Trastuzumab improves tumor perfusion and vascular delivery of cytotoxic therapy in a murine model of HER2+ breast cancer: preliminary results

Abstract

To employ in vivo imaging and histological techniques to identify and quantify vascular changes early in the course of treatment with trastuzumab in a murine model of HER2+ breast cancer. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was used to quantitatively characterize vessel perfusion/permeability (via the parameter K (trans) ) and the extravascular extracellular volume fraction (v e ) in the BT474 mouse model of HER2+ breast cancer (N = 20) at baseline, day one, and day four following trastuzumab treatment (10 mg/kg). Additional cohorts of mice were used to quantify proliferation (Ki67), microvessel density (CD31), pericyte coverage (α-SMA) by immunohistochemistry (N = 44), and to quantify human VEGF-A expression (N = 29) throughout the course of therapy. Longitudinal assessment of combination doxorubicin ± trastuzumab (N = 42) tested the hypothesis that prior treatment with trastuzumab will increase the efficacy of subsequent doxorubicin therapy. Compared to control tumors, trastuzumab-treated tumors exhibited a significant increase in K (trans) (P = 0.035) on day four, indicating increased perfusion and/or vessel permeability and a simultaneous significant increase in v e (P = 0.01), indicating increased cell death. Immunohistochemical and ELISA analyses revealed that by day four the trastuzumab-treated tumors had a significant increase in vessel maturation index (i.e., the ratio of α-SMA to CD31 staining) compared to controls (P < 0.001) and a significant decrease in VEGF-A (P = 0.03). Additionally, trastuzumab dosing prior to doxorubicin improved the overall effectiveness of the therapies (P < 0.001). This study identifies and validates improved perfusion characteristics following trastuzumab therapy, resulting in an improvement in trastuzumab-doxorubicin combination therapy in a murine model of HER2+ breast cancer. This data suggests properties of vessel maturation. In particular, the use of DCE-MRI, a clinically available imaging method, following treatment with trastuzumab may provide an opportunity to optimize the scheduling and improve delivery of subsequent cytotoxic therapy.

Keywords: Adriamycin; Angiogenesis; BT474; Herceptin; Immunohistochemistry; MRI; Normalization.

Fig. 1

The temporal relationship between treatments, imaging, and histological evaluation for trastuzumab are shown for experiments one and two. Experiment one, a DCE-MRI study evaluated longitudinal changes in vascularity between trastuzumab-treated and control BT474 HER2+ breast cancer tumor-bearing mice until day four after initiating treatment (day zero). Experiment two utilized this same mouse model for an extended time through 10 days and evaluated cellular and vascular changes with histological analysis to confirm alterations observed in experiment one

Fig. 2

a Displays representative longitudinal parametric maps of K^trans (overlaid on T₂-weighted anatomical images at the imaging time points) in the treated (first column) or control (second column) animals from experiment one. b Treated tumors exhibited group increases in K^trans from day zero to day four (P = 0.028). Compared to control tumors, the trastuzumab-treated tumors had a significantly larger K^trans value on day four (P = 0.035). c ROC analysis on day four between treatment and control mice was 0.86 (P = 0.03)

Fig. 3

a Displays representative longitudinal, parametric maps of v_e (overlaid on T₂-weighted anatomical images at each imaging time point) in treated (first column) or control (second column) animals from experiment one. b Treated tumors exhibited group increases in v_e from day zero to day one (P = 0.04) and to day four (P = 0.002). (C) ROC analysis on day four between treatment and control mice was 0.91 (P = 0.01)

Fig. 4

Trastuzumab-treated tumors present significantly decreased tumor size compared to control tumors in experiment two (N = 4 per group) (a). On day 10, there is a significant difference in tumor volume (mm³) between the groups (P = 0.03), confirming response to trastuzumab. Representative images of Ki67 histological staining reveals qualitative differences in percentage of viable cells undergoing proliferation on day seven (b). In addition, tumors from treated animals exhibited a significantly lower percent of proliferation, as stained by Ki67, compared to control (vehicle-treated) mice, on day ten (P < 0.001) (c). Important to note, on day zero, the control and treated groups (a, c) are the same cohort of mice

Fig. 5

Representative images of α-SMA and CD31 staining are shown for treated and control tumors on day four (a). Corresponding quantitative histological analysis revealed that trastuzumab-treated tumors simultaneously show a significant increase in microvessel density (CD31 staining, P < 0.01) (b) and an increase in α-SMA (pericyte coverage, P < 0.0001) (c) on day four. d Presents the overall increase in the “vessel maturation index” (ratio of α-SMA to CD31 staining) that was seen in treated tumors compared to controls (P = 0.0009). Important to note, on day zero, the control and treated groups are the same cohort of mice (b, c, d)

Fig. 6

Quantification of VEGF-A concentration (pg/mL) by ELISA is shown for treated and control tumors over a seven-day period following treatment. On day four, the ELISA assay reveals a significant decrease in VEGF in treated tumors compared to controls (P = 0.03)

Fig. 7

Tumor growth curves and response to treatment over 61 days. Treatment was initiated on day 35 and mice were treated with either saline, doxorubicin alone, trastuzumab alone, doxorubicin 24 h prior to trastuzumab or trastuzumab 24 h prior to doxorubicin, or doxorubicin given simultaneously with both doses of trastuzumab (doxorubicin + trastuzumab). a Displays all groups, while (b–d) highlight important comparisons within those groups. b Displays the control group and single agent drug groups; trastuzumab significantly decreases tumor volume compared to both saline and doxorubicin alone (P < 0.0001). c Reveals that the order of dosing (while keeping the same total amount of drug given constant) plays a significant role in tumor response to treatment (P < 0.0001). d Shows that trastuzumab given prior to one dose of doxorubicin is not significantly different than two doses of doxorubicin administered simultaneously with trastuzumab (P = 0.84)