Abstract P6-01-03: Trastuzumab-induced hypoxia changes in a HER2+ murine model of breast cancer
Authors: Sorace AG , Quarles CC , Yankeelov TE 
Published: May 2016

Abstract
Introduction: The primary goal of this study is to quantitatively map the changes in perfusion and hypoxia in response to trastuzumab treatment through imaging and immunohistochemical studies in a murine model of HER2+ breast cancer. Correlating quantitative imaging data with pathological validation of vessel architecture will identify the temporal changes in vascular function, which will enable optimizing the order and timing of multi-modal (i.e., trastuzumab, chemo- and radiation) therapy, leading to significantly improved anticancer response.
Experimental Design: Mice were implanted subcutaneously with BT474 breast cancer cells (1×107) and randomly assorted into groups: treated (10 mg/kg trastuzumab) or control (saline). After tumors reached ∼225 mm3, animals (n = 20) were imaged with dynamic contrast enhanced-MRI (7.0 T MRI) before treatment (day 0), and 24 hours after each treatment (day 1 and day 4). Pharmacokinetic parameters, Ktrans and ve, were extracted. Subgroups of animals were sacrificed for histology between days 0 through 7 (n = 36). Tumor sections were paraffin-embedded and stained with CA-IX, CD31, α-SMA, Ki67 and H&E. Slides were scanned in high resolution (20×) and quantitatively analyzed with Leica SCN400 software. Another cohort of animals (n = 32) was utilized to identify longitudinal changes in functional vascular (Hoechst 33342) and hypoxia (pimonidazole) through immunofluorescence. Agents were injected prior to sacrifice between days 0 through 7 and tumors were immediately frozen for processing.
Results: Treated tumors exhibited a significant increase in Ktrans (p = 0.03) on day 4 compared to controls, indicative of heightened vessel perfusion and/or permeability. Additionally on day 4, treated tumors exhibited a significant increase in ve (p = 0.01), the extravascular extracellular volume fraction, indicating increased cell death. Significant decreases in Ki67 proliferation staining (p = 0.02) and tumor volume at day 7 (p = 0.04) in the treated group confirmed tumor response to trastuzumab. Immunohistochemical analyses revealed treated tumors have a significant decrease in CD31 microvessel density staining (p = 0.01), with a simultaneous significant increase in α-SMA pericyte coverage staining (p = 0.05) on day 4; thus, there was an overall increase in the "vessel maturation index" (ratio of α-SMA to CD31 staining) compared to controls (p = 0.01). CA-IX staining demonstrated increased hypoxia in the control group compared to treated, showing significant increases on day 3 (p = 0.03) and day 7 (p = 0.002). Qualitative differences were noted between control and treated groups for both functional vasculature and pimonidazole hypoxia fluorescent staining.
Conclusion: Increased intratumoral vascular delivery (Ktrans) with a simultaneous increase in vessel maturation (immunohistochemistry) are exhibited on day 4 post trastuzumab treatment. Additionally, decreased hypoxia is revealed in treated tumors compared to controls. Hypoxic tumor cells show resistance to both radiation and chemotherapy, therefore temporarily improving the tumor's functional vasculature and decreasing hypoxia during trastuzumab treatment has potential to enhance the effectiveness of these combination therapies.
Citation Format: Sorace AG, Quarles CC, Yankeelov TE. Trastuzumab-induced hypoxia changes in a HER2+ murine model of breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-01-03.