Antitumor effects of eribulin depend on modulation of the tumor microenvironment by vascular remodeling in mouse models

Ken Ito, Shusei Hamamichi, Takanori Abe, Tsuyoshi Akagi, Hiroshi Shirota, Satoshi Kawano, Makoto Asano, Osamu Asano, Akira Yokoi, Junji Matsui, Izumi O Umeda, Hirofumi Fujii, Ken Ito, Shusei Hamamichi, Takanori Abe, Tsuyoshi Akagi, Hiroshi Shirota, Satoshi Kawano, Makoto Asano, Osamu Asano, Akira Yokoi, Junji Matsui, Izumi O Umeda, Hirofumi Fujii

Abstract

We previously reported that eribulin mesylate (eribulin), a tubulin-binding drug (TBD), could remodel tumor vasculature (i.e. increase tumor vessels and perfusion) in human breast cancer xenograft models. However, the role of this vascular remodeling in antitumor effects is not fully understood. Here, we investigated the effects of eribulin-induced vascular remodeling on antitumor activities in multiple human cancer xenograft models. Microvessel densities (MVD) were evaluated by immunohistochemistry (CD31 staining), and antitumor effects were examined in 10 human cancer xenograft models. Eribulin significantly increased MVD compared to the controls in six out of 10 models with a correlation between enhanced MVD levels and antitumor effects (R2 = 0.54). Because of increased MVD, we next used radiolabeled liposomes to examine whether eribulin treatment would result in increased tumoral accumulation levels of these macromolecules and, indeed, we found that eribulin, unlike vinorelbine (another TBD) enhanced them. As eribulin increased accumulation of radiolabeled liposomes, we postulated that this treatment might enhance the antitumor effect of Doxil (a liposomal anticancer agent) and facilitate recruitment of immune cells into the tumor. As expected, eribulin enhanced antitumor activity of Doxil in a post-erlotinib treatment H1650 (PE-H1650) xenograft model. Furthermore, infiltrating CD11b-positive immune cells were significantly increased in multiple eribulin-treated xenografted tumors, and natural killer (NK) cell depletion reduced the antitumor effects of eribulin. These findings suggest a contribution of the immune cells for antitumor activities of eribulin. Taken together, our results suggest that vascular remodeling induced by eribulin acts as a microenvironment modulator and, consequently, this alteration enhanced the antitumor effects of eribulin.

Keywords: Eribulin; liposome; natural killer cell; tubulin dynamics inhibitor; vascular remodeling.

© 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Figures

Figure 1
Figure 1
A critical factor associated with vascular remodeling effect induced by eribulin in multiple human cancer xenograft models. Increase of microvessel density (MVD) was defined as a parameter for vascular remodeling effect of eribulin. Eribulin (ERI, 1.5 mg/kg) was given to mice at day 0 by tail vein injection. (a) Antitumor effects of eribulin in 10 human cancer xenograft models (n = 6 for each group). (b) MVD at 7 days after treatment of eribulin in 10 human cancer xenograft models (n = 3). (c) Representative immunohistochemical staining with CD31 (tumor vessel) and alpha‐smooth muscle actin (α‐SMA) (pericyte) in H460 and H1975 xenografted tumors. Majority of vessels were not covered by pericytes. Brown, CD31, red, α‐SMA. (d) Correlation between enhancement of vascular remodeling (MVD) and antitumor effects of eribulin. *P < 0.05.
Figure 2
Figure 2
Vascular remodeling effect of eribulin, but not vinorelbine, in human cancer cell xenograft models. Eribulin (ERI) and vinorelbine (VNR) were given by tail vein injection with Q4D2 schedule. Two days after the last administration of the anticancer agents, tumors were harvested and stained with CD31 and alpha‐smooth muscle actin (α‐SMA) antibodies. (a) Representative immunohistochemical staining with CD31 and α‐SMA in the HSAEC_4T53RD (HSAEC RD) and H1650 xenografted tumors. Brown, CD31, red, α‐SMA. (b) MVD in the xenografted tumor (n = 4). *P < 0.05.
Figure 3
Figure 3
Comparison of antitumor effects of tubulin targeted agents, eribulin and vinorelbine in the xenograft models. In vivo antitumor activities of two tubulin inhibitors (n = 6, each group). Eribulin (ERI) at 2 mg/kg and vinorelbine (VNR) at 15 mg/kg (both maximum tolerable doses [MTD]) were given to mice by tail vein injection (Q4D2 schedule). Graphs show antitumor effects of eribulin and vinorelbine in the HSAEC_4T53RD (HSAEC RD), H1650, and post‐erlotinib treatment H1650 (PE‐H1650) xenograft models. Experiments were carried out twice independently.
Figure 4
Figure 4
Eribulin treatment increased accumulation of PEGylated liposomes and enhanced antitumor effects of Doxil (Janssen Pharmaceutical K.K.) in the post‐erlotinib treatment H1650 (PE‐H1650) xenograft models. (a) Accumulation levels of liposomes in the tumor were measured by radiolabeled liposomes. Graph shows accumulation of 111In‐labeled PEGylated liposomes in the tumor. The radiolabeled liposomes were injected 3 days after giving drugs. Muscle was collected as a negative control. (b) In vivo antitumor activities of tubulin‐targeted agents and Doxil with several regimens in the PE‐H1650 xenograft model (n = 6, each group). *P < 0.05, eribulin compared with other groups. ERI, eribulin; VNR, vinorelbine.
Figure 5
Figure 5
Contribution of immune cells for antitumor activities of eribulin. Two days after the last administration of the anticancer agents, tumors were harvested and stained with CD11b antibody. (a) Representative images of infiltrating host immune cells into the non‐small‐cell lung cancer (NSCLC) xenografted tumor. Red, CD11b+ immune cells (monocyte, NK etc.); blue, Hoechst staining (nucleus). (b) Graph shows CD11b+ area in the xenografted tumor. (c) Comparison of antitumor effects of eribulin with or without NK cell depletion in a LOX melanoma xenograft model (n = 7). ERI, eribulin; NK, natural killer cells; VNR, vinorelbine. *P < 0.05.
Figure 6
Figure 6
Role of eribulin as a tumor microenvironment modulator. Merits of the vascular remodeling effect are summarized in the schema. Our cumulative experimental results demonstrate that eribulin functions as a microenvironment modulator through increased drug accumulation (e.g. small molecule by reduced interstitial fluid pressure [IFP] and large molecule by enhanced permeability and retention [EPR] effect), increased infiltrating immune cells, as well as reduced hypoxic region. Collectively, these findings illustrate the significance of eribulin‐induced vascular remodeling effect for cancer treatment in multiple mouse xenograft models. EMT, epithelial to mesenchymal transition.

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