Combining IL-2-based immunotherapy with commensal probiotics produces enhanced antitumor immune response and tumor clearance

Abstract

Background: Interleukin-2 (IL-2) serves as a pioneer of immunotherapeutic agent in cancer treatment. However, there is a considerable proportion of patients who cannot benefit from this therapy due to the limited clinical responses and dose-limiting toxicities. Mounting evidence indicates that commensal microbiota shapes the outcome of cancer immunotherapies. In this study, we aim to investigate the enhancing effect of Akkermansia muciniphila (AKK), a beneficial commensal microbe receiving considerable attentions, on the antitumor efficacy of IL-2 and explore the underlying molecular mechanism.

Methods: Colorectal carcinoma patient-derived tumor tissues were used to evaluate the therapeutic efficacy of combination treatment. AKK was orally delivered to B16F10 and CT26 tumor-bearing mice along with systemic IL-2 treatment. Flow cytometry was carried out to analyze the tumor immune microenvironment. The molecular mechanism of the enhanced therapeutic efficacy was explored by RNA-seq and then verified in tumor-bearing mice.

Results: Combined treatment with IL-2 and AKK showed a stronger antitumor efficacy in colorectal cancer patient-derived tumor tissues. Meanwhile, the therapeutic outcome of IL-2 was significantly potentiated by oral administration of AKK in subcutaneous melanoma and colorectal tumor-bearing mice, resulting from the strengthened antitumor immune surveillance. Mechanistically, the antitumor immune response elicited by AKK was partially mediated by Amuc, derived from the outer membrane protein of AKK, through activating toll-like receptor 2 (TLR2) signaling pathway. Besides, oral supplementation with AKK protected gut barrier function and maintained mucosal homeostasis under systemic IL-2 treatment.

Conclusion: These findings propose that IL-2 combined with AKK is a novel therapeutic strategy with prospecting application for cancer treatment in clinical practice.

Keywords: combination; drug therapy; immunotherapy; lymphocytes; tumor microenvironment; tumor-infiltrating.

Figure 1

Schematic illustration of the combined treatment of IL-2 and AKK in tumor suppression. AKK restores the therapeutic efficacy of IL-2 to trigger a stronger antitumor immune response, which is initiated from the activation of TLR2 signaling pathway via its outer membrane protein Amuc. Moreover, AKK improves the integrity of intestinal barrier and gut microbiota homeostasis in IL-2-treated mice, probably due to the crosstalk between AKK and gut commensal microbiota. AKK, Akkermansia muciniphila; CTL, cytotoxic T lymphocyte; DC, dendritic cell; IL-2, interleukin 2; TLR, toll-like receptor.

Figure 2

Effects of combination treatment of IL-2 and AKK in ex vivo tumor tissues isolated from patients with CRC. Tumor tissues were dissociated into small pieces, digested and filtrated to generate single-cell suspensions. The cell suspensions were treated with AKK and IL-2 in combination or individually. (A) Schematic illustration of combination treatment of IL-2 and AKK in CRC patient-derived ex vivo tumor tissues. (B) Tumor cells were collected and stained with FITC-conjugated Annexin-V and PI for apoptosis detection by flow cytometry. (C–E) Representative flow cytometry analysis of CD8⁺/CD4⁺ ratio in CD3⁺ T cells (C), activated DCs (D) and cytotoxic effector T cells (E) in tumor-infiltrating lymphocytes isolated from patients with CRC. (F–I) Percentage of apoptosis tumor cells among different groups (F), ratio of CD8⁺/CD4⁺ in CD3⁺ T cells (G), CD80⁺ CD86⁺ in CD11c⁺ cells (H) and IFN-γ⁺ CD8⁺ in CD3⁺ T cells (I). All data are shown as mean±SD (n=3) (**p<0.01). AKK, Akkermansia muciniphila; APC, allophycocyanin; CRC, colorectal cancer; CTL, cytotoxic T lymphocyte; DC, dendritic cell; FITC, fluorescein isothiocyanate; IFN, interferon; IL-2, interleukin-2; PBS, phosphate-buffered saline; PE-PI, phycoerythrin-propidium iodide.

Figure 3

Antitumor efficacy of combination treatment of IL-2 and AKK in CT26 and B16F10 tumor-bearing mice. (A, C) Tumor growth in CT26 (A) and B16F10 (C) tumor-bearing mice (n=6). (B, D) Kaplan-Meier survival rate of CT26 (B) and B16F10 (D) tumor-bearing mice after different treatments (n=8). (E, F) Tumor weight in CT26 tumor-bearing mice (E) and B16F10 tumor-bearing mice (F) at the end of the experiment (n=6). All data are shown as mean±SD (*p<0.05, **p<0.01). AKK, Akkermansia muciniphila; IL-2, interleukin-2.

Figure 4

Alterations of tumor immune microenvironment in CT26 tumor-bearing mice receiving combination therapy of IL-2 and AKK. (A) Representative flow cytometry analysis of CTLs in tumor-draining lymph nodes. (B) Representative flow cytometry analysis of Tregs in tumor-draining lymph nodes. (C, D) Proportions of IFN-γ⁺ CD8⁺ in CD3⁺ T cells (C) and Foxp3⁺ CD25⁺ in CD4⁺ T cells (D). (E–H) ELISA measurement of IFN-γ (E) and IL-2 (F) in the homogenates of tumor tissues. ELISA measurement of TNF-α (G) and TGF-β (H) in the serum. (I) Percentage of side population cells in tumor tissues of B16F10 tumor-bearing mice at the end of tumor growth inhibition experiments. (J, K) Relative colony size (J) and number (K) of tumor spheroids on the fifth day after the tumor cells were seeded into the soft 3D fibrin gels. The tumor cells were collected and digested from tumor tissues of CT26 tumor-bearing mice receiving different treatments. All data are shown as mean±SD (n=6) (*p<0.05, **p<0.01). AKK, Akkermansia muciniphila; APC, allophycocyanin; IFN, interferon; IL-2, interleukin-2; TGF-β, transforming growth factor-β; TNF-α, tumor necrosis factor-α.

Figure 5

Antitumor effects of Amuc and its combination with IL-2 in CT26 tumor-bearing mice. (A) Tumor growth in mice treated with the pasteurized AKK. (B) Tumor growth in mice treated with the culture supernatant of AKK. (C) Tumor growth in mice treated with IL-2 and Amuc. (D) Tumor growth in mice treated with IL-2 and AKK bound with Amuc-specific antibody. (E) Proportions of IFN-γ⁺ CD8⁺ in CD3⁺ T cells in tumor-draining lymph nodes. (F) Proportions of Foxp3⁺ CD25⁺ in CD4⁺ T cells in tumor-draining lymph nodes. (G) Representative flow cytometry analysis of the CTLs in tumor-draining lymph nodes. (H) Representative flow cytometry analysis of Tregs in tumor-draining lymph nodes. All data are shown as mean±SD (n=6) (*p<0.05, **p<0.01). AKK, Akkermansia muciniphila; APC, allophycocyanin; CTL, cytotoxic T lymphocyte; IFN, interferon; IL-2, interleukin-2.

Figure 6

Mechanism study of Amuc in inducing antitumor-specific immune responses. (A–C) The involvement of immune responses in the tumor-infiltrating lymphocytes treated with Amuc. (A) 3D-principal coordinate analysis (3D-PCoA) analysis of the gene expression profiles. (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the identified differentially expressed genes. (C) Gene ontology (GO) enrichment analysis of the identified differentially expressed genes involved in the immune functions. (D–G) The involvement of TLR2 pathway in the antitumor effects of Amuc in tumor-bearing mice, BLP acts as a TLR1/TLR2 agonist while CU-CPT22 acts as a TLR1/TLR2 antagonist. (D) Tumor growth under different treatment in CT26 tumor-bearing mice (n=6). (E) The proportions of IFN-γ⁺ CD8⁺ in CD3⁺ T cells in tumor-draining lymph nodes. (F) The proportions of Foxp3⁺ CD25⁺ in CD4⁺ T cells in tumor-draining lymph nodes. (G) The proportions of CD11c⁺ MHC-II⁺ cells in tumor-draining lymph nodes. All data are shown as mean±SD (*p<0.05, **p<0.01). BLP, bacterial lipoprotein; ECM, extracellular matrix; FDR, false discovery rate; IFN, interferon; MHC, major histocompatibility complex; NF-κB, nuclear factor-κB; PBS, phosphate-buffered saline; TLR2, toll-like receptor 2.

Figure 7

Oral administration of AKK improved gut barrier function and commensal microbiota homeostasis under IL-2 treatment in tumor-bearing mice. (A) Representative images of H&E and AB-PAS staining of the colon tissues and immunofluorescence staining of the mucus in the small intestine. (B) 3D-Principal coordinate analysis (3D- PCoA) of fecal samples (Bray-Curtis distances), followed by Adonis test (*p<0.05). (C) Observed richness (Sobs) index. (D) Shannon diversity index. All data are shown as mean±SD (n=6) (*p<0.05, **p<0.01). AB-PAS, alcian blue-periodic acid Schiff; AKK, Akkermansia muciniphila; DAPI, 4’,6-diamidino-2-phenylindole; IL-2, interleukin 2; OTU, operational taxonomic unit.