Augmentation of Nab-Paclitaxel Chemotherapy Response by Mechanistically Diverse Antiangiogenic Agents in Preclinical Gastric Cancer Models

Abstract

Gastric adenocarcinoma (GAC) remains the third most common cause of cancer-related deaths worldwide. Systemic chemotherapy is commonly recommended as a fundamental treatment for metastatic GAC; however, standard treatment has not been established yet. Angiogenesis plays a crucial role in the progression and metastasis of GAC. We evaluated therapeutic benefits of mechanistically diverse antiangiogenic agents in combination with nab-paclitaxel, a next-generation taxane, in preclinical models of GAC. Murine survival studies were performed in peritoneal dissemination models, whereas tumor growth studies were performed in subcutaneous GAC cell-derived or patient-derived xenografts. The mechanistic evaluation involved IHC and Immunoblot analysis in tumor samples. Nab-paclitaxel increased animal survival that was further improved by the addition of antiangiogenic agents ramucirumab (or its murine version DC101), cabozantinib and nintedanib. Nab-paclitaxel combination with nintedanib was most effective in improving animal survival, always greater than 300% over control. In cell-derived subcutaneous xenografts, nab-paclitaxel reduced tumor growth while all three antiangiogenic agents enhanced this effect, with nintedanib demonstrating the greatest inhibition. Furthermore, in GAC patient-derived xenografts the combination of nab-paclitaxel and nintedanib reduced tumor growth over single agents alone. Tumor tissue analysis revealed that ramucirumab and cabozantinib only reduced tumor vasculature, whereas nintedanib in addition significantly reduced tumor cell proliferation and increased apoptosis. Effects of nab-paclitaxel, a promising chemotherapeutic agent for GAC, can be enhanced by new-generation antiangiogenic agents, especially nintedanib. The data suggest that nab-paclitaxel combinations with multitargeted antiangiogenic agents carry promising potential for improving clinical GAC therapy. Mol Cancer Ther; 17(11); 2353-64. ©2018 AACR.

Figure 1:. Antiangiogenic agents improve animal survival benefits of nab-paclitaxel.

A. Animal survival study in the MKN-45 GAC cell-derived peritoneal dissemination model. B. Animal survival study in the KATO-III GAC cell-derived peritoneal dissemination model. Ten days after tumor cell injection in NOD/SCID mice, treatment was started with nab-paclitaxel, ramucirumab, cabozantinib and nintedanib for 2 weeks. The curve represents the animal survival time from the beginning of therapy. Statistical group differences in survival time were calculated using log-rank testing.

Figure 2:. Antiangiogenic agents enhance nab-paclitaxel antitumor effects in GAC cell-derived xenografts.

Tumor growth inhibition in MKN-45 GAC cell-derived subcutaneous xenografts (A, B, C) or in SNU-5 GAC cell-derived subcutaneous xenografts (D, E, F). Ten days after tumor cell injection when all mice had a measurable tumor, mice were treated with nab-paclitaxel, ramucirumab, cabozantinib and nintedanib for 2 weeks. A, D. Tumor size was measured twice a week using calipers and plotted. B, E. Net growth in tumor size was calculated by subtracting tumor volume on the first treatment day from that on the final day. C, F. Mean tumor weight was calculated from final day tumor weights in each group and presented as a Box plot. Data are representative of mean values ± standard deviation from 5 mice per group. Statistical analysis was performed by one-way ANOVA for multiple group comparison and Student’s t-test for the individual group comparison.

Figure 3:. Antiangiogenic agents and nab-paclitaxel: Antitumor effects in MKN-45 cell-derived xenografts and in patient-derived xenografts.

Tumor-bearing NOD/SCID mice were treated with DC101 and nab-paclitaxel in MKN-45 GAC cell-derived subcutaneous xenografts, n=5 (A, B, C, D) or with nintedanib and nab-paclitaxel in GAC patient-derived xenografts, n=3 (E, F). When all mice had a measurable tumor, they were treated for 2 weeks. A, E. Tumor size was measured twice a week using calipers and plotted. B, F. Net growth in tumor size was calculated by subtracting tumor volume on the first treatment day from that on the final day. Data are representative of mean values ± standard deviation. C. Mean tumor weight was calculated from final day tumor weights in each group and presented as a Box plot. D. The curve represents the animal survival time from the beginning of therapy. Statistical group differences in survival time were calculated using log-rank testing.

Figure 4:. Antiangiogenic agents and nab-paclitaxel: effects on tumor cell proliferation and apoptosis.

Tumor tissue sections obtained from the MKN-45 subcutaneous xenograft study after 2-week treatment with nab-paclitaxel, ramucirumab, cabozantinib, and nintedanib, were used for the IHC analysis. A. Tissue sections were immunostained with Ki67 antibody to determine tumor cell proliferation. Ki67-positive cells were counted in five different high-power fields. B. Tumor cell apoptosis was measured by staining tumor tissue section with TUNEL procedure. TUNEL-positive apoptotic cells were counted in five different high-power fields. For both immunostaining experiments, slides were photographed under a fluorescent microscope and the data are expressed as the mean ± standard deviation.

Figure 5:. Antiangiogenic agents and nab-paclitaxel: effects on microvessel density and marker proteins. A.

Tumor tissue sections obtained from the MKN-45 subcutaneous xenograft study after 2-week treatment were used for evaluating intratumoral microvessel density. Tumor sections were incubated with anti-endomucin antibody and slides were photographed under a fluorescent microscope. Endomucin positive vessels were calculated within a microscopic HPF in a blinded manner and the data are expressed as the mean ± standard deviation. B. MKN-45 GAC cell monolayers were treatment with nab-paclitaxel, ramucirumab, cabozantinib or nintedanib, cell lysates were prepared after 16 hours incubation and analyzed by immunoblotting. The intensity of bands was quantitated by densitometry and is represented in the bar graph after normalizing values with corresponding total protein expression or GAPDH expression.

Figure 6:. Antiangiogenic agents and nab-paclitaxel: In vitro GAC cell proliferation.

GAC cells (AGS, SNU-1, SNU-5, SNU-16, MKN-45, and KATO-III) were plated on 96-well plates and treated with nab-paclitaxel, ramucirumab, cabozantinib or nintedanib. WST-1 reagent (10 μl) was added to each well after 72-hour incubation followed by additional incubation for 2 hours. The absorbance at 450 nm was measured using a microplate reader. The resulting number of viable cells was calculated by measuring the absorbance of color produced in each well. Data are the mean ± SD of triplicate determinations.