Beta-glucan-induced inflammatory monocytes mediate antitumor efficacy in the murine lung

Abstract

β-Glucan is a naturally occurring glucose polysaccharide with immunostimulatory activity in both infection and malignancy. β-Glucan's antitumor effects have been attributed to the enhancement of complement receptor 3-dependent cellular cytotoxicity, as well as modulation of suppressive and stimulatory myeloid subsets, which in turn enhances antitumor T cell immunity. In the present study, we demonstrate antitumor efficacy of yeast-derived β-glucan particles (YGP) in a model of metastatic-like melanoma in the lung, through a mechanism that is independent of previously reported β-glucan-mediated antitumor pathways. Notably, efficacy is independent of adaptive immunity, but requires inflammatory monocytes. YGP-activated monocytes mediated direct cytotoxicity against tumor cells in vitro, and systemic YGP treatment upregulated inflammatory mediators, including TNFα, M-CSF, and CCL2, in the lungs. Collectively, these studies identify a novel role for inflammatory monocytes in β-glucan-mediated antitumor efficacy, and expand the understanding of how this immunomodulator can be used to generate beneficial immune responses against metastatic disease.

Keywords: Anti-tumor; Beta-glucan; Cytotoxicity; Immune therapy; Inflammatory monocytes.

Fig. 1

β-Glucan mediates antitumor efficacy in a model of metastatic melanoma. a Experimental scheme: C57BL/6J mice were given β-glucan (YGP) i.p. on days − 7 and − 4, and challenged with 3 × 10⁵ B16.F10 i.v. on day 0. Tumor burden was evaluated at various timepoints up to 14 days after tumor challenge; b Lung photos at day 14; c Tyrosinase qPCR of total lung RNA of lungs shown in b, plus non-tumor bearing control lungs not pictured; d Mice treated as in a. Tyrosinase qPCR of total lung RNA 2 h after tumor inoculation (as in a), plus non-tumor bearing control lungs; e Tyrosinase qPCR of total lung RNA 48 h after tumor inoculation (as in a), plus non-tumor bearing control lungs

Fig. 2

β-Glucan induces an early expansion of myeloid cells in the lung. Mice were treated as in Fig. 1a, and lungs were harvested at the indicated time point; the right lung was perfused and processed into single-cell suspensions and stained for flow cytometry. a Gating strategy to identify CD4 and CD8 T cells (live singlet CD45⁺CD11b⁺CD45⁺CD4⁺ or 8⁺), neutrophils (live singlet CD45⁺CD11b⁺Ly6C⁻Ly6G⁺), inflammatory monocytes (live singlet CD45⁺CD11b⁺Ly6C^hiLy6G⁻), and Ly6C⁻ Mo/Mφ (live singlet CD45⁺CD11b⁺Ly6C⁻Ly6G⁻); b Total right lobe numbers of the indicated populations, without tumor inoculation; c Total right lobe numbers of each population at 2 h post B16 i.v. tumor challenge; d Total right lobe numbers of each population at 14 days post-tumor challenge. Each panel includes representative data from one of several similar experiments with comparable results

Fig. 3

β-Glucan stimulates inflammatory mediator expression in the lung and circulation. Mice were treated with β-glucan as in Fig. 1a, and plasma and total lung protein were harvested at day 0 (no tumor), and 24 h post B16 challenge. Samples were analyzed using a Millipore mouse multiplex kit on the Luminex platform. Normalized protein concentrations shown for a TNFα; b CCL-2; c IL-6; d G-CSF; e M-CSF; and f IL-10. Measurements were normalized to total protein values obtained from a BCA total protein assay

Fig. 4

β-Glucan-mediated protection against metastatic melanoma does not require adaptive immunity, NK cells or granulocytes. Mice were treated as in Fig. 1a, and lungs were harvested 14 days after i.v. B16 tumor challenge; a Lung photos from RAG^−/− mice; b Tyrosinase qPCR of total lung RNA from RAG^−/− lungs shown in a; c C57BL/6J mice were treated as in Fig. 1a, with some also given αNK1.1 Ab i.p. on days − 6 and − 2; d Tyrosinase qPCR of total lung RNA from lungs shown in c; e C57BL/6J mice were treated as in Fig. 1a, with some also given αLy6G Ab on days − 8, -6, -4, and − 2; f Tyrosinase qPCR of total lung RNA from lungs shown in e

Fig. 5

Inflammatory monocytes are required for β-glucan’s efficacy against metastatic melanoma, and β-glucan enhances monocyte cytotoxicity against B16 in vitro. Mice were treated as in Fig. 1a, and lungs were harvested at day 14 post B16 i.v. a Day 14 lung photos from WT C57BL/6J or CCR2^−/− mice treated as in Fig. 1a, b Tyrosinase qPCR of total lung RNA of lungs shown in a; c WT C57BL/6J or CCR2.DTR^+/− mice were treated as in Fig. 1a, with the CCR2.DTR mice also receiving DT i.p. on days − 8, − 6, and − 4. Day 14 lung photos shown; d Tyrosinase qPCR of total lung RNA from lungs shown in c; e BM-derived monocytes were isolated and stimulated with either RPMI or β-glucan for 24 h, followed by a 3-day resting period, activated with LPS for 24 h, then plated with B16-luciferase for 24 h at a monocyte:B16 ratio of 100:1. After 24 h of co-culture luciferin was added and luminescence intensity determined. See methods for % lysis calculation