M2 Monocytes Research Articles

Preoperative Immune Cell Dysregulation Accompanies Ovarian Cancer Patients into the Postoperative Period.

Ovarian cancer (OC) poses a significant global health challenge with high mortality rates, emphasizing the need for improved treatment strategies. The immune system's role in OC progression and treatment response is increasingly recognized, particularly regarding peripheral blood mononuclear cells (PBMCs) and cytokine production. This study aimed to investigate PBMC subpopulations (T and B lymphocytes, natural killer cells, monocytes) and cytokine production, specifically interleukin-1 beta (IL-1β), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12 (IL-12), and tumor necrosis factor alpha (TNFα), in monocytes of OC patients both preoperatively and during the early postoperative period. Thirteen OC patients and 23 controls were enrolled. Preoperatively, OC patients exhibited changes in PBMC subpopulations, including decreased cytotoxic T cells, increased M2 monocytes, and the disbalance of monocyte cytokine production. These alterations persisted after surgery with subtle additional changes observed in PBMC subpopulations and cytokine expression in monocytes. Considering the pivotal role of these altered cells and cytokines in OC progression, our findings suggest that OC patients experience an enhanced pro-tumorigenic environment, which persists into the early postoperative period. These findings highlight the impact of surgery on the complex interaction between the immune system and OC progression. Further investigation is needed to clarify the underlying mechanisms during this early postoperative period, which may hold potential for interventions aimed at improving OC management.

The protective effects of alamandine on angiotensin II-induced cardiac dysfunction and remodeling in rats

Alamandine (Ala), a relatively recent addition to the renin-angiotensin system (RAS), is a heptapeptide sharing structural similarities with angiotensin-(1-7). This peptide has garnered significant attention due to its distinctive antihypertensive, vasodilatory, and antifibrotic effects. In this study, we sought to determine whether Ala acts as a counter-regulatory agent against angiotensin II-induced cardiac hypertrophy, fibrosis, and inflammation in rats. Male SD rats (n=6 in each group) received a two-week subcutaneous infusion of vehicle (saline), angiotensin II (ANG II, 150 ng/kg/min), or a combination of ANG II and Ala (50 ng/kg/min) via mini osmotic pumps. Left ventricular (LV) function was evaluated through echocardiography. Histological analysis of cardiac hypertrophy and fibrosis was conducted using Hematoxylin Eosin and Trichome-Mason staining, respectively. Compared to the vehicle group, treatment with Ala significantly (*p<0.05) attenuated ANG II-induced cardiac mass (659.27 ± 54.26* vs. 824.48 ± 40.38 mg) with reduced LV thickness and end-diastolic volume. Ala treatment also improved cardiac function, as indicated by reduced global longitudinal strain and myocardial performance index. Histological images revealed that Ala treatment markedly reduced cardiomyocyte area (383.63* ± 7.20 vs 490.59 ± 13.13 μm²) and fibrosis area (5.22* ± 0.93 vs. 9.64 ± 0.36%) in rats treated with ANG II, along with reduced mRNA expression of pro-hypertrophic markers such as brain natriuretic peptide (4.29 ± 0.50* vs 7.53 ± 0.68, fold change) and β-myosin heavy chain (6.11 ± 0.61* vs 10.29 ± 1.02), as well as pro-fibrosis markers collagen 1 (5.72 ± 0.59* vs 10.56 ± 1.69), fibronectin (2.18 ± 0.34* vs 4.69 ± 0.70), and α-smooth muscle actin (2.14 ± 0.42* vs 3.85 ± 0.06), when compared to the rats treated with ANG II alone. Flow cytometry analysis also showed that Ala treatment significantly reduced ANG II-induced immune responses in the heart by reducing immune cell populations including CD4, CD8 T cells, B cells and NK cells, and increasing presence of anti-inflammatory M2 monocytes. Additionally, the mRNA levels of interlukin-6, CD 68, and chemokine monocyte chemoattractant protein-1 were significantly decreased in Ala-treated rats. Furthermore, treatment with Ala reversed ANG II-induced alterations in the mRNA expression of angiotensin-converting enzyme (ACE, 2.05 ± 0.16* vs. 3.08 ± 0.25), angiotensin type 1 receptor (1.45 ± 0.22* vs. 2.56 ± 0.31), and ACE 2 (1.08 ± 0.10** vs. 0.38 ± 0.06) in the LV of the heart. Taken together, these findings demonstrate that alamandine exerts a protective effect against ANG II-induced cardiac dysfunction, hypertrophy, fibrosis, and inflammation, potentially acting as a protective component of the RAS to counteract the adverse effects of ANG II. Alamandine might be a potential therapeutic agent in the treatment of cardiovascular disorders. Supported by NIH grants R01 HL139521 & 155091. 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.

NOD/Scid IL2Rγnull Mice Reconstituted with PBMCs from Patients with Atopic Dermatitis or Psoriasis Vulgaris Reflect the Respective Phenotype

NOD/Scid IL2Rγnull (NSG) mice reconstituted with peripheral blood mononuclear cells (PBMC) donated by patients with ulcerative colitis or Crohn’s disease highly reflect the respective pathological phenotype. To determine if these findings could be applicable to atopic dermatitis (AD) and psoriasis vulgaris (PV), PBMCs isolated from AD and PV patients were first subjected to immunological profiling. Subsequently, NSG mice were reconstituted with these PBMCs. Hierarchical clustering and network analysis revealed a distinct profile of AD and PV patients with activated CD4+ T cells (CD69, CD25) occupying a central position in the AD network and CD4+ CD134+ cells acting as the main hub in the PV network.Following dermal application of DMSO, both NSG-AD and NSG-PV mice exhibited increased clinical, skin and histological scores. Immuno-histochemical analysis, frequencies of splenic human leukocytes, and cytokine expression levels indicated that CD4+ CD69+ cells, M1 and TSLPR-expressing monocytes, switched B cells and MCP-3 were the driving factors of inflammation in NSG-AD mice. In contrast, inflammation in NSG-PV mice was characterized by an increase in fibroblasts in the epidermis, frequencies of CD1a-expressing monocytes and IL-17 levels. Therefore, the pathological phenotypes of NSG-AD and NSG-PV mice differ and partially reflect the respective human disease.

XRCC1: a potential prognostic and immunological biomarker in LGG based on systematic pan-cancer analysis.

X-ray repair cross-complementation group 1 (XRCC1) is a pivotal contributor to base excision repair, and its dysregulation has been implicated in the oncogenicity of various human malignancies. However, a comprehensive pan-cancer analysis investigating the prognostic value, immunological functions, and epigenetic associations of XRCC1 remains lacking. To address this knowledge gap, we conducted a systematic investigation employing bioinformatics techniques across 33 cancer types. Our analysis encompassed XRCC1 expression levels, prognostic and diagnostic implications, epigenetic profiles, immune and molecular subtypes, Tumor Mutation Burden (TMB), Microsatellite Instability (MSI), immune checkpoints, and immune infiltration, leveraging data from TCGA, GTEx, CELL, Human Protein Atlas, Ualcan, and cBioPortal databases. Notably, XRCC1 displayed both positive and negative correlations with prognosis across different tumors. Epigenetic analysis revealed associations between XRCC1 expression and DNA methylation patterns in 10 cancer types, as well as enhanced phosphorylation. Furthermore, XRCC1 expression demonstrated associations with TMB and MSI in the majority of tumors. Interestingly, XRCC1 gene expression exhibited a negative correlation with immune cell infiltration levels, except for a positive correlation with M1 and M2 macrophages and monocytes in most cancers. Additionally, we observed significant correlations between XRCC1 and immune checkpoint gene expression levels. Lastly, our findings implicated XRCC1 in DNA replication and repair processes, shedding light on the precise mechanisms underlying its oncogenic effects. Overall, our study highlights the potential of XRCC1 as a prognostic and immunological pan-cancer biomarker, thereby offering a novel target for tumor immunotherapy.

Inhibition of MET Tyrosine Kinase Provides a Strategy for Targeting the Inflammatory Tumor Microenvironment

Background: In acute myeloid leukemia (AML), age-associated inflammation may contribute to leukemia progression and remodel the tumor microenvironment (TME). Macrophages, crucial components of the TME, play a critical role in modulating T cell immune response against leukemia cells. However, macrophage polarization to an M2 phenotype, known as tumor-associated macrophages (TAMs), have an immunosuppressive action and interplay with leukemia cells by promoting their survival, immune evasion, and chemotherapy resistance. MET, a hepatocyte growth factor (HGF) receptor, has been demonstrated to contribute to shifting macrophages to TAMs. Hence, we hypothesize that inhibiting Met and, consequently, HGF action and signaling could be a potential strategy to target the inflammatory TME and improve AML clinical outcomes. Aims: Here,we analyze the MET gene expression in leukemia patients mononuclear cells and the impact on clinical outcomes and we also assess the contribution of a specific MET inhibitor, B2000 (Brigham University), to leukemia progression by investigating the in vitro, ex vivo and in vivo effects with focus on the inflammatory TME. Method s: MET mRNA expression data were obtained from TCGA AML study, performed in cells isolated from 290 AML patients (median age 61 years [range:18-87]). Human peripheral blood and bone marrow (BM) mononuclear cells (BMNC/PBMC) (n=10) were obtained from healthy donors (HD). HD-PBMC were used for the isolation of monocytes. Human leukemia cell lines (OCI-AML3, MOLM-13) and BMNC from HD and AML patients (n=10) were treated with B2000 and apoptosis rates were evaluated. To obtain monocyte-derived macrophages (MDMs), monocytes were stimulated with GM-CSF/LPS/IFN-y or M-CSF/IL-4, for M1 or M2 polarization, respectively, and treated with B2000 in monocultures and cocultures. For in viv o analyses, BALB/c mice received a single intraperitoneal injection of lipopolysaccharide (LPS) to provoke an inflammatory shock and were treated with B2000. The analysis of the immune response and the characterization of inflammatory cells was performed by flow cytometry. Results: High MET expression in FAB M1, M3 and M7 AML patients ( P<.0001) was observed. B2000 was effective in killing leukemia cell lines ( P<.0001) and primary BMNCs from AML patients ( P<.005) without affecting HD-BMNC. HD-PBMC-derived monocytes treated with B2000 had increased classical monocyte population (CD14highCD16-) ( P<.001). B2000 also caused a significant apoptosis increase in leukemia cells cocultured with monocytes, without modulating the monocytes population ( P<.0001). To confirm whether monocytes could acquire a tumoricidal phenotype, monocytes were pre-treated with B2000 and, then, cocultured with leukemia cells. B2000-programmed monocytes enhanced leukemia cell apoptosis ( P<.001). The analysis of MDMs revealed a shorter population of M2 MDMs (HLA-DR-CD206+) and increased population of M1 MDMs (HLA-DR+CD80+) in B2000-treated MDMs when compared with the vehicle treatment ( P<.0001). To validate these findings, we evaluated B2000 action in the inflammatory TME by co-culturing leukemia cells with MDMs and results showed that apoptosis was increased in leukemia cells in the presence of B2000 compound ( P<.01). The in vivo analysis in an inflammatory mice model showed increased frequency of M1 monocytes (CD11b+F4/80+CD80+) and decreased M2 (CD11b+F4/80+CD206+) in B2000-treated ( P<.05). Accordingly, B2000 restored lymphocyte B ( P<.05) and T ( P<.05) levels in the BM, spleen and peripheral blood, decreasing the inflammatory burden. Conclusion: We demonstrate the pre-clinical efficacy of a MET inhibitor, B2000, in enhancing anti-tumor immunity in vitro, ex vivo and in vivo by promoting M1 macrophage polarization and tumoricidal phenotype, contributing to inhibition of leukemia cell expansion and inducing the specific leukemia apoptosis. B2000 also acted on restoring lymphocyte levels and, consequently, reducing inflammatory stress. Our findings highlight an important role of this novel MET-inhibitor onto the inflammatory TME, representing an important agent against hematological neoplasms with a novel immunomodulatory effect in directly reducing leukemia cell proliferation as well as promoting TAMs anti-tumoral effector functions. Funding: FAPESP #2017/21801-2, #2019/25247-5, #2021/05320-0; CNPq #303405/2018-0.

Abstract P120: Effect Of Alamandine On Isoproterenol-induced Left Ventricular Dysfunction, Cardiac Remodeling And Immune Responses In Rat

Alamandine, a newly identified derivative of angiotensin II has been shown to exert antihypertensive and vasodilative actions, and may represent a novel component of cardioprotective axis of renin angiotensin system. We sought to determine the protective effects of alamandine on isoproterenol (ISO) induced cardiac remodeling and dysfunction in rats. Male SD rats (n=6 in each group) received a two-week subcutaneous infusion of vehicle, ISO (10 mg/kg/day) or a combination of ISO and alamandine (50 ng/kg/min). Compared with vehicle group, the ratio of heart/body weight (mg/g) in ISO treated rats was significantly (*P<0.05) elevated (3.93 ± 0.18 vs. 3.07 ± 0.08*), which was markedly attenuated by alamandine (3.50 ± 0.08*). Echocardiographic assessment showed that alamandine improved ISO-induced left ventricular (LV) dysfunction evidenced by reducing LV thickness (1.68 ± 0.07* vs 1.90 ± 0.02 mm), end diastolic and systolic volume. Rats treated with alamandine had reduced E/E' ratio (15.16 ± 1.04* vs 26.81 ± 3.79) and increased E'/A' ratio (1.09 ± 0.05 vs 0.57 ± 0.07 ** P<0.01), indicating the improved diastolic cardiac function. The reduced myocardial performance index (0.74 ± 0.06* vs. 1.24 ± 0.15) in alamandine treated rats demonstrated an improvement of global cardiac dysfunction. Histological analysis also revealed that alamandine attenuated ISO induced increase in cardiomyocyte size (372.46 ± 11.13** vs 551.99 ± 11.79 μm 2 ) and myocardial fibrosis (1.39 ± 0.16 ** vs 4.57 ± 0.35), along with reduced mRNA expression of hypertrophy biomarkers atrial natriuretic peptide (2.45 ± 0.71** vs 8.08 ± 0.74), brain natriuretic peptide (2.86 ± 0.35 ** vs 12.53 ± 1.12), beta myosin heavy chain (1.71 ± 0.25* vs 5.38 ± 0.95) and fibrosis genes – collagen-1 (1.97 ± 0.45* vs 4.25 ± 0.6), fibronectin (2.04 ± 0.32** vs 5.05 ± 0.74), α-smooth muscle actin (1.53 ± 0.1** vs 2.71 ± 0.17). Flow cytometry analysis unveiled that alamandine reduced neutrophil, CD8 T cells and M1 monocytes, but increased M2 monocytes of left ventricle, implying the reduction of inflammation of the heart. These data suggest that alamandine plays a cardioprotective role in improving cardiac hypertrophy and fibrosis, probably via an immune mechanism to alleviate inflammatory responses during cardiac remodeling.

Molecular subtyping of acute myeloid leukemia through ferroptosis signatures predicts prognosis and deciphers the immune microenvironment.

Acute myeloid leukemia (AML) is one of the most aggressive hematological malignancies with a low 5-year survival rate and high rate of relapse. Developing more efficient therapies is an urgent need for AML treatment. Accumulating evidence showed that ferroptosis, an iron-dependent form of programmed cell death, is closely correlated with cancer initiation and clinical outcome through reshaping the tumor microenvironment. However, understanding of AML heterogeneity based on extensive profiling of ferroptosis signatures remains to be investigated yet. Herein, five independent AML transcriptomic datasets (TCGA-AML, GSE37642, GSE12417, GSE10358, and GSE106291) were obtained from the GEO and TCGA databases. Then, we identified two ferroptosis-related molecular subtypes (C1 and C2) with distinct prognosis and tumor immune microenvironment (TIME) by consensus clustering. Patients in the C1 subtype were associated with favorable clinical outcomes and increased cytotoxic immune cell infiltration, including CD8+/central memory T cells, natural killer (NK) cells, and non-regulatory CD4+ T cells while showing decreased suppressive immune subsets such as M2 macrophages, neutrophils, and monocytes. Functional enrichment analysis of differentially expressed genes (DEGs) implied that cell activation involved in immune response, leukocyte cell-cell adhesion and migration, and cytokine production were the main biological processes. Phagosome, antigen processing and presentation, cytokine-cytokine receptor interaction, B-cell receptor, and chemokine were identified as the major pathways. To seize the distinct landscape in C1 vs. C2 subtypes, a 5-gene prognostic signature (LSP1, IL1R2, MPO, CRIP1, and SLC24A3) was developed using LASSO Cox stepwise regression analysis and further validated in independent AML cohorts. Patients were divided into high- and low-risk groups, and decreased survival rates were observed in high- vs. low-risk groups. The TIME between high- and low-risk groups has a similar scenery in C1 vs. C2 subtypes. Single-cell-level analysis verified that LSP1 and CRIP1 were upregulated in AML and exhausted CD8+ T cells. Dual targeting of these two markers might present a promising immunotherapeutic for AML. In addition, potential effective chemical drugs for AML were predicted. Thus, we concluded that molecular subtyping using ferroptosis signatures could characterize the TIME and provide implications for monitoring clinical outcomes and predicting novel therapies.

M1 monocytes as a predictor of short-term progression of carotid atherosclerosis in asymptomatic middle-aged patients

M1 monocytes as a predictor of short-term progression of carotid atherosclerosis in asymptomatic middle-aged patients

Supplementation with EPA and DHA omega-3 fatty acids improves peripheral immune cell mitochondrial dysfunction and inflammation in subjects with obesity

Omega-3 fatty acids (w-3 FA) have anti-inflammatory effects and improve mitochondrial function. Nonetheless, little is known about their effect on mitochondrial bioenergetics of peripheral blood mononuclear cells (PBMCs) in individuals with obesity. Thus, this study aimed to determine the mitochondrial bioenergetics status and cell subset composition of PBMCs during obesity, before and after 1 month supplementation with w-3 FA. We performed a case-control study with twelve women with normal BMI (lean group) and 19 with grade 2 obesity (obese group), followed by a before-after prospective study where twelve subjects with obesity received a 1 month intervention with 5.25 g of w-3 FA (3.5 g eicosapentaenoic (EPA) and 1.75 g docosahexaenoic (DHA) acids), and obtained PBMCs from all participants. Mitochondrial bioenergetic markers, including basal and ATP-production associated respiration, proton leak, and nonmitochondrial respiration, were higher in PBMCs from the obese group vs. the lean group. The bioenergetic health index (BHI), a marker of mitochondrial function, was lower in the obese vs. the lean group. In addition, Th1, Th2, Th17, CD4+ Tregs, CD8+ Tregs, and Bregs, M1 monocytes and pDCreg cells were higher in PBMCs from the obese group vs. the lean group. The w-3 FA intervention improved mitochondrial function, mainly by decreasing nonmitochondrial respiration and increasing the reserve respiratory capacity and BHI. The intervention also reduced circulating pro-inflammatory and anti-inflammatory lymphocyte and monocytes subsets in individuals with obesity. The mitochondrial dysfunction of PBMCs and the higher proportion of peripheral pro-inflammatory and anti-inflammatory immune cells in subjects with obesity, improved with 1 month supplementation with EPA and DHA.

Low-dose decitabine promotes M2 macrophage polarization in patients with primary immune thrombocytopenia via enhancing KLF4 binding to PPARγ promoter.

The first-line therapy is effective for the treatment of primary immune thrombocytopenia (ITP); however, maintaining the long-term responses remains challenging. Low-dose decitabine (DAC) has been adopted to treat refractory ITP, while its role in macrophage polarization has not been fully understood. We aimed to investigate the mechanistic role of DAC in M2 macrophage polarization and evaluated its therapeutic effect in ITP. The M2 monocytes were identified by flow cytometry from peripheral blood mononuclear cells in healthy controls (HCs) and ITP patients. The expression of PPARγ, Arg-1, DNMT3b and NLRP3, together with IL-10 plasma levels was measured to examine its function. Bisulfite-sequencing PCR was used to evaluate the methylation status of PPARγ promoter, and the binding affinity of KLF4 was measured by Cut&Tag. A sh-PPARγ THP-1 cell line was created to verify if low-dose DAC-modulated M2 macrophage polarization was PPARγ-dependent. The passive ITP models were used to investigate the therapeutic effects of low-dose DAC and its role in modulating polarization and immunomodulatory function of macrophages. NLRP3 inflammasome and reactive oxygen species were also tested to understand the downstream of PPARγ. The M2 monocytes with impaired immunoregulation were observed in ITP. After high-dose dexamethasone (HD-DXM) treatment, M2 monocytes increased significantly with the elevated expression of PPARγ, Arg-1 and IL-10 in CR patients. Low-dose DAC promoted M2 macrophage polarization in a PPARγ-dependent way via demethylating the promoter of PPARγ, especially the KLF4 binding sites. Low-dose DAC alleviated ITP mice by restoring the M1/M2 balance and fine-tuning immunomodulatory function of macrophages. The downstream of the PPARγ modulation of M2 macrophage polarization might physiologically antagonize NLRP3 inflammasome. Low-dose DAC promoted M2 macrophage polarization due to the demethylation within the promoter of PPARγ, thus enhanced the KLF4 binding affinity in ITP.

M1 And M2 Monocytes In Concentrated Bone Marrow Aspirate As Predictive Biomarkers For Clinical Response After Foot And Ankle Procedures

M1 And M2 Monocytes In Concentrated Bone Marrow Aspirate As Predictive Biomarkers For Clinical Response After Foot And Ankle Procedures

Characterization of MCL-1 in patients with colorectal cancer (CRC): Expression, molecular profiles, and outcomes.

3085 Background: Myeloid cell leukemia 1 (MCL-1) is a member of the BCL-2 protein family and is anti-apoptotic/pro-survival in function. Dysregulation of MCL-1 expression has been reported in several solid tumors, including lung and breast cancer. In CRC, MCL-1 has been associated with resistance to chemotherapeutic drugs and multi-kinase inhibitor regorafenib. Our study aimed to characterize the molecular features associated with MCL-1 gene expression in CRC. Methods: 28,576 CRC samples were analyzed by Caris Life Sciences (Phoenix, AZ) with WTS (Illumina NovaSeq) and NextGen DNA sequencing (NextSeq, 592 Genes and NovaSEQ, WES). MCL-1 expression was stratified by quartiles where top quartile transcripts per million (TPM) were considered high (Q4) and bottom quartile low (Q1). Cell infiltration (CI) in the tumor microenvironment (TME) was estimated by RNA deconvolution analysis using QuantiSEQ. Interferon-gamma and T-cell inflamed signatures were also calculated from RNA data. X2 and Fisher-Exact tests were used, and statistical significance was determined as a P-value adjusted for multiple comparisons ( q < 0.05). Real world survival was obtained from insurance claims data and Kaplan-Meier estimates were calculated for molecularly defined patients. Results: MCL-1 expression was significantly increased in rectal tumors compared to other primary tumor sites (median TPM [mTPM] 20.8 vs 18.9 vs 18.7, in rectal vs left-sided vs right-sided, respectively, q < 0.0001). MCL-1 high tumors were associated with increased rate of PD-L1 IHC levels (Q1 vs Q4: 4.2% vs 9.7% [all CRC]; 3.7% vs 8.9% [pMMR/MSS], q < 0.0001), while MCL-1-low showed marginally increased rates of TMB-high (8.0% vs 7.0% [all CRC]; 3.3% vs 2.6% [pMMR/MSS], P < 0.05). MCL-1-high expression was associated with a higher T-cell inflamed signature and IFN score ( q < 0.0001). MCL-1 high was associated with higher mutation rates of CDKN2A, BRCA2, KRAS and GNAS, while lower mutation rates of TP53, PIK3CA, PTEN, BRAF, APC, FBXW7, AMER1, SOX9, and SMAD2 and copy number amplifications in several genes ( q < 0.05). MCL-1 high TME had higher CI of M1 and M2 macrophages, monocytes, B cells, NK cells, and T-reg infiltration, while dendritic cells and CD4+ T-cell were lower ( q < 0.05). Patients with high MCL-1 expressing CRC had longer survival (Q4 vs Q1: 33.5 vs 24.8 months, HR 0.72, 95% CI 0.67-0.77, P < 0.0001) and longer time-on-treatment with regorafenib although with limited clinical benefit (Q4 vs Q1: 2.03 vs 1.06 months, HR: 0.67, 95% CI: 0.50-0.89, P = 0.005). Conclusions: Our data show a strong correlation between distinct immune biomarkers, TME cell infiltration and MCL-1 expression in CRC. Furthermore, increased tumor MCL-1 expression improved patient prognosis and treatment outcomes. These findings suggest a key clinical role for MCL-1 as an important modulator of anti-tumor immunity and TME and a potential biomarker in CRC.

Abstract 6676: Exploratory biomarker analyses in phase 1 study of regorafenib, ipilimumab, and nivolumab (RIN) in chemotherapy resistant microsatellite stable (MSS) metastatic colorectal cancer (mCRC)

Abstract Background: We recently reported an objective response rate of 36.4% in a phase I trial of RIN in MSS mCRC patients without liver metastases, while no response was noted in patients with liver metastases. Here we characterized kinetics in local and systemic immune responses associated with clinical outcomes, liver metastasis, and immune related toxicity during RIN treatment. Methods: Patients with chemotherapy-resistant MSS mCRC received RIN at (R) at 80 mg QD x 21 days in 4-week(w) cycle, (I) at 1mg/kg Q6w and (N) at 240 mg Q 2w (n=29). Needle biopsies were collected at baseline (n=8) and 4 weeks (n=6) after treatment. Blood samples were collected at baseline (n=29), 4 weeks (n=29) and 8 weeks (n=25) after treatment. Alterations in the tumor microenvironment (TME) were characterized by RNA sequencing. We analyzed the immune cell composition and activities in the blood, including 90 subpopulations of leukocytes and 29 activation/suppression/proliferation markers, with high-dimensional spectral flow cytometry. Results: The clinical responses are positively correlated with the induction of local anti-tumor immunity. Patients with better clinical outcomes exhibited higher levels of lymphocyte infiltration, chemokine production, and antigen-presentation potential in the local TME following one cycle of RIN treatment. The duration of progression-free survival (PFS) was correlated with a higher baseline STING signal in the TME. In the peripheral blood, the responders have more circulating CD4 T cells, B cells, and cDC1, while expressing lower immune activation markers, especially in T cells with type 1 immunity at baseline. At 4 weeks, immune activation and proliferation markers were higher in responders than non-responders. Interestingly, immune effectors (non-Tregs, CD45RA− CD8 T cells, NKT cells, and M1 monocytes) in the blood were proportionally reduced in responders at 4 weeks, coincident with their enrichment in the local TME. We also compared peripheral immune signatures between patients with and without liver metastases, liver metastases (n=7) were associated with a significantly lower ratio of CD4/CD8 T cells in the blood, a phenomenon of immunosenescence, both at baseline and after RIN treatment. Lastly, the occurrence of immune-related toxicities was closely correlated with the induction of immune activities in both acellular and cellular components in the circulation. Conclusions: Clinical benefit of RIN was correlated to more robust immune activation of the TME and peripheral blood following one cycle of treatment. A higher baseline STING signal in the TME was associated with longer PFS. Liver metastasis was associated with a lower ratio of peripheral CD4/CD8 T cells which may contribute to the resistance to immunotherapy in those patients. Citation Format: Jian Ye, Chongkai Wang, Colt Egelston, Weihua Guo, Peter P. Lee, Marwan Fakih. Exploratory biomarker analyses in phase 1 study of regorafenib, ipilimumab, and nivolumab (RIN) in chemotherapy resistant microsatellite stable (MSS) metastatic colorectal cancer (mCRC). [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6676.

Abstract 79: Study of tumor microenvironment of ovarian clear cell carcinoma

Abstract Background: Clear cell ovarian carcinoma (CCOC) is characterized by a distinct histologic and molecular profile, and associated with very poor responses to standard treatment consisting of surgery and carboplatin:taxol chemotherapy. CCOC is chemo-resistant at the time of diagnosis and response to chemotherapy in the recurrent setting is less than 10%. Literature suggests a potential role for immune checkpoint inhibitors (ICI). However, only a subgroup of patients (20%) responds to ICI and little is known about the mechanisms of response and resistance to therapy. We postulate that a better understanding of CCOC tumor microenvironment (TME) could help predict patients’ response to ICI. Objective: The aim of this study is to investigate the relationship between TME immune infiltrate and clinical/outcome in 22 CCOC cases and identify subsets of CCOC who may benefit from immunotherapy. Material & Methods: We characterized the immune landscape of 11 early and 11 advanced CCOC through a multiplex IHC Discovery Platform. Spatial single-cell proteomics analyses (cyclic-IF) and spatially-resolved RNAseq in 10 CCOC cases using an OC tissue microarray (TMA) were performed. Results were corelated with clinical and treatment outcome. Results: The percent of CD8, CD4, CD20 B cells Tregs, PD1, PDL1, monocytes and M2 monocytes and myeloid cells was significantly higher in advanced than early-stage cancers. Recurrent cancers were more immunosuppressive than cases with no recurrence. Tumor infiltrate was dense in 4 cases. Four patients with early-stage disease had a high number of CD8 naïve cells and experienced no recurrence. TME analysis of single case of advanced CCOC (before and after chemotherapy), revealed that hot tumor changed to cold tumor, suggesting the resistance to treatment. Cyclic IF analyses identified 3 tumor phenotype groups in a subset of 10 CCOC present on the OC TMA. The majority of CCOC had high expression of CCNE and high PI3K-AKT-mTor activity, low expression of hormone receptors (AR, ERa, PRg) and low cell cycle activity. Some tumors were also high for HER2. The stromal compartment was enriched in collagen VI, aSMA and PDGFR. The immune monitoring of several of those samples also revealed an immunosuppressive microenvironment, including the presence of M2 macrophages and expression of immune checkpoint proteins PD-L1 and B7-H4. Conclusion: Our study shows that various phenotypes of CCOC are defined by the cancer cell profiles and TME content. Importantly, a strong immunosuppressive microenvironment was detected in many samples, suggesting a potential response to ICI. Furthermore, we detected the expression of several therapeutic targets (MAPK, CCNE, PI3K-AKT-mTOR, HSP90, HER2), including oncogenic signaling pathways (RTK, MAPK). Different tumor phenotypes identified across our CCOC samples suggest that clear cell carcinoma could be subclassified into subtypes that should be treated differently. Citation Format: Tanja Pejovic, Sonali Joshi, Shawn Campbell, Dhanir Tailor, Joanna Pucilowska, Benjamin Tate, Pierre-Valérien Abate, Korina Mouzakitis, Marilyne Labrie, Elizabeth Munro, Jenna Emerson, Sanjay V. Malhotra. Study of tumor microenvironment of ovarian clear cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 79.

Coinfection with influenza virus and non-typeable Haemophilus influenzae aggregates inflammatory lung injury and alters gut microbiota in COPD mice.

Acute exacerbation of chronic obstructive pulmonary disease (AECOPD) is associated with high mortality rates. Viral and bacterial coinfection is the primary cause of AECOPD. How coinfection with these microbes influences host inflammatory response and the gut microbiota composition is not entirely understood. We developed a mouse model of AECOPD by cigarette smoke exposure and sequential infection with influenza H1N1 virus and non-typeable Haemophilus influenzae (NTHi). Viral and bacterial titer was determined using MDCK cells and chocolate agar plates, respectively. The levels of cytokines, adhesion molecules, and inflammatory cells in the lungs were measured using Bio-Plex and flow cytometry assays. Gut microbiota was analyzed using 16S rRNA gene sequencing. Correlations between cytokines and gut microbiota were determined using Spearman's rank correlation coefficient test. Coinfection with H1N1 and NTHi resulted in more severe lung injury, higher mortality, declined lung function in COPD mice. H1N1 enhanced NTHi growth in the lungs, but NTHi had no effect on H1N1. In addition, coinfection increased the levels of cytokines and adhesion molecules, as well as immune cells including total and M1 macrophages, neutrophils, monocytes, NK cells, and CD4 + T cells. In contrast, alveolar macrophages were depleted. Furthermore, coinfection caused a decline in the diversity of gut bacteria. Muribaculaceae, Lactobacillus, Akkermansia, Lachnospiraceae, and Rikenella were further found to be negatively correlated with cytokine levels, whereas Bacteroides was positively correlated. Coinfection with H1N1 and NTHi causes a deterioration in COPD mice due to increased lung inflammation, which is correlated with dysbiosis of the gut microbiota.

Abstract P2-20-12: Abundance of tumor-infiltrating lymphocytes is associated with pathologic complete response to neoadjuvant chemotherapy but not with survival in ER-positive/HER2-negative breast cancer

Abstract INTRODUCTION: Abundance of tumor infiltrating lymphocytes (TILs) is well known to be associated with achievement of pathologic complete response (pCR) after neoadjuvant chemotherapy and is a surrogate of better survival in triple negative breast cancer (TNBC). However, the association remains unclear in ER-positive/HER2-negative, which is the most common subtype in breast cancer. Further, pathological quantification of TILs is known for its ambiguity. We hypothesized that abundance of TILs, quantified by transcriptomic signatures, in ER-positive/HER2-negative is associated with different biology and is not a surrogate of survival unlike in TNBC. METHODS: A total of 6035 primary breast cancer patients from three cohorts (TCGA, METABRIC, and SCAN-B) with full transcriptome and clinical data were analyzed. Lymphocyte fractionation in the tumor microenvironment of a bulk tumor was estimated from the transcriptome using two different deconvolution tools, CIBERSORTx and xCell. RESULTS: First, the correlation between TIL score measured histologically on TCGA patients and gene expression of CD3D, CD3E, CD3G, CD8A, CD4, and total lymphocytes, and total T-cells measured by xCell or CIBERSORTx were analyzed. Total sum of lymphocytes by xCell correlated the strongest, thus it was adopted as the TIL score. The TIL score strongly correlated with CD3G, CD3E, CD3D, CD8A, and CD4 gene expressions in both TCGA and GSE96058 cohorts (all r > 0.48). Among the infiltrated cells; M1 and M2 macrophages, dendritic cells, mast cells and monocytes were all higher, and microvascular endothelial cells and pericytes were lower in TIL score high tumors in ER-positive/HER2-negative (all p < 0.01) where M2 and mast cells were not in HER2+ and TNBC in both 2 cohorts. Enrichment of immune related gene sets IL2/Stat5 signaling, IL6/Jak/Stat3 signaling, inflammatory response, and Complement were less in high TIL group of ER-positive/HER2-negativethan HER2 and TNBC. In agreement, cytolytic activity was lower in TIL high of ER-positive/HER2-negative compared from TNBC. On the other hand, high TIL group in ER-positive/HER2-negative had significantly higher intratumor heterogeneity, homologous recombination deficiency, mutation burden, and neoantigens, which were not the case in the other subtypes. In agreement, all the cell proliferation-related gene sets; E2F targets, G2M checkpoint, mitotic spindle, Myc targets v1 and v2, were more strongly enriched in high TIL group of ER-positive/HER2-negative compared from HER2+ and TNBC. MKI67 gene expression and Nottingham histological Grade were uniformly elevated with high TIL regardless of subtypes. In three independent neoadjuvant cohorts, we found that pathologic complete response rates of high TIL group. On the other hand, high TIL group did not show any survival benefit compared with low TIL group in ER-positive/HER2-negative, unlike in HER2+ or TNBC. CONCLUSION: High tumor-infiltrating lymphocytes in ER-positive/HER2-negative breast cancer was associated with less immune response and higher genetic alteration and cell proliferation compared to the other subtypes, leading to better response of neoadjuvant chemotherapy, but no benefit in survival outcome. Citation Format: Rongrong Wu, Masayuki Nagahashi, Masanori Oshi, Yasuo Miyoshi, Takashi Ishikawa, Kazuaki Takabe. Abundance of tumor-infiltrating lymphocytes is associated with pathologic complete response to neoadjuvant chemotherapy but not with survival in ER-positive/HER2-negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-20-12.

Peripheral Immunophenotype in IgG4-Related Disease and Its Association with Clinical Phenotypes and Disease Activity.

Diverse immune cell subsets have been described in IgG4-related disease (IgG4-RD). If there is a different immunophenotype according to clinical phenotype and activity status is not known. Levels of IL-4-, IL-13-, IL-5-, and IL-21-producing CD4+ T cells (Th2 subsets), CD4+ cytotoxic T lymphocytes (CD4+CTLs), T helper 9 cells, T follicular helper cells (Tfh; Tfh1/Tfh2/Tfh17/Tf regulatory [Tfr]), Foxp3+ regulatory T cells, Type 1 regulatory T cells (Tr1), T helper 3 regulatory cells (Th3), IL-10-producing regulatory B cells (Bregs), IL-10-expressing regulatory plasmacytoid dendritic (pDC IL-10+) cells, and M1 and M2 monocytes were determined by flow cytometry in 43 IgG4-RD patients and 12 controls. All immune subsets were higher in patients vs. controls. CD4+/IL-4+, CD4+/IL-5+, CD4+CTLs, Tfh2, Tfh17, Tfr, and M1 monocyte cell number was different among IgG4-RD clinical phenotypes. The pancreato-hepato-biliary phenotype was characterized by a higher CD4+CTLs, Tfh17, Tfh2, and Tfr and lower M1 cell number. An increased CD4+CTLs and Th3 cell number distinguished the head and neck-limited phenotype, while the retroperitoneal/aortic and Mikulicz/systemic phenotypes were characterized by increased Th2 subsets. Tfh17, Tr1, Th3, pDC, M1, and M2 monocytes were augmented in active patients. In summary, the clinical heterogeneity of IgG4-RD might be driven by the participation of different immunophenotypes and, consequently, by a different fibroinflammatory process.

Non-Canonical WNT/Wnt5a Pathway Activity in Circulating Monocytes of Untreated Psoriatic Patients: An Exploratory Study of Its Association with Inflammatory Cytokines and Cardiovascular Risk Marker-ADAMTS7

The leading cause of death in psoriasis is cardiovascular disease. The determinants that induce the increase in this risk are not known. The systemic inflammatory process is dependent on lymphocytes and monocytes, as has been proposed. However, adaptation modules such as mTOR have recently been mentioned as having a role. Other factors, such as WNT and its non-canonical WNT5a-inducing pathway, are relevant in inflammation, cell migration, and neoangiogenesis. Thus, we studied circulating monocytes from untreated severe psoriatic patients and characterized inflammatory cytokines, chemokines, mTOR activity, and the cardiovascular risk marker ADAMTS7. Peripheral blood from ten severely psoriatic patients (Psoriasis severity index greater than 10) was extracted and age- and sex-matched with healthy subjects. Surface and intracellular flow cytometry were performed for cytokine, chemokine receptors, and mTOR activity. ADAMTS7 was measured using ELISA. Psoriatic patients had a higher frequency of WNT5a+ cells in monocytes, which also had higher levels of IL-1β, IL-6, CXCR3, CCR2, and phosphorylated S6R protein. We found that M1 monocytes are dominant in the WNT5a+ cell group, and intracellular levels of WNT5a were also augmented. Levels of WNT5a were correlated with ADAMTS7, a blood marker related to the pathogenesis of atheromatosis. WNT5a could be relevant to the cardiovascular risk of psoriatic patients considering its association with higher levels of inflammatory cytokines, chemokine receptors and the pro-atherogenic profile of circulating monocytes.

New Markers for Cardiovascular Disease in Psoriatic Patients: Preliminary Study on Monocyte Phenotype, ADAMTS7, and mTOR Activity

Psoriasis is a skin disease with occasional involvement of non-cutaneous territories. Beyond the usual, cardiovascular events are more frequent in these patients and correlate only partially with disease activity, suggesting the presence of other unknown factors. We selected ten psoriatic patients without treatment in the last year and matched them for age and gender with eleven healthy subjects. Ficoll-extracted mononuclear cells were analyzed with flow cytometry for monocyte surface phenotype markers, intracellular NFκB/inflammasome-dependent interleukins, and chemotaxis receptor CXCR3. Using ELISA, patient serum was evaluated for ADAMTS7 and CXCL10. Inflammatory M1 monocytes showed higher levels of IL-1β and IL-6 in psoriatic patients. M2 monocytes also showed higher levels of intracellular inflammatory cytokines. Nevertheless, IL-6 values were higher compared to other monocytes and IL-1β. The mTORC activation markers ADAMTS7 and S6Rp were higher in psoriatic patients than in healthy controls. In psoriatic patients, serum levels of ADAMTS7 were elevated, and M2 monocytes showed a distinct inflammatory response with higher relative levels of NFκB-dependent IL-6 and less activity of the CXCR3-CXCL10 chemotactic pathway. These data suggest pathways with potential markers for prediction and early detection of cardiovascular risk in psoriatic patients.

EPCO-18. UNSUPERVISED MACHINE LEARNING ON TUMOR IMMUNE TRANSCRIPTOMIC DATA REVEALS DISTINCT IMMUNOLOGIC SUBTYPES OF GLIOBLASTOMA

Abstract Glioblastoma (GBM) has demonstrated a limited therapeutic response to single-agent immunotherapies, highlighting the need for a better understanding of GBM tumor immune phenotypes to inform targeted immunologic treatments. To elucidate GBM immunologic phenotypes we performed unsupervised machine learning on microarray transcriptomic data from 815 newly diagnosed GBM patients. We utilized immune genes only and performed k-means clustering for k=2-10 with within-cluster sum of squares and silhouette methods used to identify the optimal number of clusters. Identified subtypes were further characterized using xCell to estimate the tumor infiltration of 64 immune and stromal cell types. Two primary immunologic subtypes were identified: subtype 1 (423 patients) and subtype 2 (392 patients). There was no difference in patient age (mean age 58 years) or gender between subtypes. Subtype 1 was primarily classical subtype GBMs (50.5%, p< 0.001), while subtype 2 was composed of primarily mesenchymal subtype GBMs (74.2%). Relative to subtype 1, subtype 2 was characterized by high expression of immune checkpoint and co-stimulatory proteins, including CD86, OX40L, IDO1, and LAG3. Subtype 2 also had increased activation of immune genes associated with cytolytic activity and antigen processing. In addition, subtype 2 had increased infiltration of M2 macrophages, monocytes, dendritic cells, and neutrophils while subtype 1 had higher infiltration of CD8 naïve T cells. Multivariate logistic regression confirmed that subtype 2 was characterized by monocytes, M1 and M2 macrophages, neutrophils, and dendritic cells while subtype 1 was characterized by CD8 naïve T cells, regulatory T cells, and naive B cells. Subtype 2 had reduced progression-free survival, but there was no difference in overall survival between the two subtypes. In summary, unsupervised machine learning of GBM immune transcriptomic data identified two immune phenotypes, one of which was more immunologically active than the other, potentially informing future personalized immunotherapy treatments.