AXL/WRNIP1 Mediates Replication Stress Response and Promotes Therapy Resistance and Metachronous Metastasis in HER2+ Breast Cancer.

Therapy resistance and metastatic progression are primary causes of cancer-related mortality. Disseminated tumor cells possess adaptive traits that enable them to reprogram their metabolism, maintain stemness, and resist cell death, facilitating their persistence to drive recurrence. The survival of disseminated tumor cells also depends on their ability to modulate replication stress in response to therapy while colonizing inhospitable microenvironments. In this study, we discovered that the nuclear translocation of AXL, a TAM receptor tyrosine kinase, and its interaction with WRNIP1, a DNA replication stress response factor, promotes the survival of HER2+ breast cancer cells that are resistant to HER2-targeted therapy and metastasize to the brain. In preclinical models, knocking down or pharmacologically inhibiting AXL or WRNIP1 attenuated protection of stalled replication forks. Furthermore, deficiency or inhibition of AXL and WRNIP1 also prolonged metastatic latency and delayed relapse. Together, these findings suggest that targeting the replication stress response, which is a shared adaptive mechanism in therapy-resistant and metastasis-initiating cells, could reduce metachronous metastasis and enhance the response to standard-of-care therapies. SIGNIFICANCE: Nuclear AXL and WRNIP1 interact and mediate replication stress response, promote therapy resistance, and support metastatic progression, indicating that targeting the AXL/WRNIP1 axis is a potentially viable therapeutic strategy for breast cancer.

Identification of a core EMT signature that separates basal-like breast cancers into partial- and post-EMT subtypes.

Epithelial-mesenchymal transition (EMT) is a cellular plasticity program critical for embryonic development and tissue regeneration, and aberrant EMT is associated with disease including cancer. The high degree of plasticity in the mammary epithelium is reflected in extensive heterogeneity among breast cancers. Here, we have analyzed RNA-sequencing data from three different mammary epithelial cell line-derived EMT models and identified a robust mammary EMT gene expression signature that separates breast cancers into distinct subgroups. Most strikingly, the basal-like breast cancers form two subgroups displaying partial-EMT and post-EMT gene expression patterns. We present evidence that key EMT-associated transcription factors play distinct roles at different stages of EMT in mammary epithelial cells.

Refining the Role of Pyruvate Dehydrogenase Kinases in Glioblastoma Development.

Glioblastoma (GB) are the most frequent brain cancers. Aggressive growth and limited treatment options induce a median survival of 12-15 months. In addition to highly proliferative and invasive properties, GB cells show cancer-associated metabolic characteristics such as increased aerobic glycolysis. Pyruvate dehydrogenase (PDH) is a key enzyme complex at the crossroads between lactic fermentation and oxidative pathways, finely regulated by PDH kinases (PDHKs). PDHKs are often overexpressed in cancer cells to facilitate high glycolytic flux. We hypothesized that targeting PDHKs, by disturbing cancer metabolic homeostasis, would alter GB progression and render cells vulnerable to additional cancer treatment. Using patient databases, distinct expression patterns of PDHK1 and PDHK2 in GB tissues were obvious. To disturb protumoral glycolysis, we modulated PDH activity through the genetic or pharmacological inhibition of PDHK in patient-derived stem-like spheroids. Striking effects of PDHKs inhibition using dichloroacetate were observed in vitro on cell morphology and metabolism, resulting in increased intracellular ROS levels and decreased proliferation and invasion. In vivo findings confirmed a reduction in tumor size and better survival of mice implanted with PDHK1 and PDHK2 knockout cells. Adding a radiotherapeutic protocol further resulted in a reduction in tumor size and improved mouse survival in our model.

AXL targeting restores PD-1 blockade sensitivity of STK11/LKB1 mutant NSCLC through expansion of TCF1+ CD8 T cells.

Mutations in STK11/LKB1 in non-small cell lung cancer (NSCLC) are associated with poor patient responses to immune checkpoint blockade (ICB), and introduction of a Stk11/Lkb1 (L) mutation into murine lung adenocarcinomas driven by mutant Kras and Trp53 loss (KP) resulted in an ICB refractory syngeneic KPL tumor. Mechanistically this occurred because KPL mutant NSCLCs lacked TCF1-expressing CD8 T cells, a phenotype recapitulated in human STK11/LKB1 mutant NSCLCs. Systemic inhibition of Axl results in increased type I interferon secretion from dendritic cells that expanded tumor-associated TCF1+PD-1+CD8 T cells, restoring therapeutic response to PD-1 ICB in KPL tumors. This was observed in syngeneic immunocompetent mouse models and in humanized mice bearing STK11/LKB1 mutant NSCLC human tumor xenografts. NSCLC-affected individuals with identified STK11/LKB1 mutations receiving bemcentinib and pembrolizumab demonstrated objective clinical response to combination therapy. We conclude that AXL is a critical targetable driver of immune suppression in STK11/LKB1 mutant NSCLC.

Intrinsic Differences in Spatiotemporal Organization and Stromal Cell Interactions Between Isogenic Lung Cancer Cells of Epithelial and Mesenchymal Phenotypes Revealed by High-Dimensional Single-Cell Analysis of Heterotypic 3D Spheroid Models.

The lack of inadequate preclinical models remains a limitation for cancer drug development and is a primary contributor to anti-cancer drug failures in clinical trials. Heterotypic multicellular spheroids are three-dimensional (3D) spherical structures generated by self-assembly from aggregates of two or more cell types. Compared to traditional monolayer cell culture models, the organization of cells into a 3D tissue-like structure favors relevant physiological conditions with chemical and physical gradients as well as cell-cell and cell-extracellular matrix (ECM) interactions that recapitulate many of the hallmarks of cancer in situ. Epidermal growth factor receptor (EGFR) mutations are prevalent in non-small cell lung cancer (NSCLC), yet various mechanisms of acquired resistance, including epithelial-to-mesenchymal transition (EMT), limit the clinical benefit of EGFR tyrosine kinase inhibitors (EGFRi). Improved preclinical models that incorporate the complexity induced by epithelial-to-mesenchymal plasticity (EMP) are urgently needed to advance new therapeutics for clinical NSCLC management. This study was designed to provide a thorough characterization of multicellular spheroids of isogenic cancer cells of various phenotypes and demonstrate proof-of-principle for the applicability of the presented spheroid model to evaluate the impact of cancer cell phenotype in drug screening experiments through high-dimensional and spatially resolved imaging mass cytometry (IMC) analyses. First, we developed and characterized 3D homotypic and heterotypic spheroid models comprising EGFRi-sensitive or EGFRi-resistant NSCLC cells. We observed that the degree of EMT correlated with the spheroid generation efficiency in monocultures. In-depth characterization of the multicellular heterotypic spheroids using immunohistochemistry and high-dimensional single-cell analyses by IMC revealed intrinsic differences between epithelial and mesenchymal-like cancer cells with respect to self-sorting, spatiotemporal organization, and stromal cell interactions when co-cultured with fibroblasts. While the carcinoma cells harboring an epithelial phenotype self-organized into a barrier sheet surrounding the fibroblasts, mesenchymal-like carcinoma cells localized to the central hypoxic and collagen-rich areas of the compact heterotypic spheroids. Further, deep-learning-based single-cell segmentation of IMC images and application of dimensionality reduction algorithms allowed a detailed visualization and multiparametric analysis of marker expression across the different cell subsets. We observed a high level of heterogeneity in the expression of EMT markers in both the carcinoma cell populations and the fibroblasts. Our study supports further application of these models in pre-clinical drug testing combined with complementary high-dimensional single-cell analyses, which in turn can advance our understanding of the impact of cancer-stroma interactions and epithelial phenotypic plasticity on innate and acquired therapy resistance in NSCLC.

Dissecting the Role of AXL in Cancer Immune Escape and Resistance to Immune Checkpoint Inhibition.

The development and implementation of Immune Checkpoint Inhibitors (ICI) in clinical oncology have significantly improved the survival of a subset of cancer patients with metastatic disease previously considered uniformly lethal. However, the low response rates and the low number of patients with durable clinical responses remain major concerns and underscore the limited understanding of mechanisms regulating anti-tumor immunity and tumor immune resistance. There is an urgent unmet need for novel approaches to enhance the efficacy of ICI in the clinic, and for predictive tools that can accurately predict ICI responders based on the composition of their tumor microenvironment. The receptor tyrosine kinase (RTK) AXL has been associated with poor prognosis in numerous malignancies and the emergence of therapy resistance. AXL is a member of the TYRO3-AXL-MERTK (TAM) kinase family. Upon binding to its ligand GAS6, AXL regulates cell signaling cascades and cellular communication between various components of the tumor microenvironment, including cancer cells, endothelial cells, and immune cells. Converging evidence points to AXL as an attractive molecular target to overcome therapy resistance and immunosuppression, supported by the potential of AXL inhibitors to improve ICI efficacy. Here, we review the current literature on the prominent role of AXL in regulating cancer progression, with particular attention to its effects on anti-tumor immune response and resistance to ICI. We discuss future directions with the aim to understand better the complex role of AXL and TAM receptors in cancer and the potential value of this knowledge and targeted inhibition for the benefit of cancer patients.

Human Organotypic Airway and Lung Organoid Cells of Bronchiolar and Alveolar Differentiation Are Permissive to Infection by Influenza and SARS-CoV-2 Respiratory Virus.

The ongoing coronavirus disease 2019 (COVID-19) pandemic has led to the initiation of unprecedented research efforts to understand the pathogenesis mediated by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). More knowledge is needed regarding the cell type-specific cytopathology and its impact on cellular tropism. Furthermore, the impact of novel SARS-CoV-2 mutations on cellular tropism, alternative routes of entry, the impact of co-infections, and virus replication kinetics along the respiratory tract remains to be explored in improved models. Most applied virology models are not well suited to address the remaining questions, as they do not recapitulate the histoarchitecture and cellular composition of human respiratory tissues. The overall aim of this work was to establish from single biopsy specimens, a human adult stem cell-derived organoid model representing the upper respiratory airways and lungs and explore the applicability of this model to study respiratory virus infection. First, we characterized the organoid model with respect to growth pattern and histoarchitecture, cellular composition, and functional characteristics. Next, in situ expression of viral entry receptors, including influenza virus-relevant sialic acids and SARS-CoV-2 entry receptor ACE2 and TMPRSS2, were confirmed in organoids of bronchiolar and alveolar differentiation. We further showed successful infection by pseudotype influenza A H7N1 and H5N1 virus, and the ability of the model to support viral replication of influenza A H7N1 virus. Finally, successful infection and replication of a clinical isolate of SARS-CoV-2 were confirmed in the organoids by TCID50 assay and immunostaining to detect intracellular SARS-CoV-2 specific nucleocapsid and dsRNA. The prominent syncytia formation in organoid tissues following SARS-CoV-2 infection mimics the findings from infected human tissues in situ. We conclude that the human organotypic model described here may be particularly useful for virology studies to evaluate regional differences in the host response to infection. The model contains the various cell types along the respiratory tract, expresses respiratory virus entry factors, and supports successful infection and replication of influenza virus and SARS-CoV-2. Thus, the model may serve as a relevant and reliable tool in virology and aid in pandemic preparedness, and efficient evaluation of antiviral strategies.

C/EBPB-dependent adaptation to palmitic acid promotes tumor formation in hormone receptor negative breast cancer.

Epidemiological studies have established a positive association between obesity and the incidence of postmenopausal breast cancer. Moreover, it is known that obesity promotes stem cell-like properties of breast cancer cells. However, the cancer cell-autonomous mechanisms underlying this correlation are not well defined. Here we demonstrate that obesity-associated tumor formation is driven by cellular adaptation rather than expansion of pre-existing clones within the cancer cell population. While there is no correlation with specific mutations, cellular adaptation to obesity is governed by palmitic acid (PA) and leads to enhanced tumor formation capacity of breast cancer cells. This process is governed epigenetically through increased chromatin occupancy of the transcription factor CCAAT/enhancer-binding protein beta (C/EBPB). Obesity-induced epigenetic activation of C/EBPB regulates cancer stem-like properties by modulating the expression of key downstream regulators including CLDN1 and LCN2. Collectively, our findings demonstrate that obesity drives cellular adaptation to PA drives tumor initiation in the obese setting through activation of a C/EBPB dependent transcriptional network.

AXL targeting by a specific small molecule or monoclonal antibody inhibits renal cell carcinoma progression in an orthotopic mice model.

AXL tyrosine kinase activation enhances cancer cell survival, migration, invasiveness, and promotes drug resistance. AXL overexpression is typically detected in a high percentage of renal cell carcinomas (RCCs) and is strongly associated with poor prognosis. Therefore, AXL inhibition represents an attractive treatment option in these cancers. In this preclinical study, we investigated the antitumor role of a highly selective small molecule AXL inhibitor bemcentinib (BGB324, BerGenBio), and a newly developed humanized anti-AXL monoclonal function blocking antibody tilvestamab, (BGB149, BerGenBio), in vitro and an orthotopic RCC mice model. The 786-0-Luc human RCC cells showed high AXL expression. Both bemcentinib and tilvestamab significantly inhibited AXL activation induced by Gas6 stimulation in vitro. Furthermore, tilvestamab inhibited the downstream AKT phosphorylation in these cells. The 786-0-Luc human RCC cells generated tumors with high Ki67 and vimentin expression upon orthotopic implantation in athymic BALB/c nude mice. Most importantly, both bemcentinib and tilvestamab inhibited the progression of tumors induced by the orthotopically implanted 786-0 RCC cells. Remarkably, their in vivo antitumor effectiveness was not significantly enhanced by concomitant administration of a multi-target tyrosine kinase inhibitor. Bemcentinib and tilvestamab qualify as compounds of potentially high clinical interest in AXL overexpressing RCC.

Phosphatidylserine receptors enhance SARS-CoV-2 infection.

Phosphatidylserine (PS) receptors enhance infection of many enveloped viruses through virion-associated PS binding that is termed apoptotic mimicry. Here we show that this broadly shared uptake mechanism is utilized by SARS-CoV-2 in cells that express low surface levels of ACE2. Expression of members of the TIM (TIM-1 and TIM-4) and TAM (AXL) families of PS receptors enhance SARS-CoV-2 binding to cells, facilitate internalization of fluorescently-labeled virions and increase ACE2-dependent infection of SARS-CoV-2; however, PS receptors alone did not mediate infection. We were unable to detect direct interactions of the PS receptor AXL with purified SARS-CoV-2 spike, contrary to a previous report. Instead, our studies indicate that the PS receptors interact with PS on the surface of SARS-CoV-2 virions. In support of this, we demonstrate that: 1) significant quantities of PS are located on the outer leaflet of SARS-CoV-2 virions, 2) PS liposomes, but not phosphatidylcholine liposomes, reduced entry of VSV/Spike pseudovirions and 3) an established mutant of TIM-1 which does not bind to PS is unable to facilitate entry of SARS-CoV-2. As AXL is an abundant PS receptor on a number of airway lines, we evaluated small molecule inhibitors of AXL signaling such as bemcentinib for their ability to inhibit SARS-CoV-2 infection. Bemcentinib robustly inhibited virus infection of Vero E6 cells as well as multiple human lung cell lines that expressed AXL. This inhibition correlated well with inhibitors that block endosomal acidification and cathepsin activity, consistent with AXL-mediated uptake of SARS-CoV-2 into the endosomal compartment. We extended our observations to the related betacoronavirus mouse hepatitis virus (MHV), showing that inhibition or ablation of AXL reduces MHV infection of murine cells. In total, our findings provide evidence that PS receptors facilitate infection of the pandemic coronavirus SARS-CoV-2 and suggest that inhibition of the PS receptor AXL has therapeutic potential against SARS-CoV-2.

AXL Inhibition Represents a Novel Therapeutic Approach in BCR-ABL Negative Myeloproliferative Neoplasms.

BCR-ABL negative myeloproliferative neoplasms (MPNs) consist of essential thrombocythemia, polycythemia vera, and myelofibrosis. The majority of patients harbor the JAK2-activating mutation V617F. JAK2 inhibitors were shown to reduce symptom burden and splenomegaly in MPN patients. However, treatment options are limited after failure of JAK2 inhibitors. AXL, a member of the TAM family of receptor tyrosine kinases, mediates survival and therapy resistance of different myeloid cancers including acute myeloid leukemia and chronic myeloid leukemia. We studied the relevance of AXL as a target in MPN using primary patient cells and preclinical disease models. We found that AXL is abundantly activated in MPN cells and that its ligand growth arrest-specific gene 6 is upregulated in MPN patients. Pharmacologic and genetic blockade of AXL impaired viability, decreased proliferation and increased apoptosis of MPN cells. Interestingly, ruxolitinib treatment induced increased phosphorylation of AXL indicating that activation of AXL might mediate resistance to ruxolitinib. Consistently, the AXL inhibitor bemcentinib exerted additive effects with ruxolitinib via impaired STAT3, STAT5, and AKT signaling. Both agents had activity when employed alone and exerted an additive effect on survival and splenomegaly in vivo. Moreover, bemcentinib treatment normalized red blood cell count and hemoglobin levels in vivo. Thus, our data indicate that AXL inhibition represents a novel treatment option in MPN warranting clinical investigation.

Association of AXL and PD-L1 Expression with Clinical Outcomes in Patients with Advanced Renal Cell Carcinoma Treated with PD-1 Blockade.

PURPOSE: A minority of patients currently respond to single-agent immune-checkpoint blockade (ICB), and strategies to increase response rates are urgently needed. AXL is a receptor tyrosine kinase commonly associated with drug resistance and poor prognosis in many cancer types, including in clear-cell renal cell carcinoma (ccRCC). Recent experimental cues in breast, pancreatic, and lung cancer models have linked AXL with immune suppression and resistance to antitumor immunity. However, its role in intrinsic and acquired resistance to ICB remains largely unexplored. EXPERIMENTAL DESIGN: In this study, tumoral expression of AXL was examined in ccRCC specimens from 316 patients who were metastatic receiving the PD-1 inhibitor nivolumab in the GETUG AFU 26 NIVOREN trial after failure of antiangiogenic therapy. We assessed associations between AXL and patient outcomes following PD-1 blockade, as well as the relationship with various markers, including PD-L1; VEGFA; the immune markers CD3, CD8, CD163, and CD20; and the mutational status of the tumor-suppressor gene von Hippel-Lindau (VHL). RESULTS: Our results show that high AXL-expression level in tumor cells is associated with lower response rates and a trend to shorter progression-free survival following anti-PD-1 treatment. AXL expression was strongly associated with tumor-PD-L1 expression, especially in tumors with VHL inactivation. Moreover, patients with tumors displaying concomitant PD-L1 expression and high AXL expression had the worst overall survival. CONCLUSIONS: Our findings propose AXL as candidate factor of resistance to PD-1 blockade, and provide compelling support for screening both AXL and PD-L1 expression in the management of advanced ccRCC.See related commentary by Hahn et al., p. 6619.

Axl-inhibitor bemcentinib alleviates mitochondrial dysfunction in the unilateral ureter obstruction murine model.

Renal fibrosis is a progressive histological manifestation leading to chronic kidney disease (CKD) and associated with mitochondrial dysfunction. In previous work, we showed that Bemcentinib, an Axl receptor tyrosine kinase inhibitor, reduced fibrosis development. In this study, to investigate its effects on mitochondrial dysfunction in renal fibrosis, we analysed genome-wide transcriptomics data from a unilateral ureter obstruction (UUO) murine model in the presence or absence of bemcentinib (n = 6 per group) and SHAM-operated (n = 4) mice. Kidney ligation resulted in dysregulation of mitochondria-related pathways, with a significant reduction in the expression of oxidative phosphorylation (OXPHOS), fatty acid oxidation (FAO), citric acid cycle (TCA), response to reactive oxygen species and amino acid metabolism-related genes. Bemcentinib treatment increased the expression of these genes. In contrast, AKT/PI3K signalling pathway genes were up-regulated upon UUO, but bemcentinib largely inhibited their expression. At the functional level, ligation reduced mitochondrial biomass, which was increased upon bemcentinib treatment. Serum metabolomics analysis also showed a normalizing amino acid profile in UUO, compared with SHAM-operated mice following bemcentinib treatment. Our data suggest that mitochondria and mitochondria-related pathways are dramatically affected by UUO surgery and treatment with Axl-inhibitor bemcentinib partially reverses these effects.

Phosphatidylserine Receptors Enhance SARS-CoV-2 Infection: AXL as a Therapeutic Target for COVID-19.

Phosphatidylserine (PS) receptors are PS binding proteins that mediate uptake of apoptotic bodies. Many enveloped viruses utilize this PS/PS receptor mechanism to adhere to and internalize into the endosomal compartment of cells and this is termed apoptotic mimicry. For viruses that have a mechanism(s) of endosomal escape, apoptotic mimicry is a productive route of virus entry. We evaluated if PS receptors serve as cell surface receptors for SARS-CoV-2 and found that the PS receptors, AXL, TIM-1 and TIM-4, facilitated virus infection when low concentrations of the SARS-CoV-2 cognate receptor, ACE2, was present. Consistent with the established mechanism of PS receptor utilization by other viruses, PS liposomes competed with SARS-CoV-2 for binding and entry. We demonstrated that this PS receptor enhances SARS-CoV-2 binding to and infection of an array of human lung cell lines and is an under-appreciated but potentially important host factor facilitating SARS-CoV-2 entry.

AXL Is a Key Factor for Cell Plasticity and Promotes Metastasis in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDA), a leading cause of cancer-related death in the United States, has a high metastatic rate, and is associated with persistent immune suppression. AXL, a member of the TAM (TYRO3, AXL, MERTK) receptor tyrosine kinase family, is a driver of metastasis and immune suppression in multiple cancer types. Here we use single-cell RNA-sequencing to reveal that AXL is expressed highly in tumor cells that have a mesenchymal-like phenotype and that AXL expression correlates with classic markers of epithelial-to-mesenchymal transition. We demonstrate that AXL deficiency extends survival, reduces primary and metastatic burden, and enhances sensitivity to gemcitabine in an autochthonous model of PDA. PDA in AXL-deficient mice displayed a more differentiated histology, higher nucleoside transporter expression, and a more active immune microenvironment compared with PDA in wild-type mice. Finally, we demonstrate that AXL-positive poorly differentiated tumor cells are critical for PDA progression and metastasis, emphasizing the potential of AXL as a therapeutic target in PDA. IMPLICATIONS: These studies implicate AXL as a marker of undifferentiated PDA cells and a target for therapy.

Blocking Aerobic Glycolysis by Targeting Pyruvate Dehydrogenase Kinase in Combination with EGFR TKI and Ionizing Radiation Increases Therapeutic Effect in Non-Small Cell Lung Cancer Cells.

Increased glycolytic activity is a hallmark of cancer initiation and progression and is often observed in non-small cell lung cancer (NSCLC). Pyruvate dehydrogenase (PDH) complex acts as a gatekeeper between glycolysis and oxidative phosphorylation, and activation of PDH is known to inhibit glycolytic activity. As part of a standard therapeutic regimen, patients with NSCLC harboring oncogenic mutations in the epidermal growth factor receptor (EGFR) are treated with EGFR tyrosine kinase inhibitors (EGFR TKIs). Independent of good initial response, development of resistance to this therapy is inevitable. In the presented work, we propose that inhibition of glycolysis will add to the therapeutic effects and possibly prevent development of resistance against both EGFR TKIs and ionizing radiation in NSCLC. Analysis of transcriptome data from two independent NSCLC patient cohorts identified increased expression of pyruvate dehydrogenase kinase 1 (PDHK1) as well as upregulated expression of genes involved in glucose metabolism in tumors compared to normal tissue. We established in vitro models of development of resistance to EGFR TKIs to study metabolism and determine if targeting PDHK would prevent development of resistance to EGFR TKIs in NSCLC cells. The PDHK1 inhibitor dichloroacetate (DCA) in combination with EGFR TKIs and/or ionizing radiation was shown to increase the therapeutic effect in our NSCLC cell models. This mechanism was associated with redirected metabolism towards pyruvate oxidation and reduced lactate production, both in EGFR TKI sensitive and resistant NSCLC cells. Using DCA, the intracellular pool of pyruvate available for lactic fermentation becomes limited. Consequently, pyruvate is redirected to the mitochondria, and reinforces mitochondrial activity. Addition of DCA to cell culture deacidifies the extracellular microenvironment as less lactate is produced and excreted. In our study, we find that this redirection of metabolism adds to the therapeutic effect of EGFR TKI and ionizing radiation in NSCLC.

High-dimensional immunotyping of tumors grown in obese and non-obese mice.

Obesity is a disease characterized by chronic low-grade systemic inflammation and has been causally linked to the development of 13 cancer types. Several studies have been undertaken to determine whether tumors evolving in obese environments adapt differential interactions with immune cells and whether this can be connected to disease outcome. Most of these studies have been limited to single-cell lines and tumor models and analysis of limited immune cell populations. Given the multicellular complexity of the immune system and its dysregulation in obesity, we applied high-dimensional suspension mass cytometry to investigate how obesity affects tumor immunity. We used a 36-marker immune-focused mass cytometry panel to interrogate the immune landscape of orthotopic syngeneic mouse models of pancreatic and breast cancer. Unanchored batch correction was implemented to enable simultaneous analysis of tumor cohorts to uncover the immunotypes of each cancer model and reveal remarkably model-specific immune regulation. In the E0771 breast cancer model, we demonstrate an important link to obesity with an increase in two T-cell-suppressive cell types and a decrease in CD8 T cells.

AXL Is a Driver of Stemness in Normal Mammary Gland and Breast Cancer.

The receptor tyrosine kinase AXL is associated with epithelial plasticity in several solid tumors including breast cancer and AXL-targeting agents are currently in clinical trials. We hypothesized that AXL is a driver of stemness traits in cancer by co-option of a regulatory function normally reserved for stem cells. AXL-expressing cells in human mammary epithelial ducts co-expressed markers associated with multipotency, and AXL inhibition abolished colony formation and self-maintenance activities while promoting terminal differentiation in vitro. Axl-null mice did not exhibit a strong developmental phenotype, but enrichment of Axl + cells was required for mouse mammary gland reconstitution upon transplantation, and Axl-null mice had reduced incidence of Wnt1-driven mammary tumors. An AXL-dependent gene signature is a feature of transcriptomes in basal breast cancers and reduced patient survival irrespective of subtype. Our interpretation is that AXL regulates access to epithelial plasticity programs in MaSCs and, when co-opted, maintains acquired stemness in breast cancer cells.

Inhibiting the GAS6/AXL axis suppresses tumor progression by blocking the interaction between cancer-associated fibroblasts and cancer cells in gastric carcinoma.

BACKGROUND: The effects of cancer-associated fibroblasts (CAF) on the progression of gastric carcinoma (GC) has recently been demonstrated. However, agents targeting the interaction between CAF and GC cells have not been applied in a clinical setting. Here, we examined if inhibition for Axl receptor tyrosine kinase (AXL) can suppress CAF-induced aggressive phenotype in GC. METHODS: We investigated the function of CAF-derived growth arrest-specific 6 (GAS6), a major ligand of AXL, on the migration and proliferation of GC cells. The effect of the AXL inhibitor, BGB324, on the CAF-induced aggressive phenotype of GC cells was also investigated. In addition, we performed immunohistochemistry to examine the expression of phosphorylated AXL protein in 175 GC tissues and evaluated its correlation with the prognosis. RESULTS: The qPCR and western blot analysis showed that GAS6 expression was higher in CAF relative to other cells. We found that co-culture with CAF increased the phosphorylation of AXL (P-AXL), differentiation into a mesenchymal-like phenotype, and cell survival in GC cell lines. When the expression of AXL was genetically inhibited in GC cells, the effect of CAF was reduced. BGB324, a small molecule inhibitor of AXL, suppressed the effects of CAF on GC cell lines. In GC tissues, high levels of P-AXL were significantly associated with poor overall survival (P = 0.022). CONCLUSIONS: We concluded that CAF are a major source of GAS6 and that GAS6 promotes an aggressiveness through AXL activation in GC. We suggested that an AXL inhibitor may be a novel agent for GC treatment.