Vascular priming enhances chemotherapeutic efficacy against head and neck cancer

Abstract

Purpose: The need to improve chemotherapeutic efficacy against head and neck squamous cell carcinomas (HNSCC) is well recognized. In this study, we investigated the potential of targeting the established tumor vasculature in combination with chemotherapy in head and neck cancer.

Methods: Experimental studies were carried out in multiple human HNSCC xenograft models to examine the activity of the vascular disrupting agent (VDA) 5,6-dimethylxanthenone-4-acetic acid (DMXAA) in combination with chemotherapy. Multimodality imaging (magnetic resonance imaging, bioluminescence) in conjunction with drug delivery assessment (fluorescence microscopy), histopathology and microarray analysis was performed to characterize tumor response to therapy. Long-term treatment outcome was assessed using clinically-relevant end points of efficacy.

Results: Pretreatment of tumors with VDA prior to administration of chemotherapy increased intratumoral drug delivery and treatment efficacy. Enhancement of therapeutic efficacy was dependent on the dose and duration of VDA treatment but was independent of the chemotherapeutic agent evaluated. Combination treatment resulted in increased tumor cell kill and improvement in progression-free survival and overall survival in both ectopic and orthotopic HNSCC models.

Conclusion: Our results show that preconditioning of the tumor microenvironment with an antivascular agent primes the tumor vasculature and results in enhancement of chemotherapeutic delivery and efficacy in vivo. Further investigation into the activity of antivascular agents in combination with chemotherapy against HNSCC is warranted.

Keywords: Angiogenesis; Head and neck squamous cell carcinoma; Vascular disrupting agents; Vascular targeting.

Figure 1. Impact of vascular priming on drug delivery

(A) Fluorescence microscopic images of subcutaneous FaDu tumors treated with Doxorubicin alone (Free Dox) or VDA (DMXAA 25 mg/kg, i.p.) in combination with Doxorubicin administration (VDA + Dox). (B) Bar graph shows quantification of fluorescent intensity levels from confocal microscopy images. A total of 14 fields were analyzed from 3 tumors per group (**p <0.01).

Figure 2. MRI and histopathologic assessment of HNSCC response to combination treatment

(A) Panel of images represent axial T2-weighted images of mice showing subcutaneous FaDu tumors from control, VDA alone (DMXAA 25 mg/kg, i.p.), chemotherapy alone (CPT-11 50 mg/kg, i.p.) and combination groups (n=6 tumors per group). Images were acquired 48 hours post 2^nd dose of treatment. (B) Bar graph shows tumor volumes calculated from multislice T2-weighted images of animals in all four groups (*p<0.05, ***p<0.001). (C) Panel of images represents photomicrographs of hematoxylin & eosin (H&E) and Ki67 stained sections of tumors from control and treatment groups (n = 3 per group). Whole tumor sections are also shown adjacent to the 10x magnified images. Quantitative estimates of necrosis (D) and Ki67 staining (E) showed increased necrosis and decreased in tumor proliferation following combination treatment compared to controls and either monotherapy (*p<0.05, **p<0.01***p<0.001).

Figure 2. MRI and histopathologic assessment of HNSCC response to combination treatment

(A) Panel of images represent axial T2-weighted images of mice showing subcutaneous FaDu tumors from control, VDA alone (DMXAA 25 mg/kg, i.p.), chemotherapy alone (CPT-11 50 mg/kg, i.p.) and combination groups (n=6 tumors per group). Images were acquired 48 hours post 2^nd dose of treatment. (B) Bar graph shows tumor volumes calculated from multislice T2-weighted images of animals in all four groups (*p<0.05, ***p<0.001). (C) Panel of images represents photomicrographs of hematoxylin & eosin (H&E) and Ki67 stained sections of tumors from control and treatment groups (n = 3 per group). Whole tumor sections are also shown adjacent to the 10x magnified images. Quantitative estimates of necrosis (D) and Ki67 staining (E) showed increased necrosis and decreased in tumor proliferation following combination treatment compared to controls and either monotherapy (*p<0.05, **p<0.01***p<0.001).

Figure 2. MRI and histopathologic assessment of HNSCC response to combination treatment

(A) Panel of images represent axial T2-weighted images of mice showing subcutaneous FaDu tumors from control, VDA alone (DMXAA 25 mg/kg, i.p.), chemotherapy alone (CPT-11 50 mg/kg, i.p.) and combination groups (n=6 tumors per group). Images were acquired 48 hours post 2^nd dose of treatment. (B) Bar graph shows tumor volumes calculated from multislice T2-weighted images of animals in all four groups (*p<0.05, ***p<0.001). (C) Panel of images represents photomicrographs of hematoxylin & eosin (H&E) and Ki67 stained sections of tumors from control and treatment groups (n = 3 per group). Whole tumor sections are also shown adjacent to the 10x magnified images. Quantitative estimates of necrosis (D) and Ki67 staining (E) showed increased necrosis and decreased in tumor proliferation following combination treatment compared to controls and either monotherapy (*p<0.05, **p<0.01***p<0.001).

Figure 3. In vivo efficacy of vascular disruption in combination with chemotherapy against subcutaneous FaDu tumor xenografts

Change in tumor volumes of individual animals in control (A), VDA (B; DMXAA 25 mg/kg, i.p, once a week for 3 weeks), chemotherapy (C; Docetaxel 20 mg/kg, i.p. once a week for 3 weeks) and combination groups (n=8-14 mice per group) (D) over the 60-day monitoring period. Kaplan-Meier survival analysis of animals in all 4 groups is also shown (E). Combination treatment conferred a significant survival benefit compared to control (p<0.0001), VDA alone (p<0.05) and chemotherapy alone (p<0.01) (n=8-14 mice per group).

Figure 4. Antitumor activity of combination treatment against orthotopic FaDu tumors

(A) Bioluminescent images of control and VDA-treated mice bearing orthotopic FaDu tumors at baseline, 1hr and 24hrs post VDA treatment (DMXAA 25mg/kg × 1 dose) (n=3 mice per group). (B) Reported flux values over time for control and VDA treated mice. A significant increase in flux was observed at 1 hour (p<0.001) followed by a reduction at 24 hours hours post VDA treatment (p<0.001). (C) T2-weighted images of mice bearing orthotopic FaDu tumors (arrows) acquired once a week during the eight week monitoring period. Significant tumor growth inhibition following combination treatment compared to single agent treatment and controls. Kaplan-Meier plots showing progression free survival (D) and overall survival (E) of animals from all four groups. Combination treatment resulted in a significant increase in OS of animals compared to VDA alone (p<0.001) and chemotherapy alone (p<0.05) (n=5-9 mice per group).

Figure 5. Response of patient tumor-derived HNSCC xenografts to combined VDA-chemotherapy

Hematoxylin and eosin stained sections of patient tumor-derived HNSCC xenografts 24 hours post treatment with VDA alone (25 mg/kg, i.p.) or in combination with chemotherapy (Docetaxel, 10 mg/kg, i.p.). Images shown are at 10x magnification. Extensive necrosis was seen following combination treatment compared to VDA alone or chemotherapy alone.