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Abstract Plasmacytoid dendritic cells (pDCs) are multifaceted immune cells executing various innate immunological functions. Their first line of defence consists in type I interferons (I-IFN) production upon nucleic acids sensing through endosomal Toll-like receptor (TLR) 7- and 9-dependent signalling pathways. Type I IFNs are a class of proinflammatory cytokines that have context-dependent functions on cancer immunosurveillance and immunoediting. In the last few years, different studies have reported that pDCs are also able to sense cytosolic DNA through cGAS–STING (stimulator of interferon genes) pathway eliciting a potent I-IFN production independently of TLR7/9. Human pDCs are also endowed with direct effector functions via the upregulation of TRAIL and production of granzyme B, the latter modulated by cytokines abundant in cancer tissues. pDCs have been detected in a wide variety of human malignant neoplasms, including virus-associated cancers, recruited by chemotactic stimuli. Although the role of pDCs in cancer immune surveillance is still uncompletely understood, their spontaneous activation has been rarely documented; moreover, their presence in the tumor microenvironment (TME) has been associated with a tolerogenic phenotype induced by immunosuppressive cytokines or oncometabolites. Currently tested treatment options can lead to pDCs activation and disruption of the immunosuppressive TME, providing a relevant clinical benefit. On the contrary, the antibody–drug conjugates targeting BDCA-2 on immunosuppressive tumor-associated pDCs (TA-pDCs) could be proposed as novel immunomodulatory therapies to achieve disease control in patients with advance stage hematologic malignancies or solid tumors. This Review integrate recent evidence on the biology of pDCs and their pharmacological modulation, suggesting their relevant role at the forefront of cancer immunity.

Abstract Background Mucosal Melanomas (MM) are highly aggressive neoplasms arising from mucosal melanocytes. Current treatments offer a limited survival benefit for patients with advanced MM; moreover, the lack of pre-clinical cellular systems has significantly limited the understanding of their immunobiology. Methods Five novel cell lines were obtained from patient-derived biopsies of MM arising in the sino-nasal mucosa and designated as SN-MM1-5. The morphology, ultrastructure and melanocytic identity of SN-MM cell lines were validated by transmission electron microscopy and immunohistochemistry. Moreover, in vivo tumorigenicity of SN-MM1-5 was tested by subcutaneous injection in NOD/SCID mice. Molecular characterization of SN-MM cell lines was performed by a mass-spectrometry proteomic approach, and their sensitivity to PI3K chemical inhibitor LY294002 was validated by Akt activation, measured by pAkt(Ser473) and pAkt(Thr308) in immunoblots, and MTS assay. Results This study reports the validation and functional characterization of five newly generated SN-MM cell lines. Compared to the normal counterpart, the proteomic profile of SN-MM is consistent with transformed melanocytes showing a heterogeneous degree of melanocytic differentiation and activation of cancer-related pathways. All SN-MM cell lines resulted tumorigenic in vivo and display recurrent structural variants according to aCGH analysis. Of relevance, the microscopic analysis of the corresponding xenotransplants allowed the identification of clusters of MITF-/CDH1-/CDH2 + /ZEB1 + /CD271 + cells, supporting the existence of melanoma-initiating cells also in MM, as confirmed in clinical samples. In vitro, SN-MM cell lines were sensitive to cisplatin, but not to temozolomide. Moreover, the proteomic analysis of SN-MM cell lines revealed that RICTOR, a subunit of mTORC2 complex, is the most significantly activated upstream regulator, suggesting a relevant role for the PI3K-Akt-mTOR pathway in these neoplasms. Consistently, phosphorylation of NDRG1 and Akt activation was observed in SN-MM, the latter being constitutive and sustained by PTEN loss in SN-MM2 and SN-MM3. The cell viability impairment induced by LY294002 confirmed a functional role for the PI3K-Akt-mTOR pathway in SN-MM cell lines. Conclusions Overall, these novel and unique cellular systems represent relevant experimental tools for a better understanding of the biology of these neoplasms and, as an extension, to MM from other sites.

IntroductionPlasmacytoid dendritic cells (pDCs) infiltrate a large set of human cancers. Interferon alpha (IFN-α) produced by pDCs induces growth arrest and apoptosis in tumor cells and modulates innate and adaptive immune cells involved in anti-cancer immunity. Moreover, effector molecules exert tumor cell killing. However, the activation state and clinical relevance of pDCs infiltration in cancer is still largely controversial. In Primary Cutaneous Melanoma (PCM), pDCs density decreases over disease progression and collapses in metastatic melanoma (MM). Moreover, the residual circulating pDC compartment is defective in IFN-α production.MethodsThe activation of tumor-associated pDCs was evaluated by in silico and microscopic analysis. The expression of human myxovirus resistant protein 1 (MxA), as surrogate of IFN-α production, and proximity ligation assay (PLA) to test dsDNA-cGAS activation were performed on human melanoma biopsies. Moreover, IFN-α and CXCL10 production by in vitro stimulated (i.e. with R848, CpG-A, ADU-S100) pDCs exposed to melanoma cell lines supernatants (SN-mel) was tested by intracellular flow cytometry and ELISA. We also performed a bulk RNA-sequencing on SN-mel-exposed pDCs, resting or stimulated with R848. Glycolytic rate assay was performed on SN-mel-exposed pDCs using the Seahorse XFe24 Extracellular Flux Analyzer.ResultsBased on a set of microscopic, functional and in silico analyses, we demonstrated that the melanoma milieu directly impairs IFN-α and CXCL10 production by pDCs via TLR-7/9 and cGAS-STING signaling pathways. Melanoma-derived immunosuppressive cytokines and a metabolic drift represent relevant mechanisms enforcing pDC-mediated melanoma escape.DiscussionThese findings propose a new window of intervention for novel immunotherapy approaches to amplify the antitumor innate immune response in cutaneous melanoma (CM).

Standardized treatment options are lacking for patients with unresectable or multifocal follicular dendritic cell sarcoma (FDCS) and disease-related mortality is as high as 20%. Applying whole-genome sequencing (WGS) in one case and whole-exome sequencing (WES) in additional twelve cases, this study adds information on the molecular landscape of FDCS, expanding knowledge on pathobiological mechanisms and identifying novel markers of potential theragnostic significance. Massive parallel sequencing showed high frequency of mutations on oncosuppressor genes, particularly in RB1, CARS and BRCA2 and unveiled alterations on homologous recombination DNA damage repair-related genes in 70% (9/13) of cases. This indicates that patients with high-stage FDCS may be eligible for poly ADP ribose polymerase inhibition protocols. Low tumor mutational burden was confirmed in this study despite common PDL1 expression in FDCS arguing on the efficacy of immune checkpoint inhibitors. CDKN2A deletion, detected by WGS and confirmed by fluorescence in situ hybridization in 41% of cases (9/22) indicates that impairment of cell cycle regulation may sustain oncogenesis in FDCS. Absence of mutations in the RAS/RAF/MAPK pathway and lack of clonal hematopoiesis-related mutations in FDCS sanction its differences from dendritic cell-derived neoplasms of hematopoietic derivation. WGS and WES in FDCS provides additional information on the molecular landscape of this rare tumor, proposing novel candidate genes for innovative therapeutical approaches to improve survival of patients with multifocal disease.

Abstract Prostate cancer (PCa) is a leading cause of death in the male population commonly treated with androgen deprivation therapy that often relapses as androgen-independent and aggressive castration-resistant prostate cancer (CRPC). Ferroptosis is a recently described form of cell death that requires abundant cytosolic labile iron to promote membrane lipid peroxidation and which can be induced by agents that inhibit the glutathione peroxidase-4 activity such as RSL3. Exploiting in vitro and in vivo human and murine PCa models and the multistage transgenic TRAMP model of PCa we show that RSL3 induces ferroptosis in PCa cells and demonstrate for the first time that iron supplementation significantly increases the effect of RSL3 triggering lipid peroxidation, enhanced intracellular stress and leading to cancer cell death. Moreover, the combination with the second generation anti-androgen drug enzalutamide potentiates the effect of the RSL3 + iron combination leading to superior inhibition of PCa and preventing the onset of CRPC in the TRAMP mouse model. These data open new perspectives in the use of pro-ferroptotic approaches alone or in combination with enzalutamide for the treatment of PCa.

Abstract Adenoid cystic carcinoma (ACC) of salivary gland is a slowly growing tumor showing a propensity for delayed recurrence, with decreased survival rates. The identification of poor prognosis patients may help in defining molecular-based targeted strategies in this rare disease orphan of new treatments. Through a gene expression microarray-based approach followed by GSE functional analysis the expression profile of 46 primary untreated ACC samples and of ACC (h-TERT) tumor cells was analyzed. Patients who experienced early relapse showed enrichment in proliferation-related gene sets, including the G2-M checkpoint, E2F and myc targets, and in gene sets related to IFN signaling and aberrant proteostasis (FDR

Gastrointestinal graft-versus-host disease (GvHD) is a major cause of mortality and morbidity following allogeneic bone marrow transplantation (allo-BMT). Chemerin is a chemotactic protein that recruits leukocytes to inflamed tissues by interacting with ChemR23/CMKLR1, a chemotactic receptor expressed by leukocytes, including macrophages. During acute GvHD, chemerin plasma levels were strongly increased in allo-BM-transplanted mice. The role of the chemerin/CMKLR1 axis in GvHD was investigated using Cmklr1-KO mice. WT mice transplanted with an allogeneic graft from Cmklr1-KO donors (t-KO) had worse survival and more severe GvHD. Histological analysis demonstrated that the gastrointestinal tract was the organ mostly affected by GvHD in t-KO mice. The severe colitis of t-KO mice was characterized by massive neutrophil infiltration and tissue damage associated with bacterial translocation and exacerbated inflammation. Similarly, Cmklr1-KO recipient mice showed increased intestinal pathology in both allogeneic transplant and dextran sulfate sodium–induced colitis. Notably, the adoptive transfer of WT monocytes into t-KO mice mitigated GvHD manifestations by decreasing gut inflammation and T cell activation. In patients, higher chemerin serum levels were predictive of GvHD development. Overall, these results suggest that CMKLR1/chemerin may be a protective pathway for the control of intestinal inflammation and tissue damage in GvHD.

Abstract Objectives Laryngeal squamous cell carcinomas (LSCCs) typically have an excellent prognosis for stage I tumors but a significant risk of locoregional and distant recurrence for intermediate to advanced disease. This study will investigate the clinical relevance of the tumor microenvironment in a large cohort of treatment‐naïve patients affected by stage II–IV LSCC. Methods Whole slide‐based digital pathology analysis was applied to measure six immune cell populations identified by immunohistochemistry (IHC) staining for CD3, CD8, CD20, CD66b, CD163 and CD38. Survival analysis was performed by Cox proportional hazards models and unsupervised hierarchical clustering using the k‐means method. Double IHC staining and in‐situ hybridisation by RNAscope allowed further analysis of a protumoral B cell population. Results A cohort of 98 patients was enrolled and analysed. The cluster of immune‐infiltrated LSCCs demonstrated a significantly worse disease‐specific survival rate. We also discovered a new association between high CD20+ B cells and a greater risk of distant recurrence. The phenotypic analysis of infiltrating CD20+ B cells showed a naïve (BCL6−CD27−Mum1−) regulatory phenotype, producing TGFβ but not IL10, according to an active TGFβ pathway, as proved by positive pSMAD2 staining. Conclusion The identification of regulatory B cells in the context of LSCC, along with the activation of the TGFβ pathway, could provide the basis for new trials investigating the efficacy of already available molecules targeting the TGFβ pathway in the treatment of LSCC.

UNC5B is a Netrin-1 receptor expressed in endothelial cells that in the absence of ligand induces apoptosis. Here the authors identify an UNC5B splicing isoform that is insensitive to the pro-survival ligand Netrin-1 and is required for apoptosis-dependent blood vessel development.

Colorectal carcinoma (CRC) represents a lethal disease with heterogeneous outcomes. Only patients with mismatch repair (MMR) deficient CRC showing microsatellite instability and hyper-mutated tumors can obtain clinical benefits from current immune checkpoint blockades; on the other hand, immune- or target-based therapeutic strategies are very limited for subjects with mismatch repair proficient CRC (CRCpMMR). Here, we report a comprehensive typing of immune infiltrating cells in CRCpMMR. We also tested the expression and interferon-γ-modulation of PD-L1/CD274. Relevant findings were subsequently validated by immunohistochemistry on fixed materials. CRCpMMR contain a significantly increased fraction of CD163+ macrophages (TAMs) expressing TREM2 and CD66+ neutrophils (TANs) together with decrease in CD4−CD8−CD3+ double negative T lymphocytes (DNTs); no differences were revealed by the analysis of conventional and plasmacytoid dendritic cell populations. A fraction of tumor-infiltrating T-cells displays an exhausted phenotype, co-expressing PD-1 and TIM-3. Remarkably, expression of PD-L1 on fresh tumor cells and TAMs was undetectable even after in vitro stimulation with interferon-γ. These findings confirm the immune suppressive microenvironment of CRCpMMR characterized by dense infiltration of TAMs, occurrence of TANs, lack of DNTs, T-cell exhaustion, and interferon-γ unresponsiveness by host and tumor cells. Appropriate bypass strategies should consider these combinations of immune escape mechanisms in CRCpMMR.

Angiogenesis is crucial for cancer progression. While several anti-angiogenic drugs are in use for cancer treatment, their clinical benefits are unsatisfactory. Thus, a deeper understanding of the mechanisms sustaining cancer vessel growth is fundamental to identify novel biomarkers and therapeutic targets. Alternative splicing (AS) is an essential modifier of human proteome diversity. Nevertheless, AS contribution to tumor vasculature development is poorly known. The Neuro-Oncological Ventral Antigen 2 (NOVA2) is a critical AS regulator of angiogenesis and vascular development. NOVA2 is upregulated in tumor endothelial cells (ECs) of different cancers, thus representing a potential driver of tumor blood vessel aberrancies. Here, we identified novel AS transcripts generated upon NOVA2 upregulation in ECs, suggesting a pervasive role of NOVA2 in vascular biology. In addition, we report that NOVA2 is also upregulated in ECs of gastric cancer (GC), and its expression correlates with poor overall survival of GC patients. Finally, we found that the AS of the Rap Guanine Nucleotide Exchange Factor 6 (RapGEF6), a newly identified NOVA2 target, is altered in GC patients and associated with NOVA2 expression, tumor angiogenesis, and poor patient outcome. Our findings provide a better understanding of GC biology and suggest that AS might be exploited to identify novel biomarkers and therapeutics for anti-angiogenic GC treatments.

Acquisition of dying tumor cell-associated antigens is an essential step for the initiation of anti-tumor immune response by conventional type 1 dendritic cells (cDC1). Here the authors show that the loss of TIM4 expression in lung tumor associated cDC1 is associated with less efficient uptake of cell associated antigens and reduction of CD8 + T cell activation in advanced lung tumors.

Crohn’s disease results from transmural inflammation in the gut, but analyses of local immune populations are still lacking. Here, the authors show, by combining multiple single-cell approaches, that intraepithelial and lamina propria T cells are heterogenous, show unique phenotypes, and exhibit altered subsets upon inflammation.

Histopathologic assessment of high-risk endometrial cancer (EC) suffers from intersubject variability and poor reproducibility. The pragmatic classification in four molecular subgroups helps to overcome these limits, showing a significant prognostic value. The “no specific molecular profile” (NSMP) is the most heterogeneous EC subgroup, requiring further characterization to better guide its clinical management. DNA sequencing of POLE exonuclease domain and immunohistochemistry for PMS2, MSH6, and p53 were performed in order to stratify a cohort of 94 high-risk EC patients in the four molecular subgroups. Moreover, a panel of seven additional biomarkers was tested. Patients were found to be 16% POLE-mutated, 36% mismatch repair-deficient, 27% p53-abnormal, and 21% NSMP. In the multivariable model, molecular groups confirmed their significant association with disease-specific survival and progression-free survival, with p53-abnormal and NSMP endometrial cancer characterized by poor outcomes. Among the additional evaluated biomarkers, L1CAM was the only one with a significant prognostic value within the NSMP subgroup. NSMP/L1CAM-positive patients experienced the worst outcome and were “early-relapsing” after platinum-based chemotherapy, with a significantly shorter platinum-free interval compared to L1CAM-negative patients. L1CAM appears to be a promising candidate as a prognostic and predictive biomarker in the high-risk NSMP subgroup, which is actually known to lack specific molecular markers.

Macrophages are the most abundant immune cells of the tumor microenvironment (TME) and have multiple important functions in cancer. During tumor growth, both tissue-resident macrophages and newly recruited monocyte-derived macrophages can give rise to tumor-associated macrophages (TAMs), which have been associated with poor prognosis in most cancers. Compelling evidence indicate that the high degree of plasticity of macrophages and their ability to self-renew majorly impact tumor progression and resistance to therapy. In addition, the microenvironmental factors largely affect the metabolism of macrophages and may have a major influence on TAMs proliferation and subsets functions. Thus, understanding the signaling pathways regulating TAMs self-renewal capacity may help to identify promising targets for the development of novel anticancer agents. In this review, we focus on the environmental factors that promote the capacity of macrophages to self-renew and the molecular mechanisms that govern TAMs proliferation. We also highlight the impact of tumor-derived factors on macrophages metabolism and how distinct metabolic pathways affect macrophage self-renewal.

Ovarian carcinomas (OCs) are poorly immunogenic and immune checkpoint inhibitors (ICIs) have offered a modest benefit. In this study, high CD3+ T-cells and CD163+ tumor-associated macrophages (TAMs) densities identify a subgroup of immune infiltrated high-grade serous carcinomas (HGSCs) with better outcomes and superior response to platinum-based therapies. On the contrary, in most clear cell carcinomas (CCCs) showing poor prognosis and refractory to platinum, a high TAM density is associated with low T cell frequency. Immune infiltrated HGSC are characterized by the 30-genes signature (OC-IS30) covering immune activation and IFNγ polarization and predicting good prognosis (n = 312, TCGA). Immune infiltrated HGSC contain CXCL10 producing M1-type TAM (IRF1+pSTAT1Y701+) in close proximity to T-cells. A fraction of these M1-type TAM also co-expresses TREM2. M1-polarized TAM were barely detectable in T-cell poor CCC, but identifiable across various immunogenic human cancers. Single cell RNA sequencing data confirm the existence of a tumor-infiltrating CXCL10+IRF1+STAT1+ M1-type TAM overexpressing antigen processing and presentation gene programs. Overall, this study highlights the clinical relevance of the CXCL10+IRF1+STAT1+ macrophage subset as biomarker for intratumoral T-cell activation and therefore offers a new tool to select patients more likely to respond to T-cell or macrophage-targeted immunotherapies.

Pulmonary fibrosis is a progressive scarring disease of the lungs, characterized by inflammation, fibroblast activation, and deposition of extracellular matrix. The long pentraxin 3 (PTX3) is a member of the pentraxin family with non-redundant functions in innate immune responses, tissue repair, and haemostasis. The role played in the lungs by PTX3 during the fibrotic process has not been elucidated. In this study, the impact of PTX3 expression on lung fibrosis was assessed in an intratracheal bleomycin (BLM)-induced murine model of the disease applied to wild type animals, transgenic mice characterized by endothelial overexpression and stromal accumulation of PTX3 (Tie2-PTX3 mice), and genetically deficient Ptx3−/− animals. Our data demonstrate that PTX3 is produced during BLM-induced fibrosis in wild type mice, and that PTX3 accumulation in the stroma compartment of Tie2-PTX3 mice limits the formation of fibrotic tissue in the lungs, with reduced fibroblast activation and collagen deposition, and a decrease in the recruitment of the immune infiltrate. Conversely, Ptx3-null mice showed an exacerbated fibrotic response and decreased survival in response to BLM treatment. These results underline the protective role of endogenous PTX3 during lung fibrosis and pave the way for the study of novel PTX3-derived therapeutic approaches to the disease.

The endothelin-1 (ET-1) receptors were recently found to mediate pro-survival functions in multiple myeloma (MM) cells in response to autocrine ET-1. This study investigated the effectiveness of macitentan, a dual ET-1 receptor antagonist, in MM treatment, and the mechanisms underlying its activities. Macitentan affected significantly MM cell (RPMI-8226, U266, KMS-12-PE) survival and pro-angiogenic cytokine release by down-modulating ET-1-activated MAPK/ERK and HIF-1α pathways, respectively. HIF-1α silencing abrogated the ET-1 mediated induction of genes encoding for pro-angiogenic cytokines such as VEGF-A, IL-8, Adrenomedullin, and ET-1 itself. Upon exposure to macitentan, MM cells cultured in the presence of the hypoxia-mimetic agent CoCl2, exogenous ET-1, or CoCl2 plus ET-1, down-regulated HIF-1α and the transcription and release of downstream pro-angiogenic cytokines. Consistently, macitentan limited significantly the basal pro-angiogenic activity of RPMI-8226 cells in chorioallantoic membrane assay. In xenograft mouse models, established by injecting NOG mice either via intra-caudal vein with U266 or subcutaneously with RPMI-8226 cells, macitentan reduced effectively the number of MM cells infiltrating bone marrow, and the size and microvascular density of subcutaneous MM tumors. ET-1 receptors targeting by macitentan represents an effective anti-proliferative and anti-angiogenic therapeutic approach in preclinical settings of MM.

Abstract Objectives The role of tumor‐associated neutrophils (TANs) in the nodal spread of cancer cells remains unexplored. The present study evaluates the occurrence and clinical significance of human nodal TANs. Methods The relevance, derivation, phenotype and interactions of nodal TANs were explored via a large immunohistochemical analysis of carcinoma‐draining lymph nodes, and their clinical significance was evaluated on a retrospective cohort of oral squamous cell carcinomas (OSCC). The tumor‐promoting function of nodal TAN was probed in the OSCC TCGA dataset combining TAN and epithelial‐to‐mesenchymal transition (EMT) signatures. Results The pan‐carcinoma screening identified a consistent infiltration (59%) of CD66b+ TANs in tumor‐draining lymph nodes (TDLNs). Microscopic findings, including the occurrence of intra‐lymphatic conjugates of TANs and cancer cells, indicate that TANs migrate through lymphatic vessels. In vitro experiments revealed that OSCC cell lines sustain neutrophil viability and activation via release of GM‐CSF. Moreover, by retrospective analysis, a high CD66b+ TAN density in M‐TDLNs of OSCC (n = 182 patients) predicted a worse prognosis. The analysis of the OSCC‐TCGA dataset unveiled that the expression of a set of neutrophil‐specific genes in the primary tumor (PT) is highly associated with an EMT signature, which predicts nodal spread. Accordingly, in the PT of OSCC cases, CD66b+TANs co‐localised with PDPN+S100A9− EMT‐switched tumor cells in areas of lymphangiogenesis. The pro‐EMT signature is lacking in peripheral blood neutrophils from OSCC patients, suggesting tissue skewing of TANs. Conclusion Our findings are consistent with a novel pro‐tumoral TAN compartment that may promote nodal spread via EMT, through the lymphatics.

Background: . The occurrence of trans-placental transmission of severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) infection remains highly debated. Placental positivity for SARS-CoV-2 has been reported in selected cases, but infection or virus-associated disease of fetal tissues or newborns remains to be demonstrated. Methods: We screened for SARS-CoV-2 spike (S) protein expression placentas from 101 women who delivered between February 7 and May 15, 2020, including 15 tested positive for SARS-CoV-2 RNA, 34 tested negative, and 52 not evaluated as they did not meet testing criteria (32), or delivered before COVID-19 pandemic declaration (20). Immunostain for SARS-CoV-2 nucleocapsid (N) was performed in the placentas of all COVID-19 positive women. One placenta resulted positive for the SARS-CoV-2 S and N proteins, which was further studied by RNA-in situ hybridization and RT-PCR for S transcripts, and by electron microscopy. A comprehensive immunohistochemical and immunofluorescence analysis of the placental inflammatory infiltrate completed the investigations. Findings: SARS-CoV-2 S and N proteins were strongly expressed in the placenta of a COVID-19 pregnant woman whose newborn tested positive for viral RNA and developed COVID-19 pneumonia soon after birth. SARS-CoV-2 antigens, RNA and/or particles morphologically consistent with coronavirus were identified in villous syncytiotrophoblast, endothelial cells, fibroblasts, in maternal macrophages, and in Hofbauer cells and fetal intravascular mononuclear cells. The placenta intervillous inflammatory infiltrate consisted of neutrophils and monocyte-macrophages expressing activation markers. Absence of villitis was associated with an increase in the number of Hofbauer cells, which expressed PD-L1. Scattered neutrophil extracellular traps (NETs) were identified by immunofluorescence. Interpretation: We provide first-time evidence for maternal-fetal transmission of SARS-CoV-2, likely propagated by circulating virus-infected fetal mononuclear cells. Placenta infection was associated with recruitment of maternal inflammatory cells in the intervillous space, without villitis. PD-L1 expression in syncytiotrophoblast and Hofbaeur cells, together with limited production of NETs, may have prevented immune cell-driven placental damage, ensuring sufficient maternal-fetus nutrient exchanges.

High-grade serous ovarian carcinoma (HGSOC) usually spreads directly into the peritoneal cavity following a transcoelomic dissemination route, although distant hematogenous metastasis exist and have been reported. However, no tumor markers can currently predict the risk of distant metastases in HGSOC. Claudins, belonging to tight-junction proteins, are dysregulated in HGSOC and functionally related to cancer progression. Here we analyzed claudin-3, -4, and -7 expression as potential markers of distant metastases. Using quantitative RT-PCR and immunohistochemistry we assessed the expression of claudins in primary HGSOC tissues, normal ovarian, and normal fallopian tube epithelia and correlated it with clinicopathological features, including the site of metastasis and the route of dissemination. Gene set enrichment analysis was performed on microarray-generated gene expression data to investigate key pathways in patients with distant metastases. We found the overall expression level of claudin-3, -4, and -7 mRNA decreased in HGSOC compared to normal tubal epithelium, currently considered the potential site of origin of many HGSOC. The reduced expression of claudin-7 is significantly associated with the development of distant metastases (p = 0.016), mainly by hematogenous route (p = 0.025). In patients with diminished expression of claudin-7, immunohistochemical staining revealed a heterogeneous pattern of membranous staining with discontinuous expression of claudin-7 along the cell border, indicative of a dischoesive architecture. The estimated reduction in the probability of distant disease is of 39% per unit increase in the level of claudin-7 (p = 0.03). Genes involved in epithelial to mesenchymal transition, hypoxia, and angiogenesis processes resulted strongly associated to hematogenous recurrence. Our data suggest a potential role of claudin-7 in discriminating distant metastatic events in HGSOC patients. The quantification of its expression levels could be a useful tool to identify patient deserving a personalized follow-up in terms of clinical and radiological assessment.

BRAF is one of the most common mutated kinases detected in human cancer, particularly in cases of primary cutaneous melanomas (PCM). Mutations of the BRAF proto-oncogene, at the p.V600 codon, has been detected in more than 50% of primary and metastatic melanoma cells in clinical samples. In addition to the most frequent BRAF p.V600E mutation, corresponding to the single base pair substitution c.1799T>A, rarer mutations, within and outside the V600 codon, have been described. Expectedly, BRAF and MEK inhibitors (or their combination) have been poorly explored as potential therapeutic strategies in metastatic melanomas harboring this rare mutation. By using a set of sequencing techniques and immunohistochemistry, this work reports the genomic and clinical features of two melanoma patients showing a rare complex mutation affecting codon V600 and K601 of the BRAF gene, leading to a V600E2; K601I change. Specifically, these two patients show a distinct clinical behavior and significantly differ in their responses to BRAF and MEK inhibitors. Indeed, although this treatment has proven to be effective and safe in both cases, the observed variability between the two patients resulted as a direct consequence of the baseline extent of brain involvement, intracranial treatment failure as well as on the PTEN status.

Oral cavity squamous cell carcinoma (OSCC) is a common head and neck cancer characterized by a poor prognosis associated with locoregional or distant failure. Among the predictors of prognosis, a dense infiltration of adaptive immune cells is protective and associated with improved clinical outcomes. However, few tools are available to integrate immune contexture variables into clinical settings. By using digital microscopy analysis of a large retrospective OSCC cohort (n = 182), we explored the clinical significance of tumor-infiltrating CD8+ T-cells. To this end, CD8+ T-cells counts were combined with well-established clinical variables and peripheral blood immune cell parameters. Through variable clustering, five metavariables (MV) were obtained and included descriptors of nodal (NODALMV) and primary tumor (TUMORMV) involvement, the frequency of myeloid (MYELOIDMV) or lymphoid (LYMPHOIDMV) peripheral blood immune cell populations, and the density of tumor-infiltrating CD8+ T-cells (TI-CD8MV). The clinical relevance of the MV was evaluated in the multivariable survival models. The NODALMV was significantly associated with all tested outcomes (p < 0.001), the LYMPHOIDMV showed a significant association with the overall, disease-specific and distant recurrence-free survival (p < 0.05) and the MYELOIDMV with the locoregional control only (p < 0.001). Finally, TI-CD8MV was associated with distant recurrence-free survival (p = 0.029). Notably, the performance in terms of survival prediction of the combined effect of NODALMV and immune metavariables (LYMPHOIDMV, MYELOIDMV and TI-CD8MV) was superior to the TNM stage for most of the outcomes analyzed. These findings indicate that the analysis of the baseline host immune features are promising tools to complement clinical features, in stratifying the risk of recurrences.

A subset of placentas from pregnant women having the SARS-CoV-2 infection have been found to be infected with the coronavirus using molecular pathology methods including immunohistochemistry and RNA in situ hybridization. These infected placentas can demonstrate several unusual findings which occur together—chronic histiocytic intervillositis, trophoblast necrosis and positive staining of the syncytiotrophoblast for SARS-CoV-2. They frequently also have increased fibrin deposition, which can be massive in some cases. Syncytiotrophoblast is the most frequent fetal-derived cell type to be positive for SARS-CoV-2. It has recently been shown that in a small number of infected placentas, villous stromal macrophages, termed Hofbauer cells, and villous capillary endothelial cells can also stain positive for SARS-CoV-2. This report describes a placenta from a pregnant woman with SARS-CoV-2 that had chronic histiocytic intervillositis, trophoblast necrosis, increased fibrin deposition and positive staining of the syncytiotrophoblast for SARS-CoV-2. In addition, molecular pathology testing including RNAscope and immunohistochemistry for SARS-CoV-2 and double-staining immunohistochemistry using antibodies to E-cadherin and GATA3 revealed that cytotrophoblast cells stained intensely for SARS-CoV-2. All of the cytotrophoblast cells that demonstrated positive staining for SARS-CoV-2 were in direct physical contact with overlying syncytiotrophoblast that also stained positive for the virus. The pattern of cytotrophoblast staining for SARS-CoV-2 was patchy, and there were chorionic villi having diffuse positive staining of the syncytiotrophoblast for SARS-CoV-2, but without staining of cytotrophoblast. This first detailed description of cytotrophoblast involvement by SARS-CoV-2 adds another fetal cell type from infected placentas that demonstrate viral staining.

Abstract Background Epithelial ovarian cancer (EOC) is a spectrum of different diseases, which makes their treatment a challenge. Forkhead box M1 (FOXM1) is an oncogene aberrantly expressed in many solid cancers including serous EOC, but its role in non-serous EOCs remains undefined. We examined FOXM1 expression and its correlation to prognosis across the three major EOC subtypes, and its role in tumorigenesis and chemo-resistance in vitro. Methods Gene signatures were generated by microarray for 14 clear-cell and 26 endometrioid EOCs, and 15 normal endometrium snap-frozen biopsies. Validation of FOXM1 expression was performed by RT–qPCR and immunohistochemistry in the same samples and additionally in 50 high-grade serous EOCs and in their most adequate normal controls (10 luminal fallopian tube and 20 ovarian surface epithelial brushings). Correlations of FOXM1 expression to clinic-pathological parameters and patients’ prognosis were evaluated by Kaplan-Meier and Cox proportional-hazards analyses. OVCAR-3 and two novel deeply characterized EOC cell lines (EOC-CC1 and OSPC2, with clear-cell and serous subtype, respectively) were employed for in vitro studies. Effects of FOXM1 inhibition by transient siRNA transfection were evaluated on cell-proliferation, cell-cycle, colony formation, invasion, and response to conventional first- and second-line anticancer agents, and to the PARP-inhibitor olaparib. Gene signatures of FOXM1-silenced cell lines were generated by microarray and confirmed by RT-qPCR. Results A significant FOXM1 mRNA up-regulation was found in EOCs compared to normal controls. FOXM1 protein overexpression significantly correlated to serous histology (p = 0.001) and advanced FIGO stage (p = 0.004). Multivariate analyses confirmed FOXM1 protein overexpression as an independent indicator of worse disease specific survival in non-serous EOCs, and of shorter time to progression in platinum-resistant cases. FOXM1 downregulation in EOC cell lines inhibited cell growth and clonogenicity, and promoted the cytotoxic effects of platinum compounds, doxorubicin hydrochloride and olaparib. Upon FOXM1 knock-down in EOC-CC1 and OSPC2 cells, microarray and RT-qPCR analyses revealed the deregulation of several common and other unique subtype-specific FOXM1 putative targets involved in cell cycle, metastasis, DNA repair and drug response. Conclusions FOXM1 is up-regulated in all three major EOCs subtypes, and is a prognostic biomarker and a potential combinatorial therapeutic target in platinum resistant disease, irrespective of tumor histology.

Lung cancer represents an extremely diffused neoplastic disorder with different histological/molecular features. Among the different lung tumors, non-small-cell lung cancer (NSCLC) is the most represented histotype, characterized by various molecular markers, including the expression/overexpression of the fibroblast growth factor receptor-1 (FGFR1). Thus, FGF/FGFR blockade by tyrosine kinase inhibitors (TKi) or FGF-ligand inhibitors may represent a promising therapeutic approach in lung cancers. In this study we demonstrate the potential therapeutic benefit of targeting the FGF/FGFR system in FGF-dependent lung tumor cells using FGF trapping (NSC12) or TKi (erdafitinib) approaches. The results show that inhibition of FGF/FGFR by NSC12 or erdafitinib induces apoptosis in FGF-dependent human squamous cell carcinoma NCI-H1581 and NCI-H520 cells. Induction of oxidative stress is the main mechanism responsible for the therapeutic/pro-apoptotic effect exerted by both NSC12 and erdafitinib, with apoptosis being abolished by antioxidant treatments. Finally, reduction of c-Myc protein levels appears to strictly determine the onset of oxidative stress and the therapeutic response to FGF/FGFR inhibition, indicating c-Myc as a key downstream effector of FGF/FGFR signaling in FGF-dependent lung cancers.

Estrogen receptor alpha-positive (ERα+) luminal tumors are the most frequent subtype of breast cancer. Stat1−/− mice develop mammary tumors that closely recapitulate the biological characteristics of this cancer subtype. To identify transforming events that contribute to tumorigenesis, we performed whole genome sequencing of Stat1−/− primary mammary tumors and matched normal tissues. This investigation identified somatic truncating mutations affecting the prolactin receptor (PRLR) in all tumor and no normal samples. Targeted sequencing confirmed the presence of these mutations in precancerous lesions, indicating that this is an early event in tumorigenesis. Functional evaluation of these heterozygous mutations in Stat1−/− mouse embryonic fibroblasts showed that co-expression of truncated and wild-type PRLR led to aberrant STAT3 and STAT5 activation downstream of the receptor, cellular transformation in vitro, and tumor formation in vivo. In conclusion, truncating mutations of PRLR promote tumor growth in a model of human ERα+ breast cancer and warrant further investigation.

Background: Intestinal metaplasia/differentiation in primary endometrial carcinomas is an uncommon phenomenon, with only few cases described. Material and Methods: We performed a systematic review of endometrial carcinomas with intestinal metaplasia/differentiation interrogating the electronic databases Pubmed, Web of Science, and Scopus, and we reported an additional case arising in a 49-year-old woman. Results: We identified only eight patients diagnosed with endometrial carcinomas exhibiting intestinal metaplasia/differentiation, and additionally our case. Endometrial carcinomas with intestinal-type features can present in pure or mixed forms in association with usual-type endometrioid carcinomas; in mixed forms, the two neoplastic components may derive from a common neoplastic progenitor, as evidenced by the concomitant loss of MSH2 and MSH6 protein expression in our case. Disease recurrences occur in a significant fraction of the cases, including patients diagnosed in low-stage disease. Conclusions: Endometrial carcinomas with intestinal metaplasia/differentiation are rare and they may represent a more aggressive tumor variant, thus requiring a proper treatment despite the low-tumor stage. The ProMise classification should be performed also in these unusual tumors, since they can be associated with mismatch repair system defects.

The prognosis of metastatic melanoma (MM) patients has remained poor for a long time. However, the recent introduction of effective target therapies (BRAF and MEK inhibitors for BRAFV600-mutated MM) and immunotherapies (anti-CTLA-4 and anti-PD-1) has significantly improved the survival of MM patients. Notably, all these responses are highly dependent on the fitness of the host immune system, including the innate compartment. Among immune cells involved in cancer immunity, properly activated plasmacytoid dendritic cells (pDCs) exert an important role, bridging the innate and adaptive immune responses and directly eliminating cancer cells. A distinctive feature of pDCs is the production of high amount of type I Interferon (I-IFN), through the Toll-like receptor (TLR) 7 and 9 signaling pathway activation. However, published data indicate that melanoma-associated escape mechanisms are in place to hijack pDC functions. We have recently reported that pDC recruitment is recurrent in the early phases of melanoma, but the entire pDC compartment collapses over melanoma progression. Here, we summarize recent advances on pDC biology and function within the context of melanoma immunity.

Background: Urothelial bladder cancers (UBCs) are distinct in two main molecular subtypes, namely basal and luminal type. Subtypes are also diverse in term of immune contexture, providing a rationale for patient selection to immunotherapy. Methods: By digital microscopy analysis of a muscle-invasive BC (MIBC) cohort, we explored the density and clinical significance of CD66b+ tumor-associated-neutrophils (TAN) and CD3+ T cells. Bioinformatics analysis of UBC datasets and gene expression analysis of UBC cell lines were additionally performed. Results: Basal type BC contained a significantly higher density of CD66b+ TAN compared to the luminal type. This finding was validated on TCGA, GSE32894 and GSE124305 datasets by computing a neutrophil signature. Of note, basal-type MIBC display a significantly higher level of chemokines (CKs) attracting neutrophils. Moreover, pro-inflammatory stimuli significantly up-regulate CXCL1, CXCL2 and CXCL8 in 5637 and RT4 UBC cell lines and induce neutrophil chemotaxis. In term of survival, a high density of T cells and TAN was significantly associated to a better outcome, with TAN density showing a more limited statistical power and following a non-linear predicting model. Conclusions: TAN are recruited in basal type MIBC by pro-inflammatory CKs. This finding establishes a groundwork for a better understanding of the UBC immunity and its relevance.

During pregnancy, immune cells infiltrate the placenta at different stages of fetal development. NK cells and macrophages are the most predominant cell types. These immune cells play pleiotropic roles, as they control spiral artery remodeling to ensure appropriate blood supply and maintain long-term tolerance to a true allograft; yet, they must be able to mount appropriate immune defenses to pathogens that may threaten the fetus. Whether the same cell type accomplishes all these tasks or if there are dedicated subsets remains controversial. Here, we identify and characterize two distinct subsets of myeloid cells that differ in their pro-inflammatory/regulatory capacity. While one subset predominantly produces the immune-modulating cytokine IL-10, the second subset has superior capacity to secrete pro-inflammatory mediators, such as IL-1β and IL-6. The putative regulatory myeloid cells also express high levels of inhibitory receptors and their ligands, including programmed cell death 1 (PD1) ligands. Importantly, a large fraction of CD8 and CD4 cells in normal term human placenta are PD1 positive, suggesting that the PD1/PD1 ligands axis might be critical to maintain tolerance during pregnancy.

Bladder tumors are a diffuse type of cancer. Long pentraxin-3 (PTX3) is a component of the innate immunity with pleiotropic functions in the regulation of immune response, tissue remodeling, and cancer progression. PTX3 may act as an oncosuppressor in different contexts, functioning as an antagonist of the fibroblast growth factor/fibroblast growth factor receptor (FGF/FGFR) system, rewiring the immune microenvironment, or acting through mechanisms not yet fully clarified. In this study we used biopsies and data mining to assess that PTX3 is differentially expressed during the different stages of bladder cancer (BC) progression. BC cell lines, representative of different tumor grades, and transgenic/carcinogen-induced models were used to demonstrate in vitro and in vivo that PTX3 production by tumor cells decreases along the progression from low-grade to high-grade advanced muscle invasive forms (MIBC). In vitro and in vivo data revealed for the first time that PTX3 modulation and the consequent impairment of FGF/FGR systems in BC cells have a significant impact on different biological features of BC growth, including cell proliferation, motility, metabolism, stemness, and drug resistance. PTX3 exerts an oncosuppressive effect on BC progression and may represent a potential functional biomarker in BC evolution. Moreover, FGF/FGFR blockade has an impact on drug resistance and stemness features in BC.

Urothelial bladder cancer (UBC) are classified into luminal and basal subtypes showing distinct molecular features and clinical behaviour. Recent in silico data have proposed the activation on the Signal Transducer and Activator of Transcription 3 (STAT3) as relevant transcription factor in UBC. To answer this question, we have combined the retrospective analysis of clinical samples, functional assays on cell lines, interrogation of public UBC datasets and a murine model of basal-type UBC. Immunohistochemistry on a retrospective UBC cohort uncovered that STAT3 Y705 phosphorylation (pSTAT3) is significantly increased in infiltrating basal-type UBC compared to luminal UBC. In vitro, STAT3 silencing in UBC cell lines significantly reduced tumor cell viability and invasion. Gene expression profile of UBC cell lines combined with the analysis of the Cancer Genome Atlas (TCGA) and GSE32894 UBC datasets showed that increased expression of a set of STAT3 targets predicts basal-type, propensity to local progression and worse prognosis. MYC and FOSL1 represent relevant STAT3 downstream targets, as validated by their co-localization in pSTAT3+ UBC cancer cells. These findings were largely reproduced in the BBN-induced murine model of basal-type UBC. Of note, FOSL1 protein resulted strongly expressed in the non-papillary UBC pathway and FOSL1-regulated transcripts were significantly enriched in the transition from NMIBC to MIBC, as indicated by the interrogation of the GSE32894 dataset. The blockade of the STAT3 pathway might represent a novel treatment option for these neoplasms. Monitoring pSTAT3 and the downstream targets, particularly FOSL1, could provide meaningful levels of UBC stratification.

PGE2 is a lipid mediator abundantly produced in inflamed tissues that exerts relevant immunoregulatory functions. Dendritic cells (DCs) are key players in the onset and shaping of the inflammatory and immune responses and, as such, are well known PGE2 targets. By contrast, the precise role of human DCs in the production of PGE2 is poorly characterized. Here, we asked whether different ligands of Toll-like receptors (TLRs), a relevant family of pathogen-sensing receptors, could induce PGE2 in human DCs. The only active ligands were LPS (TLR4 ligand) and R848 (TLR7-8 ligand) although all TLRs, but TLR9, were expressed and functional. While investigating the molecular mechanisms hindering the release of PGE2, our experiments highlighted so far oversight differences in TLR signalling pathways in terms of MAPK and NF-κB activation. In addition, we identified that the PGE2-limiting checkpoint downstream TLR3, TLR5, and TLR7 was a defect in COX2 induction, while TLR1/2 and TLR2/6 failed to mobilize arachidonic acid, the substrate for the COX2 enzyme. Finally, we demonstrated the in vivo expression of PGE2 by myeloid CD11c+ cells, documenting a role for DCs in the production of PGE2 in human inflamed tissues.