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

• Published in: Journal of experimental & clinical cancer research Vol. 41; no. 1; pp. 1 - 326
• Main Authors: Lucarini, Valeria, Melaiu, Ombretta, D’Amico, Silvia, Pastorino, Fabio, Tempora, Patrizia, Scarsella, Marco, Pezzullo, Marco, De Ninno, Adele, D’Oria, Valentina, Cilli, Michele, Emionite, Laura, Infante, Paola, Di Marcotullio, Lucia, De Ioris, Maria Antonietta, Barillari, Giovanni, Alaggio, Rita, Businaro, Luca, Ponzoni, Mirco, Locatelli, Franco, Fruci, Doriana
• Format: Journal Article
• Language: English
• Published: London BioMed Central 17-11-2022; BMC
• Subjects: Antibodies; Cancer therapies; Chemotherapy; Drug Evaluation; Experiments; Immunity (Disease); Immunomodulation; Immunotherapy; Lymphocytes; Metastasis; Neuroblastoma; Patients; Tumor Microenvironment; Tumors; Rome Italy; Italy
• ISSN: 1756-9966; 0392-9078; 1756-9966
• DOI: 10.1186/s13046-022-02525-9

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 CD8
+
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 CD8
+
T cells and NK cells. A concomitant production of inflammatory chemokines involved in remodelling the tumor immune landscape was also detected. Interestingly, this treatment induced immune cell recruitment against PDOTS and activation of CD8
+
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.