Colony-stimulating Factor Receptor Research Articles

Long-term reprogramming of primed microglia after moderate inhibition of CSF1R signaling.

In acute neuroinflammation, microglia activate transiently, and return to a resting state later on. However, they may retain immune memory of such event, namely priming. Primed microglia are more sensitive to new stimuli and develop exacerbated responses, representing a risk factor for neurological disorders with an inflammatory component. Strategies to control the hyperactivation of microglia are, hence, of great interest. The receptor for colony stimulating factor 1 (CSF1R), expressed in myeloid cells, is essential for microglia viability, so its blockade with specific inhibitors (e.g. PLX5622) results in significant depletion of microglial population. Interestingly, upon inhibitor withdrawal, new naïve microglia repopulate the brain. Depletion-repopulation has been proposed as a strategy to reprogram microglia. However, substantial elimination of microglia is inadvisable in human therapy. To overcome such drawback, we aimed to reprogram long-term primed microglia by CSF1R partial inhibition. Microglial priming was induced in mice by acute neuroinflammation, provoked by intracerebroventricular injection of neuraminidase. After 3-weeks recovery, low-dose PLX5622 treatment was administrated for 12 days, followed by a withdrawal period of 7 weeks. Twelve hours before euthanasia, mice received a peripheral lipopolysaccharide (LPS) immune challenge, and the subsequent microglial inflammatory response was evaluated. PLX5622 provoked a 40%-50% decrease in microglial population, but basal levels were restored 7 weeks later. In the brain regions studied, hippocampus and hypothalamus, LPS induced enhanced microgliosis and inflammatory activation in neuraminidase-injected mice, while PLX5622 treatment prevented these changes. Our results suggest that PLX5622 used at low doses reverts microglial priming and, remarkably, prevents broad microglial depletion.

Heterozygous missense CSF1R variants hamper in vitro CD34+-derived dendritic cell generation but not in vivo dendritic cell development

Colony stimulating factor 1 receptor (CSF1R) is an essential receptor for both colony stimulating factor 1 (CSF1) and interleukin (IL) 34 signaling expressed on monocyte precursors and myeloid cells, including monocytes, dendritic cells (DC), and microglia. In humans, dominant heterozygous pathogenic variants in CSF1R cause a neurological condition known as CSF1R-related disorder (CSF1R-RD), typically with late onset, previously referred to as adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP). CSF1R-RD is characterized by microglia reduction and altered monocyte function; however, the impact of pathogenic CSF1R variants on the human DC lineage remains largely unknown.We previously reported that cord blood CD34+ stem cell-derived DCs generated in vitro originate specifically from CSF1R expressing precursors. In this study, we examined the DC lineage of four unrelated patients with late-onset CSF1R-RD who carried heterozygous missense CSF1R variants (c.2330G>A, c.2375C>A, c.2329C>T, and c.2381T>C) affecting different amino acids in the protein tyrosine kinase domain of CSF1R. CD34+ stem cells and CD14+ monocytes were isolated from peripheral blood and subjected to an in vitro culture protocol to differentiate towards conventional DCs and monocyte-derived DCs, respectively. Flow cytometric analysis revealed that monocytes from patients with late-onset CSF1R-RD were still able to differentiate into monocyte-derived DCs in vitro, whereas the ability of CD34+ stem cells to differentiate into conventional DCs was impaired. Strikingly, the peripheral blood of patients contained all naturally occurring DC subsets. We conclude that the in vitro abrogation of DC-development in patients with heterozygous pathogenic missense CSF1R variants does not translate to an impairment in DC development in vivo and speculate that CSF1R signalling in vivo is compensated, which needs further study.

Nemo-like kinase blocks myeloid differentiation by targeting tumor suppressor C/EBPα in AML.

CCAAT/enhancer-binding protein α (C/EBPα), a key myeloid transcription factor, drives myeloid differentiation from blast cells by regulating the expression of granulocyte colony stimulating factor receptor and C/EBPε as required for promoting granulocyte differentiation. Here, we show that serine/threonine-protein kinase NLK, also known as Nemo-like kinase, physically associates with C/EBPα and phosphorylates it at multiple sites, including Ser21, Thr226, Thr230 and S234, leading to its ubiquitin-mediated degradation. Individual phospho-point mutants of C/EBPα could be phosphorylated by NLK, but a mutant with all phosphorylatable residues replaced by alanine resisted phosphorylation and degradation by NLK, as did the single point mutants. Furthermore, although ectopic expression of NLK enhanced phosphorylation of C/EBPα levels, it markedly inhibited total C/EBPα protein levels. Conversely, NLK depletion inhibited endogenous C/EBPα phosphorylation but enhanced its total protein levels in several acute myeloid leukemia (AML) cell lines and in peripheral blood mononuclear cells isolated from number of AML patient samples. Importantly, NLK depletion in peripheral blood mononuclear cells from primary AML patients not only restored C/EBPα protein levels, but also induced myeloid differentiation, suggesting that NLK could be therapeutically targeted to restore C/EBPα to resolve differentiation arrest in AML.

Macrophage colony-stimulating factor and its role in the tumor microenvironment: novel therapeutic avenues and mechanistic insights.

The tumor microenvironment is a complex ecosystem where various cellular and molecular interactions shape the course of cancer progression. Macrophage colony-stimulating factor (M-CSF) plays a pivotal role in this context. This study delves into the biological properties and functions of M-CSF in regulating tumor-associated macrophages (TAMs) and its role in modulating host immune responses. Through the specific binding to its receptor colony-stimulating factor 1 receptor (CSF-1R), M-CSF orchestrates a cascade of downstream signaling pathways to modulate macrophage activation, polarization, and proliferation. Furthermore, M-CSF extends its influence to other immune cell populations, including dendritic cells. Notably, the heightened expression of M-CSF within the tumor microenvironment is often associated with dismal patient prognoses. Therefore, a comprehensive investigation into the roles of M-CSF in tumor growth advances our comprehension of tumor development mechanisms and unveils promising novel strategies and approaches for cancer treatment.

Abstract 6648: Low-intensity pulsed ultrasound enhances the presentation of brain-derived antigens by facilitating the migration of antigen-presenting cells from the brain to the periphery

Abstract Gliomas, the most common primary brain tumors, remain largely incurable despite advancements in surgical and adjuvant therapies. While immunotherapies offer promising avenues, their efficacy has been limited by formidable anatomical barriers, such as the blood-brain barrier (BBB), which restricts the migration of immune cells into the brain and impedes the efficient drainage of brain-derived antigens, establishing an immune-privileged environment. Consequently, neoplastic formations within the brain often escape detection by the peripheral immune system, complicating therapeutic interventions. The application of low-intensity pulsed ultrasound with microbubbles (LIPU/MB) has emerged as a potential solution, enabling the transient opening of the BBB. This facilitates drug transport and immune cell trafficking, including Chimeric Antigen Receptor (CAR) T cells, into brain tumors. However, challenges such as antigen-loss escape persist with CAR T cells. One proposed solution involves promoting antigen/epitope spreading by inducing the presentation of brain (tumor) antigens to the systemic immune system. To investigate this, we conducted intravital imaging using Colony-stimulating factor receptor 1 (CSFR1) and CX3CR1 reporter mice, visualizing antigen-presenting cell locomotion in the brain before and after LIPU/MB treatment using two-photon microscopy. Within one hour after LIPU/MB, we observed the migration of CSFR1+ and CX3CR1+ immune cells from the brain parenchyma and perivascular space into the bloodstream, a phenomenon absent prior to LIPU/MB treatment. Beyond that, flow cytometry identified CD45intermediateCD11b+-CX3CR1+-TMEM119+-P2RY12+microglia-like' cells in the blood. To further assess the immunological effects of LIPU/MB on brain antigen presentation, we utilized transgenic mice expressing a minigene encoding four immunogenic peptides under the glial fibrillary acidic protein (GFAP) promoter. LIPU/MB treatment was associated with increased proliferation and priming of adoptively transferred CNS antigen-specific naïve T cells in cervical lymph nodes, ultimately homing into the CNS, indicating enhanced antigen presentation. Furthermore, in a subset of glioblastoma (GBM) patients undergoing LIPU/MB combined with doxorubicin and anti-PD-1 blocking antibodies, CD11b+CD14+P2YR12+ microglia were detected in blood samples during cycles 3-6 of treatment. This increase was absent in blood samples from healthy donors.Collectively, our findings suggest that LIPU/MB represents a novel approach to augment brain-antigen presentation in the periphery, holding potential implications for improving the immunotherapeutic management of gliomas.[M.G., V.A.A., A.M.S., and H.O. contributed equally to this work.] Citation Format: Marco Gallus, Victor Andres Arrieta, Atsuro Saijo, Pavlina Chuntova, Akane Yamamichi, Jeffrey Haegelin, Su Phyu, Lan Phung, Shayan A. Amir-Kabirian, Senthilnath Lakshmanachetty, Li Chen, Karl Habashy, Andrew Gould, Christina Amidei, Catalina Lee Chang, Roger Stupp, Michael Canney, Hideho Okada. Low-intensity pulsed ultrasound enhances the presentation of brain-derived antigens by facilitating the migration of antigen-presenting cells from the brain to the periphery [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 6648.

Gene Expression of CSF3R/CD114 Is Associated with Poorer Patient Survival in Glioma.

Gliomas comprise most cases of central nervous system (CNS) tumors. Gliomas afflict both adults and children, and glioblastoma (GBM) in adults represents the clinically most important type of malignant brain cancer, with a very poor prognosis. The cell surface glycoprotein CD114, which is encoded by the CSF3R gene, acts as the receptor for the granulocyte colony stimulating factor (GCSF), and is thus also called GCSFR or CSFR. CD114 is a marker of cancer stem cells (CSCs), and its expression has been reported in several cancer types. In addition, CD114 may represent one among various cases where brain tumors hijack molecular mechanisms involved in neuronal survival and synaptic plasticity. Here, we describe CSF3R mRNA expression in human gliomas and their association with patient prognosis as assessed by overall survival (OS). We found that the levels of CSF3R/CD114 transcripts are higher in a few different types of gliomas, namely astrocytoma, pilocytic astrocytoma, and GBM, in comparison to non-tumoral neural tissue. We also observed that higher expression of CSF3R/CD114 in gliomas is associated with poorer outcome as measured by a shorter OS. Our findings provide early evidence suggesting that CSF3R/CD114 shows a potential role as a prognosis marker of OS in patients with GBM.

Abstract 116: Increased Collagen I/Collagen III Ratio is Associated With Hemorrhage in Brain Arteriovenous Malformations in Human and Mouse

Background and purpose: Brain arteriovenous malformation (bAVM) is an important risk factor for hemorrhagic stroke in young adults, but the underlying mechanisms are unclear. Increase of Col I/Col III ratio enhances vessel wall stiffness and renders vessel less resistant to changes of blood flow and pressure. Hypothesis: Col I/Col III ratio is increased in bAVM vessels, which is associated with bAVM hemorrhage. Method: Human bAVMs were collected from HHT and sporadic bAVM patients. Temporal lobe specimens from epilepsy patients were used as controls. Three mouse models were used: 1) PdgfbicreER; Alk1 f/f ; 2) PdgfbicreER; Eng f/f ; 3) Alk 1 f/f . Mice in models 1 and 2 have Pdgfb promoter driving, tamoxifen (TM) inducible cre expression in endothelial cells (ECs). BAVMs were induced by intra-brain injection of AAV-VEGF to induce angiogenesis and i.p. injection of TM to delete Alk1 or Eng in ECs. In model 3, bAVMs were by intra-brain injection of AAV-VEGF and an adenoviral vector expressing Cre to delete Alk1 gene focally. Colony stimulating factor receptor inhibitor (PLX5622) was given to Alk1 f/f mice 8 week after model induction when bAVMs have fully developed to transiently deplete microglia. Vascular Col I and Col III levels were analyzed by immunostaining using anti- CD31 and anti-Col I or Col II antibodies. Brain AVM bleeding was detected by Prussian blue staining, and RNA-Sequencing used to study gene profiles. Results: Col I and Col III levels and the Col I/Col III ratio were higher in the vessels of human HHT and sporadic bAVM than controls. Col I/Col III ratio was higher in ruptured than unruptured sporadic bAVM. RNA-seq data shown that Col I and Co III mRNA levels were higher in Eng deficient bAVMs than normal brain angiogenic regions. Col I and Col III levels were higher on Alk1 and Eng deficient bAVMs compared to normal vessels. Col I/Col III ratio was also increased in the Alk1 deficient bAVMs. Depletion of microglia by PLX5622 reduced Col I and Col III levels and reduced microhemorrhage of Alk1 deficient bAVMs. Conclusion: Col I, Col III and Col I/Col III ratio are increased on human and mouse bAVM vessels, which is associated with bAVM hemorrhage. Depletion of microglia reduces Col I and Col III and hemorrhage in bAVMs in mice.

Validation of CSF-1 receptor (CD115) staining for analysis of murine monocytes by flow cytometry.

CD115, the receptor for colony stimulating factor 1, is essential for survival and differentiation of monocytes and macrophages and is therefore frequently used to define monocyte subsets and their progenitors in immunological assays. However, CD115 surface expression and detection by flow cytometry is greatly influenced by cell isolation and processing methods, organ source, and disease context. In a systematic analysis of murine monocytes, we define experimental conditions that preserve or limit CD115 surface expression and staining by flow cytometry. We also find that, independent of conditions, CD115 surface levels are consistently lower in Ly6Clo monocytes than in Ly6Chi monocytes, with the exception of Ly6Clo monocytes in the bone marrow. Furthermore, in contrast to IL-34, the presence of colony stimulating factor 1 impairs CD115 antibody staining in a dose-dependent manner, which, in a model of ischemic kidney injury with elevated levels of colony stimulating factor 1, influenced quantification of kidney monocytes. Thus, staining and experimental conditions affect quantitative and qualitative analysis of monocytes and may influence experimental conclusions.

Abstract A072: Ghrelin deletion reduces mammary tumor growth and enhances response to immunotherapy

Abstract Ghrelin is a peptide hormone, primarily produced in the stomach, best known for its role as a regulator of nutrient sensing, appetite, and meal initiation. However, increasing evidence supports a more complex and nuanced role for ghrelin in a diverse array of biological processes, including cancer. Ghrelin’s effects on breast cancer development and progression are not clear, as the literature detailing its effects is conflicting. Several studies in breast cancer survivors have concluded that higher expression of ghrelin is correlated with better recurrence-free and breast cancer-specific survival. However, additional clinical and foundational science studies have suggested greater complexity for ghrelin’s role in breast cancer development as a splice variant of ghrelin, namely In1-ghrelin, is associated with increased tumor proliferation and reduced disease-free survival of breast cancer patients. Thus, the current study sought to determine the effects of ghrelin deletion within mammary tumor cells (67NR) on subsequent tumor growth. Specifically, we hypothesized that ghrelin deletion would significantly alter tumor growth. Adult female mice received bilateral orthotopic injections of the nonmetastatic murine breast carcinoma cell line 67NR or one of two CRISPR generated ghrelin deleted cell lines 1G9 or 2G2 (derived from the 67NR cell line). Ghrelin deletion significantly reduced tumor volume and tumor mass by ~60% and ~55%, respectively. Additionally, ghrelin deletion significantly increased median survival duration by ~30%. When examining changes within the tumor microenvironment we observed a significant increase in macrophage colonization in ghrelin knockouts relative to the parental 67NR tumors. Notably, the percent F4/80 staining within the tumor positively correlated with length of survival. To determine the role of these F4/80 cells on tumor progression, we ablated these macrophages by feeding chow containing the colony stimulating factor receptor 1 inhibitor, PLX5622, for the entirety of the study. Ablating macrophages significantly increased tumor volume and tumor mass and rendered the previous beneficial effects of ghrelin deletion null, suggesting that the positive effects of ghrelin deletion may be due to actions on macrophages. Lastly, because of the increased macrophage infiltration within ghrelin knockout tumors, we sought to determine their repose to an immunotherapy targeting macrophages, namely anti-CD47. Notably, ghrelin deleted tumors displayed a positive response to the immunotherapy and further reduction in tumor growth (~80% reduction in tumor volume compared to 67NR tumors). Whereas, 67NR tumors were unresponsive to the immunotherapy. These data add to the literature detailing the complex effects of ghrelin on tumor growth and suggest that ghrelin may be a druggable target to slow tumor development. Citation Format: William H Walker II, Brittany D Elliott, Claire O Kisamore, Randy J Nelson, A. Courtney DeVries. Ghrelin deletion reduces mammary tumor growth and enhances response to immunotherapy [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A072.

CXCR2-Blocking Has Context-Sensitive Effects on Rat Glioblastoma Cell Line Outgrowth (S635) in an Organotypic Rat Brain Slice Culture Depending on Microglia-Depletion (PLX5622) and Dexamethasone Treatment

In glioblastoma (GBM), the interplay of different immune cell subtypes, cytokines, and/or drugs shows high context-dependencies. Interrelations between the routinely applied dexamethasone (Dex) and microglia remain elusive. Here, we exploited rat organotypic brain slice co-cultures (OBSC) to examine the effects on a rat GBM cell line (S635) outgrowth resulting from the presence of Dex and pretreatment with the colony-stimulating factor receptor 1 (CSF1-R) inhibitor PLX5622: in native OBSC (without PLX5622-pretreatment), a diminished S635 spheroid outgrowth was observable, whereas Dex-treatment enhanced outgrowth in this condition compared to PLX5622-pretreated OBSC. Screening the supernatants of our model with a proteome profiler, we found that CXCL2 was differentially secreted in a Dex- and PLX5622-dependent fashion. To analyze causal interrelations, we interrupted the CXCL2/CXCR2-axis: in the native OBSC condition, CXCR2-blocking resulted in increased outgrowth, in combination with Dex, we found potentiated outgrowth. No effect was found in the PLX5622-pretreated. Our method allowed us to study the influence of three different factors—dexamethasone, PLX5622, and CXCL2—in a well-controlled, simplified, and straight-forward mechanistic manner, and at the same time in a more realistic ex vivo scenario compared to in vitro studies. In our model, we showed a GBM outgrowth enhancing synergism between CXCR2-blocking and Dex-treatment in the native condition, which was levelled by PLX5622-pretreatment.

PLUME: A single-arm phase II trial evaluating the combination of pembrolizumab and lenvatinib in metastatic uveal melanoma (mUM) patients (pts) previously treated or not with tebentafusp.

TPS9613 Background: Up to 50% of pts with uveal melanoma (UM) develop metastases, mainly hepatic, with poor overall survival and limited treatment options as response rate to first-line immune checkpoint inhibitors (ICI) is low in mUM pts. Tebentafusp, a bispecific T-cell engager, was recently approved in this setting but only in HLA A*02-01+ mUM patients. Lenvatinib is a tyrosine kinase inhibitor targeting multiple growth factor receptors including vascular endothelial growth factor receptors (VEGFRs), fibroblast growth factor receptors (FGFRs) and colony-stimulating factor receptor (CSF1-R), which are key pathways in UM. Lenvatinib showed an immune-modulating effect in preclinical models. The combination demonstrated synergic clinical anti-tumor activity in various tumor types including microsatellite-stable endometrial cancers, kidney cancers and ICI-resistant skin melanomas. We hypothesize that combining pembrolizumab with lenvatinib in mUM may lead to an improved immune response in mUM patients. Methods: Fifty-four, ICI-naïve, metastatic UM pts will be enrolled in this monocentric, single-arm phase II trial at Institut Curie, Paris, France. Because previous exposure to tebentafusp might increase the activity of subsequent ICI, the combination will be assessed in two independent cohorts: cohort 1 with HLA A*02-01neg, tebentafusp-naive patients (22 assessable patients), and cohort 2 with HLA A*02-01pos pts previously treated with tebentafusp (28 assessable patients). Up to 54 patients may be included according to an 8% drop-out rate. Each participant will receive pembrolizumab (200 mg Q3W, maximum of 35 cycles) plus lenvatinib (starting at 20mg QD) until reaching a discontinuation criterion. Liver MRI and chest-abdomen-pelvis CT will be performed every 9 weeks until progressive disease. Primary objective is to evaluate the progression-free survival (PFS) after nine cycles of treatment (27 weeks) as assessed by the investigator using the RECIST v1.1. Secondary objectives include PFS according to immune RECIST (iRECIST), overall survival, overall response rate, safety and tolerability, quality of life. Inclusion period started mid-July 2022 for two years. To date, twenty-four (21 in cohort 1, 3 in cohort 2) of planned 54 pts have been enrolled in six months. Biospecimens (longitudinal blood samples and tumor tissue at inclusion) will be collected during the study to allow biomarker research. Clinical trial information: NCT05282901 .

OA05 Harnessing high throughput multiplex immunoassays and protein association networks to identify diagnostic protein biomarkers of rheumatoid arthritis-cardiovascular disease (RA-CVD) multimorbid axis

Abstract Background/Aims People with rheumatoid arthritis (RA) have higher mortality rates compared to the general population, primarily due to excess cardiovascular (CV) disease (CVD). The earliest stages of RA are associated with CV abnormalities but biomarkers to identify CVD in early RA are lacking. We aimed to identify focused protein biomarkers that associate with cardiac magnetic resonance imaging (CMR) abnormalities in a treatment-naïve, new-onset RA trial cohort and the predominant inflammatory and metabolic pathways involved in RA-CVD. Methods CADERA (Coronary Artery Disease in Early RA) was a bolt-on study to a parent RCT in an early RA inception cohort. 81 treatment-naïve early RA patients underwent CMR at baseline as part of CADERA. Proximity extension immunoassay (OIink) was used to measure normalised protein expression across inflammation, cardiovascular II/III and cardiometabolic panels in CADERA patients. Bayesian mixed effects linear regression was used to identify significant proteins associated with CMR parameters of myocardial oedema/fibrosis and vascular stiffness at baseline. Then, an expanded protein-protein interaction (PPI) network using above proteins was created using an induced network approach (String-DB) and k-means clustering applied to identify protein clusters that were subsequently subjected to Gene ontology (GO) and KEGG enrichment analysis. Results Of 340 proteins analysed using Olink, 108 proteins were associated with CMR markers of myocardial fibrosis/oedema (64 proteins) and vascular stiffness (44 proteins) Table 1 highlights the top 5 proteins for each CMR parameter. K-means clustering of the expanded PPI network identified 4 clusters with roles in vascular endothelial growth factor receptor binding and JAK-STAT signalling pathway (cluster 1); IL27 receptor binding and NF-kappa B signalling pathway (cluster 2); Macrophage CSF receptor binding and ErbB signalling pathway (cluster 3); and IL10 receptor activity and complement and coagulation cascades (cluster 4). Conclusion This is the first study to identify protein biomarkers of RA-CVD, an area of unmet clinical need, using a combination of targeted high throughput immunoassays, Bayesian and network analyses. Targeted techniques such as Olink may help identify potential diagnostic biomarkers of RA-CVD. Pathway analysis provides insight into the inter-relationship between inflammation and metabolic mediators of RA-CVD axis. Disclosure R. Shukla: None. N. Black: None. D. Plant: None. C. Miller: None. S. Plein: None. M.H. Buch: None.

Pexidartinib synergize PD-1 antibody through inhibiting treg infiltration by reducing TAM-derived CCL22 in lung adenocarcinoma.

There is a crosstalk between Tumor-associated macrophages (TAM) and tumor-infiltrating T cells in tumor environment. TAM could inhibit the activity of cytotoxic T cells; TAM could also regulate the composition of T cells in tumor immune environment. The combination therapy for TAM and tumor infiltrated T cells has been widely noticed, but the crosstalk between TAM and tumor infiltrated T cells remains unclear in the process of combination therapy. We treated lung adenocarcinoma tumor models with pexidartinib, which targets macrophage colony stimulating factor receptor (M-CSFR) and c-kit tyrosine kinase, to inhibited TAM. Pexidartinib inhibited the ratio of macrophages in the tumor and also altered macrophage polarization. In addition to reprogram TAM, pexidartinib also changed the composition of tumor-invasive T cells. After pexidartinib treatment, the total number of T cells, CD8+ T cells and Treg cells were all decreased, the ratio of CD8+T/Treg increased significantly. According to the analysis of cytokines and chemokines during the treatment of pexidartinib, CCL22, as a chemokine for Treg recruitment, significantly decreased after the treatment of pexidartinib. Base on the above observation, the combination of pexidartinib and PD-1 antibody were used in the treatment of lung adenocarcinoma subcutaneous tumor model, the combination therapy has significantly improved the efficacy of tumor treatment compared with the monotherapy. Meanwhile, compared with pexidartinib monotherapy, the combination treatment further switches the polarization status of tumor-associated macrophages. In summary, our results showed that the combination of pexidartinib and PD-1 antibody showed a synergy and significantly improved the anti-tumor efficacy, through pexidartinib increasing CD8T/Treg ratio by reducing TAM-derived CCL22.

Immunohistochemical analysis of expression of VEGFR2, KIT, PDGFR-β, and CDK4 in canine urothelial carcinoma.

Urothelial carcinomas (UCs), also known as transitional cell carcinomas, are the most common canine urinary tract neoplasms. Tyrosine kinases (TKs) are enzymes that tightly regulate cell growth and differentiation through phosphorylation. Receptor TK (RTK) inhibitors are currently used to treat UCs. Toceranib phosphate (Palladia; Pfizer) is an RTK inhibitor that blocks the activity of vascular endothelial growth factor receptor 2 (VEGFR2), platelet-derived growth factor receptor-alpha and -beta (PDGFR-α, -β), FMS-like tyrosine kinase 3, stem cell factor receptor (KIT, kinase inhibitor targeting), and colony stimulating factor receptor. To better understand UCs and validate treatment targets, we performed immunohistochemical staining for RTKs, as well as a novel target, cyclin-dependent kinase 4 (CDK4, a central regulator of the mammalian cell cycle), on formalin-fixed, paraffin-embedded tissues from bladder biopsies from 17 dogs with UCs, 17 dogs with cystitis (diseased controls), and 8 normal dogs (negative controls). Although immunohistochemical scores could not be extrapolated to prognostic value, response to treatment, and outcome of patients with UC, we demonstrated expression of PDGFR-β and VEGFR2 in UCs; all UC samples staining positively for VEGFR2. Minimal positive staining for KIT was noted in the tumor samples. CDK4 staining intensity was significantly weaker in UCs compared with normal and cystitis bladder samples. The intense staining of VEGFR2 in UC cells suggested that VEGFR2 may be of prognostic and/or therapeutic value in dogs with UC. Overexpression of VEGFR2 in UC cells validates this receptor as a treatment target in UC.

Effects of autochthonous strains mixture on gut microbiota and metabolic profile in cobia (Rachycentroncanadum)

The fish immune system is a topic or subject that offers a unique understanding of defensive system evolution in vertebrate heredity. While gut microbiota plays several roles in fish: well-being, promoting health and growth, resistance to bacterial invasion, regulation of energy absorption, and lipid metabolism. However, studies on fish gut microbiota face practical challenges due to the large number of fish varieties, fluctuating environmental conditions, and differences in feeding habits. This study was carried out to evaluate the impacts of supplemented three autochthonous strains, Bacillus sp. RCS1, Pantoeaagglomerans RCS2, and Bacilluscereus RCS3 mixture diet on cobia fish (Rachycentroncanadum). Also, chromatography, mass spectrometry and high throughput sequencing were combined to explore composition and metabolite profile of gut microbiota in juvenile cobia fed with supplemented diet. In the trial group, juvenile cobia received diets supplemented with 1 × 1012 CFU mL−1 autochthonous strains for ten weeks and a control diet without supplementation. Juvenile cobia receiving diets supplementation exhibited significantly improved growth than those without additives (control). Haematological indices, such as red blood cells, white blood cells, corpuscular haemoglobin concentration, mean corpuscular volume, haemoglobin, and mean corpuscular haemoglobin, were higher in the supplemented group. Similarly, digestive enzymes (trypsin, lipase, amylase, pepsin and cellulose, activities) activities were higher in supplemented diet with an indigenous isolates mixture. Serum biochemical parameters albumin, globulin, and total protein were significantly higher, while triglyceride, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and cholesterol showed no significant difference. On the other hand, glucose was significantly (P < 0.05) higher in the group without supplementation. On gene expression in the midgut, Immunoglobulin, Colony-stimulating factor receptor 1, major histocompatibility complex 1 were up-regulated by native isolates while T cell receptor beta, and Major histocompatibility complex 2 showed no significant difference. Gut bacterial composition was altered in fish receiving supplemented diet with autochthonous strains. Metabolomics also revealed that some metabolic pathways were considerably enriched in fish fed with supplemented diet; pathway analysis based on Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment revealed that differentially expressed metabolites were involved in galactose metabolism, tryptophan metabolism, carbohydrate digestion and absorption, purine metabolism, and ABC transporters. Functional analysis of bacterial community showed that differences in enriched metabolic pathways generally comprised carbohydrate and its metabolites, nucleotide and its metabolites, amino acid and its metabolites, heterocyclic compounds, and tryptamines, cholines, pigments. The current investigation results showed that autochthonous strains mixture has significantly enhanced the growth, survival, and innate and adaptive immunities of juvenile cobia.

Oral Cancer Cells Release Vesicles that Cause Pain.

Oral cancer pain is attributed to the release from cancers of mediators that sensitize and activate sensory neurons. Intraplantar injection of conditioned media (CM) from human tongue cancer cell line HSC-3 or OSC-20 evokes nociceptive behavior. By contrast, CM from noncancer cell lines, DOK, and HaCaT are non-nociceptive. Pain mediators are carried by extracellular vesicles (EVs) released from cancer cells. Depletion of EVs from cancer cell line CM reverses mechanical allodynia and thermal hyperalgesia. CM from non-nociceptive cell lines become nociceptive when reconstituted with HSC-3 EVs. Two miRNAs (hsa-miR-21-5p and hsa-miR-221-3p) are identified that are present in increased abundance in EVs from HSC-3 and OSC-20 CM compared to HaCaT CM. The miRNA target genes suggest potential involvement in oral cancer pain of the toll like receptor 7 (TLR7) and 8 (TLR8) pathways, as well as signaling through interleukin 6 cytokine family signal transducer receptor (gp130, encoded by IL6ST) and colony stimulating factor receptor (G-CSFR, encoded by CSF3R), Janus kinase and signal transducer and activator of transcription 3 (JAK/STAT3). These studies confirm the recent discovery of the role of cancer EVs in pain and add to the repertoire of algesic and analgesic cancer pain mediators and pathways that contribute to oral cancer pain.

Abstract 1528: Molecular mechanisms of TSLP as a therapy for CRLF2 B-Cell acute lymphoblastic leukemia

Abstract B-cell acute lymphoblastic leukemia (B-ALL) is the most common type of leukemia in children. B-ALL characterized by cytokine receptor-like factor 2 (CRLF2) overexpression (CRLF2 B-ALL) has a survival rate of &amp;lt;30% and is the highest risk sub-group of B-ALL in both adults and children. CRLF2 is a receptor component for the cytokine thymic stromal lymphopoietin (TSLP). TSLP plays a role in the survival and proliferation of B-cell precursors, thus explaining the oncogenic role of increased CRLF2 signaling in CRLF2 B-ALL. To our surprise, we found that high-levels of TSLP eliminated leukemia cells in patient-derived xenograft (PDX) models of CRLF2 B-ALL. CRLF2 and IL-7 receptor-alpha (IL-7Ra) form the heterodimer type-I cytokine receptor for TSLP cytokine. Binding of TSLP to its CRLF2 receptor complex induces JAK-STAT5 and PI3K-AKT pathway signals. TSLP shares the IL-7Ra with Interleukin 7 (IL-7) which has a heterodimer receptor consisting of IL-7Ra and the common gamma chain. High-levels of IL-7 (50 ng/ml) have been shown to induce IL-7Ra internalization and degradation in T-cells. We hypothesize that high-level TSLP induces internalization and degradation of IL-7Ra leading to CRLF2 signal inhibition, death of CRLF2 B-ALL cells and the anti-leukemia effects that we have observed in PDX mice. To test this hypothesis, we treated CRLF2 B-ALL cell lines with different TSLP concentrations to observe the effect of TSLP on its receptor and CRLF2 signaling. Flow cytometry data showed that continuous or a pulse of high-dose TSLP induced a loss of surface IL-7Ra expression for up to 24 hours. Phosphorylation assays showed that cells cultured with high-dose TSLP were unresponsive to subsequent TSLP-induced phosphorylation events (pSTAT5 and pRPS6), indicating CRLF2 signal inhibition. In conclusion, high-dose TSLP induces loss of (IL-7Ra) and inhibition of CRLF2 signaling. These results suggest that TSLP exerts its anti-leukemia effects by shutting down CRLF2-mediated signals possibly via the loss of the IL-7Ra receptor component. Citation Format: Hossam Alkashgari, Cornelia Stoian, Caleb Ruiz-Jimenez, Jacqueline Coats, Carlos A. Casiano, Sinisa Dovat, Kimberly J. Payne. Molecular mechanisms of TSLP as a therapy for CRLF2 B-Cell acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1528.

The Primary Microglial Leukodystrophies: A Review.

Primary microglial leukodystrophy or leukoencephalopathy are disorders in which a genetic defect linked to microglia causes cerebral white matter damage. Pigmented orthochromatic leukodystrophy, adult-onset orthochromatic leukodystrophy associated with pigmented macrophages, hereditary diffuse leukoencephalopathy with (axonal) spheroids, and adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) are different terms apparently used to designate the same disease. However, ALSP linked to dominantly inherited mutations in CSF1R (colony stimulating factor receptor 1) cause CSF-1R-related leukoencephalopathy (CRP). Yet, recessive ALSP with ovarian failure linked to AARS2 (alanyl-transfer (t)RNA synthase 2) mutations (LKENP) is a mitochondrial disease and not a primary microglial leukoencephalopathy. Polycystic membranous lipomembranous osteodysplasia with sclerosing leukoencephalopathy (PLOSL; Nasu–Hakola disease: NHD) is a systemic disease affecting bones, cerebral white matter, selected grey nuclei, and adipose tissue The disease is caused by mutations of one of the two genes TYROBP or TREM2, identified as PLOSL1 and PLOSL2, respectively. TYROBP associates with receptors expressed in NK cells, B and T lymphocytes, dendritic cells, monocytes, macrophages, and microglia. TREM2 encodes the protein TREM2 (triggering receptor expressed on myeloid cells 2), which forms a receptor signalling complex with TYROBP in macrophages and dendritic cells. Rather than pure microglial leukoencephalopathy, NHD can be considered a multisystemic “immunological” disease.

CSF-1 maintains pathogenic but not homeostatic myeloid cells in the central nervous system during autoimmune neuroinflammation.

Abstract The receptor for colony stimulating factor 1 (CSF-1R) is important for the survival and function of myeloid cells that mediate pathology during experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). CSF-1 and IL-34, the ligands of CSF-1R, have similar bio-activities but distinct tissue and context-dependent expression patterns, suggesting that they have different roles. This could be the case in EAE, given that CSF-1 expression is upregulated in the CNS, while IL-34 remains constitutively expressed. We found that targeting CSF-1 with neutralizing antibody halted ongoing EAE, with efficacy superior to CSF-1R inhibitor BLZ945, whereas IL-34 neutralization had no effect, suggesting that pathogenic myeloid cells were maintained by CSF-1. Both anti-CSF-1 and BLZ945 treatment greatly reduced the number of monocyte-derived cells and microglia in the CNS. However, anti-CSF-1 selectively depleted inflammatory microglia and monocytes in inflamed CNS areas, whereas BLZ945 depleted virtually all myeloid cells, including quiescent microglia, throughout the CNS. Anti-CSF-1 treatment reduced the size of demyelinated lesions and microglial activation in the grey matter. Lastly, we found that bone marrow-derived immune cells were the major mediators of CSF-1R-dependent pathology, while microglia played a lesser role. Our findings suggest that targeting CSF-1 could be effective in ameliorating MS pathology, while preserving the homeostatic functions of myeloid cells, thereby minimizing risks associated with ablation of CSF-1R-dependent cells. National Multiple Sclerosis Society (RG-1803-30491) National Institutes of Health T32 training grant (T32AI134646, NIAID)

CSF-1 maintains pathogenic but not homeostatic myeloid cells in the central nervous system during autoimmune neuroinflammation

The receptor for colony stimulating factor 1 (CSF-1R) is important for the survival and function of myeloid cells that mediate pathology during experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). CSF-1 and IL-34, the ligands of CSF-1R, have similar bioactivities but distinct tissue and context-dependent expression patterns, suggesting that they have different roles. This could be the case in EAE, given that CSF-1 expression is up-regulated in the CNS, while IL-34 remains constitutively expressed. We found that targeting CSF-1 with neutralizing antibody halted ongoing EAE, with efficacy superior to CSF-1R inhibitor BLZ945, whereas IL-34 neutralization had no effect, suggesting that pathogenic myeloid cells were maintained by CSF-1. Both anti–CSF-1 and BLZ945 treatment greatly reduced the number of monocyte-derived cells and microglia in the CNS. However, anti–CSF-1 selectively depleted inflammatory microglia and monocytes in inflamed CNS areas, whereas BLZ945 depleted virtually all myeloid cells, including quiescent microglia, throughout the CNS. Anti–CSF-1 treatment reduced the size of demyelinated lesions and microglial activation in the gray matter. Lastly, we found that bone marrow–derived immune cells were the major mediators of CSF-1R–dependent pathology, while microglia played a lesser role. Our findings suggest that targeting CSF-1 could be effective in ameliorating MS pathology, while preserving the homeostatic functions of myeloid cells, thereby minimizing risks associated with ablation of CSF-1R–dependent cells.