Infiltration Of Myeloid-derived Suppressor Cells Research Articles

Siwu decoction suppress myeloid-derived suppressor cells through tumour cells necroptosis to inhibit hepatocellular carcinoma

BackgroundHuman hepatocellular carcinoma (HCC) acquired resistance to anti-cancer agents due to the presence of immunosuppressive tumour microenvironment (TME) established by the interaction between tumour cells and immune populations. New treatment targeting the interaction is urgently needed and clinically beneficial to patients with HCC. This study aims to explore the anti-tumour effect of a Traditional Chinese Medicine formula Siwu Decoction (SWD) and its potential mechanism. Materials and methodsThe chemical profile of SWD was determined by high-performance liquid chromatography coupled with mass spectrometry. In vitro and in vivo effects of SWD in regressing HCC were assessed. The role of myeloid-derived suppressor cells (MDSCs) in mediating SWD-induced HCC inhibition was determined by adoptive transfer assay. The regulation of SWD-induced interaction between HCC cells and MDSCs was also confirmed both in vitro and in vivo. ResultsSWD dose-dependent inhibited the HCC growth and lung metastasis in an orthotopic growth tumour in mice, without significant toxicity and adverse side effect. SWD induced necroptosis in HCC cells, but did not directly inhibit in vitro culture of MDSCs, instead, SWD-treated HCC cell culture supernatant suppressed MDSCs by inducing its cell apoptosis. The necroptotic response of HCC cells can also suppress the MDSCs population in the TME without reducing circulating MDSCs infiltration into the tumours. Adoptive transfer of MDSCs recovered tumour growth and lung metastasis of HCC in SWD-treated mice. In HCC cells, SWD induced a necroptotic response, and blockade of necroptotic response in HCC cells recovered the MDSCs population in vitro and in vivo, and restored tumour growth and lung metastasis in SWD-treated mice. A combination of SWD improves the anti-HCC efficacy of sorafenib without inducing adverse side effects. Albiflorin, the effective compound of SWD, its anti-HCC manner has been verified to be consistent with that of SWD. ConclusionOur study observed for the first time that SWD can suppress HCC by regulating MDSCs through necroptosis of tumour cells in the TME. The main effective compound of SWD, albiflorin can be a potential adjuvant therapy in the clinical management of human HCC.

Effects of the subtypes of apolipoprotein E on immune inhibition and prognosis in patients with Hepatocellular Carcinoma

PurposeTo investigate whether prognosis of patients with hepatocellular carcinoma (HCC) is affected by the abundance and subgroups of myeloid-derived suppressor cells (MDSCs) as well as subtypes and expression of apolipoprotein E (apoE).Methods31 HCC patients were divided into three groups according to blood total apoE level for detecting the abundance of immunoregulatory cells by flow cytometry. Tumour tissue microarrays from 360 HCC patients were evaluated about the abundance and subgroups of MDSCs and the expression of apoE2, apoE3, apoE4 by immunofluorescence staining and immunohistochemistry staining. Survival analysis by means of univariate, multivariate COX regression and Kaplan-Meier methods of the 360 patients was performed based on clinical and pathological examinations along with 10 years’ follow-up data.ResultsThe lower apoE group presented higher abundance of MDSCs in the peripheral blood of HCC patients than higher apoE group. The abundance of monocyte-like MDSCs (M-MDSCs) was higher in the apoE low level group than high level group (p = 0.0399). Lower H-score of apoE2 (HR = 6.140, p = 0.00005) and higher H-score of apoE4 (HR = 7.001, p = 0.009) in tumour tissue were significantly associated with shorter overall survival (OS). The higher infiltration of polymorphonuclear granulocyte-like MDSCs (PMN-MDSCs, HR = 3.762, p = 0.000009) and smaller proportion of M-MDSCs of total cells (HR = 0.454, p = 0.006) in tumour tissue were independent risk factors for shorter recurrence-free survival (RFS).ConclusionThe abundance of MDSCs in HCC patients’ plasma negatively correlates with the level of apoE. The expression of apoE4 in HCC tissue indicated a poor prognosis while apoE2 might be a potential protective factor.

Increase in GPIHBP1 expression in advanced stage colorectal cancer indicates poor immune surveillance.

Glycosylphosphatidylinositol (GPI)-anchored high-density lipoprotein-binding protein 1 (GPIHBP1) plays a crucial role in fatty acid metabolism, which is involved in the progression of colorectal cancer (CRC). The aim of this study was to determine the expressional variations of GPIHBP1 in CRC at different stages and to verify whether this protein affects the shaping of the immune microenvironment of cancer cells. Variations of GPIHBP1 messenger RNA (mRNA) levels were first analysed using The Cancer Genome Atlas (TCGA) database. Protein levels of GPIHBP1 in cancer nest cells, stromal cells or surrounding normal tissues from 68 patients with CRC were checked by immunohistochemistry. Infiltration of immune cells such as macrophages, myeloid-derived suppressor cells (MDSCs), CD8+ and CD56+ cells was parallelly stained in the same tissues. Ectopic GPIHBP1 expressed colonic tumour cells were transplanted into the back of mice. Tumour growth and immune cell infiltrations were also observed. Compared with those in healthy tissues, GPIHBP1 mRNA and protein levels decreased in the patients with CRC at Dukes A-B stage but gradually increased in the patients at Dukes C-D stage. GPIHBP1 in foci or stroma was positively correlated with recruited macrophages or MDSCs and negatively correlated with recruited CD8+, CD56+ or granzyme+ cells. The mice injected with GPIHBP1 overexpression cells bore large tumours. Histological analysis confirmed the infiltration of many macrophages and MDSCs but less CD8+ T or CD56+ cells. The increased expression of GPIHBP1 is involved in the progression of CRC. High GPIHBP1 level of advanced CRC indicates efficient immune evasion in tumour microenvironment.

KRAS mutation subtypes responded differently to combination immunotherapy via disturbing myeloid-derived suppressor cells in non-small cell lung cancer.

8635 Background: The combination of immune checkpoint inhibitors (ICIs) and chemotherapy has shown high efficacy in non-small cell lung cancer (NSCLC) patients, but further exploration of the predictive biomarkers is needed. KRAS is the most common driver oncogene in patients with NSCLC. However, the influence of different KRAS mutation subtypes on immunotherapy response remains controversial. We aimed to evaluate the value of KRAS mutation subtypes for predicting response to immunotherapy in NSCLC patients and to further analyze the related tumor immune microenvironment (TiME) phenotypes. Methods: In this retrospective study, clinical data were extracted from the medical records of patients with advanced NSCLC treated with ICIs and whose available molecular analysis included KRAS mutation information between January 2017 and December 2022. NSCLC tissue samples were collected for TiME analysis. Differences in immune cell subsets between different groups were analyzed using the Mann‒Whitney U test. Clinical characteristics, ICI treatment efficacy, progression-free survival (PFS), and overall survival (OS) were analyzed using multivariate Cox models. Results: A total of 1913 eligible KRAS-mutated NSCLC patients were screened, and 137 patients who received systemic immunotherapy were enrolled (108 males vs 29 females), with a median age of 62 (range, 39-84) years. Among them, 126 (92.0%) patients had adenocarcinoma, 99 (72.3%) patients were systemic therapy naïve, and 124 (90.5%) patients received combined immunotherapy. Among the patients with KRAS mutations, 48 (35.0%) had G12C, 20 (14.6%) had G12V, 27 (19.7%) had G12D and 42 (30.7%) had other subtypes. PFS was significantly longer in the G12C subgroup than in the other mutation subgroup (median PFS: 11.3 vs 6.8 months, P = 0.001). The median PFS times in the G12C/G12V subgroup and the other groups were 11.1 months and 6.5 months, respectively ( P= 0.0005). Multivariate analysis of the immunotherapy cohort revealed that G12C/G12V was an independent predictor of PFS ( P = 0.003, hazard ratio (HR): 2.021, 95% CI: 1.265-3.227). Further TiME analysis suggested that the infiltration of myeloid-derived suppressor cells (MDSCs, CD33+CD11b+) in the tumor and stromal areas in the G12C/G12V subgroup was significantly lower than that in the other subgroup (tumor area, P= 0.045; stroma area, P= 0.015). However, no significant difference in PD-L1 expression was found between the G12C/G12V group and the other groups. Conclusions: KRAS G12C and G12V were associated with favorable combined ICI treatment efficacy in patients with NSCLC. Patients with G12C or G12V mutations had longer PFS and lower infiltration of MDSCs. KRAS mutation subtypes are potential predictive biomarkers disturbed by MDSCs for combination therapy in NSCLC patients.

Identification of PSMD11 as a novel cuproptosis- and immune-related prognostic biomarker promoting lung adenocarcinoma progression.

Due to the unfavorable prognosis associated with lung adenocarcinoma (LUAD), the development of targeted therapies and immunotherapies is essential. Cuproptosis, an emerging form of regulated cell death, is implicated in mitochondrial metabolism and is induced by copper ions. This study aimed to explore the prognostic value of cuproptosis- and immune-related genes (CIRGs) in LUAD. We used The Cancer Genome Atlas database to develop a prognostic prediction model for LUAD patients based on eight CIRGs. Using Cox regression analysis, we determined that the CIRG signature is a reliable, independent prognostic factor. We further identified PSMD11 as a critical CIRG and performed immunohistochemistry to study the protein expression levels of PSMD11 in LUAD tissues. We also investigated the impact of PSMD11 on the biological behavior of lung cancer cell lines. We found that patients with low PSMD11 expression levels displayed an improved prognosis compared with those with high PSMD11 expression levels. Overexpression of PSMD11 enhanced proliferation, migration, invasion, and tumor growth of lung carcinoma cell line A549, while PSMD11 knockdown diminished proliferation, migration, invasion, and tumor growth of lung carcinoma cell line PC9. Additionally, we discovered that PSMD11 expression was positively correlated with the infiltration of myeloid-derived suppressor cells and the increased expression of immunosuppressive molecules. These findings suggest that PSMD11 may serve as a valuable prognostic biomarker and therapeutic target for LUAD.

Chronic Microglia Activation Is Suffcient to Induce Significant Increase in Pulmonary Pressure

Introduction: Microglia, the primary resident immune cells of the brain, can be activated very early in disease. Our previous studies have indicated that microglia activation and neuroinflammation in autonomic brain areas were associated with pulmonary hypertension (PH). Furthermore, we also demonstrated that the attenuation of neuroinflammation by either pharmacological inhibition of microglia with minocycline or genetic disruption of neuro-microglia interaction by CX3CR1 depletion led to protection against pulmonary hypertension. Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) plays a crucial role in controlling inflammatory responses via gene expression regulation. NF-κB signaling in microglia triggers a distinct neuroinflammatory response with increased migration, phagocytosis, and release of proinflammatory cytokines such as IL-6, IL-1β and TNF-α. Thus, in this study we investigated if targeted microglia activation was suffcient to initiate the cascade of events that may contribute to the development of pulmonary hypertension. Study Objective: This study was designed to test the hypothesis that chronic microglia activation is suffcient to induce pulmonary hypertension Methods To evaluate the specific contribution of microglia pro-inflammatory activation to modulate pulmonary pressure, we used a mouse line with inducible expression of the Cre-recombinase directed to the Cx3cr1 promoter crossed with IKKcaflox-flox mice. The breeding scheme CX3CR1creER/+: IKKcaflox-flox allows the Cre-recombination events, as induced by tamoxifen administration, to occur in many myeloid cell types. However, when recombination is followed by at least six weeks of time for the repopulation of cells with high turnover rates, this protocol promotes a relatively selective activation of NF-κB signaling on microglia. First, CX3CR1creER/+:IKKcaflox-flox mice (n=14) were injected with tamoxifen or oil (control) (i.p.) daily at 150 mg/kg body weight for six consecutive days. After 8 weeks, invasive RVSP measurement was performed, and tissues were harvested for following analysis. Briefly, a Millar catheter is inserted into the right ventricle. Ventricle pressures and ECG were recorded and analyzed. PH phenotype also included the measurement of the Fulton index (RV:LV+S) and histological and flow cytometry analyses of lung. Data was collected in two separate experiments with similar results. Results: Activation of NF-κB on microglia was suffcient to induce a slight but significant increase in right ventricular systolic pressure (RVSP), surrogate of pulmonary pressure (RVSP: Tamoxifen: 26.23 ± 0.5861 mmHg; Oil: 23.16 ± 0.4339 mmHg (p = 0.001)), and increased right heart contractility (+dP/dt: Tamoxifen: 1586± 90.18 mmHg/s; Oil: 1250 ± 65.39 mmHg/s (p = 0.0092). Interestingly, activation of NF-κB on microglia also induced a significant increase in lung infiltration of monocytic myeloid-derived suppressor cells (Mo-MDSC, CD11b+Ly6ChiLy6G−) and granulocytic-like MDSCs (PMN-MDSC, CD11b+Ly6Clo Ly6G+). Conclusion: Activation of NF-κB on microglia was suffcient to induce a significant increase in pulmonary pressure and increase lung infiltration of myeloid-derived suppressor cells associated with smooth muscle cell proliferation in pulmonary hypertension. K99 HL165026, R01 HL142776. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Early administration of Wumei Wan inhibit myeloid-derived suppressor cells via PI3K/Akt pathway and amino acids metabolism to prevent colitis-associated colorectal cancer

Ethnopharmacological relevanceWumei Wan (WMW), a traditional Chinese medicine prescription, has been proved to be effective in treating Colitis-associated colorectal cancer (CAC), but it has not been proven to be effective in different stages of CAC. Aim of the studyThe purpose of our study is to investigate the therapeutic effect and mechanism of WMW on the progression of CAC. Materials and methodsAzioximethane (AOM) and dextran sulfate sodium (DSS) were used to treat mice for the purpose of establishing CAC models. WMW was administered in different stages of CAC. The presentative chemical components in WMW were confirmed by LC-MS/MS under the optimized conditions. The detection of inflammatory cytokines in the serum and colon of mice were estimated by qRT-PCR and ELISA. The changes of T cells and myeloid-derived suppressor cells (MDSCs) in each group were detected by flow cytometry. The metabolic components in serum of mice were detected by UPLC-MS/MS. Expression of genes and proteins were detected by eukaryotic transcriptomics and Western blot to explore the key pathway of WMW in preventing CAC. ResultsWMW had significant effect on inhibiting inflammatory responses and tumors during the early development stage of CAC when compared to other times. WMW increased the length of mice's colons, reduced the level of IL-1β, IL-6, TNF-α in colon tissues, and effectively alleviated colonic inflammation, and improved the pathological damage of colon tissues. WMW could significantly reduce the infiltration of MDSCs in the spleen, increase CD4+ T cells and CD8+ T cells in the spleen of CAC mice, and effectively reform the immune microenvironment in CAC mice.Transcriptomics analysis revealed that 2204 genes had different patterns of overlap in the colon tissues of mice between control group, AOM + DSS group, and early administration of WMW group. And KEGG enrichment analysis showed that PI3K/Akt signaling pathway, ECM-receptor interaction, IL-17 signaling pathway, MAPK signaling pathway, pancreatic secretion, thermogenesis, and Rap1 signaling pathway were all involved.The serum metabolomics results of WMW showed that the metabolic compositions of the control group, AOM + DSS group and the early stage of WMW were different, and 42 differential metabolites with the opposite trends of changes were screened. The metabolic pathways mainly included pyrimidine metabolism, glycine, serine and threonine metabolism, tryptophan metabolism, and purine metabolism. And amino acids and related metabolites may play an important role in WMW prevention of CAC. ConclusionWMW can effectively prevent the occurrence and development of CAC, especially in the initial stage. WMW can reduce the immune infiltration of MDSCs in the early stage. Early intervention of WMW can improve the metabolic disorder caused by AOM + DSS, especially correct the amino acid metabolism. PI3K/Akt signaling pathway was inhabited in early administration of WMW, which can regulate the amplification and function of MDSCs.

Radiotherapy Enhances Metastasis Through Immune Suppression by Inducing PD-L1 and MDSC in Distal Sites.

Radiotherapy (RT) is a widely employed anticancer treatment. Emerging evidence suggests that RT can elicit both tumor-inhibiting and tumor-promoting immune effects. The purpose of this study is to investigate immune suppressive factors of radiotherapy. We used a heterologous two-tumor model in which adaptive concomitant immunity was eliminated. Through analysis of PD-L1 expression and myeloid-derived suppressor cells (MDSC) frequencies using patient peripheral blood mononuclear cells and murine two-tumor and metastasis models, we report that local irradiation can induce a systemic increase in MDSC, as well as PD-L1 expression on dendritic cells and myeloid cells, and thereby increase the potential for metastatic dissemination in distal, nonirradiated tissue. In a mouse model using two distinct tumors, we found that PD-L1 induction by ionizing radiation was dependent on elevated chemokine CXCL10 signaling. Inhibiting PD-L1 or MDSC can potentially abrogate RT-induced metastasis and improve clinical outcomes for patients receiving RT. Blockade of PD-L1/CXCL10 axis or MDSC infiltration during irradiation can enhance abscopal tumor control and reduce metastasis.

Bioinformatics-based screening and analysis of the key genes involved in the influence of antiangiogenesis on myeloid-derived suppressor cells and their effects on the immune microenvironment.

This study aimed to screen differentially expressed genes (DEGs) involved in the influence of antiangiogenic therapy on myeloid-derived suppressor cell (MDSC) infiltration and investigate their mechanisms of action. Data on DEGs after the action of antiangiogenic drugs in a pan-cancer context were obtained from the Gene Expression Omnibus (GEO) database. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using the clusterProfiler package in R software. Single-sample gene set enrichment analysis was performed using the gene set variation analysis package to evaluate the levels of immune cells and the activity of immune-related pathways. The relationships of DEGs with the infiltration levels of MDSCs and specific immune cell subpopulations were investigated via gene module analysis. The top 10 key genes were subsequently obtained from PPI network analysis using the cytoHubba plugin of the Cytoscape platform. When the DEGs of the four datasets were intersected, a DEG in the intersection of three datasets and 12 DEGs in the intersection of two datasets were upregulated, and 28 DEGs in the intersection of two datasets were downregulated. GO and KEGG pathway enrichment analyses revealed that the DEGs were associated with multiple important signaling pathways closely related to tumor onset and development, including cell differentiation, cell proliferation, the cell cycle, and immune responses. Most downregulated genes in lung adenocarcinoma (LUAD) were positively correlated with MDSC expression. Only MGP was negatively correlated; the correlation between CACNG6 and MDSC expression was statistically insignificant. In lung squamous cell carcinoma (LUSC), the relationships of PMEPA1, PCDH7, NEURL1B, and CACNG6 with MDSC expression were statistically insignificant; MGP was negatively correlated with MDSC expression. The top 10 key genes with the highest degree scores obtained using the cytoHubba plugin of Cytoscape were AURKB, RRM2, BUB1, NUSAP1, PRC1, TOP2A, NCAPH, CENPA, KIF2C, and CCNA2. Most of these genes were upregulated in LUAD and associated with immune cell infiltration and prognosis in tumors. An analysis of the relationships between DEGs and infiltration by other specific immune cells revealed the presence of consistent patterns in the downregulated genes, which exhibited positive correlations with the levels of Th2 cells, γδ T cells, and CD56dim NK cells, and negative correlations with other infiltrating immune cells. Antiangiogenic therapy may regulate MDSC infiltration through multiple important signaling pathways closely associated with tumor onset and development, such as cell differentiation, cell proliferation, the cell cycle, and immune responses. Antiangiogenic drugs may exert effects by affecting various types of infiltrating cells associated with immune suppression.

Abstract 4989: Targeting IL-1β mitigates radiation induced MDSC expansion & improves survival in glioblastoma

Abstract Background: Our previous research demonstrated that radiation therapy (RT) for glioblastoma (GBM) induces aberrant myelopoiesis, increasing production of myeloid derived suppressor cells (MDSCs) and exacerbating T-cell suppression. However, the precise biological mechanisms underlying this phenomenon remain unclear. This study aims to elucidate the role of the pro-inflammatory cytokine interleukin-1beta (IL-1β) in RT-induced myelopoiesis and its correlation with poor prognosis in GBM patients. Methods: We conducted an extensive analysis of circulatory immune cells and pro-inflammatory cytokines in GBM patients treated with chemoradiotherapy. Using an orthotopic mouse model of GBM, we investigated the impact of IL-1β on myelopoiesis and survival. Functional assays were employed to examine MDSC expansion. Multi-color flow cytometry was used for immune profiling. Results: We found that RT elevated the plasma IL-1β concentration in both GBM patients and GBM-bearing mice, which also corresponded with an increase of MDSC in blood. In the murine GBM model, RT appeared to promote hematopoietic stem (HSPC) differentiation towards myelopoiesis. This led to increased MDSC precursor cells in the bone marrow, elevated circulating MDSCs in blood, and more infiltration of MDSCs in the tumor microenvironment (TME). Similar effects were observed in GBM-bearing mice following the administration of exogenous IL-1β. In contrast, inhibition of IL-1β using anti-IL-1β antibody during RT led to reduced myelopoiesis. When anti-IL-1β antibody was combined with functional inhibition of MDSCs using phosphodiesterase-5 inhibitor-tadalafil during RT, we observed decreased MDSC infiltration and increased CD8+ T cell infiltration in the TME. The combination approach also extended the survival of GBM-bearing mice after RT longer than either drug alone. Conclusions: Irradiation of GBM appears to induce IL-1β to promote aberrant myelopoiesis and MDSC expansion. A multi-pronged approach to inhibit MDSC production using IL-1β inhibitor and MDSC function using tadalafil during RT may be a promising approach to overcome the immune-suppressive TME of GBM. Citation Format: Subhajit Ghosh, Jiayi Huang, David DeNardo, Dennis Hallahan, Jerry Jaboin, Dinesh Thotala. Targeting IL-1β mitigates radiation induced MDSC expansion & improves survival in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4989.

Abstract 5361: Aberrant glycosylation in mPDAC is associated with increased epithelial-mesenchymal transition and recruitment of immunosuppressive cells

Abstract The tumor-associated glycan Tn antigen (GalNAc-α-O-Ser/Thr) is associated with poor prognosis in various solid tumors. However, the mechanisms that Tn antigen promotes tumorigenesis are incompletely understood. Here, we generated Tn antigen-expressing murine pancreatic ductal adenocarcinoma (mPDAC) tumor cell lines through CRISPR/Cas9-mediated knockout of the C1galt1c1 gene. Tn antigen expressing mPDAC cell lines exhibited increased in vitro proliferation and migration compared to parental controls. RNA sequencing revealed that loss of C1galt1c1 in mPDAC cells correlated with increased expression of genes associated with epithelial-mesenchymal transition, myeloid recruitment, and extracellular matrix deposition. Consequently, knock-down of elevated genes using CRISPR interference dampened in vitro cell proliferation of C1galt1c1 knockout mPDAC cells to the growth rate of parental controls. Additionally, C1galt1c1 knockout mPDAC cells implanted into mice led to accelerated tumor growth and increased tumor infiltration of myeloid-derived suppressor cells and neutrophils as determined by flow cytometry and scRNAseq. Interestingly, surface expression of a binding partner of Tn antigen, macrophage galactose-type lectin 2 (Mgl2), was altered in the immune infiltrate of C1galt1c1 knockout tumors suggesting a role for the Mgl2-Tn interaction in tumor progression. Mgl2-deficient mice exhibit increased T effector cells in the draining lymph nodes of tumors highlighting a potential mechanism for promoting the influx of immune cells in ordinarily cold tumors. Together, our data indicate that truncated O-glycans expressed commonly on tumor cells promotes tumorigenesis through creating an immunosuppressive microenvironment in mPDAC. Citation Format: Shawna Brookens, David Degaramo, Kiesha Wilson, Wilder Condori Obregon, Prakash Nagarkatti, Mitzi Nagarkatti, Avery Posey Jr.. Aberrant glycosylation in mPDAC is associated with increased epithelial-mesenchymal transition and recruitment of immunosuppressive cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5361.

Candida albicans-myeloid cells-T lymphocytes axis in the tumor microenvironment of oral tumor-bearing mice

Candida albicans (C. albicans) is associated with the development of oral cancer. Here, we report the altered tumor microenvironment in oral tumor-bearing mice caused by C. albicans infection. Single-cell RNA sequencing showed that C. albicans infection influenced the tumor microenvironment significantly. Specifically, C. albicans infection reduced the CD8+ T cells but increased the IL-17A+ CD4+ T cells and IL-17A+ γδ T cells in oral tumor. The neutralization of IL-17A or TCR γ/δ alleviated the tumor progression caused by C. albicans infection. Additionally, C. albicans infection promoted the infiltration of myeloid-derived suppressor cells (MDSCs) into tumor, especially polymorphonuclear (PMN)-MDSCs, which infiltration was reduced after the neutralization of CCL2. Thus, our findings reveal the myeloid cells-T lymphocytes axis in oral tumor microenvironment with C. albicans infection, which helps to understand the mechanisms for C. albicans promoting oral cancer from the perspective of immune microenvironment.

Clinicopathological significance of microsatellite instability and immune escape mechanism in patients with gastric solid-type poorly differentiated adenocarcinoma.

In gastric solid-type poorly differentiated adenocarcinoma (PDA), the role of microsatellite instability and immune escape mechanism remains unclear. The current study aimed to elucidate the clinical significance of mismatch repair (MMR) status, genome profile, C-X-C motif chemokine receptor 2 (CXCR2) expression, and myeloid-derived suppressor cell (MDSC) infiltration in solid-type PDA. In total, 102 primary solid-type PDA cases were retrieved, and classified into 46 deficient-MMR (dMMR) and 56 proficient-MMR (pMMR) cases based on immunohistochemistry (IHC) and polymerase chain reaction-based molecular testing results. The mRNA expression profiles (NanoString nCounter Assay) of stage-matched dMMR (n = 6) and pMMR (n = 6) cases were examined. The CXCR2 expression and MDSC infiltration (CD11b- and CD33-positive cells) were investigated via IHC in all solid-type PDA cases. mRNA analysis revealed several differentially expressed genes and differences in biological behavior between the dMMR (n = 46) and pMMR (n = 56) groups. In the multivariate analysis, the dMMR status was significantly associated with a longer disease-free survival (hazard ratio = 5.152, p = 0.002) and overall survival (OS) (hazard ratio = 5.050, p = 0.005). CXCR2-high expression was significantly correlated with a shorter OS in the dMMR group (p = 0.018). A high infiltration of CD11b- and CD33-positive cells was significantly correlated with a shorter OS in the pMMR group (p = 0.022, 0.016, respectively). dMMR status can be a useful prognostic predictor, and CXCR2 and MDSCs can be novel therapeutic targets in patients with solid-type PDA.

Abstract B087: Targeting myeloid derived suppressor cell migration decreases tumor progression in a murine model of ovarian cancer

Abstract Introduction: High grade serous ovarian, fallopian tube and primary peritoneal carcinoma (HGSC) has an immunosuppressive and tumor promoting immune microenvironment. In this study, we have targeted the migration of myeloid derived suppressor cells (MDSC) through chemical inhibition of the CXCR2 chemotactic receptor to reverse the tumor-induced immunosuppression.Methods: ID8 cells with a p53 mutation (ID8-P53null) were used as a murine model of HGSC. ID8-p53null cells were injected intraperitoneally into immune intact C57/Bl6 mice, with 10 mice per group. After 7 days, mice were treated with cisplatin (cis, 1mg/kg, i.p., weekly), a CXCR2 inhibitor (CXCR2i, 4mg/kg, i.p., daily) or a combination of the 2 treatments. Necropsy was performed on day 29. The tumors were weighed, and the number of metastatic sites was quantified. The spleen and the tumor were resected, digested and flow cytometry was performed to evaluate the effects of treatment on the tumor immune microenvironment (TIME) as well as any systemic changes (evaluated by immune cells in the spleen). Granulocytic MDSC (G-MDSC) and monocytic MDSCs (M-MDSC) were defined as CD11b+Ly6G+,Ly6Clo and CD11b+Ly6G−Ly6Chi, respectively. One-way ANOVA with Tukey multiple comparisons was used for statistics with p<0.05 as the cut off for significance. Results: Tumor weights were significantly less in cis treated vs control mice (0.064g vs 0.220g, p <0.001) and CXCR2i treated vs control mice (0.171g vs 0.220g, p=.003). There was no additive effect of the CXCR2i to the cis treatment (cis 0.064g vs cis + CXCR2i 0.049g, p=0.62). The number of metastatic sites as a measurement of dissemination also decreased with cis or CXCR2i (number of sites: control: 47 vs cis: 12.7 vs CXCR2i: 32.5 vs cis + CXCR2i: 10.2). When the TIME was evaluated by flow cytometry, there were no significant differences in the immune populations including T-cells, NK cells, myeloid cells, and macrophages. However, there was a trend for cis to increase both the G-MDSC and M-MDSC populations and this effect was inhibited with the combination treatment of cis and CXCR2i. For the G-MDSC population control: 0.6% vs cis: 0.9% vs cis+CXCR2i: 0.5% and M-MDSC population control 3.4% vs cis: 6.8% vs cis+CXCR2i: 2.6%. Conclusion: CXCR2 is a chemotactic receptor present on MDSCs. We targeted this receptor to decrease the migration and infiltration of MDSCs into the ovarian cancer TIME. The CXCR2i decreased tumor progression in the ID8-p53null model of ovarian cancer. The TIME was evaluated by flow cytometry, and there was a trend for cis to increase the suppressive MDSC populations and this increase was inhibited by the CXCR2i. These exciting data suggest that traditional chemotherapy may lead to a more immunosuppressive TIME, which could have implications for future recurrence and treatment. Blocking the migration of immunosuppressive cells into the TIME may have a long-term impact on survival in HGSC. Survival studies in this murine model of HGSC are currently underway. Citation Format: Nicole Marjon, Railey Mikeska, Elizabeth R. Woodruff, Ritsuko Iwanaga, Tomomi Yamamoto, Benjamin G. Bitler. Targeting myeloid derived suppressor cell migration decreases tumor progression in a murine model of ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B087.

Sexual-biased necroinflammation is revealed as a predictor of bevacizumab benefit in glioblastoma.

Glioblastoma (GBM) is a highly malignant brain tumor that affects men more often than women. In addition, the former shows a poorer survival prognosis. To date, the reason for this sex-specific aggressiveness remains unclear. Therefore, the aim of this study is to investigate tumor processes that explain these sex differences. This was a retrospective study of GBM patients which was stratified according to sex. A cohort with 73 tumors was analyzed with immunohistochemistry, RNA-seq and RT-qPCR to characterize differences in vascular and immunological profiles. Transcriptomic profiling, gene set enrichment analysis, and pathway enrichment analysis were used for discovering molecular pathways predominant in each group. We further investigated the therapeutic effect of bevacizumab (vascular endothelial growth factor A (VEGFA) blocking antibody) in a retrospective GBM cohort (36 tumors) based on sex differences. We found that under hypoxic tumor conditions, 2 distinct tumor immuno-angiogenic ecosystems develop linked to sex differences and ESR1 expression is generated. One of these subgroups, which includes male patients with low ESR1 expression, is characterized by vascular fragility associated with the appearance of regions of necrosis and high inflammation (called necroinflamed tumors). This male-specific tumor subtype shows high inflammation related to myeloid-derived suppressor cells infiltration. Using this stratification, we identified a possible group of patients who could respond to bevacizumab (BVZ) and revealed a genetic signature that may find clinical applications as a predictor of those who may benefit most from this treatment. This study provides a stratification based on the sexual differences in GBM, which associates the poor prognosis with the presence of immunosuppressive myeloid cells in the necrotic areas. This new stratification could change the current prognosis of GBM and identifies those who respond to BVZ treatment.

DeepRisk network: an AI-based tool for digital pathology signature and treatment responsiveness of gastric cancer using whole-slide images.

Digital histopathology provides valuable information for clinical decision-making. We hypothesized that a deep risk network (DeepRisk) based on digital pathology signature (DPS) derived from whole-slide images could improve the prognostic value of the tumor, node, and metastasis (TNM) staging system and offer chemotherapeutic benefits for gastric cancer (GC). DeepRisk is a multi-scale, attention-based learning model developed on 1120 GCs in the Zhongshan dataset and validated with two external datasets. Then, we assessed its association with prognosis and treatment response. The multi-omics analysis and multiplex Immunohistochemistry were conducted to evaluate the potential pathogenesis and spatial immune contexture underlying DPS. Multivariate analysis indicated that the DPS was an independent prognosticator with a better C-index (0.84 for overall survival and 0.71 for disease-free survival). Patients with low-DPS after neoadjuvant chemotherapy responded favorably to treatment. Spatial analysis indicated that exhausted immune clusters and increased infiltration of CD11b+CD11c+ immune cells were present at the invasive margin of high-DPS group. Multi-omics data from the Cancer Genome Atlas-Stomach adenocarcinoma (TCGA-STAD) hint at the relevance of DPS to myeloid derived suppressor cells infiltration and immune suppression. DeepRisk network is a reliable tool that enhances prognostic value of TNM staging and aid in precise treatment, providing insights into the underlying pathogenic mechanisms.

The Combination of Anti-CD47 Antibody with CTLA4 Blockade Enhances Anti-Tumor Immunity in Non-Small Cell Lung Cancer via Normalization of Tumor Vasculature and Reprogramming of the Immune Microenvironment.

In solid tumors, the formidable anti-tumor impact resulting from blocking the "don't eat me" signal, arising from CD47-SIRPα interaction, is constrained, especially compared to its efficacy in hematopoietic malignancies. Activating macrophage anti-tumor activity not only necessitates the inhibition of the "don't eat me" signal, but also the activation of the "eat me" (pre-phagocyte) signal. Intriguingly, the cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) antibody (Ab) has been identified to stimulate Fc receptor-mediated active phagocytes in the tumor microenvironment, thereby generating "eat me" signals. This study postulates that concurrently targeting CD47 and CTLA4 could intensify the anti-tumor effects by simultaneously blocking the "don't eat me" signal while triggering the "eat me" signal. The experimental data from this investigation confirm that the combined targeting of CD47 and CTLA4 enhances immunity against solid tumors in LLC cell-transplanted tumor-bearing mice. This effect is achieved by reducing myeloid-derived suppressor cell infiltration while increasing the presence of effector memory CD8+ T cells, NK1.1+ CD8+ T cells, and activated natural killer T cells. Meanwhile, combination therapy also alleviated anemia. Mechanistically, the anti-CD47 Ab is shown to upregulate CTLA4 levels in NSCLC cells by regulating Foxp1. Furthermore, targeting CD47 is demonstrated to promote tumor vascular normalization through the heightened infiltration of CD4+ T cells. These findings suggest that the dual targeting of CD47 and CTLA4 exerts anti-tumor effects by orchestrating the "eat me" and "don't eat me" signals, reshaping the immune microenvironment, and fostering tumor vascular normalization. This combined therapeutic approach emerges as a potent strategy for effectively treating solid tumors.

Abstract B024: A histological data-driven analysis of the hypoxic microenvironment of preclinical murine bladder tumors

Abstract Hypoxia is common in many solid tumors, including bladder cancer (BC). Preclinical murine models that mimic relevant molecular and phenotypical characteristics of clinical BC were used to analyze the immune cell composition within the hypoxic tumor microenvironment. The experimental protocols involve intravesical installations, adoptively transferred T cells, and chemotherapy and were designed to recreate therapeutic procedures used in a recently open feasibility clinical trial of intravesical adoptive cell therapy (ACT) for BC patients. We analyzed 18 murine BC tissues from a treatment protocol that included intravesical installment of MB49-OVA tumor cells on day 0, gemcitabine treatment on day 10, and/or intravesical transfer of OT-I transgenic T cells on day 14, in addition to untreated controls and normal bladder tissues. The OT-I cells mimic the T cells used in clinical ACT. The collected tissues were sliced, and consecutive sections were stained for vasculature, tumor, and immune cells: CD8+, CD4+, and myeloid-derived suppressor cells (MDSCs). Subsequently, these histology images were co-registered, segmented, and preprocessed to identify the tumor boundaries, select viable vasculature, resolve cell overlaps, and annotate bladder cavities. A multi-cell off-lattice hybrid agent-based model that combines discrete cells and vasculature with continuous oxygen kinetics was developed to recreate the differential oxygenation in the tumor microenvironment. In all tissues, the hypoxic microenvironment was more prevalent in the tumor regions than in the nontumor regions due to differences between the irregular tumor vasculature vs. normal vasculature. The T cell and MDSC infiltration was increased in the tumors treated with gemcitabine (as mono- or combination therapy). Despite the significant hypoxia level in these tumors, only small proportions of CD8+s and CD4+s were hypoxic, as the majority resided in the well-oxygenated portions of the tumor. High infiltration of CD8+ cells was observed in tumors treated with OT-I cells compared to the controls. However, only a small proportion were hypoxic, and most CD8+s and MDSCs were well-oxygenated. The CD8+s were located far from the intravesical source, with some cells near the vasculature. Hypoxic native CD8+s in untreated tissues were observed inside the tumors, and most well-oxygenated CD8+s were near the tumor borders. The CD8+s also resided in the well-oxygenated regions in the normal tissues. Our reconstructed tissue oxygenation based on histology images revealed that the hypoxic tumor microenvironment hinders T cell accumulation in treated and untreated tumors. Such quantitative analyses may encourage designing more effective hypoxia-mediated therapies. Citation Format: Awino Maureiq E. Ojwang, Anjun Hu, McKenzie Williams, Sarah Bazargan, Joseph O. Johnson, Shari Pilon-Thomas, Katarzyna A. Rejniak. A histological data-driven analysis of the hypoxic microenvironment of preclinical murine bladder tumors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr B024.

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

In this study, we investigated infection-related tumor growth, focusing on myeloid-derived suppressor cells (MDSCs) in clinical and experimental settings. 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. 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. Long-lasting enhanced MDSCs associated with infection may contribute to infection-related tumor progression.

Exosome-derived tRNA fragments tRF-GluCTC-0005 promotes pancreatic cancer liver metastasis by activating hepatic stellate cells

Early metastasis is the primary factor in the very poor prognosis of pancreatic ductal adenocarcinoma (PDAC), with liver metastasis being the most common form of distant metastasis in PDAC. To investigate the mechanism of PDAC liver metastasis, we found that PDAC cells can promote the formation of pre-metastatic niches (PMNs) through exosomes to facilitate liver metastasis in the early stage. In our study, hepatic stellate cells (HSCs) were treated with PDAC-derived exosomes (PDAC-exo), and the activation of HSCs was detected. A novel transfer RNA-derived fragment, the tRF-GluCTC-0005 was obtained by small RNA sequencing from serum exosomes of PDAC patients. Bioinformatics analysis and RNA pull-down assays revealed the interaction between WDR1 and tRF-GluCTC-0005. A KPC transgenic mouse model and an AAV-mediated sh-WDR1 mouse model were used to detect the mechanism of liver metastasis in vivo. Finally, the dual luciferase reporter assay, protein mutation truncation assay, Co-IP assay, and flow cytometry assay were used to explore the molecular mechanism in HSCs activation and PMNs formation. We found that the tRF-GluCTC-0005 in exosomes binds to the 3’ untranslated region of the mRNA of the WDRl in HSCs and increases mRNA stability. The N-terminals of WDR1 bind to the YAP protein directly, inhibit YAP phosphorylation, and promote the expression of YAP transcription factors. The tRF-GluCTC-0005 in PDAC-exo significantly recruits myeloid-derived suppressor cells (MDSCs) in the liver, creating a PMNs immunosuppressive microenvironment and further advancing liver metastasis from PDAC. Our results suggest that the key of PDAC liver metastasis is the activation of HSCs through upregulation of WDR1 by tRF-GluCTC-0005 in exosomes, which mediates the infiltration of MDSCs to form PMNs.