Quantifying the effects of combination trastuzumab and radiation therapy in human epidermal growth factor receptor 2 positive breast cancer

Abstract

Background: Trastuzumab, a clinical antibody targeted to the human epidermal growth factor receptor 2 (HER2), has been shown to sensitize cells to radiation in vitro. Current studies lack longitudinal evaluation of cellular response and in vivo data is limited. The purpose of this study is to quantify the effects of combination trastuzumab and radiation therapy in vitro and in vivo over time to determine if there is a synergistic interaction. Methods: EGFP expressing BT474, SKBR3 and MDA-MB-231 cell lines were treated with 0.1 ng/ml of trastuzumab, 5 or 10 Gy of radiation, or combination treatment, and imaged using fluorescence live cell microscopy for one week. The Bliss independence model was used to quantify the effects of combination treatment. HER2+ tumor bearing mice (female NU/J) (N=34) were treated with saline, 10 mg/kg of trastuzumab, 5 or 10 Gy of radiation, or combination treatment. Tumor size was measured three times per week for four weeks via caliper measurements. Additional mice (N=13) were treated with 10 mg/kg of trastuzumab, 5 Gy of radiation, or combination treatment. Tumors were harvested at one week and evaluated with immunohistochemistry for inflammation (CD45), vascularity (CD31 and α-SMA), and hypoxia (pimonidazole). Results: Altering the order of therapies did not significantly affect BT474 cell proliferation in vitro (P>0.05). The interaction index calculations revealed additive effects of trastuzumab and radiation treatment in all three cell lines in vitro. In vivo results revealed significant differences in tumor response between mice treated with 5 and 10 Gy single agent radiation (P < 0.001); however, no difference was seen in the combination groups when trastuzumab was added to the radiation regimen (P=0.56), indicating a lower dose of radiation could be used without decreasing therapeutic efficacy. Histology results revealed increases in inflammation (CD45+) in mice receiving trastuzumab (P<0.05). Conclusions: Longitudinal evaluation of cell proliferation in vitro showed additive effects of combination therapy. In vivo results show a potential to achieve the same efficacy of treatment with reduced radiation when also administering trastuzumab. Further evaluation of tumor microenvironmental alterations during treatment could identify mechanisms of increased therapeutic efficacy in this regimen.

Quantifying Tumor Heterogeneity via MRI Habitats to Characterize Microenvironmental Alterations in HER2+ Breast Cancer

“…We observed significant correlations between the percent tumor volume of HV-HC habitats and histological measures of vascularity (CD31 and vascular maturation index) and macrophage infiltration (CD45+, F4/80+). These findings provide further evidence of the relationship between vasculature and macrophage infiltration observed in previous studies [ 29 , 30 , 31 ]; future work should explore habitat imaging in syngeneic mouse models with intact immune systems to further understand the relationship between habitats and immune infiltrates. Additionally, the LV-HC habitat, a subregion of increased cellular density and decreased vascular perfusion, was found to positively correlate with the percent area of pimonidazole-stained regions.…”