Strategies for overcoming tumor resistance to immunotherapy: Harnessing the power of radiation therapy.

Immune checkpoint inhibitors (ICI) have revolutionized cancer treatment; yet their efficacy remains variable across patients. This review delves into the intricate interplay of tumor characteristics contributing to resistance against ICI therapy and suggests that combining with radiotherapy (RT) holds promise. Radiation, known for its ability to trigger immunogenic cell death and foster an in situ vaccination effect, may counteract these resistance mechanisms, enhancing ICI response and patient outcomes. However, particularly when delivered at high-dose, it may trigger immunosuppressive mechanism and consequent side-effects. Notably, low-dose radiotherapy (LDRT), with its capacity for tumor reprogramming and reduced side effects, offers the potential for widespread application. Preclinical and clinical studies have shown encouraging results in this regard.

Establishment of a murine hepatocellular carcinoma model by hydrodynamic injection and characterization of the immune tumor microenvironment.

Hepatocellular carcinoma (HCC) is one of the most prevalent malignant neoplasms. Current treatments for HCC, such as tyrosine kinase inhibitors, have limited efficacy, highlighting the urgent need for better therapies. Immunotherapies, including anti-programmed death receptor 1 (PD-1) and anti-Cytotoxic T-lymphocyte associated protein 4 (CTLA-4), and more recently, the combination of anti-PD-L1 and anti-vascular endothelial growth factor (VEGF) monoclonal antibodies, have shown efficacy against HCC, resulting in Food and Drug Administration (FDA) approval. However, these immunotherapies only show efficiency in a small proportion of patients, meaning there is a great need to improve and optimize treatments against HCC. Accurate animal models that mimic human HCC are necessary to help better understand the nature of these tumors, which in turn will allow the development and testing of new treatments. Existing pre-clinical HCC models can be divided into non-genetic and genetic models. Non-genetic models involve implanting human or murine HCC cell lines or inducing tumors using chemical compounds or dietary modifications. These models have limitations, including slow tumor development and a lack of resemblance to human HCC. Genetic models, on the other hand, manipulate gene expression to induce HCC in mice and provide a better understanding of the effects of specific genes on tumor development. One method commonly used to generate HCC is hydrodynamic tail vein injection (HTVI), which consists of the delivery of oncogenes directly to the liver, resulting in expression and subsequent hepatocyte transformation. Usually, Sleeping Beauty transposase-containing plasmids are used to achieve stable and long-term gene expression. Once the HCC tumor is generated, and a proper tumor microenvironment (TME) is established, it is important to study the immune compartment of the TME, which plays a crucial role in HCC development and response to treatment. Techniques like flow cytometry can be used to analyze the immune cell populations in HCC tumors and assess their impact on tumor development and survival in mice. In this article, we thoroughly describe an example of the methodology to successfully generate HCC murine models via HTVI, and we propose a way to characterize the immune TME by flow cytometry.

Opportunities and challenges of low-dose radiation to enable immunotherapy efficacy.

Therapeutic monoclonal antibodies blocking different immune checkpoints, have demonstrated efficacy against a wide variety of solid tumors. The exclusion or absence of lymphocytes within the tumor microenvironment (TME) is one of the main resistance mechanisms to immune checkpoint inhibitor (ICI)-based therapies. Therefore, there is a growing interest in identifying novel approaches to promote T cell infiltration on immune-deserted (cold) and immune-excluded tumors to turn them into inflamed (hot) tumors. Here, we provide a comprehensive overview of the recently published studies showing the potential of low-dose radiation (LDRT) to reprogram the TME to allow and promote T-cell infiltration and thus, improve currently approved ICI-based therapies.

Identification of HLA class I-restricted immunogenic neoantigens in triple negative breast cancer.

Immune checkpoint inhibitor (ICI)-based immunotherapy in triple negative breast cancer (TNBC) is achieving limited therapeutic results, requiring the development of more potent strategies. Combination of ICI with vaccination strategies would enhance antitumor immunity and response rates to ICI in patients having poorly infiltrated tumors. In heavily mutated tumors, neoantigens (neoAgs) resulting from tumor mutations have induced potent responses when used as vaccines. Thus, our aim was the identification of immunogenic neoAgs suitable as vaccines in TNBC patients. By using whole exome sequencing, RNAseq and HLA binding algorithms of tumor samples from a cohort of eight TNBC patients, we identified a median of 60 mutations/patient, which originated a putative median number of 98 HLA class I-restricted neoAgs. Considering a group of 27 predicted neoAgs presented by HLA-A*02:01 allele in two patients, peptide binding to HLA was experimentally confirmed in 63% of them, whereas 55% were immunogenic in vivo in HLA-A*02:01+ transgenic mice, inducing T-cells against the mutated but not the wild-type peptide sequence. Vaccination with peptide pools or DNA plasmids expressing these neoAgs induced polyepitopic T-cell responses, which recognized neoAg-expressing tumor cells. These results suggest that TNBC tumors harbor neoAgs potentially useful in therapeutic vaccines, opening the way for new combined immunotherapies.

Gemcitabine-mediated depletion of immunosuppressive dendritic cells enhances the efficacy of therapeutic vaccination.

Vaccination using optimized strategies may increase response rates to immune checkpoint inhibitors (ICI) in some tumors. To enhance vaccine potency and improve thus responses to ICI, we analyzed the gene expression profile of an immunosuppressive dendritic cell (DC) population induced during vaccination, with the goal of identifying druggable inhibitory mechanisms. RNAseq studies revealed targetable genes, but their inhibition did not result in improved vaccines. However, we proved that immunosuppressive DC had a monocytic origin. Thus, monocyte depletion by gemcitabine administration reduced the generation of these DC and increased vaccine-induced immunity, which rejected about 20% of LLC-OVA and B16-OVA tumors, which are non-responders to anti-PD-1. This improved efficacy was associated with higher tumor T-cell infiltration and overexpression of PD-1/PD-L1. Therefore, the combination of vaccine + gemcitabine with anti-PD-1 was superior to anti-PD-1 monotherapy in both models. B16-OVA tumors benefited from a synergistic effect, reaching 75% of tumor rejection, but higher levels of exhausted T-cells in LLC-OVA tumors co-expressing PD-1, LAG3 and TIM3 precluded similar levels of efficacy. Our results indicate that gemcitabine is a suitable combination therapy with vaccines aimed at enhancing PD-1 therapies by targeting vaccine-induced immunosuppressive DC.

The role of dendritic cells in the immune niche of the peritoneum.

Dendritic cells (DCs) are professional antigen presenting cells that play an important role in the induction of T cell responses. Different subsets (cDC1s, cDC2s, pDCs, and moDCs) were described based on the expression of different surface markers and functions. In the context of peritoneum, DCs are also a key population cell orchestrating immune responses against pathogens, malignant cells and tissue-damage. Furthermore, they play an important role in the promotion of an anti-inflammatory microenvironment, which is necessary to maintain tolerance and adipocyte homeostasis. The aim of this review is to summarize the current knowledge of the functional and phenotypic features of peritoneal DCs and shed some light on the importance of these cells within this unique cavity and its associated components: the omentum, the mesentery and gut-associated lymphoid tissue (GALT).

Overcoming T cell dysfunction in acidic pH to enhance adoptive T cell transfer immunotherapy.

The high metabolic activity and insufficient perfusion of tumors leads to the acidification of the tumor microenvironment (TME) that may inhibit the antitumor T cell activity. We found that pharmacological inhibition of the acid loader chloride/bicarbonate anion exchanger 2 (Ae2), with 4,4'-diisothiocyanatostilbene-2,2'-disulfonicacid (DIDS) enhancedCD4+ andCD8+ T cell function upon TCR activation in vitro, especially under low pH conditions. In vivo, DIDS administration delayed B16OVA tumor growth in immunocompetent mice as monotherapy or when combined with adoptive T cell transfer of OVA-specificT cells. Notably, genetic Ae2 silencing in OVA-specificT cells improvedCD4+/CD8+ T cell function in vitro as well as their antitumor activity in vivo. Similarly, genetic modification of OVA-specificT cells to overexpress Hvcn1, a selectiveH+ outward current mediator that prevents cell acidification, significantly improved T cell function in vitro, even at low pH conditions. The adoptive transfer of OVA-specificT cells overexpressing Hvcn1 exerted a better antitumor activity in B16OVA tumor-bearingmice. Hvcn1 overexpression also improved the antitumor activity of CAR T cells specific for Glypican 3 (GPC3) in mice bearing PM299L-GPC3tumors. Our results suggest that preventing intracellular acidification by regulating the expression of acidifier ion channels such as Ae2 or alkalinizer channels like Hvcn1 in tumor-specificlymphocytes enhances their antitumor response by making them more resistant to the acidic TME.

Therapeutic Vaccines against Hepatocellular Carcinoma in the Immune Checkpoint Inhibitor Era: Time for Neoantigens?

Immune checkpoint inhibitors (ICI) have been used as immunotherapy for hepatocellular carcinoma (HCC) with promising but still limited results. Identification of immune elements in the tumor microenvironment of individual HCC patients may help to understand the correlations of responses, as well as to design personalized therapies for non-responder patients. Immune-enhancing strategies, such as vaccination, would complement ICI in those individuals with poorly infiltrated tumors. The prominent role of responses against mutated tumor antigens (neoAgs) in ICI-based therapies suggests that boosting responses against these epitopes may specifically target tumor cells. In this review we summarize clinical vaccination trials carried out in HCC, the available information on potentially immunogenic neoAgs in HCC patients, and the most recent results of neoAg-based vaccines in other tumors. Despite the low/intermediate mutational burden observed in HCC, data obtained from neoAg-based vaccines in other tumors indicate that vaccines directed against these tumor-specific antigens would complement ICI in a subset of HCC patients.

Neoantigens as potential vaccines in hepatocellular carcinoma.

BACKGROUND: Neoantigens, new immunogenic sequences arising from tumor mutations, have been associated with response to immunotherapy and are considered potential targets for vaccination. Hepatocellular carcinoma (HCC) is a moderately mutated tumor, where the neoantigen repertoire has not been investigated. Our aim was to analyze whether tumors in HCC patients contain immunogenic neoantigens suitable for future use in therapeutic vaccination. METHODS: Whole-exome sequencing and RNAseq were performed in a cohort of fourteen HCC patients submitted to surgery or liver transplant. To identify mutations, single-nucleotide variants (SNV) originating non-synonymous changes that were confirmed at the RNA level were analyzed. Immunogenicity of putative neoAgs predicted by HLA binding algorithms was confirmed by using in vitro HLA binding assays and T-cell stimulation experiments, the latter in vivo, by immunizing HLA-A*02.01/HLA-DRB1*01 (HHD-DR1) transgenic mice, and in in vitro, using human lymphocytes. RESULTS: Sequencing led to the identification of a median of 1217 missense somatic SNV per patient, narrowed to 30 when filtering by using RNAseq data. A median of 13 and 5 peptides per patient were predicted as potential binders to HLA class I and class II molecules, respectively. Considering only HLA-A*02.01- and HLA-DRB1*01-predicted binders, 70% demonstrated HLA-binding capacity and about 50% were immunogenic when tested in HHD-DR1 mice. These peptides induced polyfunctional T cells that specifically recognized the mutated but not the wild-type sequence as well as neoantigen-expressing cells. Moreover, coimmunization experiments combining CD8 and CD4 neoantigen epitopes resulted in stronger CD8 T cell responses. Finally, responses against neoantigens were also induced in vitro using human cells. CONCLUSION: These results show that mutations in HCC tumors may generate immunogenic neoantigens with potential applicability for future combinatorial therapeutic strategies.

Inhibition of adjuvant-induced TAM receptors potentiates cancer vaccine immunogenicity and therapeutic efficacy.

Analyzing immunomodulatory elements operating during antitumor vaccination in prostate cancer patients and murine models we identified IL-10-producing DC as a subset with poorer immunogenicity and clinical efficacy. Inhibitory TAM receptors MER and AXL were upregulated on murine IL-10+ DC. Thus, we analyzed conditions inducing these molecules and the potential benefit of their blockade during vaccination. MER and AXL upregulation was more efficiently induced by a vaccine containing Imiquimod than by a poly(I:C)-containing vaccine. Interestingly, MER expression was found on monocyte-derived DC, and was dependent on IL-10. TAM blockade improved Imiquimod-induced DC activation in vitro and in vivo, resulting in increased vaccine-induced T-cell responses, which were further reinforced by concomitant IL-10 inhibition. In different tumor models, a triple therapy (including vaccination, TAM inhibition and IL-10 blockade) provided the strongest therapeutic effect, associated with enhanced T-cell immunity and enhanced CD8+ T cell tumor infiltration. Finally, MER levels in DC used for vaccination in cancer patients correlated with IL-10 expression, showing an inverse association with vaccine-induced clinical response. These results suggest that TAM receptors upregulated during vaccination may constitute an additional target in combinatorial therapeutic vaccination strategies.

Cold-Inducible RNA Binding Protein as a Vaccination Platform to Enhance Immunotherapeutic Responses Against Hepatocellular Carcinoma.

Therapies based on immune checkpoint inhibitors (ICPI) have yielded promising albeit limited results in patients with hepatocellular carcinoma (HCC). Vaccines have been proposed as combination partners to enhance response rates to ICPI. Thus, we analyzed the combined effect of a vaccine based on the TLR4 ligand cold-inducible RNA binding protein (CIRP) plus ICPI. Mice were immunized with vaccines containing ovalbumin linked to CIRP (OVA-CIRP), with or without ICPI, and antigen-specific responses and therapeutic efficacy were tested in subcutaneous and orthotopic mouse models of liver cancer. OVA-CIRP elicited polyepitopic T-cell responses, which were further enhanced when combined with ICPI (anti-PD-1 and anti-CTLA-4). Combination of OVA-CIRP with ICPI enhanced ICPI-induced therapeutic responses when tested in subcutaneous and intrahepatic B16-OVA tumors, as well as in the orthotopic PM299L HCC model. This effect was associated with higher OVA-specific T-cell responses in the periphery, although many tumor-infiltrating lymphocytes still displayed an exhausted phenotype. Finally, a new vaccine containing human glypican-3 linked to CIRP (GPC3-CIRP) induced clear responses in humanized HLA-A2.01 transgenic mice, which increased upon combination with ICPI. Therefore, CIRP-based vaccines may generate anti-tumor immunity to enhance ICPI efficacy in HCC, although blockade of additional checkpoint molecules and immunosuppressive targets should be also considered.

When Cancer Vaccines Go Viral.

Induction of antitumor responses by vaccines requires strong immunogens. Heterologous viral prime/boost immunization with the BN-CV301 vaccine promotes activation of immune responses that provide a clinical benefit to patients with cancer. This viral platform may be used to harbor different antigens and prime tumor immunity potentially useful for combinatorial strategies.See related article by Gatti-Mays et al., p. 4933.