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

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.

Monocyte and Macrophage in Neuroblastoma: Blocking Their Pro-Tumoral Functions and Strengthening Their Crosstalk with Natural Killer Cells

“…As already discussed above, IL-6, released by mononuclear phagocytes upon NB conditioning, and TGF-β1, produced by both immune and tumor cells, inhibit the IL-2-mediated activation of NK cells, through the activation of the STAT3 and SMAD2/3 pathways and suppression of IFN-γ, granzymes, and perforin release. This is in line with the well-documented regulatory role of TGF-β in NK cell activation that emerged from several studies [ 32 , 76 ] supporting the development in NB of preclinical [ 77 ] and clinical studies with combining immunotherapies including the block of the TGF-β activity. Importantly, in NK cells, TGF-β also decreased the expression of activating receptors involved in NB recognition and modified the chemokine receptor repertoire, likely hampering the NK cell recruitment at the tumor sites [ 78 ].…”

Repurposing Anthracycline Drugs as Potential Antibiotic Candidates and Potentiators to Tackle Multidrug-Resistant Pathogens

“…Similarly, noticeable DNA damage in MX-treated bacteria was also observed in RPMI. The efficacy of low-dose MX in conjunction with TGF-β and PD-1 blockade has been demonstrated as a chemotherapeutic intervention for effectively managing tumor growth through the modulation of intratumoral immune cell composition and abundance . Interestingly, a recent study also investigated the immunological activity of MX and reported that MX promoted macrophage recruitment and enhanced the expression of pro-inflammatory cytokines at the wound sites, thus augmenting intracellular bacterial killing .…”

“…The efficacy of low-dose MX in conjunction with TGF-β and PD-1 blockade has been demonstrated as a chemotherapeutic intervention for effectively managing tumor growth through the modulation of intratumoral immune cell composition and abundance. 34 Interestingly, a recent study also investigated the immunological activity of MX and reported that MX promoted macrophage recruitment and enhanced the expression of proinflammatory cytokines at the wound sites, thus augmenting intracellular bacterial killing. 35 Consistently, our study also found that MX stimulated the upregulation of inflammatory cytokines and triggered an immune response, ultimately leading to the successful eradication of pathogenic bacteria in vivo.…”

Trial watch: chemotherapy-induced immunogenic cell death in oncology

“…Lucarini et al (Bambino Gesù Children’s Hospital, Rome, Italy) investigated the combination strategy of mitoxantrone and anti-transforming growth factor beta (TGFB1) with programmed cell death-1 (PD-1, also known as PDCD1 ) blockade in neuroblastoma mouse models. They found that the low dose of mitoxantrone by itself was already able to increase IFNγ and granzyme B (GZMB) in CD8 + T cells, which were further increased upon combination with anti-TGFβ and anti-PD-1 blockades 218 . Several papers also revealed a novel connection between anti-cancer agents and the ICD pathway 125 .…”