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The Vipr2-selective Antagonist Ks-133 Changes Macrophage Polarization and Exerts Potent Anti-tumor Effects as a Single Agent and in Combination With an Anti-pd-1 Antibody

Kotaro Sakamoto, Wararat Kittikulsuth, Eijiro Miyako, Akumwami Steeve, Rika Ishimura, Shinsaku Nakagawa, Yukio Ago, Akira Nishiyama

Abstract

We have previously demonstrated that KS-133 is a specific and potent antagonist of vasoactive intestinal peptide receptor 2 (VIPR2). We have also shown that vasoactive intestinal peptide–VIPR2 signaling affects the polarity and activation of tumor-associated macrophages, which is another strategy for cancer immunotherapy apart from the activation of effector T cells. In this study, we aimed to examine whether the selective blockade of VIPR2 by KS-133 changes the polarization of macrophages and induces anti-tumor effects. In the presence of KS-133, genetic markers indicative of tumor-aggressive M1-type macrophages were upregulated, and conversely, those of tumor-supportive M2-type macrophages were downregulated. Daily subcutaneous administration of KS-133 tended to suppress the growth of CT26 tumors (murine colorectal cancer-derived cells) implanted subcutaneously in Balb/c mice. To improve the pharmacological efficacy and reduce the number of doses, we examined a nanoformulation of KS-133 using the US Food and Drug Administration-approved pharmaceutical additive surfactant Cremophor® EL. KS-133 nanoparticles (NPs) were approximately 15 nm in size and stable at 4°C after preparation. Meanwhile, KS-133 was gradually released from the NPs as the temperature was increased.

Introduction

Vasoactive intestinal peptide receptor 2 (VIPR2) is a class-B G protein-coupler receptor that participates in various physiological function by interacting with its ligand vasoactive intestinal peptide (VIP). VIP–VIPR2 signaling has attracted attention as a drug target in the fields of central nervous system diseases, oncology, and immunity. In 2021, we generated 13-mer bicyclic peptide KS-133 as a potent and selective antagonist to VIPR2 [1]. KS-133 is resistant to protease degradation, suppresses the phosphorylation of CREB (a downstream signal of VIPR2) in brain tissues and exerts pharmacological effects in a mouse model of psychiatric disorders [1]. Several lines of evidence support associations of VIPR2 with cancer and immunity. For example, VIP–VIPR2 signaling regulates tumor cell migration [2], and inhibition of VIPR signaling promotes CD8+ T cell proliferation and immune function [3–5]. In addition, VIP is released from tumor and immune cells, and it regulates immune reactions such as anti-inflammatory activities through interactions with VIPR1 and VIPR2 [6].

Materials and method

The detailed method and DNA primer sequences were described previously [7]. RAW264.7 murine macrophage-like cells and CT26.WT murine colorectal cancer cells (CRL-2638) were purchased from RIKEN BRC (Tsukuba, Japan) and American Type Cell Collection (Manassas, VA, USA), respectively. CT26 cells (4 × 106) were grown in T75 flask with 20 mL of RPMI medium containing 10% FBS for 3 days. The culture medium (CT26-CM) was collected and centrifuged at 400 g for 5 min. RAW264.7 cells (2.4 × 105) were incubated in 24-well plate in the presence or absence of KS-133 (KS-133 concentration = 1, 3, or 10 μM, DMSO concentration = 0.1%) with 20% CT26-CM/DMEM for 3 days. During the incubation, medium containing KS-133 was replaced every day. mRNA extraction was performed using ISOGEN (Nippon Gene, Tokyo, Japan) on day 3 after CT26-CM incubation, and cDNA was prepared. The gene expression of M1 markers (TNF-α, iNOS, CXCL10), M2 markers (Mrc-1, IL-1rn, CCL22), and β-actin was analyzed by real-time polymerase chain reaction (PCR) using a 7300 Fast Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific, MA, USA,) and Light Cycler Fast Start DNA Master kit (Applied Biosystems).

Results

It has been reported that when RAW264.7 cells are incubated in the presence of the culture supernatant of CT26 cells, M1-type macrophage marker mRNA expression is decreased, and that of M2-type macrophages is increased [7]. Using this assay system, we evaluated whether KS-133 affect the mRNA expression of TNFα, iNOS, and CXCL10 as M1-type macrophage markers and Mrc-1, IL-1Rn, and CCL-22 as M2-type macrophage markers. As presented in Fig 1, KS-133 (10 μM) significantly enhanced the mRNA expression level of iNOS and CXCL10 (p < 0.05). Conversely, KS-133 significantly decreased the mRNA expression of Mrc-1 (p < 0.05). Therefore, VIPR2 inhibition by KS-133 changes the macrophage polarity toward M1.

Discussion

In this study, we demonstrated for the first time that KS-133, a VIPR2-selective antagonistic peptide, increases M1-type macrophage markers and decreases M2-type macrophage markers in vitro, exhibits anti-tumor efficacy in vivo, and enhances the pharmacological efficacy of an anti-PD-1 antibody. We previously reported that non-specific VIPR inhibitor, VIPhyb increased the expression of the M1-type macrophage markers TNF, iNOS, and CXCL10 and decreased that of the M2-type macrophage marker Mrc-1 but not of IL-1Rn or CCL22 [7]. Furthermore, knockdown of VIPR2 by siRNA decreased the expression of the M2-type macrophage markers Mrc-1, IL-1Rn, and CCL22 without affecting M1-type macrophage marker expression [7]. In the present study, KS-133 increased the expression of the M1-type macrophage marker iNOS / CXCL10 and decreased that of the M2-type macrophage marker Mrc-1 but had no effect on IL-1Rn or CCL22 expression. These data indicate that KS-133 changes the polarity of macrophages from M2 to M1 by inhibiting VIPR2.

Acknowledgments

We would like to thank Yui Ikemi, Tatsunori Miyaoka, and Daisuke Iwasaki for technical assistance with the experiments in pharmacokinetic study.

Citation: Sakamoto K, Kittikulsuth W, Miyako E, Steeve A, Ishimura R, Nakagawa S, et al. (2023) The VIPR2-selective antagonist KS-133 changes macrophage polarization and exerts potent anti-tumor effects as a single agent and in combination with an anti-PD-1 antibody. PLoS ONE 18(7): e0286651. https://doi.org/10.1371/journal.pone.0286651

Editor: Dominique Heymann, Universite de Nantes, FRANCE

Received: February 5, 2023; Accepted: May 17, 2023; Published: July 5, 2023

Copyright: © 2023 Sakamoto et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All data generated for this study are contained in the paper.

Funding: This work was partially supported by grants from JSPS KAKENHI to YA [20H03392, 21K19714] and Tokyo Biochemical Research Foundation in the form of grant to YA. This work was technically supported in part by the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research, BINDS) from AMED in the form of a grant [JP21am0101123 (Support No. 2917)] and Research Support Project for Life Science and Drug Discovery (BINDS) in the form of a grant from AMED [JP22ama121052 (Support No. 4300)] to YA. This research was also supported in part by AMED in the form of a grant to YA [JP22ym0126809].

Competing interests: The authors have read the journal’s policy and have the following competing interests: Kotaro Sakamoto is a full-time employee of Ichimaru Pharcos Co. Ltd. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

 

Source: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0286651#ack

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