Combined mitoxantrone and anti-TGFβ treatment with PD-1 blockade enhances antitumor immunity by remodelling the tumor immune landscape in neuroblastoma

(2022) 41:326 Lucarini et al. J Exp Clin Cancer Res https://doi.org/10.1186/s13046-022-02525-9 Open Access RESEARCH Combined mitoxantrone and anti‑TGFβ treatment with PD‑1 blockade enhances antitumor immunity by remodelling the tumor immune landscape in neuroblastoma Valeria Lucarini1†, Ombretta Melaiu1,2†, Silvia D’Amico1, Fabio Pastorino3, Patrizia Tempora1, Marco Scarsella4, Marco Pezzullo5, Adele De Ninno6, Valentina D’Oria7, Michele Cilli8, Laura Emionite8, Paola Infante9, Lucia Di Marcotullio9, Maria Antonietta De Ioris1, Giovanni Barillari2, Rita Alaggio10, Luca Businaro6, Mirco Ponzoni3, Franco Locatelli1,11 and Doriana Fruci1*    Abstract Background: Poor infiltration of functioning T cells renders tumors unresponsive to checkpoint-blocking immunotherapies. Here, we identified a combinatorial in situ immunomodulation strategy based on the administration of selected immunogenic drugs and immunotherapy to sensitize poorly T-cell-infiltrated neuroblastoma (NB) to the host antitumor immune response. Methods: 975A2 and 9464D NB cell lines derived from spontaneous tumors of TH-MYCN transgenic mice were employed to study drug combinations able of enhancing the antitumor immune response using in vivo and ex vivo approaches. Migration of immune cells towards drug-treated murine-derived organotypic tumor spheroids (MDOTS) were assessed by microfluidic devices. Activation status of immune cells co-cultured with drug-treated MDOTS was evaluated by flow cytometry analysis. The effect of drug treatment on the immune content of subcutaneous or orthotopic tumors was comprehensively analyzed by flow-cytometry, immunohistochemistry and multiplex immunofluorescence. The chemokine array assay was used to detect soluble factors released into the tumor microenvironment. Patient-derived organotypic tumor spheroids (PDOTS) were generated from human NB specimens. Migration and activation status of autologous immune cells to drug-treated PDOTS were performed. Results: We found that treatment with low-doses of mitoxantrone (MTX) recalled immune cells and promoted C D8+ T and NK cell activation in MDOTS when combined with TGFβ and PD-1 blockade. This combined immunotherapy strategy curbed NB growth resulting in the enrichment of a variety of both lymphoid and myeloid immune cells, especially intratumoral dendritic cells (DC) and IFNγ- and granzyme B-expressing C D8+ T cells and NK cells. A concomitant production of inflammatory chemokines involved in remodelling the tumor immune landscape was also † Valeria Lucarini and Ombretta Melaiu have contributed equally to this work, joint first authors, and co-first authors. *Correspondence: 1 Department of Paediatric Haematology/Oncology and of Cell and Gene Therapy, Bambino Gesù Children’s Hospital, IRCCS, 00146 Rome, Italy Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Lucarini et al. J Exp Clin Cancer Res (2022) 41:326 Page 2 of 19 detected. Interestingly, this treatment induced immune cell recruitment against PDOTS and activation of C D8+ T cells and NK cells. Conclusions: Combined treatment with low-dose of MTX and anti-TGFβ treatment with PD-1 blockade improves antitumor immunity by remodelling the tumor immune landscape and overcoming the immunosuppressive microenvironment of aggressive NB. Keywords: Neuroblastoma, Immunotherapy, Tumor Microenvironment, Immunomodulation, Drug Evaluation Background Immune checkpoint inhibitors (ICI) have shown impressive clinical results against a variety of highly aggressive tumors, although durable benefits have only been observed in a limited fraction of patients [1–3]. The main reason for this non-response is the lack of functional tumor-infiltrating T cells [4]. Given the great potential of ICI, it is of paramount importance to improve their efficacy by populating less infiltrated tumors with functional immune cells. Compelling evidence shows that in order to function, T cells required a favourable immune microenvironment in which other immune actors play a crucial role [5]. Indeed, tumors that respond to immunotherapy are enriched not only in T cells, but also in other immune cell populations, including conventional type 1 dendritic cells (DC1) and natural killer (NK) cells, which, by interacting with each other, create an environment suitable for the priming and expansion of tumor-specific T cells [5]. Consistently, similarly to C D8+ T cells, intratumoral DC1 and NK cells have also been associated with a strong antitumor response and favourable clinical outcome in various tumors [6]. The development of novel approaches that can restore the function of the entire immune cycle is therefore of crucial importance to increase the number of patients who can benefit from immunotherapy. In this regard, several chemotherapeutic treatments have been shown to recall immune cells in the tumor microenvironment (TME) through different mechanisms [7]. For examples, some chemotherapeutics such as doxorubicin (DX), mitoxantrone (MTX), oxaliplatin (OXP), and cyclophosphamide, when administrated at low doses are able to induce immunogenic cell death (ICD) by promoting DC activation, antigen presentation and priming of tumor-specific CD8+ T cells [7–10]. However, an increase in intratumoral immune cell infiltrate may not be conclusive because of the multiple mechanisms adopted by tumors to prevent patients’ immune system from targeting and eliminating their own tumor cells [3]. Recently, transcriptional profiling of melanoma and metastatic urothelial carcinoma patients unresponsive to anti-PD-1 therapy revealed an enrichment in pathways associated to TGFβ [11, 12], a cytokine produced by the TME that reduces immune cell recall within the tumor and inhibits the function of both T cells and NK cells [13]. Consistently, recent use of TGFβblocking antibodies has been shown to overcome resistance to anti-PD-1 thera (...truncated)