Tumor-associated neutrophils (TANs) in human carcinoma-draining lymph nodes: a novel TAN compartment.

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.

Plasmacytoid Dendritic Cell Impairment in Metastatic Melanoma by Lactic Acidosis.

The introduction of targeted therapies and immunotherapies has significantly improved the outcome of metastatic melanoma (MM) patients. These approaches rely on immune functions for their anti-melanoma response. Plasmacytoid dendritic cells (pDCs) exhibit anti-tumor function by production of effector molecules, type I interferons (I-IFNs), and cytokines. Tissue and blood pDCs result compromised in MM, although these findings are still partially conflicting. This study reports that blood pDCs were dramatically depleted in MM, particularly in patients with high lactate dehydrogenase (LDH) and high tumor burden; the reduced pDC frequency was associated with poor overall survival. Circulating pDCs resulted also in significant impairment in interferon alpha (IFN-α) and C-X-C motif chemokine 10 (CXCL10) production in response to toll-like receptor (TLR)-7/8 agonists; on the contrary, the response to TLR-9 agonist remained intact. In the BRAFV600+ subgroup, no recovery of pDC frequency could be obtained by BRAF and MEK inhibitors (BRAFi; MEKi), whereas their function was partially rescued. Mechanistically, in vitro exposure to lactic acidosis impaired both pDC viability and function. In conclusion, pDCs from MM patients were found to be severely impaired, with a potential role for lactic acidosis. Short-term responses to treatments were not associated with pDC recovery, suggesting long-lasting effects on their compartment.

Human Plasmacytoid Dendritic Cells and Cutaneous Melanoma.

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.

Collapse of the Plasmacytoid Dendritic Cell Compartment in Advanced Cutaneous Melanomas by Components of the Tumor Cell Secretome.

Melanoma is an immunogenic neoplasm infiltrated by T cells, although these adaptive T cells usually fail to eradicate the tumor. Plasmacytoid dendritic cells (PDCs) are potent regulators of the adaptive immune response and can eliminate melanoma cells via TLR-mediated effector functions. The PDC compartment is maintained by progressively restricted bone marrow progenitors. Terminally differentiated PDCs exit the bone marrow into the circulation, then home to lymph nodes and inflamed peripheral tissues. Infiltration by PDCs is documented in various cancers. However, their role within the melanoma immune contexture is not completely known. We found that in locoregional primary cutaneous melanoma (PCM), PDC infiltration was heterogeneous, occurred early, and was recurrently localized at the invasive margin, the site where PDCs interact with CD8+ T cells. A reduced PDC density was coupled with an increased Breslow thickness and somatic mutations at the NRAS p.Q61 codon. Compared with what was seen in PCM, high numbers of PDCs were found in regional lymph nodes, as also identified by in silico analysis. In contrast, in metastatic melanoma patients, PDCs were mostly absent in the tumor tissues and were significantly reduced in the circulation, particularly in the advanced M1c group. Exposure of circulating PDCs to melanoma cell supernatant (SN-mel) depleted of extracellular vesicles resulted in significant PDC death. SN-mel exposure also resulted in a defect of PDC differentiation from CD34+ progenitors. These findings indicate that soluble components released by melanoma cells support the collapse of the PDC compartment, with clinical implications for refining TLR agonist-based trials.

Phosphocaveolin-1 enforces tumor growth and chemoresistance in rhabdomyosarcoma.

Caveolin-1 (Cav-1) can ambiguously behave as either tumor suppressor or oncogene depending on its phosphorylation state and the type of cancer. In this study we show that Cav-1 was phosphorylated on tyrosine 14 (pCav-1) by Src-kinase family members in various human cell lines and primary mouse cultures of rhabdomyosarcoma (RMS), the most frequent soft-tissue sarcoma affecting childhood. Cav-1 overexpression in the human embryonal RD or alveolar RH30 cells yielded increased pCav-1 levels and reinforced the phosphorylation state of either ERK or AKT kinase, respectively, in turn enhancing in vitro cell proliferation, migration, invasiveness and chemoresistance. In contrast, reducing the pCav-1 levels by administration of a Src-kinase inhibitor or through targeted Cav-1 silencing counteracted the malignant in vitro phenotype of RMS cells. Consistent with these results, xenotransplantation of Cav-1 overexpressing RD cells into nude mice resulted in substantial tumor growth in comparison to control cells. Taken together, these data point to pCav-1 as an important and therapeutically valuable target for overcoming the progression and multidrug resistance of RMS.

Caveolin 1 is a marker of poor differentiation in Rhabdomyosarcoma.

Caveolins consist of three different membrane scaffolding proteins that play a variety of processes in different tissues. In skeletal muscle caveolins are differentially distributed, with Caveolin 1 (Cav-1) being uniquely expressed in satellite cells and Caveolin 3 (Cav-3) in mature myofibers. Rhabdomyosarcoma (RMS) represents the most common childhood soft-tissue sarcoma arising from mesenchimal precursors which fail to undergo proper commitment to muscle lineage. Cav-3 has been proposed as a marker of RMS with a high degree of differentiation, while biological significance of Cav-1 expression in RMS is still a matter of debate. In the present study we show that Cav-1 is predominantly expressed in the embryonal RMS histotype, as further confirmed by transcript and protein analysis in different in vitro human RMS cell lines. Immature cell phenotype of human embryonal RD line, carrying spontaneous activating RAS mutations, was significantly associated to ERK MAPK signalling pathway and featured by high Cav-1 levels, whereas pharmacological attenuation of the ERK pathway, improving cell differentiation, lead to Cav-1 down-regulation. Overall, these data place Cav-1 as a valuable marker of diagnosis for RMS characterised by low degree of differentiation.

Intractable epilepsy secondary to cyclosporine toxicity in children undergoing allogeneic hematopoietic bone marrow transplantation.

The long-term evolution to intractable epilepsy in children treated with cyclosporine administered for graft-versus-host-disease after hematopoietic stem cell transplantation was evaluated. In a group of 185 children treated with cyclosporine after bone marrow transplantation, 15 (8%) presented with acute seizures that were generalized in 7 and focal in 7 and had absence status in 1. Electroencephalography (EEG) and neuroimaging showed predominant abnormalities in the occipital regions. One patient died shortly after the seizure; in seven cases, seizures remitted, whereas relapses were observed in seven others. After the first year, seizures persisted chronically in four cases and evolved to intractable epilepsy. Focal temporal epilepsy was diagnosed in three cases, whereas in the fourth case, a multifocal epilepsy was observed. Magnetic resonance imaging (MRI) detected mesial temporal sclerosis in all of these cases. The risk factors associated with evolution to epilepsy included lower age at transplantation (3-5 years), more than one relapsing seizure in the first year after transplantation, and longer treatment with cyclosporine. Not only can cyclosporine cause acute central nervous system toxicity, it can also determine intractable epilepsy associated with mesial temporal sclerosis.