinib plus DC101 (orange); trastuzumab plus lapatinib (brown); and the triple combination of trastuzumab, lapatinib, and DC101 (cyan) (n = 6–9 mice). RLU/s, Relative Light Units per second.

 

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