Improved tumor vascular function following high-dose epidermal growth factor receptor tyrosine kinase inhibitor therapy

Abstract

Purpose: To determine if inhibitors of the human growth factor receptor (HER) family can be used to enhance tumor vascular permeability and perfusion and optimize the efficacy of cytotoxic chemotherapeutics. Poor tumor vascular function limits the delivery and efficacy of cancer chemotherapeutics and HER family tyrosine kinases mediate tumor-endothelial signaling in both of these compartments.

Materials and methods: BT474 human breast cancer tumors were established in mice and the biologic effects of the HER tyrosine kinase inhibitor (TKI) gefitinib on tumor vascular function was determined by dynamic contrast-enhanced MRI (DCE-MRI), and on tumor vascular architecture and perfusion by immunofluorescence microscopy.

Results: A brief dose of gefitinib enhances the antitumor activity of paclitaxel in vivo but not in cell culture, suggesting that its chemoenhancing activity involves the in vivo microenvironment. A brief high dose of gefitinib induces a decrease in endothelial transfer constant (Kps) and a concomitant increase in tumor fractional plasma volume (fPV). These changes are accompanied by a rapid reduction in tumor volume, likely due to decreased tumor edema, and modestly improved tumor vascular architecture and perfusion on microscopy.

Conclusion: These data suggest that HER family TKIs have the potential to optimize the tumor microenvironment for delivery of cytotoxic chemotherapeutics.

Figure 1

60 mice bearing equal sized BT474 tumors were randomized into 6 groups and treated according to 6 treatment arms as indicated. Paclitaxel was administered at the fixed standard dose of 30 mg/kg given once per week. A) control arm received vehicle oral gavage daily, B) daily gefitinib+paclitaxel arm received gefitinib at 60 mg/kg daily. This is the MTD for daily gefitinib in this combination. C) Single agent daily gefitinib arm received gefitinib at 150 mg/kg daily. This is the MTD for single agent gefitinib. D) Paclitaxel alone arm received vehicle oral gavage daily. E) pulse gefitinib+paclitaxel arm received gefitinib for two days each week at 250 mg/kg followed by paclitaxel on the third day. This is the MTD for gefitinib in this combination. F) The pulse gefitinib alone arm received gefitinib for two days each week at 250 mg/kg. This arm is a no-paclitaxel control for arm E.

Figure 2

BT474 cells were seeded in 10cm dishes and the following day treated with the indication concentrations of paclitaxel concomitantly with 5uM gefitinib or DMSO (dimethyl sulfoxide) vehicle control. After 24 hours, cells were harvested and apoptotic cells were identified and quantitated by FACS analysis of annexin V binding as described in methods. Annexin V binding identifies apoptotic cells.

Figure 3

Mice bearing BT474 tumors of approximately equal size were treated according to 3 treatment arms. A) A schematic displaying the treatments administered in the three arms. The pulse-treatment arm consisted of 6 mice receiving gefitinib for only two days at 1000 mg/kg/day. The continuous-treatment arm consisted of 6 mice receiving gefitinib daily at 150 mg/kg/day. The control arm consisted of four mice receiving vehicle by oral gavage. All mice underwent DCE-MRI studies at baseline and on day 3 and on day 8 as depicted. B) Transendothelial permeability (Kps) values were calculated for each of the three treatment arms at the three studied timepoints. Y axis reflects Kps in ml/100g/min. B) Fractional plasma volume (fPV) was calculated for each of the three treatment arms at the three studied timepoints. Y axis reflects fPV as a fraction of 1. C) Tumor volume in voxel numbers is shown for each of the three treatment arms at the three studied timepoints. Error bars; SD.

Figure 4

Eight mice bearing BT474 tumors of approximately equal size were divided into two groups and treated with 1000 mg/kg gefitinib for two days or control gavage and on the third day the tumor vasculature was studied by lectin-perfusion in vivo staining and by anti-CD31 immunostaining of tumor sections as described in methods. Lectin perfusion staining is in green; anti-CD31 staining is in red.

Figure 4

Eight mice bearing BT474 tumors of approximately equal size were divided into two groups and treated with 1000 mg/kg gefitinib for two days or control gavage and on the third day the tumor vasculature was studied by lectin-perfusion in vivo staining and by anti-CD31 immunostaining of tumor sections as described in methods. Lectin perfusion staining is in green; anti-CD31 staining is in red.