CCL2 Levels Research Articles

Aflatoxin B1 and Epstein–Barr virus-induced CCL22 expression stimulates B cell infection

Epstein-Barr Virus (EBV) infects more than 90% of the adult population worldwide. EBV infection is associated with Burkitt lymphoma (BL) though alone is not sufficient to induce carcinogenesis implying the involvement of co-factors. BL is endemic in African regions faced with mycotoxins exposure. Exposure to mycotoxins and oncogenic viruses has been shown to increase cancer risks partly through the deregulation of the immune response. A recent transcriptome profiling of B cells exposed to aflatoxin B1 (AFB1) revealed an upregulation of the Chemokine ligand 22 (CCL22) expression although the underlying mechanisms were not investigated. Here, we tested whether mycotoxins and EBV exposure may together contribute to endemic BL (eBL) carcinogenesis via immunomodulatory mechanisms involving CCL22. Our results revealed that B cells exposure to AFB1 and EBV synergistically stimulated CCL22 secretion via the activation of Nuclear Factor-kappa B pathway. By expressing EBV latent genes in B cells, we revealed that elevated levels of CCL22 result not only from the expression of the latent membrane protein LMP1 as previously reported but also from the expression of other viral latent genes. Importantly, CCL22 overexpression resulting from AFB1-exposure in vitro increased EBV infection through the activation of phosphoinositide-3-kinase pathway. Moreover, inhibiting CCL22 in vitro and in humanized mice in vivo limited EBV infection and decreased viral genes expression, supporting the notion that CCL22 overexpression plays an important role in B cell infection. These findings unravel new mechanisms that may underpin eBL development and identify novel pathways that can be targeted in drug development.

Role of Galectin-3 in intervertebral disc degeneration: an experimental study

BackgroundThis study investigated the role of Galectin-3 in the degeneration of intervertebral disc cartilage.MethodsThe patients who underwent lumbar spine surgery due to degenerative disc disease were recruited and divided into Modic I, Modic II, and Modic III; groups. HE staining was used to detect the pathological changes in endplates. The changes of Galectin-3, MMP3, Aggrecan, CCL3, and Col II were detected by immunohistochemistry, RT-PCR, and Western blot. MTT and flow cytometry were used to detect cartilage endplate cell proliferation, cell cycle, and apoptosis.ResultsWith the progression of degeneration (from Modic I to III), the chondrocytes and density of the cartilage endplate of the intervertebral disc decreased, and the collagen arrangement of the cartilage endplate of the intervertebral disc was broken and calcified. Meanwhile, the expressions of Aggrecan, Col II, Galectin-3, Aggrecan, and CCL3 gradually decreased. After treatment with Galectin-3 inhibitor GB1107, the proliferation of rat cartilage end plate cells was significantly reduced (P < 0.05). GB1107 (25 µmol/L) also significantly promoted the apoptosis of cartilage endplate cells (P < 0.05). Moreover, the percentage of cartilage endplate cells in the G1 phase was significantly higher, while that in the G2 and S phases was significantly lower (P < 0.05). Additionally, the mRNA and protein expression levels of MMP3, CCL3, and Aggrecan in rat cartilage end plate cells were lower than those in the control group.ConclusionsGalectin-3 decreases with the progression of the cartilage endplate degeneration of the intervertebral disc. Galectin-3 may affect intervertebral disc degeneration by regulating the degradation of the extracellular matrix.

Skin Barrier– and Immune Response–Related Biomarkers of Solar UVR Exposure Comparing Indoor and Outdoor Workers

Outdoor workers have increased risk of developing keratinocyte cancer due to accumulated skin damage resulting from chronic and excessive exposure to UVR. This study aims to identify potential noninvasive biomarkers to assess chronic UVR exposure. We analyzed stratum corneum biomarkers collected from 2 skin locations and 2 occupational groups with contrasting solar UVR exposure: the forehead and retroauricular skin among outdoor workers and indoor workers. Using a linear mixed model adjusting for age and skin phototype, we compared biomarkers between both skin sites in indoor and outdoor workers. We measured markers of the immune response and skin barrier, including cytokines, GFs, 15-hydroxyeicosatetraenoic acid, cis- and trans-urocanic acid, and corneocyte topography, indicated by circular nano objects. Differences between the 2 skin sites were found for cis-urocanic acid, total urocanic acid, IL-1α, IL-1RA, IL-1RA/IL-1α, IL-18, 15-hydroxyeicosatetraenoic acid, CCL4, and circular nano objects. The levels of cis-urocanic acid and CCL4 also differed between indoor and outdoor workers. These findings underscore changes in both immune response and skin barrier induced by UVR. They indicate the potential utility of stratum corneum biomarkers in detecting both chronic UVR exposure in occupational setting and aiding in the development of preventive measures.

477 Evaluating the therapeutic efficacy of combination IL-12 and trabectedin for the treatment of triple negative breast cancer

OBJECTIVES/GOALS: IL-12 has potent immune effects but the presence of myeloid-derived suppressor cells (MDSC) can inhibit IL-12-induced NK cell cytotoxicity. Thus, we hypothesized that combining IL-12 with trabectedin, an immunosuppressive myeloid cell depleting agent, would improve its therapeutic efficacy in triple negative breast cancer (TNBC). METHODS/STUDY POPULATION: Combination IL-12 and trabectedin was tested in the 4T1 mouse model of TNBC. 4T1 cells were injected into the mammary fat pad of female BALB/cj mice. When tumors reached 50 mm3, mice were randomly divided into 4 groups and treated with PBS, IL-12 (0.5 μg/mouse 3x/wk), 0.15 mg/kg trabectedin weekly or the combination. Tumor volumes were measured by calipers. Mass cytometry was performed on spleens and tumors using a 35-antibody panel. Plasma IFN-γ levels were measured by ELISA. The role of NK cells was evaluated via depletion with anti-asialo-GM1. The Luminex Discovery Assay was used to measure plasma cytokines and immunohistochemistry was performed for CD4 and CD8a. Linear/nonlinear mixed effects modeling was used for in vivo data analysis and applicable t- or ANOVA tests were used for in vitrodata analysis. RESULTS/ANTICIPATED RESULTS: Combination IL-12 and trabectedin led to a significant reduction in tumor burden compared to single-agent IL-12, trabectedin and control treatments (all p&lt;0.001), as well as higher levels of IFN-γ (all p&lt;0.04). One combination treated mouse had complete tumor regression. Splenic MDSC were significantly decreased in combination treated mice. NK depletion abrogated the effects of combination therapy. Compared to mice receiving a control antibody, NK depletion of combination treated mice led to lower levels of CCL5 (p&lt;0.01) and CXCL10 (p&lt;0.001) and significantly higher tumor burden (p=0.001). CD8+T cell levels were significantly higher in combination treated mice compared to those receiving IL-12 (p&lt;0.01), and these levels were decreased when mice were depleted of NK cells (p=0.01). DISCUSSION/SIGNIFICANCE: TNBC represents 15% of all breast cancer diagnoses and is associated with a worse prognosis compared to other subtypes. Black women are twice as likely to be diagnosed with TNBC and more likely to die from disease than White women. Thus, there is an increasing need to develop additional therapeutic options for this disease.

Characterization of Th17 tissue-resident memory cells in non-inflamed intestinal tissue of Crohn's disease patients

Crohn's disease (CD) is a chronic inflammatory disorder affecting the bowel wall. Tissue-resident memory T (Trm) cells are implicated in CD, yet their characteristics remain unclear. We aimed to investigate the transcriptional profiles and functional characteristics of Trm cells in the small bowel of CD and their interactions with immune cells. Seven patients with CD and four with ulcerative colitis as controls were included. Single-cell RNA sequencing and paired T cell receptor sequencing assessed T cell subsets and transcriptional signatures in lamina propria (LP) and submucosa/muscularis propria-enriched fractions (SM/MP) from small bowel tissue samples. We detected 58,123 T cells grouped into 16 populations, including the CD4+ Trm cells with a Th17 signature and CD8+ Trm clusters. In CD, CD4+ Trm cells with a Th17 signature, termed Th17 Trm, showed significantly increased proportions within both the LP and SM/MP areas. The Th17 Trm cluster demonstrated heightened expression of tissue-residency marker genes (ITGAE, ITGA1, and CXCR6) along with elevated levels of IL17A, IL22, CCR6, and CCL20. The clonal expansion of Th17 Trm cells in CD was accompanied by enhanced transmural dynamic potential, as indicated by significantly higher migration scores. CD-prominent Th17 Trm cells displayed an increased interferon gamma (IFNγ)-related signature possibly linked with STAT1 activation, inducing chemokines (i.e., CXCL10, CXCL8, and CXCL9) in myeloid cells. Our findings underscored the elevated Th17 Trm cells throughout the small bowel in CD, contributing to disease pathogenesis through IFNγ induction and subsequent chemokine production in myeloid cells.

Inflammatory responses in esophageal mucosa before and after laparoscopic antireflux surgery.

Currently, the primary treatment for gastroesophageal reflux is acid suppression with proton pump inhibitors, but they are not a cure, and some patients don't respond well or refuse long-term use. Therefore, alternative therapies are needed to understand the disease and develop better treatments. Laparoscopic anti-reflux surgery (LARS) can resolve symptoms of these patients and plays a significant role in evaluating esophageal healing after preventing harmful effects. Successful LARS improves typical gastroesophageal reflux symptoms in most patients, mainly by reducing the exposure time to gastric contents in the esophagus. Amelioration of the inflammatory response and a recovery response in the esophageal epithelium is expected following the cessation of the noxious attack. To explore the role of inflammatory biomolecules in LARS and assess the time required for esophageal epithelial recovery. Of 22 patients with LARS (pre- and post/5.8 ± 3.8 months after LARS) and 25 healthy controls (HCs) were included. All subjects underwent 24-h multichannel intraluminal impedance-pH monitoring and upper gastrointestinal endoscopy, during which esophageal biopsy samples were collected using endoscopic techniques. Inflammatory molecules in esophageal biopsies were investigated by reverse transcription-polymerase chain reaction and multiplex-enzyme-linked immunosorbent assay. Post-LARS samples showed significant increases in proinflammatory cytokines [interleukin (IL)-1β, interferon-γ, C-X-C chemokine ligand 2 (CXCL2)], anti-inflammatory cytokines [CC chemokine ligand (CCL) 11, CCL13, CCL17, CCL26, CCL1, CCL7, CCL8, CCL24, IL-4, IL-10], and homeostatic cytokines (CCL27, CCL20, CCL19, CCL23, CCL25, CXCL12, migration inhibitory factor) compared to both HCs and pre-LARS samples. CCL17 and CCL21 levels were higher in pre-LARS than in HCs (P < 0.05). The mRNA expression levels of AKT1, fibroblast growth factor 2, HRAS, and mitogen-activated protein kinase 4 were significantly decreased post-LARS vs pre-LARS. CCL2 and epidermal growth factor gene levels were significantly increased in the pre-LARS compared to the HCs (P < 0.05). The presence of proinflammatory proteins post-LARS suggests ongoing inflammation in the epithelium. Elevated homeostatic cytokine levels indicate cell balance is maintained for about 6 months after LARS. The anti-inflammatory response post-LARS shows suppression of inflammatory damage and ongoing postoperative recovery.

Identification and preliminary validation of differently expressed genes as candidate biomarkers associated with atherosclerosis

ObjectiveAtherosclerosis (AS) is the primary cause of deadly cardio-cerebro vascular diseases globally. This study aims to explore the key differentially expressed genes (DEGs), potentially serving as predictive biomarkers for AS. MethodsMicroarray datasets were retrieved from the GEO database for DEGs and DE-miRNAs identification. Then biological function of DEGs were elucidated based on gene ontology (GO) and KEGG pathway enrichment analysis. The protein–protein interaction (PPI) network and DEGs-DE-miRNAs network were constructed, with emphasis on hub DEGs selection and their interconnections. Additionally, receiver operating characteristic (ROC) curve analysis was performed to evaluate the diagnostic precision of hub DEGs for AS. More importantly, an AS Syrian Golden hamster model was established to validate the expression levels of hub DEGs in AS. ResultsA total of 203 DEGs and 10 DE-miRNAs were screened, with six genes were chosen as hub DEGs. These DEGs were significantly enriched in AS-related biological processes and pathways, such as immune and inflammatory response, cellular response to IL-1 and TNF, positive regulation of angiogenesis, Type I diabetes mellitus, Cytokine-cytokine receptor interaction, TLR signaling pathway. Also, these DEGs and DE-miRNAs formed a closely-interacted DE-miRNAs - DEGs - KEGG pathway network. Besides, hub DEGs presented promising diagnostic potential for AS (AUC: 0.781 ∼ 0.887). In addition, the protein expression levels of TNF-α, CXCL8, CCL4, IL-1β, CCL3 and CCR8 were significantly increased in AS group Syrian Golden hamsters. ConclusionThe identified candidate genes TNF, CXCL8, CCL4, IL1B, CCL3 and CCR8 may have the potential to serve as prognostic biomarker in diagnosing AS.

Abstract 6650: Macrophage pro-inflammatory phenotype skewing by the application of tumor treating fields (TTFields)

Abstract Introduction: Tumor Treating Fields (TTFields) are low intensity electric fields that disrupt cellular processes critical for cancer cell viability. TTFields have previously been shown to induce immunogenic cell death, promote dendritic cell maturation, and elicit a systemic anti-cancer immune response in preclinical models. The benefit of applying TTFields together with immune checkpoint inhibitors (ICI) was demonstrated in a phase 3 study in non-small cell lung carcinoma. In the current research, we investigated possible effects of TTFields on regulation of macrophage phenotype. Methods: Bone marrow-derived macrophages (BMDMs) were generated from the femurs and tibias of 5-8-week-old Balb\C mice. LPS+IFN-γ or IL-4 were applied to the BMDMs to induce polarization (M1 and M2, respectively). The BMDMs were then treated with TTFields (150 kHz, 24 h), and then examined by flow cytometry for surface expression of the macrophage biomarker F4/80 and the activation markers CD80, major histocompatibility complex class II (MHC II), inducible nitric oxide synthase (iNOS), CD206, and ARG-1. Cell supernatants were collected and analyzed by multiplexed secretion assay for secretion levels of CXCL1 (KC), IL-18, IL-23, IL-12p70, IL6, TNF-α, IL-12p40, TGF-β1, CCL22 (MDC), IL-10, IL-6, G-CSF, CCL17 (TARC), and IL-1β. Cell lysates were examined using a RhoA activation kit and Western blot to determine GEF-H1, c-Jun, and p65 phosphorylation levels. Results: BMDMs exposed to TTFields demonstrated elevated expression of the pro-inflammatory M1 markers CD80+ and MHC IIhigh, and decreased expression of the M2 markers CD206 and ARG-1. A pro-inflammatory secretion pattern was detected, with increased levels of CXCL1, IL-18, IL-23, IL-12p70, TNF-α, IL-12p40, CCL22, G-CSF, CCL17 and IL-1β. The treated cells further demonstrated RhoA activation and GEF-H1 phosphorylation, with subsequent activation of the transcription factors c-Jun and p65. Conclusions: This study reveals a novel immunomodulatory role for TTFields, promoting in vitro pro-inflammatory polarization of macrophage. Citation Format: Tal Kan, Yiftah Barsheshet, Boris Brant, Tharwat Haj Khalil, Tali Voloshin, Alexandra Volodin, Lilach Koren, Bella Koltun, Anat Klein-Goldberg, Efrat Zemer-Tov, Rom Paz, Adi Haber, Moshe Giladi, Uri Weinberg, Yoram Palti. Macrophage pro-inflammatory phenotype skewing by the application of tumor treating fields (TTFields) [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 6650.

Abstract 5526: Activation of platelet-activating factor (PAF) in high-risk melanoma patients with nodal metastasis

Abstract Background: Platelet-activating factor (PAF) is one of the lipid mediators in ultraviolet-induced immunosuppression signaling. Sentinel lymph nodes (SLNs) are the first organs to receive lymphatic drainage via afferent lymphatic channels leading directly from the primary melanoma. In patients with nodal metastasis, it has been shown that the SLN has a profoundly immunotolerant microenvironment. We sought to determine whether lymphatic fluid signals, including PAF, may be involved in modulating the SLN immune microenvironment to favor melanoma metastasis. Hypothesis: Upregulation of PAF can bind to its receptor on monocytes and activate downstream immunosuppressive chemokines, resulting in an immunosuppressed environment in SLNs that permits nodal metastasis and melanoma progression. Methods: Lymphatic fluids, plasma samples, and SLN samples were collected from patients undergoing surgical excision of melanoma with SLN biopsy under an IRB-approved protocol. Metabolic analyses were performed on lymphatic fluids from high- versus low-risk patients by two-dimensional liquid chromatography system coupled with mass spectrometry (2D LC-MS). Single cell sequencing was done on single cell suspensions from SLN samples. Protein array was used to compare the cytokine and chemokine levels in plasma samples from high- versus low-risk melanoma patients. We compared two groups: low-risk (no SLN metastasis) and high-risk (with SLN metastasis). Results: Lymphatic fluid PAF levels were 3-fold greater in high-risk melanoma patients compared with low-risk patients (p=0.0039). Macrophages were identified as the cell type with greatest PTAFR (platelet-activating factor receptor) expression by single cell sequencing. CCL17 levels in plasma samples from high-risk patients were significantly lower than in low-risk patients (p&amp;lt;0.05). These results suggest that increased PAF in lymphatic fluid may bind to PTAFR in SLN macrophages, inhibit chemokines (such as CCL17), and result in an immunosuppressive SLN environment to favor nodal metastasis and melanoma progression. Conclusion: Lymphatic fluid PAF signaling may contribute to an immunotolerant microenvironment in the SLN that allows nodal metastasis and melanoma progression through downregulation of CCL17. Further studies will investigate whether this pathway can be exploited to therapeutic advantage. Citation Format: Austin McMasters, Julia Chariker, Jae Yeon Hwang, Xinmin Yin, Xipeng Ma, Raobo Xu, Xiang Zhang, Juw Won Park, Kelly M. McMasters, Hongying Hao. Activation of platelet-activating factor (PAF) in high-risk melanoma patients with nodal metastasis [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 5526.

Hypoxia-induced miR-210-3p expression in lung adenocarcinoma potentiates tumor development by regulating CCL2 mediated monocyte infiltration.

In most cancers, tumor hypoxia downregulates the expression of C-C motif chemokine 2 (CCL2), and this downregulation has been implicated in monocyte infiltration and tumor progression; however, the molecular mechanism is not yet clear. We compared noncancerous and lung-adenocarcinoma human samples for hypoxia-inducible factor 1-alpha (HIF-1A), microRNA-210-3p (mir-210-3p), and CCL2 levels. Mechanistic studies were performed on lung adenocarcinoma cell lines and 3D tumor spheroids to understand the role of hypoxia-induced miR-210-3p in the regulation of CCL2 expression and macrophage polarization. HIF-1Α stabilization increases miR-210-3p levels in lung adenocarcinoma and impairs monocyte infiltration by inhibiting CCL2 expression. Mechanistically, miR-210-3p directly binds to the 3'untranslated region (UTR) of CCL2 mRNA and silences it. Suppressing miR-210-3p substantially downregulates the effect of hypoxia on CCL2 expression. Monocyte migration is significantly hampered in miR-210-3p mimic-transfected HIF-1A silenced cancer cells. In contrast, inhibition of miR-210-3p in HIF-1A-overexpressed cells markedly restored monocyte migration, highlighting a direct link between the miR-210-3p level and tumor monocyte burden. Moreover, miR-210-3p inhibition in 3D tumor spheroids promotes monocyte recruitment and skewing towards an antitumor M1 phenotype. Anti-hsa-miR-210-3p-locked nucleic acid (LNA) delivery in a lung tumor xenograft zebrafish model caused tumor regression, suggesting that miR-210-3p could be a promising target for immunomodulatory therapeutic strategies against lung adenocarcinoma.

Abstract 2485: A combined mregDC and Treg signature associates with antitumor efficacy of CCR4 antagonist tivumecirnon FLX475

Abstract Cancers evade immune surveillance through multiple mechanisms including the recruitment of immunosuppressive regulatory T cells (Treg). Tivumecirnon (formerly known as FLX475), a small molecule oral CCR4 antagonist under clinical evaluation, blocks the binding of CCR4 to its ligands CCL17 and CCL22 and thereby reduces Treg infiltration into the tumor microenvironment (TME). This relieves Treg-mediated immune suppression resulting in increased antitumor immunity. Our clinical study (NCT03674567) demonstrated that tivumecirnon can block the local accumulation of Treg in the TME and has clinical activity as monotherapy as well as in combination with pembrolizumab. In both mouse and human tumors, we identified a subset of tumor infiltrating dendritic cells (DC) producing high levels of CCL22, previously identified as mregDC, suggesting a potential role in the CCR4-dependent recruitment of Treg. To study the role of mregDC in the recruitment of Treg, non-clinical studies were performed. CD45+ immune cells from the TME of MB49 tumor bearing mice treated with tivumecirnon or vehicle and were analyzed by flow cytometry, scRNA-seq and CITE-seq. We confirmed that mregDC cells are the primary producer of CCL22 in the TME and that tivumecirnon treatment reduced the number of Treg cells in the TME and increased the percentage of IFN gamma and granzyme B expressing CD4+ T cells. Based on these findings we interrogated tumor biopsies from patients from our clinical study prior to (n = 33), and after approximately 6 weeks of treatment (n = 22, pre/post-treatment paired samples) with tivumecirnon +/- pembrolizumab. We performed RNAseq and compared to published biopsy data from anti-PD-1 treated patients. We found that higher mregDC + Treg signature levels at baseline significantly (p &amp;lt; 0.001) associated with response to tivumecirnon and pembrolizumab therapy. Importantly, analysis of published datasets revealed that higher baseline mregDC and Treg levels are not associated with response to anti-PD-1 monotherapy. Analysis of TCGA gene expression data demonstrated a positive correlation with mregDC and Treg cells across human tumor types, indicating a previously unknown relationship between these cell types. Further application of this mregDC and Treg signature to the TCGA dataset identified a subset of cancer types resistant to prior treatments that could potentially benefit from combinatorial treatment with tivumecirnon. Overall, this study demonstrated that inhibition of the CCR4 pathway using tivumecirnon can disrupt the immunosuppressive mregDC – Treg axis leading to enhanced antitumor activity. Furthermore, the levels of mregDC and Treg may be used as a biomarker for selection of patients more likely to respond to tivumecirnon in combination with anti-PD-1. Additional studies are required to confirm these results Citation Format: Adam Grant, Juraj Adamik, Jingtao Qiu, Jacob Wert, Molly Grandcolas, Scott Jacobson, Rakesh K. Goyal, William Ho, Shoji Ikeda, Marvin Au, Damian Trujillo, Michael Chisamore, Denise de Almeida Nagata, Mohsen S. Ghomi, Dirk G. Brockstedt, Paul D. Kassner, George Katibah. A combined mregDC and Treg signature associates with antitumor efficacy of CCR4 antagonist tivumecirnon FLX475 [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 2485.

Abstract 6819: Exploring the impact of IDH1-mutations on antitumor immunity in intrahepatic cholangiocarcinoma

Abstract Intrahepatic cholangiocarcinoma (CCA) is an aggressive biliary tract cancer that carries unfavorable prognosis. 10-20% of intrahepatic CCAs harbor a mutation in IDH1 that can be targeted with mutant IDH1-specific inhibitors. However, objective and durable responses to treatment with mutant IDH1 inhibitors are rare in CCA. Mutant IDH1 has neomorphic enzymatic activity that produces the oncometabolite D-2-hydroxyglutarate (D-2-HG). D-2-HG promotes biliary tumor formation through cancer cell-intrinsic mechanisms but is also a paracrine factor in the tumor microenvironment (TME). Our group and others have identified that IDH1-mutant CCAs are not responsive to immunotherapy treatment approaches in part due to decreased CD8+ T cell infiltration and an increase in immunosuppressive macrophage cells in the TME. To better understand the immunosuppressive features of the IDH1-mutant CCA TME, we generated an isogenic cell line panel of IDH1-mutant (MUT) and IDH1-wild type (WT) mouse cholangiocarcinoma cells in the SB1 cell line background through a CRISPR homology directed repair approach. Compared to IDH1-WT cells, IDH1-MUT cells produce high levels of D-2-HG and both cell line panels readily form tumors in immunocompetent C57BL/6 mice. Tumors formed from IDH1-mut cells have reduced CD8+ T cell infiltration compared to IDH1-WT tumors, a finding that correlates with analysis of human CCA samples. Using cytometry by time of flight (CyTOF), we identified that CD8+ T cells in IDH1-MUT tumors have decreased expression of key regulators of the antitumor immune response including EOMES, TBET, and GZMB compared to those isolated from IDH1-WT tumors. Analysis of secreted factors from these IDH1-MUT and IDH1-WT cells identified that IDH1-MUT cells release increased levels of CCL2, a chemokine shown to diminish the antitumor immune response across multiple cancer types, compared to IDH1-WT cells. Further, IDH1-MUT tumors grown in syngeneic, immunocompetent mice have increased CCL2 staining by IHC compared to IDH1-WT tumors. CCL2 expression is also increased in a preliminary analysis of IDH1-mutant human CCA primary tumors. Treatment of mice harboring orthotopic liver tumors formed from IDH1-MUT cells with a CCL2 neutralizing antibody resulted in decreased tumor size compared to isotype control. Together, our data suggest that CCL2 is a mediator of immunosuppression in the IDH1-mutant CCA TME and that modulation of CCL2 activity may improve outcomes in this rare disease subtype. Further work to understand the mechanisms through which IDH1 mutations regulate CCL2 levels in these tumors is underway. Citation Format: Emma Kartalia, James M. Leatherman, Jae W. Lee, Kiyoko Yoshima, Daniel J. Zabransky, Mark Yarchoan. Exploring the impact of IDH1-mutations on antitumor immunity in intrahepatic cholangiocarcinoma [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 6819.

Abstract 6797: CCR5/CCL4 axis in lung cancer clinicopathological significance and its therapeutic application

Abstract Lung cancer (LC) is the most prevalent type of cancer globally and the leading cause of cancer-related deaths. Based on the histology and type of treatments, LC is divided into small-cells and non-small cell lung cancer (NSCLC) carcinomas, which are more aggressive and less responsive to chemotherapy. Since LC is known to be a highly heterogeneous cell population, treatment is challenging. It has been observed that chemokine receptor-ligand interaction is essential for cancer cells homing to distant sites. Most malignancies, including LC, have established disease status indicators concerning elevated levels of CCL4, a structurally unique chemokine, and its receptor (CCR5). Therefore, treatment strategies that inhibit the LC cell's tumor environment may improve patients' quality of life. However, the CCR5/CCL4 interface in LC invasion and metastasis is poorly understood. In this study, we examined the expression and function of the CCR5/CCL4 axis in the development and invasiveness of LC patient's clinical tissues and cell lines. We conducted immunohistochemistry (IHC) dual staining of CCR5/CCL4 antibodies in LC tissues. Our findings demonstrated significantly (p &amp;lt; 0.001) higher expression of CCR5 and CCL4 in advanced-stage malignant tissues than in normal tissues. The in-vitro analysis of the human LC cell lines NCI-H1437, NCI-H1573, and NCI-H1975 revealed CCL4 activation expressing higher CCR5, determined by immunofluorescence, western blots, flow cytometry, 3D-invasion assay, and RT-qPCR methods. Further, a calcium mobilization assay confirmed the direct interaction between CCR5 and CCL4. However, treating with Maraviroc, a small molecule inhibitor of CCR5, to LC cells blocks their ability to proliferate, demonstrating the crucial role that CCR5-CCL4 chemotaxis plays in LC metastasis. Our findings show both the biological and clinical significance of CCR5 and CCL4 expression, which may serve as potential indicators for metastatic LC. Therefore, inhibiting the CCR5/CCL4 interaction may offer a more effective therapeutic approach for managing the progression of LC. Citation Format: Santosh K. Singh, Rajesh Singh. CCR5/CCL4 axis in lung cancer clinicopathological significance and its therapeutic application [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 6797.

Abstract 6799: The impact of targeting TRAF2 and NCK-interacting protein kinase on anti-tumor effect and tumor immune environment in cMYC-high small cell lung cancer

Abstract Background: Small cell lung cancer (SCLC) is a highly lethal malignancy with rapidly acquired chemotherapy resistance. While the inflamed SCLC subtype is associated with a significantly improved response to immunotherapy, the non-inflamed SCLC subtypes, encompassing most SCLC patients, experience only modest survival gains with chemo-immunotherapy. Wnt signaling pathway activation is associated with resistance to chemotherapy and an immune checkpoint inhibitor in several tumors. TRAF2 and NCK-interacting protein kinase (TNIK), which interacts with downstream effectors, TCF4/β-catenin transcriptional complex, is an essential activator of Wnt target genes. TNIK is highly expressed in several cancers, but the efficacy of TNIK inhibition in SCLC has not previously been researched. We previously demonstrated that single agent TNIK inhibitors are effective in a subset of SCLC. Here, we hypothesize that targeting TNIK will show an anti-tumor effect based on specific biomarkers and also modify the tumor immune environment in SCLC. Methods: We evaluated susceptibility to a small molecule TNIK inhibitor, NCB-0846, in 29 human-derived SCLC cell lines using 96-hour proliferation assays. To investigate efficacy biomarkers, we correlated NCB-0846 IC50 values with proteomic expression (profiled using reverse phase protein array). cMYC and FOXK1 expression was reduced by siRNA. Chemokine concentrations were determined by ELISA. Results: SCLC cell lines with high cMYC levels, were more sensitive to the TNIK inhibitor, NCB-0846 (F.C.=-2.07, p=0.01), while SCLC cell lines with high expression of TTF-1 were more resistant (F.C.=2.48, p&amp;lt;0.01). Knockdown of cMYC conferred resistance to NCB-0846 in cMYC-high cells, while NCB-0846 treatment resulted in decreased cMYC expression in a concentration-dependent manner. Additionally, NCB-0846 treatment decreased the immunosuppressive chemokine CCL2 levels in the supernatant of human-derived SCLC cell lines and GEMM-derived cell lines. The expression of FOXK1, a transcription factor of CCL2, was also reduced in cell lines treated with NCB-0846. Conclusions: These findings show that TNIK inhibition is more effective in cMYC-high SCLC, acting through downregulating cMYC levels. TNIK inhibition also decreases the production of CCL2, an immunosuppressive chemokine implicated in resistance to anti-PD-L1 immunotherapy. Together, these findings highlight the promising potential of TNIK inhibitors in cMYC-high SCLC. Additionally, suppressing CCL2 by TNIK inhibition supports the rationale for combining the TNIK inhibitor and an anti-PD-L1 antibody in the non-inflamed SCLC subtypes. Citation Format: Azusa Tanimoto, Kavya Ramkumar, Allison Stewart, Bingnan Zhang, Robert Cardnell, Shen Li, Qi Wang, Jing Wang, Carl Gay, Lauren Averett Byers. The impact of targeting TRAF2 and NCK-interacting protein kinase on anti-tumor effect and tumor immune environment in cMYC-high small cell lung cancer [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 6799.

O124: Investigating the Immuno-modulatory Effect of Adipose-derived Stem Cells in Radiation- Induced Fibrosis

Abstract Background and Introduction Radiation-induced fibrosis (RIF) is a complication of radiotherapy in the treatment of head and neck carcinoma with high patient morbidity. Autologous fat grafting is a viable method to treat RIF, and adipose-derived stem cells (ADSC) were found to be one of the main components to contribute to that effect. Method Experimental group consists of irradiated human-derived fibroblasts (iHDF) in vitro with the control group unexposed to radiation. Both groups were then inoculated with ADSC-controlled media and co-culture with ADSC, whilst levels of key immunomodulators expressed by iHDF cells were obtained and measured via enzyme-linked immunosorbent assay (ELISA) and real-time reverse transcription-polymerase chain reaction (RT-PCR). Results There is marginal decrease in CCL2 concentrations in co-cultured iHDF samples compared to untreated iHDF samples (p = 0.0006). There is a slight increase in CCL3 levels in iHDF co-culture samples compared to untreated iHDF samples (p = 0.0247). iHDF co-culture samples shows a statistically significant increase in IL-8 levels when compared to untreated samples of iHDF (p = 0.027). Col1A1 is reduced in iHDF samples when compared to control groups. Discussion CCL2 is implicated in recruitment of ADSCs to target sites. The increase in IL-8 secretion in ADSC samples is implicated in the pro-angiogenic properties of ADSC secretions to encourage healing in fibrotic tissue. Conclusion ADSC was demonstrated to have anti-fibrotic capabilities by promoting pro- angiogenic factors, however, experimental evidence points to a simultaneous increase in pro-inflammatory factors. Future works required to further investigate in vivo effects of ADSC.

Interleukin-2/anti-interleukin-2 complex attenuates inflammation in a mouse COPD model by expanding CD4+ CD25+ Foxp3+ regulatory T cells

Background and purposeChronic, nonspecific inflammation of the alveoli and airways is an important pathological feature of chronic obstructive pulmonary disease (COPD), while sustained inflammatory reactions can cause alveolar damage. Regulatory T cells (Tregs) inhibit inflammation, whereas the interleukin-2/anti-interleukin-2 complex (IL-2C) increases the number of Tregs; however, whether the IL-2C has a therapeutic role in COPD remains unknown. Therefore, this study investigated whether IL-2C alleviates lung inflammation in COPD by increasing the number of Tregs. Experimental approachA mouse COPD model was created by exposing mice to lipopolysaccharides (LPS) and cigarette smoke (CS), and the effects of IL-2C treatment on COPD were evaluated. The number of Tregs in the spleen and lung, pulmonary pathological changes, and inflammatory damage were examined through flow cytometry, histopathology, and immunofluorescence, respectively. Key resultsIL-2C increased the number of Treg cells in the spleen and lungs after exposure to CS and LPS, reduced the number of T helper 17 (Th17) cells in lung tissue, and improved the Th17/Treg balance. IL-2C decreased the number of inflammatory cells and reduced the levels of pro-inflammatory cytokines IL-6, TNF-α, IL-1β, CCL5, KC, and MCP-1 in bronchoalveolar lavage fluid and serum. IL-2C significantly reduced the pathological scores for lung inflammation, as well as decreased airway mucus secretion and infiltration of neutrophils and macrophages in the lungs. The depletion of Tregs using anti-CD25 antibodies eliminated the beneficial effects of IL-2C. Conclusions and implicationsIL-2C is a potential therapeutic agent for alleviating excessive inflammation in the lungs of patients with COPD.

Post-COVID-19 condition: systemic inflammation and low functional exercise capacity.

Post-COVID-19 condition (PCC) is characterised by a plethora of symptoms, with fatigue appearing as the most frequently reported. The alterations that drive both the persistent and post-acute disease newly acquired symptoms are not yet fully described. Given the lack of robust knowledge regarding the mechanisms of PCC we have examined the impact of inflammation in PCC, by evaluating serum cytokine profile and its potential involvement in inducing the different symptoms reported. In this cross-sectional study, we recruited 227 participants who were hospitalised with acute COVID-19 in 2020 and came back for a follow-up assessment 6-12 months after hospital discharge. The participants were enrolled in two symptomatic groups: Self-Reported Symptoms group (SR, n = 96), who did not present major organ lesions, yet reported several debilitating symptoms such as fatigue, muscle weakness, and persistent loss of sense of smell and taste; and the Self-Reported Symptoms and decreased Pulmonary Function group (SRPF, n = 54), composed by individuals with the same symptoms described by SR, plus diagnosed pulmonary lesions. A Control group (n = 77), with participants with minor complaints following acute COVID-19, was also included in the study. Serum cytokine levels, symptom questionnaires, physical performance tests and general clinical data were obtained in the follow-up assessment. SRPF presented lower IL-4 concentration compared with Control (q = 0.0018) and with SR (q = 0.030), and lower IFN-α2 serum content compared with Control (q = 0.007). In addition, SRPF presented higher MIP-1β serum concentration compared with SR (q = 0.029). SR presented lower CCL11 (q = 0.012 and q = 0.001, respectively) and MCP-1 levels (q = 0.052 for both) compared with Control and SRPF. SRPF presented lower G-CSF compared to Control (q = 0.014). Female participants in SR showed lower handgrip strength in relation to SRPF (q = 0.0082). Male participants in SR and SRPF needed more time to complete the timed up-and-go test, as compared with men in the Control group (q = 0.0302 and q = 0.0078, respectively). Our results indicate that different PCC symptom profiles are accompanied by distinct inflammatory markers in the circulation. Of particular concern are the lower muscle function findings, with likely long-lasting consequences for health and quality of life, found for both PCC phenotypes.

Transcriptomic profiling of osteoarthritis synovial macrophages reveals a tolerized phenotype compounded by a weak corticosteroid response.

It is well-known that long-term osteoarthritis prognosis is not improved by corticosteroid treatments. Here we investigate what could underlie this phenomenon by measuring the short term corticosteroid response of OA-Mf. We determined the genome-wide transcriptomic response to corticosteroids of end-stage osteoarthritic joint synovial macrophages (OA-Mf). This was compared with LPS-tolerized and β-glucan-trained circulating blood monocyte-derived macrophage models. Upon corticosteroid stimulation, the trained and tolerized macrophages significantly alter the abundance of 201 and 257 RNA transcripts, respectively. By contrast, by the same criteria, OA-Mf have a very restricted corticosteroid response of only 12 RNA transcripts. Furthermore, while metalloproteinases 1, -2, -3 and -10 expression clearly distinguish OA-Mf from both the tolerized and trained macrophage models, OA-Mf Interleukin 1 (IL1), chemokine (CXCL) and cytokine (CCL) family member profiles resemble the tolerized macrophage model, with the exception that OA-Mf show high levels of CCL20. Terminal osteoarthritis joints therefore harbor macrophages with an inflammatory state that closely resembles the tolerized macrophage state and this is compounded by a weak corticosteroid response capacity that may explain the lack of positive long-term effects of corticosteroid treatment for osteoarthritis patients.

Discovery of circulating blood biomarkers in patients with and without Modic changes of the lumbar spine: a preliminary analysis.

The following study aimed to determine the existence of blood biomarkers in symptomatic patients with or without lumbar Modic changes (MC). A cross-sectional sub-analyses of a prospective cohort was performed. Fasting blood samples were collected from patients with and without lumbar MC who had undergone spinal fusion or microdiscectomy. An 80-plex panel and CCL5/RANTES were used to assess preoperative plasma cytokine concentrations. Patient demographics and imaging phenotypes were also assessed. Thirty-one subjects were analysed (n = 18 no MC; n = 13 MC). No significant differences were found in age, sex, body mass index, smoking and alcohol history, and surgical procedure (i.e. fusion, decompression) between the two groups (p > 0.05). Several statistically significant blood biomarkers in MC patients were identified, including elevated levels of C-C Motif Chemokine Ligand 5 (CCL5, p = 0.0006), while Macrophage Migration Inhibitory Factor (MIF) was significantly lower (p = 0.009). Additionally, C-X-C Motif Chemokine Ligand 5 (CXCL5, p = 0.052), Pentraxin 3 (PTX3, p = 0.06) and Galectin-3 (Gal-3, p = 0.07) showed potential relevance. Moreover, MC patients exhibited significantly higher levels of disc degeneration (p = 0.0001) and displacement severity (p = 0.020). Based on multivariate analyses and controlling for disc degeneration/displacement, CCL5 (OR 1.02; 95% CI 1.002-1.033; p = 0.028) and MIF (OR 0.60; 95% CI 0.382-0.951; p = 0.030) were independently associated with MC patients. This "proof-of-concept" study is the first to identify specific and significantly circulating blood biomarkers associated with symptomatic patients with lumbar MC, independent of disc alterations of degeneration and/or bulges/herniations. Specifically, differences in CCL5 and MIF protein levels were significantly noted in MC patients compared to those without MC.

Targeting CCL2/CCR2 Signaling Overcomes MEK Inhibitor Resistance in Acute Myeloid Leukemia.

Emerging evidence underscores the critical role of extrinsic factors within the microenvironment in protecting leukemia cells from therapeutic interventions, driving disease progression, and promoting drug resistance in acute myeloid leukemia (AML). This finding emphasizes the need for the identification of targeted therapies that inhibit intrinsic and extrinsic signaling to overcome drug resistance in AML. We performed a comprehensive analysis utilizing a cohort of ∼300 AML patient samples. This analysis encompassed the evaluation of secreted cytokines/growth factors, gene expression, and ex vivo drug sensitivity to small molecules. Our investigation pinpointed a notable association between elevated levels of CCL2 and diminished sensitivity to the MEK inhibitors (MEKi). We validated this association through loss-of-function and pharmacologic inhibition studies. Further, we deployed global phosphoproteomics and CRISPR/Cas9 screening to identify the mechanism of CCR2-mediated MEKi resistance in AML. Our multifaceted analysis unveiled that CCL2 activates multiple prosurvival pathways, including MAPK and cell-cycle regulation in MEKi-resistant cells. Employing combination strategies to simultaneously target these pathways heightened growth inhibition in AML cells. Both genetic and pharmacologic inhibition of CCR2 sensitized AML cells to trametinib, suppressing proliferation while enhancing apoptosis. These findings underscore a new role for CCL2 in MEKi resistance, offering combination therapies as an avenue to circumvent this resistance. Our study demonstrates a compelling rationale for translating CCL2/CCR2 axis inhibitors in combination with MEK pathway-targeting therapies, as a potent strategy for combating drug resistance in AML. This approach has the potential to enhance the efficacy of treatments to improve AML patient outcomes.