GateMeClass: Gate Mining and Classification of cytometry data.

MOTIVATION: Cytometry comprises powerful techniques for analyzing the cell heterogeneity of a biological sample by examining the expression of protein markers. These technologies impact especially the field of oncoimmunology, where cell identification is essential to analyze the tumor microenvironment. Several classification tools have been developed for the annotation of cytometry datasets, which include supervised tools that require a training set as a reference (i.e. reference-based) and semisupervised tools based on the manual definition of a marker table. The latter is closer to the traditional annotation of cytometry data based on manual gating. However, they require the manual definition of a marker table that cannot be extracted automatically in a reference-based fashion. Therefore, we are lacking methods that allow both classification approaches while maintaining the high biological interpretability given by the marker table. RESULTS: We present a new tool called GateMeClass (Gate Mining and Classification) which overcomes the limitation of the current methods of classification of cytometry data allowing both semisupervised and supervised annotation based on a marker table that can be defined manually or extracted from an external annotated dataset. We measured the accuracy of GateMeClass for annotating three well-established benchmark mass cytometry datasets and one flow cytometry dataset. The performance of GateMeClass is comparable to reference-based methods and marker table-based techniques, offering greater flexibility and rapid execution times. AVAILABILITY AND IMPLEMENTATION: GateMeClass is implemented in R language and is publicly available at https://github.com/simo1c/GateMeClass.

Expression of the membrane tetraspanin claudin 18 on cancer cells promotes T lymphocyte infiltration and antitumor immunity in pancreatic cancer.

Tumors weakly infiltrated by T lymphocytes poorly respond to immunotherapy. We aimed to unveil malignancy-associated programs regulating T cell entrance, arrest, and activation in the tumor environment. Differential expression of cell adhesion and tissue architecture programs, particularly the presence of the membrane tetraspanin claudin (CLDN)18 as a signature gene, demarcated immune-infiltrated from immune-depleted mouse pancreatic tumors. In human pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer, CLDN18 expression positively correlated with more differentiated histology and favorable prognosis. CLDN18 on the cell surface promoted accrual of cytotoxic T lymphocytes (CTLs), facilitating direct CTL contacts with tumor cells by driving the mobilization of the adhesion protein ALCAM to the lipid rafts of the tumor cell membrane through actin. This process favored the formation of robust immunological synapses (ISs) between CTLs and CLDN18-positive cancer cells, resulting in increased T cell activation. Our data reveal an immune role for CLDN18 in orchestrating T cell infiltration and shaping the tumor immune contexture.

Axon guidance cue SEMA3A promotes the aggressive phenotype of basal-like PDAC.

OBJECTIVE: The dysregulation of the axon guidance pathway is common in pancreatic ductal adenocarcinoma (PDAC), yet our understanding of its biological relevance is limited. Here, we investigated the functional role of the axon guidance cue SEMA3A in supporting PDAC progression. DESIGN: We integrated bulk and single-cell transcriptomic datasets of human PDAC with in situ hybridisation analyses of patients' tissues to evaluate SEMA3A expression in molecular subtypes of PDAC. Gain and loss of function experiments in PDAC cell lines and organoids were performed to dissect how SEMA3A contributes to define a biologically aggressive phenotype. RESULTS: In PDAC tissues, SEMA3A is expressed by stromal elements and selectively enriched in basal-like/squamous epithelial cells. Accordingly, expression of SEMA3A in PDAC cells is induced by both cell-intrinsic and cell-extrinsic determinants of the basal-like phenotype. In vitro, SEMA3A promotes cell migration as well as anoikis resistance. At the molecular level, these phenotypes are associated with increased focal adhesion kinase signalling through canonical SEMA3A-NRP1 axis. SEMA3A provides mouse PDAC cells with greater metastatic competence and favours intratumoural infiltration of tumour-associated macrophages and reduced density of T cells. Mechanistically, SEMA3A functions as chemoattractant for macrophages and skews their polarisation towards an M2-like phenotype. In SEMA3Ahigh tumours, depletion of macrophages results in greater intratumour infiltration by CD8+T cells and better control of the disease from antitumour treatment. CONCLUSIONS: Here, we show that SEMA3A is a stress-sensitive locus that promotes the malignant phenotype of basal-like PDAC through both cell-intrinsic and cell-extrinsic mechanisms.

Plasmacytoid Dendritic Cell, Slan+-Monocyte and Natural Killer Cell Counts Function as Blood Cell-Based Biomarkers for Predicting Responses to Immune Checkpoint Inhibitor Monotherapy in Non-Small Cell Lung Cancer Patients.

The advent of immune checkpoint inhibitors (ICIs), for instance, programmed cell death 1 (PD-1)/PD-1 ligand 1 (PD-L1) blockers, has greatly improved the outcome of patients affected by non-small cell lung cancer (NSCLC). However, most NSCLC patients either do not respond to ICI monotherapy or develop resistance to it after an initial response. Therefore, the identification of biomarkers for predicting the response of patients to ICI monotherapy represents an urgent issue. Great efforts are currently dedicated toward identifying blood-based biomarkers to predict responses to ICI monotherapy. In this study, more commonly utilized blood-based biomarkers, such as the neutrophil-to-lymphocyte ratio (NLR) and the lung immune prognostic index (LIPI) score, as well as the frequency/number and activation status of various types of circulating innate immune cell populations, were evaluated in NSCLC patients at baseline before therapy initiation. The data indicated that, among all the parameters tested, low plasmacytoid dendritic cell (pDC), slan+-monocyte and natural killer cell counts, as well as a high LIPI score and elevated PD-L1 expression levels on type 1 conventional DCs (cDC1s), were independently correlated with a negative response to ICI therapy in NSCLC patients. The results from this study suggest that the evaluation of innate immune cell numbers and phenotypes may provide novel and promising predictive biomarkers for ICI monotherapy in NSCLC patients.

Immune checkpoint blockade therapy mitigates systemic inflammation and affects cellular FLIP-expressing monocytic myeloid-derived suppressor cells in non-progressor non-small cell lung cancer patients.

Cancer cells favor the generation of myeloid cells with immunosuppressive and inflammatory features, including myeloid-derived suppressor cells (MDSCs), which support tumor progression. The anti-apoptotic molecule, cellular FLICE (FADD-like interleukin-1ß-converting enzyme)-inhibitory protein (c-FLIP), which acts as an important modulator of caspase-8, is required for the development and function of monocytic (M)-MDSCs. Here, we assessed the effect of immune checkpoint inhibitor (ICI) therapy on systemic immunological landscape, including FLIP-expressing MDSCs, in non-small cell lung cancer (NSCLC) patients. Longitudinal changes in peripheral immunological parameters were correlated with patients' outcome. In detail, 34 NSCLC patients were enrolled and classified as progressors (P) or non-progressors (NP), according to the RECIST evaluation. We demonstrated a reduction in pro-inflammatory cytokines such as IL-8, IL-6, and IL-1ß in only NP patients after ICI treatment. Moreover, using t-distributed stochastic neighbor embedding (t-SNE) and cluster analysis, we characterized in NP patients a significant increase in the amount of lymphocytes and a slight contraction of myeloid cells such as neutrophils and monocytes. Despite this moderate ICI-associated alteration in myeloid cells, we identified a distinctive reduction of c-FLIP expression in M-MDSCs from NP patients concurrently with the first clinical evaluation (T1), even though NP and P patients showed the same level of expression at baseline (T0). In agreement with the c-FLIP expression, monocytes isolated from both P and NP patients displayed similar immunosuppressive functions at T0; however, this pro-tumor activity was negatively influenced at T1 in the NP patient cohort exclusively. Hence, ICI therapy can mitigate systemic inflammation and impair MDSC-dependent immunosuppression.

Pre-diagnostic prognostic value of leukocytes count and neutrophil-to-lymphocyte ratio in patients who develop colorectal cancer.

Introduction: Emerging evidence is pointing towards a relevant role of immunity in cancer development. Alterations in leukocytes count and neutrophil-to-lymphocyte ratio (NLR) at diagnosis of colorectal cancer (CRC) seems to predict poor prognosis, but no data is available for the pre-diagnostic values. Methods: Retrospective analysis of patients who underwent surgery for CRC at our center (2005 - 2020). 334 patients with a complete blood count dating at least 24 months prior to diagnosis were included. Changes in pre-diagnosis values of leukocytes (Pre-Leu), lymphocytes (Pre-Lymph), neutrophils (Pre-Neut), and NLR (Pre-NLR) and their correlation with overall- (OS) and cancer-related survival (CRS) were analyzed. Results: Pre-Leu, Pre-Neut and Pre-NLR showed an increasing trend approaching the date of diagnosis, while Pre-Lymph tended to decrease. The parameters were tested for associations with survival after surgery through multivariable analysis. After adjusting for potential confounding factors, Pre-Leu, Pre-Neut, Pre-Lymph and Pre-NLR resulted independent prognostic factors for OS and CRS. On sub-group analysis considering the interval between blood sampling and surgery, higher Pre-Leu, Pre-Neut, and Pre-NLR and lower Pre-Lymph were associated with worse CRS, and the effect was more evident when blood samples were closer to surgery. Conclusion: To our knowledge, this is the first study showing a significant correlation between pre-diagnosis immune profile and prognosis in CRC.

p140Cap inhibits ß-Catenin in the breast cancer stem cell compartment instructing a protective anti-tumor immune response.

The p140Cap adaptor protein is a tumor suppressor in breast cancer associated with a favorable prognosis. Here we highlight a function of p140Cap in orchestrating local and systemic tumor-extrinsic events that eventually result in inhibition of the polymorphonuclear myeloid-derived suppressor cell function in creating an immunosuppressive tumor-promoting environment in the primary tumor, and premetastatic niches at distant sites. Integrative transcriptomic and preclinical studies unravel that p140Cap controls an epistatic axis where, through the upstream inhibition of ß-Catenin, it restricts tumorigenicity and self-renewal of tumor-initiating cells limiting the release of the inflammatory cytokine G-CSF, required for polymorphonuclear myeloid-derived suppressor cells to exert their local and systemic tumor conducive function. Mechanistically, p140Cap inhibition of ß-Catenin depends on its ability to localize in and stabilize the ß-Catenin destruction complex, promoting enhanced ß-Catenin inactivation. Clinical studies in women show that low p140Cap expression correlates with reduced presence of tumor-infiltrating lymphocytes and more aggressive tumor types in a large cohort of real-life female breast cancer patients, highlighting the potential of p140Cap as a biomarker for therapeutic intervention targeting the ß-Catenin/ Tumor-initiating cells /G-CSF/ polymorphonuclear myeloid-derived suppressor cell axis to restore an efficient anti-tumor immune response.

Neutralization of NET-associated human ARG1 enhances cancer immunotherapy.

Myeloid cells can restrain antitumor immunity by metabolic pathways, such as the degradation of l-arginine, whose concentrations are regulated by the arginase 1 (ARG1) enzyme. Results from preclinical studies indicate the important role of arginine metabolism in pancreatic ductal adenocarcinoma (PDAC) progression, suggesting a potential for clinical application; however, divergent evolution in ARG1 expression and function in rodents and humans has restricted clinical translation. To overcome this dichotomy, here, we show that neutrophil extracellular traps (NETs), released by spontaneously activated neutrophils isolated from patients with PDAC, create a microdomain where cathepsin S (CTSS) cleaves human (h)ARG1 into different molecular forms endowed with enhanced enzymatic activity at physiological pH. NET-associated hARG1 suppresses T lymphocytes whose proliferation is restored by either adding a hARG1-specific monoclonal antibody (mAb) or preventing CTSS-mediated cleavage, whereas small-molecule inhibitors are not effective. We show that ARG1 blockade, combined with immune checkpoint inhibitors, can restore CD8+ T cell function in ex vivo PDAC tumors. Furthermore, anti-hARG1 mAbs increase the frequency of adoptively transferred tumor-specific CD8+ T cells in tumor and enhance the effectiveness of immune checkpoint therapy in humanized mice. Thus, this study shows that extracellular ARG1, released by activated myeloid cells, localizes in NETs, where it interacts with CTSS that in turn cleaves ARG1, producing major molecular forms endowed with different enzymatic activity at physiological pH. Once exocytosed, ARG1 activity can be targeted by mAbs, which bear potential for clinical application for the treatment of PDAC and require further exploration.

Harnessing the reverse cholesterol transport pathway to favor differentiation of monocyte-derived APCs and antitumor responses.

Lipid and cholesterol metabolism play a crucial role in tumor cell behavior and in shaping the tumor microenvironment. In particular, enzymatic and non-enzymatic cholesterol metabolism, and derived metabolites control dendritic cell (DC) functions, ultimately impacting tumor antigen presentation within and outside the tumor mass, dampening tumor immunity and immunotherapeutic attempts. The mechanisms accounting for such events remain largely to be defined. Here we perturbed (oxy)sterol metabolism genetically and pharmacologically and analyzed the tumor lipidome landscape in relation to the tumor-infiltrating immune cells. We report that perturbing the lipidome of tumor microenvironment by the expression of sulfotransferase 2B1b crucial in cholesterol and oxysterol sulfate synthesis, favored intratumoral representation of monocyte-derived antigen-presenting cells, including monocyte-DCs. We also found that treating mice with a newly developed antagonist of the oxysterol receptors Liver X Receptors (LXRs), promoted intratumoral monocyte-DC differentiation, delayed tumor growth and synergized with anti-PD-1 immunotherapy and adoptive T cell therapy. Of note, looking at LXR/cholesterol gene signature in melanoma patients treated with anti-PD-1-based immunotherapy predicted diverse clinical outcomes. Indeed, patients whose tumors were poorly infiltrated by monocytes/macrophages expressing LXR target genes showed improved survival over the course of therapy. Thus, our data support a role for (oxy)sterol metabolism in shaping monocyte-to-DC differentiation, and in tumor antigen presentation critical for responsiveness to immunotherapy. The identification of a new LXR antagonist opens new treatment avenues for cancer patients.

Thirty-two-year trends of cancer incidence by sex and cancer site in the Veneto Region from 1987 to 2019.

Background: This observational study considers the sex-specific incidence of the most incident cancers as recorded in the population-based Veneto Regional Cancer Registry over a period of more than 30 years (1987-2019). Methods: The Veneto Regional Cancer Registry collected data for the time interval 1987-2019. Significant changes in incidence trends calculated on age-standardized incidence rates (Annual Percent Change-APC) were identified by join point regression analysis. Results: Overall, the incidence trend for all cancers decreased in males and remained stable in females. In nine cancer sites, the incidence trends showed consistent differences by sex (oral cavity, esophagus, colon rectum and anus, liver, larynx, lung, cutaneous malignant melanoma, bladder, and thyroid gland). Other malignancies did not show significant sex-related differences (stomach, pancreas, biliary tract, kidney/urinary tract, central nervous system, multiple myeloma, non-Hodgkin lymphoma, and leukemia). Conclusion: In the period 1987-2019, this study revealed sex-related differences in cancer incidence trends. Over time, cancer incidence remained higher in males, with a decreasing epidemiological impact, plausibly resulting from prevention campaigns against environmental cancer risk factors, as tobacco and alcohol. Conversely, a significant decrease was not observed in the incidence trend in females. These findings contribute essential insights for profiling the epidemiological map of cancer in a large Italian population, allowing comparison with other European cancer epidemiology studies and providing updated data supporting sex-related primary and secondary cancer prevention strategies.

Targeting Inhibition of Accumulation and Function of Myeloid-Derived Suppressor Cells by Artemisinin via PI3K/AKT, mTOR, and MAPK Pathways Enhances Anti-PD-L1 Immunotherapy in Melanoma and Liver Tumors.

Despite the remarkable success and efficacy of immune checkpoint blockade (ICB) therapy such as anti-PD-L1 antibody in treating cancers, myeloid-derived suppressor cells (MDSCs) that lead to the formation of the protumor immunosuppressive microenvironment are one of the major contributors to ICB resistance. Therefore, inhibition of MDSC accumulation and function is critical for further enhancing the therapeutic efficacy of anti-PD-L1 antibody in a majority of cancer patients. Artemisinin (ART), the most effective antimalarial drug with tumoricidal and immunoregulatory activities, is a potential option for cancer treatment. Although ART is reported to reduce MDSC levels in 4T1 breast tumor model and improve the therapeutic efficacy of anti-PD-L1 antibody in T cell lymphoma-bearing mice, how ART influences MDSC accumulation, function, and molecular pathways as well as MDSC-mediated anti-PD-L1 resistance in melanoma or liver tumors remains unknown. Here, we reported that ART blocks the accumulation and function of MDSCs by polarizing M2-like tumor-promoting phenotype towards M1-like antitumor one. This switch is regulated via PI3K/AKT, mTOR, and MAPK signaling pathways. Targeting MDSCs by ART could significantly reduce tumor growth in various mouse models. More importantly, the ART therapy remarkably enhanced the efficacy of anti-PD-L1 immunotherapy in tumor-bearing mice through promoting antitumor T cell infiltration and proliferation. These findings indicate that ART controls the functional polarization of MDSCs and targeting MDSCs by ART provides a novel therapeutic strategy to enhance anti-PD-L1 cancer immunotherapy.

Unbalanced IDO1/IDO2 Endothelial Expression and Skewed Keynurenine Pathway in the Pathogenesis of COVID-19 and Post-COVID-19 Pneumonia.

Despite intense investigation, the pathogenesis of COVID-19 and the newly defined long COVID-19 syndrome are not fully understood. Increasing evidence has been provided of metabolic alterations characterizing this group of disorders, with particular relevance of an activated tryptophan/kynurenine pathway as described in this review. Recent histological studies have documented that, in COVID-19 patients, indoleamine 2,3-dioxygenase (IDO) enzymes are differentially expressed in the pulmonary blood vessels, i.e., IDO1 prevails in early/mild pneumonia and in lung tissues from patients suffering from long COVID-19, whereas IDO2 is predominant in severe/fatal cases. We hypothesize that IDO1 is necessary for a correct control of the vascular tone of pulmonary vessels, and its deficiency in COVID-19 might be related to the syndrome's evolution toward vascular dysfunction. The complexity of this scenario is discussed in light of possible therapeutic manipulations of the tryptophan/kynurenine pathway in COVID-19 and post-acute COVID-19 syndromes.

CD66b-CD64dimCD115- cells in the human bone marrow represent neutrophil-committed progenitors.

Here we report the identification of human CD66b-CD64dimCD115- neutrophil-committed progenitor cells (NCPs) within the SSCloCD45dimCD34+ and CD34dim/- subsets in the bone marrow. NCPs were either CD45RA+ or CD45RA-, and in vitro experiments showed that CD45RA acquisition was not mandatory for their maturation process. NCPs exclusively generated human CD66b+ neutrophils in both in vitro differentiation and in vivo adoptive transfer experiments. Single-cell RNA-sequencing analysis indicated NCPs fell into four clusters, characterized by different maturation stages and distributed along two differentiation routes. One of the clusters was characterized by an interferon-stimulated gene signature, consistent with the reported expansion of peripheral mature neutrophil subsets that express interferon-stimulated genes in diseased individuals. Finally, comparison of transcriptomic and phenotypic profiles indicated NCPs represented earlier neutrophil precursors than the previously described early neutrophil progenitors (eNePs), proNeus and COVID-19 proNeus. Altogether, our data shed light on the very early phases of neutrophil ontogeny.

Immune-Guided Therapy of COVID-19.

Vaccination has been a game changer in our efforts to address the coronavirus disease 2019 (COVID-19) pandemic. However, the disease might still represent a clinical crisis for several more years, in part because of the inevitable emergence of variants capable of evading the preexisting immunity. Drugs affecting viral spread will help curtail transmission, but therapeutics are needed to treat the more severe cases requiring hospitalization. A deep analysis of the evolving immune landscape of COVID-19 suggests that understanding the molecular bases of the distinct clinical stages is paramount if we are to limit the burden of inflammation, which can lead to death in frail individuals, according to age, sex, and comorbidities. Different phases can be defined using immune biomarkers and need specific therapeutic approaches, tailored to the underlying immune contexture.

Interrupting the nitrosative stress fuels tumor-specific cytotoxic T lymphocytes in pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest tumors owing to its robust desmoplasia, low immunogenicity, and recruitment of cancer-conditioned, immunoregulatory myeloid cells. These features strongly limit the success of immunotherapy as a single agent, thereby suggesting the need for the development of a multitargeted approach. The goal is to foster T lymphocyte infiltration within the tumor landscape and neutralize cancer-triggered immune suppression, to enhance the therapeutic effectiveness of immune-based treatments, such as anticancer adoptive cell therapy (ACT). METHODS: We examined the contribution of immunosuppressive myeloid cells expressing arginase 1 and nitric oxide synthase 2 in building up a reactive nitrogen species (RNS)-dependent chemical barrier and shaping the PDAC immune landscape. We examined the impact of pharmacological RNS interference on overcoming the recruitment and immunosuppressive activity of tumor-expanded myeloid cells, which render pancreatic cancers resistant to immunotherapy. RESULTS: PDAC progression is marked by a stepwise infiltration of myeloid cells, which enforces a highly immunosuppressive microenvironment through the uncontrolled metabolism of L-arginine by arginase 1 and inducible nitric oxide synthase activity, resulting in the production of large amounts of reactive oxygen and nitrogen species. The extensive accumulation of myeloid suppressing cells and nitrated tyrosines (nitrotyrosine, N-Ty) establishes an RNS-dependent chemical barrier that impairs tumor infiltration by T lymphocytes and restricts the efficacy of adoptive immunotherapy. A pharmacological treatment with AT38 ([3-(aminocarbonyl)furoxan-4-yl]methyl salicylate) reprograms the tumor microenvironment from protumoral to antitumoral, which supports T lymphocyte entrance within the tumor core and aids the efficacy of ACT with telomerase-specific cytotoxic T lymphocytes. CONCLUSIONS: Tumor microenvironment reprogramming by ablating aberrant RNS production bypasses the current limits of immunotherapy in PDAC by overcoming immune resistance.

Fatal cytokine release syndrome by an aberrant FLIP/STAT3 axis.

Inflammatory responses rapidly detect pathogen invasion and mount a regulated reaction. However, dysregulated anti-pathogen immune responses can provoke life-threatening inflammatory pathologies collectively known as cytokine release syndrome (CRS), exemplified by key clinical phenotypes unearthed during the SARS-CoV-2 pandemic. The underlying pathophysiology of CRS remains elusive. We found that FLIP, a protein that controls caspase-8 death pathways, was highly expressed in myeloid cells of COVID-19 lungs. FLIP controlled CRS by fueling a STAT3-dependent inflammatory program. Indeed, constitutive expression of a viral FLIP homolog in myeloid cells triggered a STAT3-linked, progressive, and fatal inflammatory syndrome in mice, characterized by elevated cytokine output, lymphopenia, lung injury, and multiple organ dysfunctions that mimicked human CRS. As STAT3-targeting approaches relieved inflammation, immune disorders, and organ failures in these mice, targeted intervention towards this pathway could suppress the lethal CRS inflammatory state.