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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., (© 2023. The Author(s).)

TIM4 has previously been associated with antitumor immunity, yet the pattern of expression and the function of this receptor across human cancer tissues remain poorly explored. Here we combined extensive immunolabeling of human tissues with in silico analysis of pan-cancer transcriptomic data sets to explore the clinical significance of TIM4 expression. Our results unveil that TIM4 is expressed on a fraction of cavity macrophages (CATIM4+MΦ) of carcinoma patients. Moreover, we uncover a high expression of TIM4 on macrophages of the T-cell zone of the carcinoma-associated tertiary lymphoid structures (TLSTIM4+MΦ). In silico analysis of a pan-cancer data set revealed a positive correlation between TIM4 expression and markers of B cells, effector CD8+ T cells, and a 12-chemokine signature defining tertiary lymphoid structure. In addition, TLSTIM4+MΦ were enriched in cancers displaying microsatellite instability and high CD8+ T-cell infiltration, confirming their association with immune-reactive tumors. Both CATIM4+MΦ and TLSTIM4+MΦ express FOLR2, a marker of tissue-resident MΦ. However, CATIM4+MΦ had a higher expression of the immunosuppressive molecules TREM2, IL10, and TGFβ as compared with TLSTIM4+MΦ. By analyzing a scRNA sequence data set of tumor-associated myeloid cells, we identified two TIM4+FOLR2+ clusters coherent with CATIM4+MΦ and TLSTIM4+MΦ. We defined specific gene signatures for each subset and found that the CATIM4+ MΦ signature was associated with worse patient survival. In contrast, TLSTIM4+MΦ gene signature positively correlates with a better prognosis. Together, these data illustrate that TIM4 marks two distinct macrophage populations with distinct phenotypes and tissue localization and that may have opposing roles in tumor immunity., (©2022 American Association for Cancer Research.)

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-IS 30 ) 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., Competing Interests: SG was employed by Diatech Pharmacogenetics srl and CR was employed by Dr Carola Ries Consulting. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ardighieri, Missale, Bugatti, Gatta, Pezzali, Monti, Gottardi, Zanotti, Bignotti, Ravaggi, Tognon, Odicino, Calza, Missolo-Koussou, Ries, Helft and Vermi.)

The aim of this article is to review a number of variables that may affect the cryopreservation of minimally manipulated products containing allogeneic or autologous hemopoietic progenitor cells (HPC) used for transplantation, with particular reference to processing, type and addition of cryoprotectant, cell concentration, volume, freezing procedure, cooling rate, storage, thawing, and quality management. After defining final product's requirements in compliance with norms, laws and regulations, it is crucial to define the critical control points of the process. New approaches of processing were developed in the last few years such as automatic devices for volume reduction and high cell concentration in the frozen product. DMSO at 10% final concentration is still the most used cryoprotectant for HPC cryopreservation. Although controlled rate freezing is the recommended method for HPC cryopreservation, alternative methods may be used. Last generation vapor storage vessels ensure temperature stability better than older tanks. Their use may reduce risks of cross-contamination. Finally we review advantages and disadvantages of thawing procedures that may be carried out in the laboratory or at the patient's bedside., (Copyright © 2016 Elsevier Ltd. All rights reserved.)