Comparative Study of the Immune Microenvironment in Heterotopic Tumor Models.

The tumor microenvironment (TME) is pivotal in cancer progression and the response to immunotherapy. A "hot" tumor typically contains immune cells that promote anti-tumor immunity, predicting positive prognosis. "Cold" tumors lack immune cells, suggesting a poor outlook across various cancers. Recent research has focused on converting "cold" tumors into "hot" tumors to enhance the success of immunotherapy. A prerequisite for the studies of the TME is an accurate knowledge of the cell populations of the TME. This study aimed to describe the immune TME of lung and colorectal cancer and melanoma, focusing on lymphoid and myeloid cell populations. We induced heterotopic immunocompetent tumors in C57BL/6 mice, using KP and LLC (Lewis lung carcinoma) cells for lung cancer, MC38 cells for colorectal cancer, and B16-F10 cells for melanoma. Immune cell infiltration was analyzed using multicolor flow cytometry in single-cell suspensions after tumor excision. KP cell tumors showed an abundance of neutrophils and eosinophils; however, they contained much less adaptive immune cells, while LLC cell tumors predominated in monocytes, neutrophils, and monocyte-derived dendritic cells. Monocytes and neutrophils, along with a significant T cell infiltration, were prevalent in MC38 tumors. Lastly, B16-F10 tumors were enriched in macrophages, while showing only moderate T cell presence. In conclusion, our data provide a detailed overview of the immune TME of various heterotopic tumors, highlighting the variabilities in the immune cell profiles of different tumor entities. Our data may be a helpful basis when investigating new immunotherapies, and thus, this report serves as a helpful tool for preclinical immunotherapy research design.

Cannabinoids and the endocannabinoid system in immunotherapy: helpful or harmful?

Numerous studies in various cancer models have demonstrated that ingredients of cannabis can influence tumor growth through the endocannabinoid system (ECS), a network of molecules (mediators, receptors, transporters, enzymes) that maintains homeostasis and protection in many tissues. The main constituents of the ECS are the classical cannabinoid (CB) receptors, such as CB1 and CB2, their endogenous ligands (endocannabinoids), and the endocannabinoids' synthesizing and degrading enzymes. The role of the ECS in cancer is still unclear and its effects often depend on the tumor entity and the expression levels of CB receptors. Many studies have highlighted the tumor cell-killing potential of CB1 agonists. However, cannabis is also known as an immunosuppressant and some data suggest that the use of cannabis during immunotherapy worsens treatment outcomes in cancer patients. CB receptors are widely present in immune cells, and together with monoacylglycerol lipase, the 2-arachidonoylglycerol degrading enzyme, they could be critically involved in the regulation of the immune cell profile of the tumor microenvironment (TME), and hence in tumor progression. So far, data on the impact of the ECS in the immune-TME are still vague. In this review, we discuss the current understanding of the ECS on immunoregulation during tumor growth, and how it might affect the outcome of cancer immunotherapy.

Tumor microenvironment-derived monoacylglycerol lipase provokes tumor-specific immune responses and lipid profiles.

We recently described that monoacylglycerol lipase (MGL) is present in the tumor microenvironment (TME), increasing tumor growth. In this study we compare the implications of MGL deficiency in the TME in different tumor types. We show that subcutaneous injection of KP (KrasLSL-G12D/p53fl/fl, mouse lung adenocarcinoma) or B16-F10 cells (mouse melanoma) induced tumor growth in MGL wild type (WT) and knockout (KO) mice. MGL deficiency in the TME attenuated the growth of KP cell tumors whereas tumors from B16-F10 cells increased in size. Opposite immune cell profiles were detected between the two tumor types in MGL KO mice. In line with their anti-tumorigenic function, the number of CD8+ effector T cells and eosinophils increased in KP cell tumors of MGL KO vs. WT mice whereas their presence was reduced in B16-F10 cell tumors of MGL KO mice. Differences were seen in lipid profiles between the investigated tumor types. 2-arachidonoylglycerol (2-AG) content significantly increased in KP, but not B16-F10 cell tumors of MGL KO vs. WT mice while other endocannabinoid-related lipids remained unchanged. However, profiles of phospho- and lysophospholipids, sphingomyelins and fatty acids in KP cell tumors were clearly distinct to those measured in B16-F10 cell tumors. Our data indicate that TME-localized MGL impacts tumor growth, as well as levels of 2-AG and other lipids in a tumor specific manner.

Cannabinoid receptor 2 plays a pro-tumorigenic role in non-small cell lung cancer by limiting anti-tumor activity of CD8+ T and NK cells.

Cannabinoid (CB) receptors (CB1 and CB2) are expressed on cancer cells and their expression influences carcinogenesis in various tumor entities. Cells of the tumor microenvironment (TME) also express CB receptors, however, their role in tumor development is still unclear. We, therefore, investigated the role of TME-derived CB1 and CB2 receptors in a model of non-small cell lung cancer (NSCLC). Leukocytes in the TME of mouse and human NSCLC express CB receptors, with CB2 showing higher expression than CB1. In the tumor model, using CB1- (CB1 -/-) and CB2-knockout (CB2 -/-) mice, only deficiency of CB2, but not of CB1, resulted in reduction of tumor burden vs. wild type (WT) littermates. This was accompanied by increased accumulation and tumoricidal activity of CD8+ T and natural killer cells, as well as increased expression of programmed death-1 (PD-1) and its ligand on lymphoid and myeloid cells, respectively. CB2 -/- mice responded significantly better to anti-PD-1 therapy than WT mice. The treatment further increased infiltration of cytotoxic lymphocytes into the TME of CB2 -/- mice. Our findings demonstrate that TME-derived CB2 dictates the immune cell recruitment into tumors and the responsiveness to anti-PD-1 therapy in a model of NSCLC. CB2 could serve as an adjuvant target for immunotherapy.

Monoacylglycerol lipase deficiency in the tumor microenvironment slows tumor growth in non-small cell lung cancer.

Monoacylglycerol lipase (MGL) expressed in cancer cells influences cancer pathogenesis but the role of MGL in the tumor microenvironment (TME) is less known. Using a syngeneic tumor model with KP cells (KrasLSL-G12D/p53fl/fl; from mouse lung adenocarcinoma), we investigated whether TME-expressed MGL plays a role in tumor growth of non-small cell lung cancer (NSCLC). In sections of human and experimental NSCLC, MGL was found in tumor cells and various cells of the TME including macrophages and stromal cells. Mice treated with the MGL inhibitor JZL184 as well as MGL knock-out (KO) mice exhibited a lower tumor burden than the controls. The reduction in tumor growth was accompanied by an increased number of CD8+ T cells and eosinophils. Naïve CD8+ T cells showed a shift toward more effector cells in MGL KOs and an increased expression of granzyme-B and interferon-γ, indicative of enhanced tumoricidal activity. 2-arachidonoyl glycerol (2-AG) was increased in tumors of MGL KO mice, and dose-dependently induced differentiation and migration of CD8+ T cells as well as migration and activation of eosinophils in vitro. Our results suggest that next to cancer cell-derived MGL, TME cells expressing MGL are responsible for maintaining a pro-tumorigenic environment in tumors of NSCLC.

IL-33 reduces tumor growth in models of colorectal cancer with the help of eosinophils.

In many types of cancer, presence of eosinophils in tumors correlate with an improved disease outcome. In line with this, activated eosinophils have been shown to reduce tumor growth in colorectal cancer (CRC). Interleukin (IL)-33 has recently emerged as a cytokine that is able to inhibit the development of tumors through eosinophils and other cells of the tumor microenvironment thereby positively influencing disease progress. Here, we asked whether eosinophils are involved in the effects of IL-33 on tumor growth in CRC.In models of CT26 cell engraftment and colitis-associated CRC, tumor growth was reduced after IL-33 treatment. The growth reduction was absent in eosinophil-deficient ΔdblGATA-1 mice but was restored by adoptive transfer of ex vivo-activated eosinophils indicating that the antitumor effect of IL-33 depends on the presence of eosinophils. In vitro, IL-33 increased the expression of markers of activation and homing in eosinophils, such as CD11b and Siglec-F, and the degranulation markers CD63 and CD107a. Increased expression of Siglec-F, CD11b and CD107a was also seen in vivo in eosinophils after IL-33 treatment. Viability and cytotoxic potential of eosinophils and their migration properties toward CCL24 were enhanced indicating direct effects of IL-33 on eosinophils. IL-33 treatment led to increased levels of IL-5 and CCL24 in tumors.Our data show that the presence of eosinophils is mandatory for IL-33-induced tumor reduction in models of CRC and that the mechanisms include eosinophil recruitment, activation and degranulation. Our findings also emphasize the potential use of IL-33 as an adjuvants in CRC immunotherapy. Abbreviations: AOM: azoxymethane; bmRPMI: bone marrow RPMI; CRC: colorectal cancer; CFSE: carboxyfluorescein succinimidyl ester; DSS: dextran sulfate sodium; EPX: eosinophil peroxidase; INF-γ: interferon gamma; ILC: innate lymphoid cell; IL-33: interleukin-33; IL-5: interleukin-5; MDSC: myeloid derived suppressor cells; NK cells: natural killer cells; P/S: penicillin/streptomycin; rm: recombinant mouse; T regs: regulatory T cells; TATE: tumor associated tissue eosinophilia; TNF-α: tumor necrosis factor alpha.