The effect of radiation treatment of solid tumors on neutrophil infiltration and function: a systematic review.

Radiotherapy (RT) initiates a local and systemic immune response which can induce anti-tumor immunity and improve immunotherapy efficacy. Neutrophils are among the first immune cells that infiltrate tumors after RT and are suggested to be essential for the initial anti-tumor immune response. However, neutrophils in tumors are associated with poor outcomes and RT induced neutrophil infiltration could also change the composition of the tumor microenvironment (TME) in favor of tumor progression. To improve RT efficacy for cancer patients it is important to understand the interplay between RT and neutrophils. Here, we review the literature on how RT affects the infiltration and function of neutrophils in the TME of solid tumors, using both patients studies and preclinical murine in vivo models. In general, it was found that neutrophil levels increase and reach maximal levels in the first days after RT and can remain elevated up to three weeks. Most studies report an immunosuppressive role of neutrophils in the TME after RT, caused by upregulated expression of neutrophil indoleamine 2,3-dioxygenase 1 (IDO1) and arginase 1 (ARG1), as well as neutrophil extracellular trap (NET) formation. RT was also associated with increased reactive oxygen species (ROS) production by neutrophils, which can both improve and inhibit anti-tumor immunity. In addition, multiple murine models showed improved RT efficacy when depleting neutrophils, suggesting that neutrophils have a pro-tumor phenotype after RT. We conclude that the role of neutrophils should not be overlooked when developing RT strategies and requires further investigation in specific tumor types. In addition, neutrophils can possibly be exploited to enhance RT efficacy by combining RT with neutrophil-targeting therapies.

Neogambogic acid enhances anti-PD-1 immunotherapy efficacy by attenuating suppressive function of MDSCs in pancreatic cancer.

BACKGROUND: Anti-PD-1-based immunotherapy has limited benefits in patients with pancreatic cancer. Accumulating data indicate that natural products exert antitumor activity by remodeling the tumor immune microenvironment. It has been reported that neogambogic acid (NGA), an active natural monomer extracted from Garcinia, has anti-inflammatory and antitumor effects. Nevertheless, there are few systematic studies on the antitumor efficacy and immunomodulatory effects of NGA in pancreatic cancer. METHODS: An orthotopic mouse model of pancreatic cancer was established and were treated with different doses of NGA. Tumor growth and ascites were observed. Flow cytometry and immunohistochemistry (IHC) were used to investigate the tumor immune microenvironment. CD11b+ MDSCs were infused back into mice with pancreatic cancer to observe tumor progression after NGA treatment. Bone marrow cells were induced to differentiate into MDSCs, and the effects of NGA on MDSCs were analyzed and the underlying mechanism was explored. The effects of NGA combined with an anti-PD-1 antibody on pancreatic cancer were further tested. RESULTS: NGA significantly inhibited the tumor growth and improve ascites character in pancreatic cancer model mice. Flow cytometry and IHC analysis revealed that NGA decreased the MDSCs proportion and infiltration in the tumor microenvironment. Moreover, adoptive MDSCs largely attenuated the inhibitory effect of NGA on the progression of pancreatic cancer. In addition, we showed that NGA significantly promoted apoptosis and inhibited the differentiation, migration and immunosuppressive function of MDSCs and decreased level of STAT3 and p-STAT3. Furthermore, we demonstrated that NGA synergistically enhanced the efficacy of anti-PD-1 antibodies against pancreatic cancer. CONCLUSION: NGA inhibited the progression of pancreatic cancer by inhibiting MDSCs in the tumor microenvironment, and enhanced the efficacy of anti-PD-1 therapy in the treatment of pancreatic cancer.

Enhanced ApcMin/+ adenoma formation after epithelial CUL4B deletion by recruitment of myeloid-derived suppressor cells.

Colorectal cancer (CRC) stands as a prevalent malignancy globally. A pivotal event in CRC pathogenesis involves the loss-of-function mutation in the APC gene, leading to the formation of benign polyps. Despite the well-established role of APC, the contribution of CUL4B to CRC initiation in the pre-tumorous stage remains poorly understood. In this investigation, we generated a murine model by crossing ApcMin/+ mice with Cul4bΔIEC mice to achieve specific deletion of Cul4b in the gut epithelium against an ApcMin/+ background. By employing histological methods, RNA-sequencing (RNA-seq), and flow cytometry, we assessed alterations and characterized the immune microenvironment. Our results unveiled that CUL4B deficiency in gut epithelium expedited ApcMin/+ adenoma formation. Notably, CUL4B in adenomas restrained the accumulation of tumor-infiltrating myeloid-derived suppressor cells (MDSCs). In vivo inhibition of MDSCs significantly delayed the growth of CUL4B deleted ApcMin/+ adenomas. Furthermore, the addition of MDSCs to in vitro cultured ApcMin/+; Cul4bΔIEC adenoma organoids mitigated their alterations. Mechanistically, CUL4B directly interacted with the promoter of Csf3, the gene encoding granulocyte-colony stimulating factor (G-CSF) by coordinating with PRC2. Inhibiting CUL4B epigenetically activated the expression of G-CSF, promoting the recruitment of MDSCs. These findings offer novel insights into the tumor suppressor-like roles of CUL4B in regulating ApcMin/+ adenomas, suggesting a potential therapeutic strategy for CRC initiation and progression in the context of activated Wnt signaling.

MDSCs in bone metastasis: Mechanisms and therapeutic potential.

Bone metastasis (BM) is a frequent complication associated with advanced cancer that significantly increases patient mortality. Myeloid-derived suppressor cells (MDSCs) play a pivotal role in BM progression by promoting angiogenesis, inhibiting immune responses, and inducing osteoclastogenesis. MDSCs induce immunosuppression through diverse mechanisms, including the generation of reactive oxygen species, nitric oxide, and immunosuppressive cytokines. Within the bone metastasis niche (BMN), MDSCs engage in intricate interactions with tumor, stromal, and bone cells, thereby establishing a complex regulatory network. The biological activities and functions of MDSCs are regulated by the microenvironment within BMN. Conversely, MDSCs actively contribute to microenvironmental regulation, thereby promoting BM development. A comprehensive understanding of the indispensable role played by MDSCs in BM is imperative for the development of novel therapeutic strategies. This review highlights the involvement of MDSCs in BM development, their regulatory mechanisms, and their potential as viable therapeutic targets.

The Influence of Myeloid-Derived Suppressor Cell Expansion in Neuroinflammation and Neurodegenerative Diseases.

The neuro-immune axis has a crucial function both during physiological and pathological conditions. Among the immune cells, myeloid-derived suppressor cells (MDSCs) exert a pivotal role in regulating the immune response in many pathological conditions, influencing neuroinflammation and neurodegenerative disease progression. In chronic neuroinflammation, MDSCs could lead to exacerbation of the inflammatory state and eventually participate in the impairment of cognitive functions. To have a complete overview of the role of MDSCs in neurodegenerative diseases, research on PubMed for articles using a combination of terms made with Boolean operators was performed. According to the search strategy, 80 papers were retrieved. Among these, 44 papers met the eligibility criteria. The two subtypes of MDSCs, monocytic and polymorphonuclear MDSCs, behave differently in these diseases. The initial MDSC proliferation is fundamental for attenuating inflammation in Alzheimer's disease (AD), Parkinson's disease (PD), and multiple sclerosis (MS), but not in amyotrophic lateral sclerosis (ALS), where MDSC expansion leads to exacerbation of the disease. Moreover, the accumulation of MDSC subtypes in distinct organs changes during the disease. The proliferation of MDSC subtypes occurs at different disease stages and can influence the progression of each neurodegenerative disorder differently.

Multiple treatments with human embryonic stem cell-derived mesenchymal progenitor cells preserved the fertility and ovarian function of perimenopausal mice undergoing natural aging.

OBJECTIVES: Currently, no approved stem cell-based therapies for preserving ovarian function during aging. To solve this problem, we developed a long-term treatment for human embryonic stem cell-derived mesenchymal progenitor cells (hESC-MPCs). We investigated whether the cells retained their ability to resist ovarian aging, which leads to delayed reproductive senescence. MATERIALS AND METHODS: In a middle-aged female model undergoing natural aging, we analyzed whether hESC-MPCs benefit the long-term maintenance of reproductive fecundity and ovarian reservoirs and how their transplantation regulates ovarian function. RESULTS: The number of primordial follicles and mice with regular estrous cycles were increased in perimenopausal mice who underwent multiple introductions of hESC-MPCs compared to age-matched controls. The estradiol levels in the hESC-MPCs group were restored to those in the young and adult groups. Embryonic development and live birth rates were higher in the hESC-MPC group than in the control group, suggesting that hESC-MPCs delayed ovarian senescence. In addition to their direct effects on the ovary, multiple-treatments with hESC-MPCs reduced ovarian fibrosis by downregulating inflammation and fibrosis-related genes via the suppression of myeloid-derived suppressor cells (MDSCs) produced in the bone marrow. CONCLUSIONS: Multiple introductions of hESC-MPCs could be a useful approach to prevent female reproductive senescence and that these cells are promising sources for cell therapy to postpone the ovarian aging and retain fecundity in perimenopausal women.

Pivotal role of myeloid-derived suppressor cells in infection-related tumor growth.

BACKGROUND: In this study, we investigated infection-related tumor growth, focusing on myeloid-derived suppressor cells (MDSCs) in clinical and experimental settings. PATIENTS AND METHODS: In the clinical study, a total 109 patients who underwent gastrectomy or esophagectomy were included. Blood samples were collected from a preoperative time point through 3 months after surgery, and MDSCs were analyzed using flow cytometry. In animal experiments, peritonitis model mice were created by CLP method. We investigated the number of splenic MDSCs in these mice using flow cytometry. Malignant melanoma cells (B16F10) were inoculated on the back of the mice, and tumor growth was monitored. We compared the level of MDSC infiltration around the tumor and the migration ability between CLP and sham-operated mice-derived MDSCs. Finally, we focused on PD-L1+ MDSCs to examine the effectiveness of anti-PD-L1 antibodies on tumor growth in CLP mice. RESULTS: In patients with postoperative infectious complication, MDSC number was found to remain elevated 3 months after surgery, when the inflammatory responses were normalized. CLP mice showed increased numbers of MDSCs, and following inoculation with B16F10 cells, this higher number of MDSCs was associated with significant tumor growth. CLP-mice-derived MDSCs had higher levels of accumulation around the tumor and had more enhanced migration ability. Finally, CLP mice had increased numbers of PD-L1+ MDSCs and showed more effective inhibition of tumor growth by anti-PD-L1 antibodies compared to sham-operated mice. CONCLUSION: Long-lasting enhanced MDSCs associated with infection may contribute to infection-related tumor progression.

Carbon Ion Irradiation Activates Anti-Cancer Immunity.

Carbon ion beams have the unique property of higher linear energy transfer, which causes clustered damage of DNA, impacting the cell repair system. This sometimes triggers apoptosis and the release in the cytoplasm of damaged DNA, leading to type I interferon (IFN) secretion via the activation of the cyclic GMP-AMP synthase-stimulator of interferon genes pathway. Dendritic cells phagocytize dead cancer cells and damaged DNA derived from injured cancer cells, which together activate dendritic cells to present cancer-derived antigens to antigen-specific T cells in the lymph nodes. Thus, carbon ion radiation therapy (CIRT) activates anti-cancer immunity. However, cancer is protected by the tumor microenvironment (TME), which consists of pro-cancerous immune cells, such as regulatory T cells, myeloid-derived suppressor cells, and tumor-associated macrophages. The TME is too robust to be destroyed by the CIRT-mediated anti-cancer immunity. Various modalities targeting regulatory T cells, myeloid-derived suppressor cells, and tumor-associated macrophages have been developed. Preclinical studies have shown that CIRT-mediated anti-cancer immunity exerts its effects in the presence of these modalities. In this review article, we provide an overview of CIRT-mediated anti-cancer immunity, with a particular focus on recently identified means of targeting the TME.

Assessment of myeloid-derived suppressor cell differentiation ex vivo.

Myeloid-derived suppressor cells (MDSCs) are major promoters of progression and metastasis in cancer. MDSCs inhibit the anti-tumor immune response through multiple mechanisms. The main MDSC functions in cancer are related to the inactivation of T cells and the establishment of an immunosuppressive tumor microenvironment (TME) through the production of pro-inflammatory cytokines, among other mechanisms. MDSCs are phenotypically similar to conventional myeloid cells, so their identification is challenging. Moreover, they infiltrate the tumors in limited numbers, and their purification from within the tumors is technically difficult and makes their study a challenge. Therefore, several ex vivo differentiation methods have been established. Our differentiation method leads to MDSCs that closely model tumor-infiltrating counterparts. In this protocol, MDSCs are differentiated from bone marrow precursors by incubation in differentiation medium produced by murine tumor cell lines engineered to constitutively express granulocyte-monocyte colony stimulating factor (GM-CSF). These ex vivo-generated MDSC subsets show high fidelity compared to their natural tumor-infiltrated counterparts. Moreover, the high yields of purification from these ex vivo differentiated MDSC enable their use for validation of new treatments in high-throughput assays. In this chapter we describe the engineering of a stable cell line overexpressing GM-CSF, followed by production and collection of conditioned media supporting MDSC differentiation. Finally, we detail the isolation procedure of bone marrow cells and the specific MDSC differentiation protocol.

Modulation of myeloid-derived suppressor cell functions by oral inflammatory diseases and important oral pathogens.

The oral cavity presents a diverse microbiota in a dynamic balance with the host. Disruption of the microbial community can promote dysregulation of local immune response which could generate oral diseases. Additionally, alterations in host immune system can result in inflammatory disorders. Different microorganisms have been associated with establishment and progression of the oral diseases. Oral cavity pathogens/diseases can modulate components of the inflammatory response. Myeloid-derived suppressor cells (MDSCs) own immunoregulatory functions and have been involved in different inflammatory conditions such as infectious processes, autoimmune diseases, and cancer. The aim of this review is to provide a comprehensive overview of generation, phenotypes, and biological functions of the MDSCs in oral inflammatory diseases. Also, it is addressed the biological aspects of MDSCs in presence of major oral pathogens. MDSCs have been mainly analyzed in periodontal disease and Sjögren's syndrome and could be involved in the outcome of these diseases. Studies including the participation of MDSCs in other important oral diseases are very scarce. Major oral bacterial and fungal pathogens can modulate expansion, subpopulations, recruitment, metabolism, immunosuppressive activity and osteoclastogenic potential of MDSCs. Moreover, MDSC plasticity is exhibited in presence of oral inflammatory diseases/oral pathogens and appears to be relevant in the disease progression and potentially useful in the searching of possible treatments. Further analyses of MDSCs in oral cavity context could allow to understand the contribution of these cells in the fine-tuned balance between host immune system and microorganism of the oral biofilm, as well as their involvement in the development of oral diseases when this balance is altered.

Intracellular Major Histocompatibility Complex Class II and C-X-C Motif Chemokine Ligand 10-Expressing Neutrophils Indicate the State of Anti-Tumor Activity Induced by Bacillus Calmette-Guérin.

(1) Background: Inflammatory responses induce the formation of both anti-tumor and pro-tumor neutrophils known as myeloid-derived suppressor cells (MDSCs). Intermittent intravesical infusion of Bacillus Calmette-Guérin (BCG) is an established cancer immunotherapy for non-muscle-invasive bladder cancer (NMIBC). However, the types of neutrophils induced via the inflammatory response to both tumor-bearing and BCG remain unclear. (2) Methods: We therefore analyzed neutrophil dynamics in the peripheral blood and urine of patients with NMIBC who received BCG therapy. Further, we analyzed the effects of BCG in a mouse intraperitoneal tumor model. (3) Results: BCG therapy induced the formation of CXCL10 and MHC class II-positive neutrophils in the urine of patients with NMIBC but did not induce MDSC formation. CXCL10- and MHC class II-expressing neutrophils were detected in peritoneal exudate cells formed after BCG administration. Partial neutrophil depletion using an anti-Ly6G antibody suppressed the upregulation of CXCL10 and MHC class II in neutrophils and reversed the anti-tumor activity of BCG in mouse models. (4) Conclusions: These results indicated that intracellular MHC class II- and CXCL10-expressing neutrophils indicate the state of anti-tumor activity induced via BCG. The status of neutrophils in mixed inflammation of immunosuppressive and anti-tumor responses may therefore be useful for evaluating immunological systemic conditions.

A research on the influence of G-CSF mobilization on donor's peripheral blood MDSCs and its relationship with patient prognosis.

OBJECTIVE: To discuss the effects of mobilization of healthy donors with granulocyte colony-stimulating factor (G-CSF) on the absolute values and functions of myeloid-derived suppressor cells (MDSCs) and subpopulations of M-MDSCs and P-MDSCs in their peripheral blood. In addition, this study also aims to investigate the impacts of the adoptively transferred MDSCs from the grafts to the patients on their prognosis and immune reconstitution. METHODS: The selection of 72 donors and 72 patients were conducted for allogeneic hematopoietic stem cell transplantation (allo-HSCT) from August 2022 to December 2022 at Lu Daopei Hospital in Beijing, China. Statistical calculations were performed by Wilcoxon signed-rank test, Kruskal Wallis test, χ2 test, Kaplan Meier test, and log-rank test to analyze the data. RESULTS & CONCLUSION: G-CSF induced significant amplification of MDSCs in the peripheral blood of donors in percentage and absolute values. Whether the level of P-MDSCs in patients conducted for the adoptive transfer of P - MDSCs is higher than 3.7× 107/kg or lower than 1.4× 107/kg leads to a poor prognosis of the patients. Ensuring a balanced state of MDSCs is crucial for effective immunotherapy. Transferring a high level of MDSCs from the graft to the patient's body is advantageous for the development of MDSCs while simultaneously inhibiting the proliferation of lymphocyte subgroups.

Use of Magnetotactic Bacteria as an MRI Contrast Agent for In Vivo Tracking of Adoptively Transferred Immune Cells.

PURPOSE: In vivo immune cell tracking using MRI can be a valuable tool for studying the mechanisms underlying successful cancer therapies. Current cell labeling methods using superparamagnetic iron oxide (SPIO) lack the persistence to track the fate and location of transplanted cells long-term. Magnetospirillum magneticum is a commercially available, iron-producing bacterium that can be taken up by and live harmoniously within mammalian cells as magneto-endosymbionts (MEs). MEs have shown promise as labeling agents for in vivo stem and cancer cell tracking but have yet to be evaluated in immune cells. This pilot study examined ME labeling in myeloid-derived suppressor cells (MDSCs), cytotoxic T lymphocytes (CTLs), and dendritic cells (DCs) and its effects on cell purity, function, and MRI contrast. PROCEDURES: MDSCs, CTLs, and DCs were incubated with MEs at various ME labeling ratios (MLR), and various biological metrics and iron uptake were assessed. For in vivo imaging, MDSCs were labeled overnight with either MEs or SPIO (Molday ION Rhodamine B) and injected into C3 tumor-bearing mice via tail vein injection 24 days post-implant and scanned daily with MRI for 1 week to assess cellular quantification. RESULTS: Following incubations, MDSCs contained > 0.6 pg Fe/cell. CTLs achieved Fe loading of < 0.5 pg/cell, and DCs achieved Fe loading of ~ 1.4 pg/cell. The suppressive functionality of MDSCs at 1000 MLR was not affected by ME labeling but was affected at 2000 MLR. Markers of CTL dysfunction were not markedly affected by ME labeling nor were DC markers. In vivo data demonstrated that the MDSCs labeled with MEs generated sufficient contrast to be detectable using TurboSPI, similar to SPIO-labeled cells. CONCLUSIONS: Cells can be labeled with sufficient numbers of MEs to be detectable with MRI without compromising cell viability. Care must be taken at higher concentrations of MEs, which may affect some cell types' functional activity and/or morphology. Immune cells with minimal phagocytic behavior have much lower iron content per cell after incubation with MEs vs SPIO; however, MEs can successfully be used as a contrast agent for phagocytic immune cells.

Myeloid-derived suppressor cells (MDSCs): what do we currently know about the effect they have against anti-PD-1/PD-L1 therapies?

Recent advances in cancer treatment such as PD-1/PD-L1 checkpoint inhibitors have prompted multiple research studies to determine all of the factors that influence response or failure to these new treatments. One of those identified factors is myeloid-derived suppressor cells (MDSCs). These cells were identified and described for the first time in 2007 in laboratory mice and cancer patients. Previous studies showed that a greater number of MDSCs was directly related to a greater tumour volume. There are two clearly identified subpopulations: Mononuclear-type myeloid-derived suppressor cells (M-MDSCs) and polymorphonuclear (PMN-MDSCs). These cell population subtypes play a very important role, depending on the type of cancer, since they have the particularity of expressing PD-L1, which interacts with PD-1, inhibiting the expansion of cytotoxic T lymphocytes, promoting resistance to these treatments.

Rac and Cdc42 inhibitors reduce macrophage function in breast cancer preclinical models.

Background: Metastatic disease lacks effective treatments and remains the primary cause of mortality from epithelial cancers, especially breast cancer. The metastatic cascade involves cancer cell migration and invasion and modulation of the tumor microenvironment (TME). A viable anti-metastasis strategy is to simultaneously target the migration of cancer cells and the tumor-infiltrating immunosuppressive inflammatory cells such as activated macrophages, neutrophils, and myeloid-derived suppressor cells (MDSC). The Rho GTPases Rac and Cdc42 are ideal molecular targets that regulate both cancer cell and immune cell migration, as well as their crosstalk signaling at the TME. Therefore, we tested the hypothesis that Rac and Cdc42 inhibitors target immunosuppressive immune cells, in addition to cancer cells. Our published data demonstrate that the Vav/Rac inhibitor EHop-016 and the Rac/Cdc42 guanine nucleotide association inhibitor MBQ-167 reduce mammary tumor growth and prevent breast cancer metastasis from pre-clinical mouse models without toxic effects. Methods: The potential of Rac/Cdc42 inhibitors EHop-016 and MBQ-167 to target macrophages was tested in human and mouse macrophage cell lines via activity assays, MTT assays, wound healing, ELISA assays, and phagocytosis assays. Immunofluorescence, immunohistochemistry, and flow cytometry were used to identify myeloid cell subsets from tumors and spleens of mice following EHop-016 or MBQ-167 treatment. Results: EHop-016 and MBQ-167 inhibited Rac and Cdc42 activation, actin cytoskeletal extensions, migration, and phagocytosis without affecting macrophage cell viability. Rac/Cdc42 inhibitors also reduced tumor- infiltrating macrophages and neutrophils in tumors of mice treated with EHop-016, and macrophages and MDSCs from spleens and tumors of mice with breast cancer, including activated macrophages and monocytes, following MBQ-167 treatment. Mice with breast tumors treated with EHop-016 significantly decreased the proinflammatory cytokine Interleukin-6 (IL-6) from plasma and the TME. This was confirmed from splenocytes treated with lipopolysaccharide (LPS) where EHop-016 or MBQ-167 reduced IL-6 secretion in response to LPS. Conclusion: Rac/Cdc42 inhibition induces an antitumor environment via inhibition of both metastatic cancer cells and immunosuppressive myeloid cells in the TME.

Targeting N6-methyladenosine reader YTHDF1 with siRNA boosts antitumor immunity in NASH-HCC by inhibiting EZH2-IL-6 axis.

BACKGROUND & AIMS: RNA N6-methyladenosine (m6A) reader protein YTHDF1 has been implicated in cancer; however, its role in hepatocellular carcinoma (HCC), especially in non-alcoholic steatohepatitis-associated HCC (NASH-HCC), remains unknown. Here, we investigated the functional role of YTHDF1 in NASH-HCC and its interplay with the tumor immune microenvironment. METHODS: Hepatocyte-specific Ythdf1-overexpressing mice were subjected to a NASH-HCC-inducing diet. Tumor-infiltrating immune cells were profiled with single-cell RNA-sequencing, flow cytometry, and immunostaining. The molecular target of YTHDF1 was elucidated with RNA-sequencing, m6A-sequencing, YTHDF1 RNA immunoprecipitation-sequencing, proteomics, and ribosome-profiling. Ythdf1 in NASH-HCC models was targeted by lipid nanoparticle (LNP)-encapsulated small-interfering Ythdf1. RESULTS: YTHDF1 is overexpressed in tumor tissues compared to adjacent peri-tumor tissues from patients with NASH-HCC. Liver-specific Ythdf1 overexpression drives tumorigenesis in dietary models of spontaneous NASH-HCC. Single-cell RNA-sequencing and flow cytometry revealed that Ythdf1 induced accumulation of myeloid-derived suppressor cells (MDSCs) and suppressed cytotoxic CD8+ T-cell function. Mechanistically, Ythdf1 expression in NASH-HCC cells induced the secretion of IL-6, which mediated MDSC recruitment and activation, leading to CD8+ T-cell dysfunction. EZH2 mRNA was identified as a key YTHDF1 target. YTHDF1 binds to m6A-modified EZH2 mRNA and promotes EZH2 translation. EZH2 in turn increased expression and secretion of IL-6. Ythdf1 knockout synergized with anti-PD-1 treatment to suppress tumor growth in NASH-HCC allografts. Furthermore, therapeutic targeting of Ythdf1 using LNP-encapsulated small-interfering RNA significantly increased the efficacy of anti-PD-1 blockade in NASH-HCC allografts. CONCLUSIONS: We identified that YTHDF1 promotes NASH-HCC tumorigenesis via EZH2-IL-6 signaling, which recruits and activates MDSCs to cause cytotoxic CD8+ T-cell dysfunction. YTHDF1 may be a novel therapeutic target to improve responses to anti-PD-1 immunotherapy in NASH-HCC. IMPACT AND IMPLICATIONS: YTHDF1, a N6-methyladenosine reader, is upregulated in patients with non-alcoholic steatohepatitis (NASH)-associated hepatocellular carcinoma (HCC); however, its role in modulating the tumor immune microenvironment in NASH-HCC remains unclear. Here, we show that Ythdf1 mediates immunosuppression in NASH-HCC and that targeting YTHDF1 in combination with immune checkpoint blockade elicits robust antitumor immune responses. Our findings suggest novel therapeutic targets for potentiating the efficacy of immune checkpoint blockade in NASH-HCC and provide the rationale for developing YTHDF1 inhibitors for the treatment of NASH-HCC.

Identification and validation of Birc5 as a novel activated cell cycle program biomarker associated with infiltration of immunosuppressive myeloid-derived suppressor cells in hepatocellular carcinoma.

BACKGROUND: Preclinical studies and clinical trials have demonstrated that tumor-intrinsic activation of the cell cycle program impedes anticancer immunotherapy. Identification of cell cycle-related biomarkers may provide novel therapeutic targets to augment the efficacy of immunotherapy in hepatocellular carcinoma (HCC). METHOD AND RESULTS: Based on the genes related to cell cycle program, two clusters (Cluster 1 and Cluster 2) were detected in HCC patients via non-negative matrix factorization algorithm. Multivariable-adjusted Cox regression analysis indicated that the cell cycle gene-based classification was a significant prognostic factor for predicting the clinical outcome of HCC patients. Cluster 1 showed shorter overall survival time and progression-free interval time was associated with activated cell cycle program, higher infiltration of myeloid-derived suppressor cells (MDSCs) and less sensitivity to immunotherapy. A three-gene prognostic model, including BIRC5, C8G, and SPP1, was constructed to characterize the cell cycle-based classification of HCC, which had strong robustness and a stable predictive performance. Notably, Birc5 was positively correlated with CD11b expression (a MDSC marker) in HCC tissue. Concordant high expression of Birc5 and intratumor infiltration level of MDSCs were correlated with worse prognosis of HCC patients. In vitro, hepatocellular Birc5 overexpression promoted immunosuppressive CD11b+ CD33+ HLA-DR- MDSC expansion from human peripheral blood mononuclear cells. Genetically modified animal model of liver cancer revealed that Birc5 depletion upregulated the genes related to lymphocyte-mediated immunity, natural killer cell-mediated immunity, interferon-gamma production, T-cell activation, and T-cell-mediated cytotoxicity. These results suggest an immunosuppressive function of Birc5 in HCC. CONCLUSION: Birc5 was a potential biomarker and inducer of intratumor infiltration of MDSCs, which led to T cell exclusion or dysfunction in tumor immune microenvironment, consequently resulting in reduced response to ICIs in HCC.

Human Mesenchymal Stem Cells Modified with the NS5A Gene of Hepatitis C Virus Induce a Cellular Immune Response Exceeding the Response to DNA Immunization with This Gene.

Hepatitis C virus (HCV) is one of the basic culprits behind chronic liver disease, which may result in cirrhosis and hepatocarcinoma. In spite of the extensive research conducted, a vaccine against HCV has not been yet created. We have obtained human mesenchymal stem cells (hMSCs) and used them for expressing the HCV NS5A protein as a model vaccination platform. Sixteen hMSC lines of a different origin were transfected with the pcNS5A-GFP plasmid to obtain genetically modified MSCs (mMSCs). The highest efficiency was obtained by the transfection of dental pulp MSCs. C57BL/6 mice were immunized intravenously with mMSCs, and the immune response was compared with the response to the pcNS5A-GFP plasmid, which was injected intramuscularly. It was shown that the antigen-specific lymphocyte proliferation and the number of IFN-γ-synthesizing cells were two to three times higher after the mMSC immunization compared to the DNA immunization. In addition, mMSCs induced more CD4+ memory T cells and an increase in the CD4+/CD8+ ratio. The results suggest that the immunostimulatory effect of mMSCs is associated with the switch of MSCs to the pro-inflammatory phenotype and a decrease in the proportion of myeloid derived suppressor cells. Thus, the possibility of using human mMSCs for the creation of a vaccine against HCV has been shown for the first time.

Single-cell transcriptome profiling of sepsis identifies HLA-DRlowS100Ahigh monocytes with immunosuppressive function.

BACKGROUND: Sustained yet intractable immunosuppression is commonly observed in septic patients, resulting in aggravated clinical outcomes. However, due to the substantial heterogeneity within septic patients, precise indicators in deciphering clinical trajectories and immunological alterations for septic patients remain largely lacking. METHODS: We adopted cross-species, single-cell RNA sequencing (scRNA-seq) analysis based on two published datasets containing circulating immune cell profile of septic patients as well as immune cell atlas of murine model of sepsis. Flow cytometry, laser scanning confocal microscopy (LSCM) imaging and Western blotting were applied to identify the presence of S100A9+ monocytes at protein level. To interrogate the immunosuppressive function of this subset, splenic monocytes isolated from septic wild-type or S100a9-/- mice were co-cultured with naïve CD4+ T cells, followed by proliferative assay. Pharmacological inhibition of S100A9 was implemented using Paquinimod via oral gavage. RESULTS: ScRNA-seq analysis of human sepsis revealed substantial heterogeneity in monocyte compartments following the onset of sepsis, for which distinct monocyte subsets were enriched in disparate subclusters of septic patients. We identified a unique monocyte subset characterized by high expression of S100A family genes and low expression of human leukocyte antigen DR (HLA-DR), which were prominently enriched in septic patients and might exert immunosuppressive function. By combining single-cell transcriptomics of murine model of sepsis with in vivo experiments, we uncovered a similar subtype of monocyte significantly associated with late sepsis and immunocompromised status of septic mice, corresponding to HLA-DRlowS100Ahigh monocytes in human sepsis. Moreover, we found that S100A9+ monocytes exhibited profound immunosuppressive function on CD4+ T cell immune response and blockade of S100A9 using Paquinimod could partially reverse sepsis-induced immunosuppression. CONCLUSIONS: This study identifies HLA-DRlowS100Ahigh monocytes correlated with immunosuppressive state upon septic challenge, inhibition of which can markedly mitigate sepsis-induced immune depression, thereby providing a novel therapeutic strategy for the management of sepsis.

The influence of antibody CD166 on the treatment of tumor and the immunological mechanism in mice bearing oral squamous cell carcinoma.

Background: This study aimed to investigate the influence of antibody CD166 on the inhibition of tumor and further investigate the influence on immune cells of tumor tissues in mice bearing oral squamous cell carcinoma (OSCC). Methods: The xenograft model was established through subcutaneously injection of mouse OSCCs cells. Ten mice were randomly divided into two groups. The treatment group was treated with antibody CD166 and the control group was injected with the same volume normal saline. Hematoxylin and eosin (H&E) was used to confirm the tissue histopathology of xenograft mice model. Flow cytometry was used to detect the proportion of CD3+CD8+ T cells, CD8+PD-1+ cells and CD11b+Gr-1+ myeloid-derived suppressor cells (MDSCs) cells in the tumor tissues. Results: After treatment with antibody CD166, the tumor volume and weight in xenograft mice model were significantly reduced. The result of flow cytometry showed that antibody CD166 showed no obvious influence on the proportion of CD3+CD8+ and CD8+PD-1+ T lymphocyte cells in the tumor tissues. In the antibody CD166 treatment group, the proportion of CD11b+Gr-1+ MDSCs cells in tumor tissues was 1.930%±0.5317%, which was significantly lower than that of the control group, 4.940%±0.3252% (P=0.0013). Conclusions: Antibody CD166 treatment helped reduce the proportion of CD11b+Gr-1+ MDSCs cells, and produced obvious therapeutic effect on the treatment of mice bearing OSCC.