Immunogenic Cell Death Inducers in Cancer Immunotherapy to Turn Cold Tumors into Hot Tumors

Abstract

The combination of chemotherapeutic agents with immune checkpoint inhibitors (ICIs) has revolutionized cancer treatment. However, its success is often limited by insufficient immune priming in certain tumors, including pediatric malignancies. In this report, we explore clinical trials currently investigating the use of immunogenic cell death (ICD)-inducing chemotherapies in combination with ICIs for both adult and pediatric cancers. Given the limited clinical data available for pediatric tumors, we focused on recent preclinical studies evaluating the efficacy of these combinations in neuroblastoma (NB). Finally, to address this gap, we propose an innovative strategy to assess the impact of ICD-inducing chemotherapies on antitumor immune responses in NB. Using tumor spheroids derived from a transgenic NB mouse model, we validated our previous in vivo findings concerning how anthracyclines, specifically mitoxantrone and doxorubicin, significantly enhance MHC class I surface expression, stimulate IFNγ and granzyme B production by CD8⁺ T cells and NK cells, and promote immune cell recruitment. Importantly, these anthracyclines also upregulated PD-L1 expression on NB spheroids. This screening platform yielded results similar to in vivo findings, demonstrating that mitoxantrone and doxorubicin are the most potent immunomodulatory agents for NB. These data suggest that the creation of libraries of ICD inducers to be tested on tumor spheroids could reduce the number of combinations to be tested in vivo, in line with the principles of the 3Rs. Furthermore, these results highlight the potential of chemo-immunotherapy regimens to counteract the immunosuppressive tumor microenvironment in NB, paving the way for improved therapeutic strategies in pediatric cancers. They provide compelling evidence to support further clinical investigations of these combinations to enhance outcomes for children with malignancies.

Keywords: anthracyclines; immune checkpoint inhibitors; immunogenic cell death; neuroblastoma; pediatric cancer; tumor microenvironment; tumor spheroids.

Figure 1

Chemotherapeutic drugs promote the recruitment and activation of CD8⁺ T cells and NK cells in co-cultures of NB spheroids and syngeneic splenocytes. (A) Experimental scheme. NB cells were seeded in ULA plates to form spheroids. The 4-day-old NB spheroids were either treated with the indicated compounds at different concentrations while monitoring their diameter over the next 96 hours or treated for 1 day, and then, after drug removal, were cultured with syngeneic splenocytes in ULA plates or a microfluidic device. (B) Time–course and dose–response curves of drug-treated 9464D and 975A2 NB spheroids. Spheroid’s diameter was evaluated from 0 to 96 hours. Each color corresponds to a tested concentration (e.g., red for 0.5 µM, green for 1 µM, etc.). Spheroid diameter was analyzed using ImageJ software v1.54j, NIH Image, NIH, Bethesda, MD, USA). Data are the mean ± SEM of 3 independent experiments in triplicate. (C,D) Representative flow cytometric analyses of MHC class I (C) and PD-L1 (D) cell surface expression in NB spheroids treated with the indicated drugs for 24 hours. The graphs represent the MFI ± SD of three independent experiments. Significance levels for comparison between samples were determined by two-tailed Student’s t test. (E) Flow cytometric analysis of IFNγ and granzyme B expression of CD8⁺ T cells and NK cells from splenocytes co-cultured with drug-treated NB spheroids for 24 hours. Significance levels for comparison between samples were determined by ANOVA. (F) Representative images of migration in microfluidic devices of red-labeled splenocytes recruited from drug-treated and untreated NB spheroids after 24 hours of co-culture (left). The number of splenocytes migrating toward treated and untreated NB spheroids is assessed using ImageJ software v1.54j, NIH Image, NIH, Bethesda, MD, USA). Data are shown as fold change ± SD (right). Significance levels for comparison between samples were determined by Student’s t-test. CTR, control; CDDP, cisplatin; DX, doxorubicin; IRI, irinotecan; MAFO, mafosfamide; MTX, mitoxantrone; OXP, oxaliplatin; TOPO, topotecan; VINC, vincristine; GZMB, granzyme B; IFNγ, interferon gamma.