Immunosuppression and metastasis are critical hallmarks of breast cancer, often linked to poor patient outcomes. The secreted cytokine chitinase-3-like 1 (CHI3L1) is frequently overexpressed in breast cancer samples and promotes an immunosuppressed tumor microenvironment. Notably, CHI3L1 expression is elevated in metastatic patient samples when compared with the matched primary breast tumor. To investigate its role in breast cancer metastasis, we generated an inducible genetically engineered mouse model that overexpresses CHI3L1 in the mammary epithelium. Ectopic expression of CHI3L1 in the polyomavirus middle T (PyMT) mouse model of breast cancer suppressed antitumor immune responses, accelerated mammary tumor onset, and enhanced lung metastasis. Mechanistically, elevated CHI3L1 expression in the mammary epithelium enhanced neutrophil recruitment, which subsequently degraded the extracellular matrix and increased the number of circulating tumor cells. These findings reveal a key mechanism driving metastatic dissemination and argue that therapeutically targeting Chi3l1 could enhance antitumor immunity and suppress metastasis.

Abstract Background: Lung cancer is the leading cause of cancer deaths worldwide. Non-small cell lung cancer(NSCLC) accounts for 85% of all lung cancers. The overall prevalence rate of NSCLC with epidermalgrowth factor receptor (EGFR) mutations are significantly increasing in US. Oncogenic EGFR is atransmembrane protein which gets auto-phosphorylated to cause EGFR mutations (L858R, T790M, exon-19-deletion) in exon 18-21. Tyrosine kinase inhibitors (TKIs) are effectively targeted to treat mutated EGFRlung cancer. These TKIs showed favorable responses on patient’s treatments for 9-18 months but duringthe treatment, patients acquired EGFR mutations which results into the TKIs drug resistance and at thatpoint TKIs stops its efficacy for further treatment. Thus, there is a high medical unmet need for a newtherapeutic strategy to overcome drug resistance in patients with EGFR mutations. CHI3L1 expressed bymacrophages, neutrophils, epithelial cells, smooth muscle cells, chondrocytes including other immune cells.The levels of circulating CHI3L1 are increased in many malignancies including cancers of the prostate,colon, rectum, ovary, kidney, breast, glioblastomas, malignant melanoma, and lung cancer. CHI3L1contributes to pulmonary metastasis and spread via the regulation of immune-checkpoint (ICP) molecules.Our studies showed that CHI3L1 regulates and is a potent stimulator of PD-1/PD-L1 and PD-L2. CHI3L1stimulates the EGFR physiologic ligands EGF or TGF-a, a well-defined growth factors that stimulate EGFRphosphorylation. EGFR-YAP/TAZ signaling plays a growth-promoting role in cancers harboring EGFRalterations, and that inhibition of YAP/TAZ in combination with EGFR might be beneficial to prevent TKIdrug resistance and cancer recurrence. Methods: We analyzed EGFR mutant and resistant cells using techniques qPCR, protein accumulation,immuno-pull-down assay, immunofluorescence, FACS and therapeutic effect of YAP inhibitors in-vitroand in-vivo. Results: We identified that CHI3L1 augments YAP/TAZ nuclear translocation in EGFR mutant and TKIresistant cells. Also, YAP/TAZ inhibitors and agonists (Verteporfin, K-975) able to block YAP/TAZactivation and that suppress PD-1/PD-L1 in EGFR mutant and TKI resistant cells. Conclusion: These findings led us to understand that CHI3L1 and PD-1/PD-L1 axis mediated throughactivation of Hippo-YAP/TAZ signaling pathways play an essential role in TKIs drug resistance andimmune tolerance that enhances the progression of EGFR NSCLC. Additionally, simultaneous targeting ofCHI3L1 and PD-1/PD-L1 axis employing bispecific antibody (CHI3L1xPD-1) may provide a bettertherapeutic option to overcome TKI resistance and immune tolerance of EGFR NSCLC. Citation Format: Suchitra Kamle, Bing Ma, Brianna Pham, Isabella Fish, Marlo Hulnick, Taka Sadanaga, Hanseok Jeong, Mara Hofstetter, Hina Khan, Christopher Azzoli, Katerina A. Politi, Roy S. Herbst, Chun Geun Lee, Jack Elias. Simultaneous targeting of CHI3L1 and PD-1/PD-L1 axis to overcome drug resistance and immune tolerance of EGFR non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7024.

Purpose:Chitinase-3-like-1 (CHI3L1) is a potent immune modulator implicated in tumor progression and immune suppression, including melanoma lung metastasis. Kasugamycin (KSM) has been reported as a pan-chitinase inhibitor with antifibrotic activity, but its effects on CHI3L1-driven immune regulation remain poorly defined. This study aimed to determine whether KSM suppresses CHI3L1-mediated tumor progression by modulating tumor-associated macrophage (TAM) differentiation and to elucidate the underlying molecular mechanisms. Methods:The anti-tumor effects of KSM were evaluated using a B16/F10 melanoma lung metastasis model. CHI3L1 gain-of-function approaches were used to assess specificity. Lung immune populations were analyzed by flow cytometry. Human THP-1 monocytes were used to examine CHI3L1-induced macrophage differentiation in vitro. Bulk RNA sequencing was performed on differentiated macrophages to identify downstream signaling pathways. Pharmacologic inhibition studies were conducted using the epidermal growth factor receptor (EGFR) inhibitor gefitinib to validate mechanistic links. Results:KSM treatment significantly reduced melanoma lung metastasis in a dose-dependent manner. CHI3L1 overexpression enhanced melanoma lung colony formation, which was effectively abrogated by KSM, indicating CHI3L1-specific anti-tumor activity. In melanoma-challenged lungs, KSM markedly decreased M2-like macrophages expressing CD206, CD163, and PD-L1. In vitro, CHI3L1 promoted M2 macrophage differentiation in THP-1 cells, which was strongly suppressed by KSM. Transcriptomic analysis revealed that EGFR expression was robustly induced by CHI3L1 and counter-regulated by KSM. Inhibition of EGFR signaling with gefitinib significantly attenuated CHI3L1-driven STAT3 activation and M2 macrophage polarization. Conclusion:These findings identify a previously unrecognized anti-tumor mechanism of KSM through inhibition of CHI3L1-EGFR-STAT3 signaling and suppression of M2-like TAM differentiation. KSM may therefore represent a promising immunomodulatory strategy for treating melanoma lung metastasis and other CHI3L1-driven malignancies.

CHI3L1, a chitinase-like protein, is implicated in pulmonary fibrosis, yet its mechanisms are incompletely understood. We demonstrated that CHI3L1 coordinates profibrotic macrophage activation and invasive myofibroblast differentiation, and their crosstalk. In vitro, CHI3L1 drove M2-like macrophage polarization with increased CD163, CD206, and PD-L1, and amplified TGF-β1-induced fibroblast responses, including myofibroblast transformation, migration, and invasion. Mechanistically, CHI3L1 enhanced TGF-β1 signaling through SMAD, AKT, and ERK pathways, and PD-L1 was required for CHI3L1/TGF-β1-driven myofibroblast transformation. Coculture studies further demonstrated the ability of CHI3L1 to induce profibrotic macrophage activation that enhanced myofibroblast transformation mediated via a CD44/PD-L1 axis. In vivo, following bleomycin challenge, CHI3L1-transgenic mice exhibited increased PD-L1+ M2 macrophages, PD-L1+PDGFRα+ fibroblasts, and PD-1+ immune cells compared with WT controls. Therapeutically, combined anti-CHI3L1 and anti-PD-1 antibodies, or a bispecific anti-CHI3L1-anti-PD-1 antibody, produced greater antifibrotic efficacy than monotherapy. These findings demonstrate crosstalk between CHI3L1 and the PD-1/PD-L1 pathway that promotes profibrotic macrophage activation and invasive fibroblast differentiation and support dual targeting of CHI3L1 and PD-1/PD-L1 as a promising therapeutic strategy for pulmonary fibrosis.

Chordomas are rare, highly morbid tumors arising from notochordal progenitor cells along the spinal axis, associated with severe neurological complications and high recurrence rates. Their resistance to conventional therapies and limited options beyond surgical resection and high-dose radiation underscore the urgent need for novel therapeutic targets. Publicly available preliminary RNA sequencing data from the Chordoma Foundation identified chitinase-3-like 1 (CHI3L1), a secreted glycoprotein implicated in immune checkpoint regulation and epithelial-mesenchymal transition (EMT), as a promising candidate for chordoma immunotherapy. Yet, the comprehensive function of CHI3L1 in chordoma immune response remains unclear. To evaluate its presence in chordoma, we employed RNA-based analyses alongside enzyme-linked immunosorbent assays (ELISA) on commercially available chordoma cell lines (JHC7, U-CH12, U-CH1, U-CH1-N) and human chordoma tumor specimens. Our results demonstrate elevated CHI3L1 expression in chordoma cells relative to notochordal precursors, with comparative analyses revealing higher CHI3L1 expression in the primary tumor relative to recurrent samples. These findings suggest the potential role of CHI3L1 in chordoma tumorigenesis, emphasizing its relevance as a biomarker and therapeutic target for primary tumors. Future studies are necessary to elucidate the mechanistic role of CHI3L1 in chordoma immune evasion and to explore targeted interventions that may improve patient outcomes in this aggressive cancer.

CHI3L1, a chitinase-like protein, plays a key role in the pathogenesis of pulmonary fibrosis, though the precise mechanisms remain unclear. This study explores how CHI3L1 regulates profibrotic macrophage activation and invasive myofibroblast differentiation and their interactions. In vitro , CHI3L1 induced profibrotic M2 macrophage activation and differentiation marked by increased expression of CD163, CD206, and PD-L1. CHI3L1 also enhanced TGF-β1 effects on lung fibroblasts including myofibroblast transformation, migration and tissue invasion. Mechanistically, CHI3L1 increased TGF-β1-stimulation of Smad, Akt and Erk signaling and PD-L1 played a significant role in TGF-β1/CHI3L1-stimulated myofibroblast transformation. Coculture experiment further confirmed the ability of CHI3L1 to induce profibrotic macrophage activation that enhanced myofibroblast transformation mediated via a CD44-PD-L1 axis. Following in vivo bleomycin challenge, CHI3L1 transgenic mice exhibited significantly higher levels of PD-L1+ M2 macrophages, PD-L1+/PDGFRα+ fibroblasts and increased numbers of PD-1+ and CD45+/PD-1+ cells compared to wild-type controls. Notably, combined treatment with anti-CHI3L1 and anti-PD-1 antibodies, or a bispecific anti-CHI3L1-anti-PD-1 antibody, resulted in greater inhibition of bleomycin-induced fibrosis than either antibody alone. These findings suggest that there is a stimulatory interaction between CHI3L1 and the PD-1/PD-L1 axis in promoting profibrotic macrophage activation and invasive fibroblast differentiation. The results also highlight the potential of bispecific targeting of CHI3L1 and the PD-1/PD-L1 pathway as an effective therapeutic approach for pulmonary fibrosis. ### Competing Interest Statement JAE is a cofounder of Elkurt Therapeutics and Sakonnet JAE is a cofounder of Elkurt Therapeutics and Sakonnet Biomedical which develop inhibitors of 18 glycosyl hydrolases as therapeutics. JAE, CGL and SK have composition of matter and use patents relating to antibodies against CHI3L1. CGL serves as a consultant for siRNAgen Inc., which develops RNA therapeutics. The other authors have declared that no conflict of interest exists. NIH Common Fund, PO1 HL114501 NIH Common Fund, https://ror.org/001d55x84, R01HL155558 DEPARTMENT OF DEFENCE, W81XWH-22-1-0041 National Research Foundation of Korea, https://ror.org/013aysd81, RS-2024-00405542

Supplementary Data and figure legends

e20622 Background: CHI3L1 is a member of the 18-glycosyl hydrolase gene family and is produced by a variety of cells including tumors and immune cells. It is overexpressed in several cancers and involved in cell death, innate immunity and tissue repair. Recent preclinical studies report that CHI3L1 regulates anti-tumor immune responses by inducing PD-1, PD-L1/2 and CTLA-4. Here we present the largest real-world dataset, investigating the association of tumor CHI3L1 RNA expression and clinical outcomes with immune checkpoint inhibitors in NSCLC. Methods: A total of 26,100 NSCLC specimens with paired DNA and RNA underwent gene expression profiling at Caris Life Sciences (Phoenix, AZ). Samples were stratified into quartiles based on CHI3L1 expression: top (Q4) and bottom (Q1). Tumors with known oncogenic drivers (DP) and lacking driver alterations (DN) were studied. PD-L1 expression was analyzed by IHC (22c3). Survival was calculated from claims data using Kaplan-Meier estimates as follows: survival on IO (IO-OS) from initiation of IO to last contact; and Pembrolizumab time on treatment (Pembro-ToT) from initiation to termination of Pembrolizumab. Hazard ratios (HR) and p-values were calculated using Cox proportional hazards model and log-rank test. Multiple hypothesis corrections were made where applicable (q < 0.05). Results: Compared to Q1, patients with Q4 tumors had a higher median age (70 vs 68), more females (54 vs 44%), non-smokers (5 vs 3%) and predominantly adenocarcinoma (AD) histology (67 vs 54%), all q < 0.05. Among drivers, KRAS (31 vs 25%) BRAF (5 vs 3%) and ALK alterations (6 vs 1%) were more prevalent in Q4 (all q < 0.05). Mutations in RB1 (6 vs 11%) , KEAP1 (9 vs 18%), STK11 (9 vs 16%) and TP53 (62 vs 71%, all q < 0.05) were less prevalent in Q4. In keeping with our report that CHI3L1 stimulates immune checkpoints, PD-L1+ (TPS > 50%: 37 vs 22% and 1-49%: 32 vs 26%, both q < 0.05) was more prevalent in Q4 and immune checkpoint expression ( CTLA4 , CD274 , HAVCR2 , IDO1 : 2.5-3.2 fold higher) and immune cell infiltrates (B-cells, neutrophils, M1 and M2 macrophages: 1.2-2 fold higher) were enriched in Q4 (all q < 0.05). Importantly, the enhanced expression of CHI3L1 in Q4 patients was associated with favorable IO-OS and Pembro-ToT; across AD and squamous histology and across DP and DN tumors (Table). Conclusions: CHI3L1 is a compelling biomarker in NSCLC that associates enhanced expression of immune checkpoints and tumor and microenvironment inflammation. Notably, high CHI3L1 is also associated with longer IO-OS and Pembro-ToT, likely due to the effects of CHI3L1 on tumor and microenvironment inflammation. Further studies correlating CHI3L1 expression are warranted, to establish its utility as a biomarker for OS and IO response. Survival in Q4 vs. Q1- only statistically significant associations displayed. HR IO-OS Pembro-ToT AD 0.7 0.8 SQ 0.8 0.8 KRAS+ 0.6 0.8 Driver- 0.8 0.8

Rationale: Transforming Growth Factor-β (TGF-β) is a major profibrotic cytokine implicated in the development and progression of pulmonary fibrosis. Stratifin (SFN), also known as 14-3-3σ, is a member of the 14-3-3 family of adaptor proteins and regulates various cellular processes, including proliferation, apoptosis, differentiation, and inflammation. Analysis of publicly available single-cell RNA sequencing data revealed consistently elevated expression of SFN in aberrant basaloid cells in the lungs of patients with idiopathic pulmonary fibrosis (IPF) compared to healthy controls. Preliminary studies further demonstrated a significant increase in SFN expression in the lungs of TGF-β transgenic (Tg) mice compared to wild-type controls. These findings led us to hypothesize that SFN may play a significant role in TGF-β-stimulated pulmonary fibrosis by influencing the differentiation of aberrant basaloid cells. Methods: We assessed the mRNA and protein expression levels of SFN in the lungs of wild-type and TGF-β Tg mice using real-time qPCR, Western blotting, and immunohistochemical (IHC) staining. We further investigated SFN's specific role in TGF-β-stimulated cellular and tissue responses through gene-specific siRNA silencing. Primary human bronchial epithelial (NHBE) cells and A549 cells were treated with recombinant TGF-β, and their cellular responses were evaluated. Results: TGF-β stimulation induced the expression of SFN and markers of epithelial-mesenchymal transition in NHBE and A549 cells over time. In NHBE cells, TGF-β significantly down-regulated KRT5 expression while preserving the expressions of KRT17 and p63. In A549 cells, TGF-β significantly increased KRT17 and p63 expressions, while KRT5 expression was not observed. Silencing SFN in NHBE cells enhanced KRT5 expression, while there were no significant changes in the expression of KRT17 and p63. Double IHC analysis showed that SFN expression frequently co-localized with KRT17(+) cells but not with KRT5(+) cells in the lungs of TGF-β Tg mice compared to wild-type controls. In vivo, SFN silencing in TGF-β Tg mice significantly reduced TGF-β-induced collagen accumulation in the lungs and increased the number of KRT5(+) cells. Conclusions: These findings support the hypothesis that SFN contributes to the pathogenesis of TGF-β-stimulated pulmonary fibrosis, potentially by regulating the differentiation of aberrant basaloid cells.

Abstract Breast cancer remains the most prevalent cancer and a major cause of mortality affecting women worldwide. Chemotherapy along with radiation and surgery have long been the standard of care for breast cancer patients. However, devastating side effects, resistance to treatments and relapse are major concerns for patients and physicians. With the rise of immunotherapies as a treatment option for various cancers, there is a critical need to understand the mechanisms through which cancers evade the immune system. Targeting such mechanisms holds the key to improve patient response to immunotherapy, especially in breast cancer where efficacy rates remain low. In a subset of poor outcome Triple Negative Breast Cancers, cytotoxic T cells are excluded from the tumor nest and restricted to the surrounding stroma, a phenomenon known as stromal restriction. Our data from genetically engineered mouse models and human tumors identified that the secreted cytokine Chitinase-3 like 1 (Chi3l1) promotes stromal restriction of T cells in several tumor types including breast, lung and colon. Chi3l1 ablation in pre-clinical models of breast cancer results in increased T cell infiltration into the tumor nest which delayed mammary tumorigenesis and improved response to immunotherapy. We further demonstrate that Chi3l1 promotes T cell exclusion through the direct induction and deposition of neutrophil extracellular traps (NETs) which form a barrier that restricts T cell entry. Neutrophil depletion or pharmacological disruption of NET formation abrogates Chi3l1-induced T cell exclusion and histologically phenocopies Chi3l1 ablation. Thus, our results indicate that Chi3l1 is a major immunosuppressive cytokine that promotes breast cancer progression by inducing NET formation and inhibiting T cell infiltration. Given the importance of T cell infiltration in immune elimination of nascent tumors, the future targeting of Chi3l1 should improve immunotherapy efficacy and outcomes in patients with tumors characterized by a T cell excluded microenvironment. Citation Format: Tarek Taifour, Sherif Samer Attalla, Dongmei Zuo, Yu Gu, Virginie Sanguin-Gendreau, Hailey Dall-Proud, Emilie Solymoss, Tung Bui, Hellen Kuasne, Vasilios Papavasiliou, Chun Geun Lee, Suchitra Kamle, Peter Siegel, Jack Elias, Morag Park, William Muller. Tumor Derived Chitinase-3 like 1 Induces Neutrophil Extracellular Traps that Promote T cell Exclusion and Resistance to Immunotherapies in Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-25-01.

Non-small cell lung cancer (NSCLC) accounts for 85 % of all lung cancers. In NSCLC, 10–20 % of Caucasian patients and 30–50 % of Asian patients have tumors with activating mutations in the Epidermal Growth Factor Receptor (EGFR). A high percentage of these patients exhibit favorable responses to treatment with tyrosine kinase inhibitors (TKI). Unfortunately, a majority of these patients develop therapeutic resistance with progression free survival lasting 9–18 months. The mechanisms that underlie the tumorigenic effects of EGFR and the ability of NSCLC to develop resistance to TKI therapies, however, are poorly understood. Here we demonstrate that CHI3L1 is produced by EGFR activation of normal epithelial cells, transformed epithelial cells with wild type EGFR and cells with cancer-associated, activating EGFR mutations. We also demonstrate that CHI3L1 auto-induces itself and feeds back to stimulate EGFR and its ligands via a STAT3-dependent mechanism(s). Highly specific antibodies against CHI3L1 (anti-CHI3L1/FRG) and TKI, individually and in combination, abrogated the effects of EGFR activation on CHI3L1 and the ability of CHI3L1 to stimulate the EGFR axis. Anti-CHI3L1 also interacted with osimertinib to reverse TKI therapeutic resistance and induce tumor cell death and inhibit pulmonary metastasis while stimulating tumor suppressor genes including KEAP1. CHI3L1 is a downstream target of EGFR that feeds back to stimulate and activate the EGFR axis. Anti-CHI3L1 is an exciting potential therapeutic for EGFR mutant NSCLC, alone and in combination with osimertinib or other TKIs.

Chitinase 1 (CHIT1) plays a role in the pathogenesis of pulmonary fibrosis by modulating canonical and noncanonical TGF-β signaling via interaction with TGFBRAP1 and FOXO3. These findings highlight the CHIT1/SMAD7 axis as a potential biomarker and therapeutic target of pulmonary fibrosis.

Innate immune responses such as phagocytosis are critically linked to the generation of adaptive immune responses against the neoantigens in cancer and the efferocytosis that is essential for homeostasis in diseases characterized by lung injury, inflammation, and remodeling as in chronic obstructive pulmonary disease (COPD). Chitinase 3-like-1 (CHI3L1) is induced in many cancers where it inhibits adaptive immune responses by stimulating immune checkpoint molecules (ICPs) and portends a poor prognosis. CHI3L1 is also induced in COPD where it regulates epithelial cell death. In this study, we demonstrate that pulmonary melanoma metastasis inhibits macrophage phagocytosis by stimulating the CD47-SIRPa and CD24-Siglec10 phagocytosis checkpoint pathways while inhibiting macrophage "eat me" signals from calreticulin and HMGB1. We also demonstrate that these effects on macrophage phagocytosis are associated with CHI3L1 stimulation of the SHP-1 and SHP-2 phosphatases and inhibition of the accumulation and phosphorylation of cytoskeleton-regulating nonmuscle myosin IIa. This inhibition of innate immune responses such as phagocytosis provides a mechanistic explanation for the ability of CHI3L1 to stimulate ICPs and inhibit adaptive immune responses in cancer and diseases such as COPD. The ability of CHI3L1 to simultaneously inhibit innate immune responses, stimulate ICPs, inhibit T cell costimulation, and regulate a number of other oncogenic and inflammation pathways suggests that CHI3L1-targeted therapeutics are promising interventions in cancer, COPD, and other disorders. The Journal of Immunology, 2024, 213: 1279-1291.

Abstract Chitinase 3–like 1 (Chi3l1) is a secreted protein that is highly expressed in glioblastoma. Here, we show that Chi3l1 alters the state of glioma stem cells (GSC) to support tumor growth. Exposure of patient-derived GSCs to Chi3l1 reduced the frequency of CD133+SOX2+ cells and increased the CD44+Chi3l1+ cells. Chi3l1 bound to CD44 and induced phosphorylation and nuclear translocation of β-catenin, Akt, and STAT3. Single-cell RNA sequencing and RNA velocity following incubation of GSCs with Chi3l1 showed significant changes in GSC state dynamics driving GSCs towards a mesenchymal expression profile and reducing transition probabilities towards terminal cellular states. ATAC-seq revealed that Chi3l1 increases accessibility of promoters containing a Myc-associated zinc finger protein (MAZ) transcription factor footprint. Inhibition of MAZ downregulated a set of genes with high expression in cellular clusters that exhibit significant cell state transitions after treatment with Chi3l1, and MAZ deficiency rescued the Chi3L-induced increase of GSC self-renewal. Finally, targeting Chi3l1 in vivo with a blocking antibody inhibited tumor growth and increased the probability of survival. Overall, this work suggests that Chi3l1 interacts with CD44 on the surface of GSCs to induce Akt/β-catenin signaling and MAZ transcriptional activity, which in turn upregulates CD44 expression in a pro-mesenchymal feed-forward loop. The role of Chi3l1 in regulating cellular plasticity confers a targetable vulnerability to glioblastoma. Significance: Chi3l1 is a modulator of glioma stem cell states that can be targeted to promote differentiation and suppress growth of glioblastoma.

In triple-negative breast cancer (TNBC), stromal restriction of CD8+ T cells associates with poor clinical outcomes and lack of responsiveness to immune-checkpoint blockade (ICB). To identify mediators of T cell stromal restriction, we profiled murine breast tumors lacking the transcription factor Stat3, which is commonly hyperactive in breast cancers and promotes an immunosuppressive tumor microenvironment. Expression of the cytokine Chi3l1 was decreased in Stat3-/- tumors. CHI3L1 expression was elevated in human TNBCs and other solid tumors exhibiting T cell stromal restriction. Chi3l1 ablation in the polyoma virus middle T (PyMT) breast cancer model generated an anti-tumor immune response and delayed mammary tumor onset. These effects were associated with increased T cell tumor infiltration and improved response to ICB. Mechanistically, Chi3l1 promoted neutrophil recruitment and neutrophil extracellular trap formation, which blocked T cell infiltration. Our findings provide insight into the mechanism underlying stromal restriction of CD8+ T cells and suggest that targeting Chi3l1 may promote anti-tumor immunity in various tumor types.

Innate immune responses such as phagocytosis are critically linked to the generation of adaptive immune responses against the neoantigens in cancer and the efferocytosis that is essential for homeostasis in diseases characterized by lung injury, inflammation, and remodeling as in Chronic Obstructive Pulmonary Disease (COPD). Chitinase 3-like-1 (CHI3L1) is induced in many cancers where it inhibits adaptive immune responses by stimulating immune checkpoint molecules (ICPs) and portends a poor prognosis. CHI3L1 is also induced in COPD where it regulates epithelial cell death. Here we demonstrate that pulmonary melanoma metastasis inhibits macrophage phagocytosis by stimulating the CD47-SIRPα and CD24-Siglec10 phagocytosis checkpoint pathways while inhibiting macrophage “eat me” signals from calreticulin and HMGB1. We also demonstrate that these effects on macrophage phagocytosis are mediated by CHI3L1 stimulation of the SHP-1 and SHP-2 phosphatases and the inhibition of the accumulation and phosphorylation of cytoskeleton-regulating non-muscle myosin IIa. This inhibition of innate immune responses like phagocytosis provides a mechanistic explanation for the ability of CHI3L1 to stimulate ICPs and inhibit adaptive immune responses in cancer and diseases like COPD. The ability of CHI3L1 to simultaneously inhibit innate immune responses, stimulate ICPs, inhibit T cell co-stimulation, and regulate a number of other oncogenic and inflammation pathways suggest that CHI3L1-targeted therapeutics are promising interventions in cancer, COPD and other disorders.### Competing Interest StatementJAE is a cofounder of Elkurt Therapeutics and is a founder of and stockholder of and serves on the Scientific Advisory Board for Ocean Biomedical, Inc., which develops inhibitors of 18 glycosyl hydrolases as therapeutics. CGL, CML, BM, and SK serves on consultants for Ocean Biomedical. Inc. JAE, CGL and SK have composition of matter and use patents relating to antibodies against CHI3L1. The other Brown University authors have declared that no conflict of interest exists. A.M.K.C. is a cofounder and consultant, and equity stockholder for Proterris, which develops therapeutic uses for carbon monoxide. A.M.K.C. is a consultant and equity stockholder for SPEXIS. A.M.K.C is a member for Medforth Board of Directors. A.M.K.C. has a use patent on CO and has a patent in COPD. DLD has received grant support from Bayer and has received awards from the Alpha-1 Foundation and the Doris Duke Foundation. EKS has received grant support from Bayer and Northpond Laboratories.