Citation: Di Matteo, S.; Avanzini, M.A.; Pelizzo, G.; Calcaterra, V.; Croce, S.; Spaggiari, G.M.; Theuer, C.; Zuccotti, G.; Moretta, L.; Pelosi, A.; et al. Neuroblastoma Tumor-Associated Mesenchymal Stromal Cells Regulate the Cytolytic Functions of NK Cells. Cancers 2023, 15, 19. https://doi.org/10.3390/ cancers15010019 Academic Editor: Shakeel Modak Received: 16 November 2022 Revised: 12 December 2022 Accepted: 16 December 2022 Published: 20 December 2022 Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). cancers Article Neuroblastoma Tumor-Associated Mesenchymal Stromal Cells Regulate the Cytolytic Functions of NK Cells Sabina Di Matteo 1 , Maria Antonietta Avanzini 2 , Gloria Pelizzo 3,4 , Valeria Calcaterra 5,6 , Stefania Croce 2 , Grazia Maria Spaggiari 7,8 , Charles Theuer 9 , Gianvincenzo Zuccotti 6 , Lorenzo Moretta 1 , Andrea Pelosi 1, * and Bruno Azzarone 1, * 1 Tumor Immunology Unit, Bambino Gesù Children’s Hospital, IRCCS, 00146 Rome, Italy 2 Immunology and Transplantation Laboratory, Pediatric Hematology Oncology Unit, Fondazione IRCCS Policlinico San Matteo, 27100 Pavia, Italy 3 Department of Biomedical and Clinical Science, University of Milan, 20122 Milan, Italy 4 Pediatric Surgery Department, “Vittore Buzzi” Children’s Hospital, 20154 Milan, Italy 5 Department of Internal Medicine, University of Pavia, 27100 Pavia, Italy 6 Pediatric Department, “Vittore Buzzi” Children’s Hospital, 20154 Milan, Italy 7 Department of Experimental Medicine (DIMES), University of Genoa, 16126 Genoa, Italy 8 Laboratory of Clinical and Experimental Immunology, IRCCS Istituto Giannina Gaslini, 16147 Genoa, Italy 9 Tracon Pharmaceuticals, Inc., San Diego, CA 92122, USA * Correspondence: andrea.pelosi@opbg.net (A.P.); bazzarone@hotmail.com (B.A.); Tel.: +39-0668594418(B.A.) Simple Summary: Different components of the tumor microenvironment, such as cancer-associated fibroblasts and tumoral mesenchymal stromal cells, play a major role in cancer progression, metastatic spread and resistance to chemo-immunotherapy. Neuroblastoma tumor-associated mesenchymal stromal cells (NB-TA-MSC) have been extensively characterized for their pro-tumorigenic properties, while their immunosuppressive potential, especially against NK cells, has not been investigated. Herein, we show for the first time that primary young/proliferating NB-TA-MSC, but not senescent ones, are resistant to direct cell lysis by activated NK cells, inhibiting proliferation, cytolytic activity and functional markers of freshly isolated NK cells. NB-TA-MSC express the neuroblastoma marker GD2, the most common target for NB immunotherapy; thus, they represent priority targets whose elimination is essential for improved NB immunotherapy. From a future perspective, in vivo eradica- tion or disabling of NB-TA-MSC could be achieved using monoclonal antibodies directed against targets different from GD2 or inducing their senescence. Abstract: Neuroblastoma tumor-associated mesenchymal stromal cells (NB-TA-MSC) have been extensively characterized for their pro-tumorigenic properties, while their immunosuppressive potential, especially against NK cells, has not been thoroughly investigated. Herein, we study the immune-regulatory potential of six primary young and senescent NB-TA-MSC on NK cell function. Young cells display a phenotype (CD105+/CD90+/CD73+/CD29+/CD146+) typical of MSC cells and, in addition, express high levels of immunomodulatory molecules (MHC-I, PDL-1 and PDL-2 and transcriptional-co-activator WWTR1), able to hinder NK cell activity. Notably, four of them express the neuroblastoma marker GD2, the most common target for NB immunotherapy. From a functional point of view, young NB-TA-MSC, contrary to the senescent ones, are resistant to activated NK cell-mediated lysis, but this behavior is overcome using anti-CD105 antibody TRC105 that activates antibody-dependent cell-mediated cytotoxicity. In addition, proliferating NB-TA-MSC, but not the senescent ones, after six days of co-culture, inhibit proliferation, expression of activating receptors and cytolytic activity of freshly isolated NK. Inhibitors of the soluble immunosuppressive factors L-kynurenine and prostaglandin E2 efficiently counteract this latter effect. Our data highlight the presence of phenotypically heterogeneous NB-TA-MSC displaying potent immunoregulatory properties towards NK cells, whose inhibition could be mandatory to improve the antitumor efficacy of targeted immunotherapy. Cancers 2023, 15, 19. https://doi.org/10.3390/cancers15010019 https://www.mdpi.com/journal/cancers