Supplementary Figures and Figure legends

Standard cancer therapy targets tumor cells without considering possible damage on the tumor microenvironment that could impair therapy response. In rectal cancer patients we find that inflammatory cancer-associated fibroblasts (iCAFs) are associated with poor chemoradiotherapy response. Employing a murine rectal cancer model or patient-derived tumor organoids and primary stroma cells, we show that, upon irradiation, interleukin-1α (IL-1α) not only polarizes cancer-associated fibroblasts toward the inflammatory phenotype but also triggers oxidative DNA damage, thereby predisposing iCAFs to p53-mediated therapy-induced senescence, which in turn results in chemoradiotherapy resistance and disease progression. Consistently, IL-1 inhibition, prevention of iCAFs senescence, or senolytic therapy sensitizes mice to irradiation, while lower IL-1 receptor antagonist serum levels in rectal patients correlate with poor prognosis. Collectively, we unravel a critical role for iCAFs in rectal cancer therapy resistance and identify IL-1 signaling as an attractive target for stroma-repolarization and prevention of cancer-associated fibroblasts senescence.

Fibrocytes are bone marrow–derived monocytic cells implicated in wound healing. Here, we identify their role in lung cancer progression/ metastasis. Selective manipulation of fibrocytes in mouse lung tumor models documents the central role of fibrocytes in boosting niche features and enhancing metastasis. Importantly, lung cancer patients show increased number of circulating fibrocytes and marked fibrocyte accumulation in the cancer niche. Using double and triple co-culture systems with human lung cancer cells, fibrocytes, macrophages and endothelial cells, we substantiate the central features of cancer-supporting niche: enhanced cancer cell proliferation and migration, macrophage activation, augmented endothelial cell sprouting and fibrocyte maturation. Upregulation of endothelin and its receptors are noted, and dual endothelin receptor blockade suppresses all cancer-supportive phenotypic alterations via acting on fibrocyte interaction with the cancer niche. We thus provide evidence for a crucial role of fibrocytes in lung cancer progression and metastasis, suggesting targets for treatment strategies.

Supplementary Table from Disruption of Prostaglandin E2 Signaling in Cancer-Associated Fibroblasts Limits Mammary Carcinoma Growth but Promotes Metastasis

Abstract Acute leukemias are systemic malignancies associated with a dire outcome. Because of low immunogenicity, leukemias display a remarkable ability to evade immune control and are often resistant to checkpoint blockade. Here, we discover that leukemia cells actively establish a suppressive environment to prevent immune attacks by co-opting a signaling axis that skews macrophages toward a tumor-promoting tissue repair phenotype, namely the GAS6/AXL axis. Using aggressive leukemia models, we demonstrate that ablation of the AXL receptor specifically in macrophages, or its ligand GAS6 in the environment, stimulates antileukemic immunity and elicits effective and lasting natural killer cell– and T cell–dependent immune response against naïve and treatment-resistant leukemia. Remarkably, AXL deficiency in macrophages also enables PD-1 checkpoint blockade in PD-1–refractory leukemias. Finally, we provide proof-of-concept that a clinical-grade AXL inhibitor can be used in combination with standard-of-care therapy to cure established leukemia, regardless of AXL expression in malignant cells. Significance: Alternatively primed myeloid cells predict negative outcome in leukemia. By demonstrating that leukemia cells actively evade immune control by engaging AXL receptor tyrosine kinase in macrophages and promoting their alternative priming, we identified a target which blockade, using a clinical-grade inhibitor, is vital to unleashing the therapeutic potential of myeloid-centered immunotherapy. This article is highlighted in the In This Issue feature, p. 2659

Macrophages supply iron to the breast tumor microenvironment by enforced secretion of lipocalin-2 (Lcn-2)-bound iron as well as the increased expression of the iron exporter ferroportin (FPN). We aimed at identifying the contribution of each pathway in supplying iron for the growing tumor, thereby fostering tumor progression. Analyzing the expression profiles of Lcn-2 and FPN using the spontaneous polyoma-middle-T oncogene (PyMT) breast cancer model as well as mining publicly available TCGA (The Cancer Genome Atlas) and GEO Series(GSE) datasets from the Gene Expression Omnibus database (GEO), we found no association between tumor parameters and Lcn-2 or FPN. However, stromal/macrophage-expression of Lcn-2 correlated with tumor onset, lung metastases, and recurrence, whereas FPN did not. While the total iron amount in wildtype and Lcn-2−/− PyMT tumors showed no difference, we observed that tumor-associated macrophages from Lcn-2−/− compared to wildtype tumors stored more iron. In contrast, Lcn-2−/− tumor cells accumulated less iron than their wildtype counterparts, translating into a low migratory and proliferative capacity of Lcn-2−/− tumor cells in a 3D tumor spheroid model in vitro. Our data suggest a pivotal role of Lcn-2 in tumor iron-management, affecting tumor growth. This study underscores the role of iron for tumor progression and the need for a better understanding of iron-targeted therapy approaches.

eradicates naive and treatment resistant leukemia 2 Irene Tirado-Gonzalez*, Arnaud Descot*, Devona Soetopo*, Aleksandra 3 Nevmerzhitskaya, Alexander Schäffer, Ivan-Maximilano Kur, Ewelina Czlonka, Carolin 4 Wachtel, Ioanna Tsoukala, Luise Müller, Anna-Lena Schäfer, Maresa Weitmann, Petra 5 Dinse, Emily Alberto, Michèle C. Buck, Jonathan JM. Landry, Bianka Baying, Julia 6 Slotta-Huspenina, Jenny Roesler, Patrick N. Harter, Anne-Sophie Kubasch, Jörn 7 Meinel, Eiman Elwakeel, Elisabeth Strack, Christine Tran Quang, Omar Abdel8 Wahab, Marc Schmitz, Andreas Weigert, Tobias Schmid, Uwe Platzbecker, 9 Vladimir Benes, Jacques Ghysdael, Halvard Bonig, Katharina S. Götze, Carla V. 10 Rothlin, Sourav Ghosh, Hind Medyouf 11 12

Activationof the tumor-associated stroma to support tumor growth is a common feature observed in different cancer entities. This principle is exemplified by cancer-associated fibroblasts (CAFs), which are educated by the tumor to shape its development across all stages. CAFs can alter the extracellular matrix (ECM) and secrete a variety of different molecules. In that manner they have the capability to affect activation, survival, proliferation, and migration of other stromal cells and cancer cell themselves. Alteration of the ECM, desmoplasia, is a common feature of breast cancer, indicating a prominent role for CAFs in shaping tumor development in the mammary gland. In this review, we summarize the multiple roles CAFs play in mammary carcinoma. We discuss experimental and clinical strategies to interfere with CAFs function in breast cancer. Moreover, we highlight the issues arising from CAFs heterogeneity and the need for further research to identify CAFs subpopulation(s) that can be targeted to improve breast cancer therapy.

Arachidonic acid epoxides generated by cytochrome P450 (CYP) enzymes have been linked to increased tumor growth and metastasis, largely on the basis of overexpression studies and the application of exogenous epoxides. Here we studied tumor growth and metastasis in Cyp2c44 −/− mice crossed onto the polyoma middle T oncogene (PyMT) background. The resulting PyMT 2c44 mice developed more primary tumors earlier than PyMT mice, with increased lymph and lung metastasis. Primary tumors from Cyp2c44-deficient mice contained higher numbers of tumor-associated macrophages, as well as more lymphatic endothelial cells than tumors from PyMT mice. While epoxide and diol levels were comparable in tumors from both genotypes, prostaglandin (PG) levels were higher in the PyMT Δ2c44 tumors. This could be accounted for by the finding that Cyp2c44 metabolized the PG precursor, PGH 2 to 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT), thus effectively reducing levels of effector PGs (including PGE 2 ). Next, proteomic analyses revealed an up-regulation of WD repeating domain FYVE1 (WDFY1) in tumors from PyMT Δ2c44 mice, a phenomenon that was reproduced in Cyp2c44-deficient macrophages as well as by PGE 2 . Mechanistically, WDFY1 was involved in Toll-like receptor signaling, and its down-regulation in human monocytes attenuated the LPS-induced phosphorylation of IFN regulatory factor 3 and nuclear factor-κB. Taken together, our results indicate that Cyp2c44 protects against tumor growth and metastasis by preventing the synthesis of PGE 2 . The latter eicosanoid influenced macrophages at least in part by enhancing Toll-like receptor signaling via the up-regulation of WDFY1.

Fibroblasts are the essential cellular architects of connective tissue and as such are crucial cells in contributing to organ homeostasis. While fulfilling important repair functions during tissue regeneration upon wounding, chronic fibroblast activation provokes pathological organ fibrosis and promotes neoplastic disease progression. Identifying targets that may serve to therapeutically terminate fibroblast activation is therefore desirable. Among the mediators that may be relevant in this context is the prostanoid prostaglandin E2 (PGE2) that is produced during inflammatory settings, where pathological fibrosis occurs. Here, we summarize current, in part controversial, concepts on the impact of PGE2 on fibroblast activation in fibrotic diseases including cancer, and discuss these findings in the context of the evolving concept of fibroblast heterogeneity.

A role of sphingolipids for inflammatory bowel disease and cancer is evident. However, the relative and separate contribution of sphingolipid deterioration in inflammation versus carcinogenesis for the pathophysiology of colitis-associated colon cancer (CAC) was unknown and therefore examined in this study. We performed isogenic bone marrow transplantation of inducible sphingosine-1-phosphate (S1P) lyase knockout mice to specifically modulate sphingolipids and associated genes and proteins in a compartment-specific way in a DSS/AOM mediated CAC model. 3D organoid cultures were used in vitro. S1P lyase (SGPL1) knockout in either immune cells or tissue, caused local sphingolipid accumulation leading to a dichotomic development of CAC: Immune cell SGPL1 knockout (I-SGPL −/− ) augmented massive immune cell infiltration initiating colitis with lesions and calprotectin increase. Pathological crypt remodeling plus extracellular S1P-signaling caused delayed tumor formation characterized by S1P receptor 1, STAT3 mRNA increase, as well as programmed cell death ligand 1 expression, accompanied by a putatively counter regulatory STAT1 S727 phosphorylation. In contrast, tissue SGPL1 knockout (T-SGPL −/− ) provoked immediate occurrence of epithelial-driven tumors with upregulated sphingosine kinase 1, S1P receptor 2 and epidermal growth factor receptor. Here, progressing carcinogenesis was accompanied by an IL-12 to IL-23 shift with a consecutive development of a T h 2/GATA3-driven, tumor-favoring microenvironment. Moreover, the knockout models showed distinct lymphopenia and neutrophilia, different from the full SGPL1 knockout. This study shows that depending on the initiating cellular S1P source, the pathophysiology of inflammation-induced cancer versus cancer-induced inflammation develops through separate, discernible molecular steps.

Interleukin-38 (IL-38) is a cytokine of the IL-1 family with a role in chronic inflammation. However, its main cellular targets and receptors remain obscure. IL-38 is highly expressed in the skin and downregulated in psoriasis patients. We report an investigation in cellular targets of IL-38 during the progression of imiquimod-induced psoriasis. In this model, IL-38 knockout (IL-38 KO) mice show delayed disease resolution with exacerbated IL-17-mediated inflammation, which is reversed by the administration of mature IL-38 or γδ T cell-receptor-blocking antibodies. Mechanistically, X-linked IL-1 receptor accessory protein-like 1 (IL1RAPL1) is upregulated upon γδ T cell activation to feedforward-amplify IL-17 production and is required for IL-38 to suppress γδ T cell IL-17 production. Accordingly, psoriatic IL1RAPL1 KO mice show reduced inflammation and IL-17 production by γδ T cells. Our findings indicate a role for IL-38 in the regulation of γδ T cell activation through IL1RAPL1, with consequences for auto-inflammatory disease.

: Cancer-associated fibroblasts (CAFs) in the tumor microenvironment contribute to all stages of tumorigenesis and are usually considered to be tumor-promoting cells. CAFs show a remarkable degree of heterogeneity, which is attributed to developmental origin or to local environmental niches, resulting in distinct CAF subsets within individual tumors. While CAF heterogeneity is frequently investigated in late-stage tumors, data on longitudinal CAF development in tumors are lacking. To this end, we used the transgenic polyoma middle T oncogene-induced mouse mammary carcinoma model and performed whole transcriptome analysis in FACS-sorted fibroblasts from early- and late-stage tumors. We observed a shift in fibroblast populations over time towards a subset previously shown to negatively correlate with patient survival, which was confirmed by multispectral immunofluorescence analysis. Moreover, we identified a transcriptomic signature distinguishing CAFs from early- and late-stage tumors. Importantly, the signature of early-stage CAFs correlated well with tumor stage and survival in human mammary carcinoma patients. A random forest analysis suggested predictive value of the complete set of differentially expressed genes between early- and late-stage CAFs on bulk tumor patient samples, supporting the clinical relevance of our findings. In conclusion, our data show transcriptome alterations in CAFs during tumorigenesis in the mammary gland, which suggest that CAFs are educated by the tumor over time to promote tumor development. Moreover, we show that murine CAF gene signatures can harbor predictive value for human cancer.

Interleukin-38 (IL-38) is a cytokine of the IL-1 family with a role in chronic inflammation. However, its main cellular targets and receptor(s) remain obscure. IL-38 is highly expressed in the skin and downregulated in psoriasis patients. Therefore, we investigated cellular targets of IL-38 during the progression of imiquimod-induced psoriasis. In this model, IL-38-knockout (IL-38 KO) mice showed delayed disease resolution due to exacerbated IL-17-mediated inflammation, which was reversed by administration of mature IL-38. Mechanistically, IL-38 antagonized X-linked interleukin-1 receptor accessory protein-like 1 (IL1RAPL1), which was upregulated upon γδ T cell activation to feed-forward amplify IL-17 production. Heightened activation of tumor-infiltrating γδ T cells was also observed in IL-38 KO oncogene-driven mammary carcinoma, accompanied by attenuated tumor growth. Our findings, which are supported by clinical data, highlight the connection between auto-inflammation and anti-tumor immunity and indicate IL-38 as a potential therapeutic target under these conditions.

The dichotomy of inflammation-induced cancer vs. cancer-induced inflammation is still enigmatic. Since sphingolipids are involved in both tumour cell survival and immune cell differentiation, migration and cytokine pattern we hypothesised their essential role in both mechanism of cancer development. We employed inducible S1P lyase (SGPL1) knockdown bone marrow-chimeric mice in a colitis associated colon cancer model leading to compartment-specific accumulation of sphingolipid levels. We found that, independent of the compartment of SGPL1 deficiency, haematopoietic or extra-haematopoietic, DSS/AOM-treated mice demonstrated tumours with s1pr1, stat3, and IL-6 elevation. However, compartment-specific actions of the SGPL1-S1P axis contributed both morphologically and genetically to a very distinct development of inflammation and carcinogenesis. Haematopoietic SGPL1 knockdown forced severe inflammatory colitis with delayed carcinogenesis characterised by pdcd4-down-regulation. In contrast, extra-haematopoietic SGPL1 knockdown triggered a much more rapid occurrence of epithelial-driven sphingosine kinase 1-positive tumours, exhibiting kras, egfr and IL23-p19 signatures and a weak but distinct Th2 driven immune microenvironment. Moreover, we found that SGPL1 derived from haematopoietic cells is sufficient for S1P-driven lymphocyte sequestration. In this study we demonstrate that compartment-specific sphingolipid modulation clarifies the dichotomy of inflammation-induced cancer vs. cancer-induced inflammation.

Metastasis is the primary cause of cancer death. The inflammatory tumor microenvironment contributes to metastasis, for instance, by recruiting blood and lymph vessels. Among tumor-infiltrating immune cells, tumor-associated macrophages (TAMs) take a center stage in promoting both tumor angiogenesis and metastatic spread. We found that genetic deletion of the S1P receptor 1 (S1pr1) alone in CD11bhi CD206+ TAMs infiltrating mouse breast tumors prevents pulmonary metastasis and tumor lymphangiogenesis. Reduced lymphangiogenesis was also observed in the nonrelated methylcholanthrene-induced fibrosarcoma model. Transcriptome analysis of isolated TAMs from both entities revealed reduced expression of the inflammasome component Nlrp3 in S1PR1-deficient TAMs. Macrophage-dependent lymphangiogenesis in vitro was triggered upon inflammasome activation and required both S1PR1 signaling and IL-1β production. Finally, NLRP3 expression in tumor-infiltrating macrophages correlated with survival, lymph node invasion, and metastasis of mammary carcinoma patients. Conceptually, our study indicates an unappreciated role of the NLRP3 inflammasome in promoting metastasis via the lymphatics downstream of S1PR1 signaling in macrophages.

Background Virus-induced dendritic cells (DCs) functional deficiency leads to sub-optimal initiation of adaptive immune responses and consequently chronic infection establishment. The present study reports an advanced hepatitis C virus (HCV) therapeutic vaccine model based on In vivo enrichment of DCs with barberry ethanolic crude extract (BCE) then pulsing them with HCV core protein. Methods DCs were enriched by BCE intravenous injection in BALB/c mice. Vaccine efficiency was assessed by flow cytometric analysis of splenocytes of immunized mice, cytokine profiling, cytotoxic T lymphocyte assay, and humoral immune response assessment. Results There was no significant difference in surface phenotypic characterization of splenocytes from mice immunized with non-BCE-enriched-core-pulsed DCs (iDcs-core) compared to those from mice injected with RPMI-1640 medium. However, splenocytes from mice immunized with BCE-enriched-core-pulsed DCs showed 197 % increase in CD16+ population, 33 % increase in MHCII + population, and 43 % decrease in CD3 + population. In iDCs-core group, 57.9 % greater anti-core cytotoxic T lymphocyte activity, up-regulation in interferon gamma and interleukin (IL) -12 expression, and down-regulation in IL-4 and IL-10 were recorded. Moreover, sustained specific anti-core antibodies were detected only in sera of the same group. Conclusions results indicate that BCE-enriched-core-transduced DCs may serve as a new model for immunotherapy of HCV chronic infection.

Berberine is a plant alkaloid that has several pharmacological effects such as antioxidant, antilipidemic, and anti-inflammatory effects. Nonalcoholic steatohepatitis (NASH) triggers different aspects of disorders such as impaired endogenous lipid metabolism, hypercholesterolemia, oxidative stress, and neurotoxicity. In this study, we examined the mechanism by which NASH induces neurotoxicity and the protective effect of berberine against both NASH and its associated neurotoxicity. NASH induced rats showed significant impairments in lipid metabolism with increased serum triglycerides, cholesterol, and low-density lipoprotein (LDL). The NASH induced group also demonstrated a significant oxidative stress which is characterized by increased TBARs level and decreased antioxidant capacity such as GSH and SOD levels. Moreover, the NASH induction was associated with inflammation which was demonstrated by increased TNFα and nitric oxide levels. Hyperglycemia and hyperinsulinemia were observed in the NASH induced group. Also, our results showed a significant increase in the expression of the acetylcholine esterase (AChE) and amyloid beta precursor protein (AβPP). These changes were significantly correlated with decreased insulin degrading enzyme (IDE) and beta-amyloid40 (Aβ40) and increased beta-amyloid42 (Aβ42) in the hippocampal region. Daily administration of berberine (50 mg/kg) for three weeks ameliorated oxidative stress, inflammation, hyperlipidemia, hyperglycemia, hyperinsulinemia, and the observed neurotoxicity.

Background and PurposeThe psychoactive cannabinoid Δ9‐tetrahydrocannabinol (THC) and the non‐psychoactive cannabinoid cannabidiol (CBD) can both reduce cancer progression, each through distinct anti‐tumour pathways. Our goal was to discover a compound that could efficiently target both cannabinoid anti‐tumour pathways.Experimental ApproachTo measure breast cancer cell proliferation/viability and invasion, MTT and Boyden chamber assays were used. Modulation of reactive oxygen species (ROS) and apoptosis was measured using dichlorodihydrofluorescein and annexin/propidium iodide, respectively, in combination with cell flow cytometry. Changes in protein levels were evaluated using Western analysis. Orthotopic and i.v. mouse models of breast cancer metastasis were used to test the activity of cannabinoids in vivo.Key ResultsCBD reduced breast cancer metastasis in advanced stages of the disease as the direct result of down‐regulating the transcriptional regulator Id1. However, this was associated with moderate increases in survival. We therefore screened for analogues that could co‐target cannabinoid anti‐tumour pathways (CBD‐ and THC‐associated) and discovered the compound O‐1663. This analogue inhibited Id1, produced a marked stimulation of ROS, up‐regulated autophagy and induced apoptosis. Of all the compounds tested, it was the most potent at inhibiting breast cancer cell proliferation and invasion in culture and metastasis in vivo.Conclusions and ImplicationsO‐1663 prolonged survival in advanced stages of breast cancer metastasis. Developing compounds that can simultaneously target multiple cannabinoid anti‐tumour pathways efficiently may provide a novel approach for the treatment of patients with metastatic breast cancer.