Fueling the fight against cancer: Exploring the impact of branched-chain amino acid catalyzation on cancer and cancer immune microenvironment.

Metabolism of Branched-chain amino acids (BCAAs) is essential for the nutrient necessities in mammals. Catalytic enzymes serve to direct the whole-body BCAAs oxidation which involve in the development of various metabolic disorders. The reprogrammed metabolic elements are also responsible for malignant oncogenic processes, and favor the formation of distinctive immunosuppressive microenvironment surrounding different cancers. The impotent immune surveillance related to BCAAs dysfunction is a novel topic to investigate. Here we focus on the BCAA catalysts that contribute to metabolic changes and dysregulated immune reactions in cancer progression. We summarize the current knowledge of BCAA catalyzation, highlighting the interesting roles of BCAA metabolism in the treatment of cancers.

GSDMD is associated with survival in human breast cancer but does not impact anti-tumor immunity in a mouse breast cancer model.

Inflammation plays a pivotal role in cancer development, with chronic inflammation promoting tumor progression and treatment resistance, whereas acute inflammatory responses contribute to protective anti-tumor immunity. Gasdermin D (GSDMD) mediates the release of pro-inflammatory cytokines such as IL-1ß. While the release of IL-1ß is directly linked to the progression of several types of cancers, the role of GSDMD in cancer is less clear. In this study, we show that GSDMD expression is upregulated in human breast, kidney, liver, and prostate cancer. Higher GSDMD expression correlated with increased survival in primary breast invasive carcinoma (BRCA), but not in liver hepatocellular carcinoma (LIHC). In BRCA, but not in LIHC, high GSDMD expression correlated with a myeloid cell signature associated with improved prognosis. To further investigate the role of GSDMD in anticancer immunity, we induced breast cancer and hepatoma tumors in GSDMD-deficient mice. Contrary to our expectations, GSDMD deficiency had no effect on tumor growth, immune cell infiltration, or cytokine expression in the tumor microenvironment, except for Cxcl10 upregulation in hepatoma tumors. In vitro and in vivo innate immune activation with TLR ligands, that prime inflammatory responses, revealed no significant difference between GSDMD-deficient and wild-type mice. These results suggest that the impact of GSDMD on anticancer immunity is dependent on the tumor type. They underscore the complex role of inflammatory pathways in cancer, emphasizing the need for further exploration into the multifaceted effects of GSDMD in various tumor microenvironments. As several pharmacological modulators of GSDMD are available, this may lead to novel strategies for combination therapy in cancer.

Myeloid-derived suppressor cells in cancer: Current knowledge and future perspectives.

MDSCs (myeloid-derived suppressor cells) are crucial for immune system evasion in cancer. They accumulate in peripheral blood and tumor microenvironment, suppressing immune cells like T-cells, natural killer cells and dendritic cells. They promote tumor angiogenesis and metastasis by secreting cytokines and growth factors and contribute to a tumor-promoting environment. The accumulation of MDSCs in cancer patients has been linked to poor prognosis and resistance to various cancer therapies. Targeting MDSCs and their immunosuppressive mechanisms may improve treatment outcomes and enhance immune surveillance by developing drugs that inhibit MDSC function, by preventing their accumulation and by disrupting the tumor-promoting environment. This review presents a detailed overview of the MDSC research in cancer with regulation of their development and function. The relevance of MDSC as a prognostic and predictive biomarker in different types of cancers, along with recent advancements on the therapeutic approaches to target MDSCs are discussed in detail.

Somatic mutations in tumor-infiltrating lymphocytes impact on antitumor immunity.

Immune checkpoint inhibitors (ICIs) exert clinical efficacy against various types of cancers by reinvigorating exhausted CD8+ T cells that can expand and directly attack cancer cells (cancer-specific T cells) among tumor-infiltrating lymphocytes (TILs). Although some reports have identified somatic mutations in TILs, their effect on antitumor immunity remains unclear. In this study, we successfully established 18 cancer-specific T cell clones, which have an exhaustion phenotype, from the TILs of four patients with melanoma. We conducted whole-genome sequencing for these T cell clones and identified various somatic mutations in them with high clonality. Among the somatic mutations, an SH2D2A loss-of-function frameshift mutation and TNFAIP3 deletion could activate T cell effector functions in vitro. Furthermore, we generated CD8+ T cell-specific Tnfaip3 knockout mice and showed that Tnfaip3 function loss in CD8+ T cell increased antitumor immunity, leading to remarkable response to PD-1 blockade in vivo. In addition, we analyzed bulk CD3+ T cells from TILs in additional 12 patients and identified an SH2D2A mutation in one patient through amplicon sequencing. These findings suggest that somatic mutations in TILs can affect antitumor immunity and suggest unique biomarkers and therapeutic targets.

Stromal CD4 (+) T Cell Subsets Mediate Antitumor Cytotoxic Immune Responses in Human Colorectal Carcinoma.

BACKGROUND/AIM: The local immune response in colorectal cancer is closely related to prognosis and therapeutic efficacy. In this study, histological analyses were performed to determine the phenotype of tumor-infiltrating lymphocytes (TILs) and their infiltration in the stromal and intratumoral regions, aiming to elucidate their interactions and prognostic effects. PATIENTS AND METHODS: Multiplex fluorescent labeling was performed using surgically resected colorectal cancer specimens to investigate the infiltration of CD45RO (+) TILs, which exhibit cytotoxicity, and subsets of CD4 (+) TILs, identified by their characteristic transcription factor expression. RESULTS: The degree of CD45RO (+) TIL infiltration in the entire observation field or stromal area was not associated with prognosis. However, a high degree of infiltration in the tumor nest (intratumoral area) was significantly associated with a favorable prognosis. CD4 (+) TILs and their subsets were not associated with prognosis. However, stratified analyses revealed that a high degree of infiltration of stromal CD4 (+) TILs and the subsets T helper (Th)1, Th2, Th17, and regulatory T cells is necessary for the association between high intratumoral CD45RO (+) TIL infiltration and favorable prognosis. CONCLUSION: A sufficient degree of infiltration of stromal CD4 (+) TIL subsets is required for intratumoral CD45RO (+) TILs to exert toxicity against cancer cells. This highlights the significance of stromal immune reactions in achieving effective cytotoxic immune responses in the intratumoral area and demonstrates the critical role of the spatial distribution pattern of TILs in exerting their functions.

Microbiota-induced plastic T cells enhance immune control of antigen-sharing tumors.

Therapies that harness the immune system to target and eliminate tumor cells have revolutionized cancer care. Immune checkpoint blockade (ICB), which boosts the anti-tumor immune response by inhibiting negative regulators of T cell activation1-3, is remarkably successful in a subset of cancer patients, yet a significant proportion do not respond to treatment, emphasizing the need to understand factors influencing the therapeutic efficacy of ICB4-9. The gut microbiota, consisting of trillions of microorganisms residing in the gastrointestinal tract, has emerged as a critical determinant of immune function and response to cancer immunotherapy, with multiple studies demonstrating association of microbiota composition with clinical response10-16. However, a mechanistic understanding of how gut commensal bacteria influence the efficacy of ICB remains elusive. Here we utilized a gut commensal microorganism, segmented filamentous bacteria (SFB), which induces an antigen-specific Th17 cell effector program17, to investigate how colonization with it affects the efficacy of ICB in restraining distal growth of tumors sharing antigen with SFB. We find that anti-PD-1 treatment effectively inhibits the growth of implanted SFB antigen-expressing melanoma only if mice are colonized with SFB. Through T cell receptor clonal lineage tracing, fate mapping, and peptide-MHC tetramer staining, we identify tumor-associated SFB-specific Th1-like cells derived from the homeostatic Th17 cells induced by SFB colonization in the small intestine lamina propria. These gut-educated ex-Th17 cells produce high levels of the pro-inflammatory cytokines IFN-γ and TNF-α, and promote expansion and effector functions of CD8+ tumor-infiltrating cytotoxic lymphocytes, thereby controlling tumor growth. A better understanding of how distinct intestinal commensal microbes can promote T cell plasticity-dependent responses against antigen-sharing tumors may allow for the design of novel cancer immunotherapeutic strategies.

Cracking the Codes behind Cancer Cells' Immune Evasion.

Immune evasion is a key phenomenon in understanding tumor recurrence, metastasis, and other critical steps in tumor progression. The tumor microenvironment (TME) is in constant flux due to the tumor's ability to release signals that affect it, while immune cells within it can impact cancer cell behavior. Cancer cells undergo several changes, which can change the enrichment of different immune cells and modulate the activity of existing immune cells in the tumor microenvironment. Cancer cells can evade immune surveillance by downregulating antigen presentation or expressing immune checkpoint molecules. High levels of tumor-infiltrating lymphocytes (TILs) correlate with better outcomes, and robust immune responses can control tumor growth. On the contrary, increased enrichment of Tregs, myeloid-derived suppressor cells, and M2-like anti-inflammatory macrophages can hinder effective immune surveillance and predict poor prognosis. Overall, understanding these immune evasion mechanisms guides therapeutic strategies. Researchers aim to modulate the TME to enhance immune surveillance and improve patient outcomes. In this review article, we strive to summarize the composition of the tumor immune microenvironment, factors affecting the tumor immune microenvironment (TIME), and different therapeutic modalities targeting the immune cells. This review is a first-hand reference to understand the basics of immune surveillance and immune evasion.

Targeting Tumor-Associated Macrophages with the Immune-Activating Nanomedicine for Achieving Strong Antitumor Activity with Rapid Clearance from the Body.

Toll-like receptors (TLRs) are a class of pattern recognition receptors (PRRs) crucial for the detection of infections and activation of downstream signaling pathways that lead to the production of pro-inflammatory cytokines and interferons. The TLR pathway is an attractive actively studied target pathway. Because of their strong immunostimulatory activity, TLRs are thought to be a "double-edged sword" for systemic treatment, even in the cancer field. To solve this, we have developed dextran-based TAM targeting activating conjugate (D-TAC) technology, which successfully uses tumor-associated macrophages (TAMs) to deliver the TLR7 agonist DSP-0509. We used low molecular weight dextran to target CD206 high M2-type macrophages, activate them, and induce a change in phenotype to antitumor M1-type macrophages with rapid clearance from the body and astonishing antitumor activity. We also demonstrated that the antitumor effect of our best drug candidate 5DEX-0509R is dependent on the abundance of TAMs, which is consistent with their mechanism of action. We believe that 5DEX-0509R generated by D-TAC technology can be a clinically applicable immunotherapy targeting the TLR signaling pathway.

Selective lysis of acute myeloid leukemia cells by CD34/CD3 bispecific antibody through the activation of γδ T-cells.

Despite the considerable progress in acute myeloid leukemia (AML) treatment, relapse after allogeneic hematopoietic stem cell transplantation (HSCT) is still frequent and associated with a poor prognosis. Relapse has been shown to be correlated with an incomplete eradication of CD34+ leukemic stem cells prior to HSCT. Previously, we have shown that a novel CD34-directed, bispecific T-cell engager (BTE) can efficiently redirect the T-cell effector function toward cancer cells, thus eliminating leukemic cells in vitro and in vivo. However, its impact on γδ T-cells is still unclear. In this study, we tested the efficacy of the CD34-specific BTE using in vitro expanded γδ T-cells as effectors. We showed that the BTEs bind to γδ T-cells and CD34+ leukemic cell lines and induce target cell killing in a dose-dependent manner. Additionally, γδ T-cell mediated killing was found to be superior to αß T-cell mediated cytotoxicity. Furthermore, we observed that only in the presence of BTE the γδ T-cells induced primary AML blast killing in vitro. Importantly, our results show that γδ T-cells did not target the healthy CD34intermediate endothelial blood-brain barrier cell line (hCMEC/D3) nor lysed CD34+ HSCs from healthy bone marrow samples.

A Higher Neutrophil Count Is Associated with Favorable Achievement of Treatment-Free Remission in Patients with Chronic Myeloid Leukemia Who Received Second Generation Tyrosine Kinase Inhibitor as Frontline Treatment.

BACKGROUND: ABL1 tyrosine kinase inhibitor discontinuation securely became among the therapeutic goal for chronic myeloid leukemia chronic phase patients (CML-CP). To establish successful prognostic factors for treatment-free remission (TFR), it is necessary to diagnose the patients with high-risk molecular relapse, however, a biomarker for the achievement of TFR has not been completely elucidated. Recent investigations have determined that neutrophils function crucially in cancer immunology. PATIENTS AND METHODS: The research was a multicenter retrospective observational study to examine the correlation between TFR and neutrophil counts before TKI discontinuation. The investigation included patients having Philadelphia chromosome-positive CML-CP who attempted the discontinuation of TKIs after a durable deep molecular response between January 2012 and July 2021 at four institutions in Japan. RESULTS: 118 CML-CP patients in total discontinued TKIs and an estimated 36-month TFR rate was 65.1%. 52 patients received second-generation TKIs as frontline. Higher neutrophil count (>3210/µL) at TKIs discontinuation was determined as an independent prognostic variable for TFR in patients who received second-generation TKIs as frontline [(HR, 0.235 (95%, confidence interval (CI) 0.078-0.711); p = 0.010]. CONCLUSIONS: The neutrophil-mediated immunomodulation can be a significant component for the effective achievement of TFR in CML supported by our clinical observation.

Human effector CD8+ T cells with an activated and exhausted-like phenotype control tumour growth in vivo in a humanized tumour model.

BACKGROUND: Humanized tumour models could be particularly valuable for cancer immunotherapy research, as they may better reflect human-specific aspects of the interfaces between tumour and immune system of human cancer. However, endogenous antitumour immunity in humanized models is still largely undefined. METHODS: We established an autologous humanized mouse tumour model by using NSG mice reconstituted with human immune cells from hematopoietic progenitors and tumours generated from transformed autologous human B cells. We demonstrate growth of solid lymphoid tumours after subcutaneous implantation, infiltration by endogenous human immune cells and immunocompetence of the model. FINDINGS: We found human T cell subsets described in human cancer, including progenitor exhausted (Tpex), terminally exhausted (Tex-term) and tissue-resident (TRM) cells in tumour-bearing humanized mice with accumulation of Tex-term and TRM in the tumour. In addition, we identified tumour-reactive CD8+ T cells through expression of CD137. This subpopulation of de novo arising human CD137+ CD8+ T cells displayed a highly proliferative, fully activated effector and exhausted-like phenotype with enhanced expression of activation and exhaustion markers like PD-1, CD39, CD160, TIM-3, TIGIT and TOX, the senescence marker CD57 (B3GAT1) and cytolytic effector molecules such as PRF1, GZMH and NKG7. Moreover, these CD137+ CD8+ T cells exhibited tumour-specific clonal expansion and presented signature overlap with tumour-reactive CD8+ T cells described in human cancer. We demonstrate superior anticancer activity of this activated and exhausted-like human CD8+ T cell subset by adoptive transfer experiments using recipients bearing autologous human tumours. Mice adoptively transferred with CD137+ CD8+ T cells showed reduced tumour growth and higher CD8+ T cell tumour infiltration, correlating with control of human tumours. INTERPRETATION: We established an immunocompetent humanized tumour model, providing a tool for immunotherapy research and defined effective anticancer activity of human effector CD8+ T cells with an activated and exhausted-like phenotype, supporting clinical exploration of such cells in adoptive T cell therapies. FUNDING: Swiss Cancer Research foundation.

Downregulation of HNRNPA1 induced neoantigen generation via regulating alternative splicing.

BACKGROUND: Immunotherapies effectively treat human malignancies, but the low response and resistance are major obstacles. Neoantigen is an emerging target for tumor immunotherapy that can enhance anti-tumor immunity and improve immunotherapy. Aberrant alternative splicing is an important source of neoantigens. HNRNPA1, an RNA splicing factor, was found to be upregulated in the majority of tumors and play an important role in the tumor immunosuppressive microenvironment. METHODS: Whole transcriptome sequencing was performed on shHNRNPA1 SKOV3 cells and transcriptomic data of shHNRNPA1 HepG2, MCF-7M, K562, and B-LL cells were downloaded from the GEO database. Enrichment analysis was performed to elucidate the mechanisms underlying the activation of anti-tumor immunity induced by HNRNPA1 knockdown. mRNA alternative splicing was analyzed and neoantigens were predicted by JCAST v.0.3.5 and Immune epitope database. The immunogenicity of candidate neoantigens was calculated by Class I pMHC Immunogenicity and validated by the IFN-γ ELISpot assay. The effect of shHNRNPA1 on tumor growth and immune cells in vivo was evaluated by xenograft model combined with immunohistochemistry. RESULTS: HNRNPA1 was upregulated in a majority of malignancies and correlated with immunosuppressive status of the tumor immune microenvironment. Downregulation of HNRNPA1 could induce the activation of immune-related pathways and biological processes. Disruption of HNRNPA1 resulted in aberrant alternative splicing events and generation of immunogenic neoantigens. Downregulation of HNRNPA1 inhibited tumor growth and increased CD8+ T cell infiltration in vivo. CONCLUSION: Our study demonstrated that targeting HNRNPA1 could produce immunogenic neoantigens that elicit anti-tumor immunity by inducing abnormal mRNA splicing. It suggests that HNRNPA1 may be a potential target for immunotherapy.

DeepHLApan: A Deep Learning Approach for the Prediction of Peptide-HLA Binding and Immunogenicity.

Neoantigens are crucial in distinguishing cancer cells from normal ones and play a significant role in cancer immunotherapy. The field of bioinformatics prediction for tumor neoantigens has rapidly developed, focusing on the prediction of peptide-HLA binding affinity. In this chapter, we introduce a user-friendly tool named DeepHLApan, which utilizes deep learning techniques to predict neoantigens by considering both peptide-HLA binding affinity and immunogenicity. We provide the application of DeepHLApan, along with the source code, docker version, and web-server. These resources are freely available at https://github.com/zjupgx/deephlapan and http://pgx.zju.edu.cn/deephlapan/ .

BCG priming followed by a novel interleukin combination activates Natural Killer cells to selectively proliferate and become anti-tumour long-lived effectors.

The short-lived nature and heterogeneity of Natural Killer (NK) cells limit the development of NK cell-based therapies, despite their proven safety and efficacy against cancer. Here, we describe the biological basis, detailed phenotype and function of long-lived anti-tumour human NK cells (CD56highCD16+), obtained without cell sorting or feeder cells, after priming of peripheral blood cells with Bacillus Calmette-Guérin (BCG). Further, we demonstrate that survival doses of a cytokine combination, excluding IL18, administered just weekly to BCG-primed NK cells avoids innate lymphocyte exhaustion and leads to specific long-term proliferation of innate cells that exert potent cytotoxic function against a broad range of solid tumours, mainly through NKG2D. Strikingly, a NKG2C+CD57-FcεRIγ+ NK cell population expands after BCG and cytokine stimulation, independently of HCMV serology. This strategy was exploited to rescue anti-tumour NK cells even from the suppressor environment of cancer patients' bone marrow, demonstrating that BCG confers durable anti-tumour features to NK cells.

Characterising the HLA-I immunopeptidome of plasma-derived extracellular vesicles in patients with melanoma.

Extracellular vesicles (EVs) frequently express human leukocyte antigen class I (HLA-I) molecules. The immunopeptidomes presented on EV HLA-I are being mapped to provide key information on both specific cancer-related peptides, and for larger immunopeptidomic signatures associated with disease. Utilizing HLA-I immunoisolation and mass spectrometry, we characterised the HLA-I immunopeptidome of EVs derived from the melanoma cancer cell line, ESTDAB-026, and the plasma of 12 patients diagnosed with advanced stage melanoma, alongside 11 healthy controls. The EV HLA-I immunopeptidome derived from melanoma cells features T cell epitopes with known immunogenicity and peptides derived from known tumour associated antigens (TAAs). Both T cell epitopes with known immunogenicity and peptides derived from known TAAs were also identifiable in the melanoma patient samples. Patient stratification into two distinct groups with varying immunological profiles was also observed. The data obtained in this study suggests for the first time that the HLA-I immunopeptidome of EVs derived from blood may aid in the detection of important diagnostic or prognostic biomarkers and also provide new immunotherapy targets.

Circulating subpopulations of non-cytotoxic ILCs in diffuse large B-cell lymphoma.

Non-cytotoxic innate lymphoid cells (ILCs) have been added to the list of immune cells that may contribute to the tumor microenvironment. Elevated levels of total ILCs and their subgroups have been reported in peripheral blood and tissue samples from patients with solid tumors, but their frequency in non-Hodgkin lymphomas, particularly diffuse large B-cell lymphoma (DLBCL), has not been clearly established. This study examined frequency and subset distribution in newly diagnosed DLBCL patients (nodal and extra-nodal) and compared it with blood specimens from healthy donors. The percentage of total ILCs (Lin - CD127+) was assessed by flow cytometry, as well as the four ILC subsets, defined as ILC1 (Lin - CD127 + cKit - CRTH2-), ILC2 (Lin - CD127 + cKit+/- CRTH2+), ILCp NCR- (Lin - CD127 + cKit + CRTH2- NKp46-) and NCR + ILC3 (Lin - CD127 + cKit + NKp46+). In the studied group of patients (n = 54), significantly lower levels of circulating total ILCs, ILC1, and ILCp NCR- were observed compared to the control group (n = 43). Similarly, there was a statistically significant decrease in the median frequency of NKp46 + ILC3 cells in lymphoma patients. Analysis of the ILC2 subpopulation showed no significant differences. The correlation of the distribution of individual subpopulations of ILCs with the stage and location of the tumor was also demonstrated. Our results suggest that circulating ILCs are activated and differentiated and/or differentially recruited to the lymph nodes or tumor microenvironment where they may be involved in antitumor defense. However, our observations require confirmation in functional studies.

Microphysiological systems as models for immunologically 'cold' tumors.

The tumor microenvironment (TME) is a diverse milieu of cells including cancerous and non-cancerous cells such as fibroblasts, pericytes, endothelial cells and immune cells. The intricate cellular interactions within the TME hold a central role in shaping the dynamics of cancer progression, influencing pivotal aspects such as tumor initiation, growth, invasion, response to therapeutic interventions, and the emergence of drug resistance. In immunologically 'cold' tumors, the TME is marked by a scarcity of infiltrating immune cells, limited antigen presentation in the absence of potent immune-stimulating signals, and an abundance of immunosuppressive factors. While strategies targeting the TME as a therapeutic avenue in 'cold' tumors have emerged, there is a pressing need for novel approaches that faithfully replicate the complex cellular and non-cellular interactions in order to develop targeted therapies that can effectively stimulate immune responses and improve therapeutic outcomes in patients. Microfluidic devices offer distinct advantages over traditional in vitro 3D co-culture models and in vivo animal models, as they better recapitulate key characteristics of the TME and allow for precise, controlled insights into the dynamic interplay between various immune, stromal and cancerous cell types at any timepoint. This review aims to underscore the pivotal role of microfluidic systems in advancing our understanding of the TME and presents current microfluidic model systems that aim to dissect tumor-stromal, tumor-immune and immune-stromal cellular interactions in various 'cold' tumors. Understanding the intricacies of the TME in 'cold' tumors is crucial for devising effective targeted therapies to reinvigorate immune responses and overcome the challenges of current immunotherapy approaches.

The temporal progression of lung immune remodeling during breast cancer metastasis.

Tumor metastasis requires systemic remodeling of distant organ microenvironments that impacts immune cell phenotypes, population structure, and intercellular communication. However, our understanding of immune phenotypic dynamics in the metastatic niche remains incomplete. Here, we longitudinally assayed lung immune transcriptional profiles in the polyomavirus middle T antigen (PyMT) and 4T1 metastatic breast cancer models from primary tumorigenesis, through pre-metastatic niche formation, to the final stages of metastatic outgrowth at single-cell resolution. Computational analyses of these data revealed a TLR-NFκB inflammatory program enacted by both peripherally derived and tissue-resident myeloid cells that correlated with pre-metastatic niche formation and mirrored CD14+ "activated" myeloid cells in the primary tumor. Moreover, we observed that primary tumor and metastatic niche natural killer (NK) cells are differentially regulated in mice and human patient samples, with the metastatic niche featuring elevated cytotoxic NK cell proportions. Finally, we identified cell-type-specific dynamic regulation of IGF1 and CCL6 signaling during metastatic progression that represents anti-metastatic immunotherapy candidate pathways.