Redefining the ontogeny of hyalocytes as yolk sac-derived tissue-resident macrophages of the vitreous body.

BACKGROUND: The eye is a highly specialized sensory organ which encompasses the retina as a part of the central nervous system, but also non-neural compartments such as the transparent vitreous body ensuring stability of the eye globe and a clear optical axis. Hyalocytes are the tissue-resident macrophages of the vitreous body and are considered to play pivotal roles in health and diseases of the vitreoretinal interface, such as proliferative vitreoretinopathy or diabetic retinopathy. However, in contrast to other ocular macrophages, their embryonic origin as well as the extent to which these myeloid cells might be replenished by circulating monocytes remains elusive. RESULTS: In this study, we combine transgenic reporter mice, embryonic and adult fate mapping approaches as well as parabiosis experiments with multicolor immunofluorescence labeling and confocal laser-scanning microscopy to comprehensively characterize the murine hyalocyte population throughout development and in adulthood. We found that murine hyalocytes express numerous well-known myeloid cell markers, but concomitantly display a distinct immunophenotype that sets them apart from retinal microglia. Embryonic pulse labeling revealed a yolk sac-derived origin of murine hyalocytes, whose precursors seed the developing eye prenatally. Finally, postnatal labeling and parabiosis established the longevity of hyalocytes which rely on Colony Stimulating Factor 1 Receptor (CSF1R) signaling for their maintenance, independent of blood-derived monocytes. CONCLUSION: Our study identifies hyalocytes as long-living progeny of the yolk sac hematopoiesis and highlights their role as integral members of the innate immune system of the eye. As a consequence of their longevity, immunosenescence processes may culminate in hyalocyte dysfunction, thereby contributing to the development of vitreoretinal diseases. Therefore, myeloid cell-targeted therapies that convey their effects through the modification of hyalocyte properties may represent an interesting approach to alleviate the burden imposed by diseases of the vitreoretinal interface.

Design, synthesis, biological evaluation, and in silico studies of novel pyridopyridine derivatives as anticancer candidates targeting FMS kinase.

Design and synthesis of novel 4-carboxamidopyrido[3,2-b]pyridine derivatives as novel rigid analogues of sorafenib are reported herein. The target compounds showed potent antiproliferative activities against a panel of NCI-60 cancer cell lines as well as hepatocellular carcinoma cell line. Compounds 8g and 9f were among the most promising derivatives in terms of effectiveness and safety. Therefore, they were further examined to demonstrate their ability to induce apoptosis and alter cell cycle progression in hepatocellular carcinoma cells. The most potent compounds were tested against a panel of kinases that indicated their selectivity against FMS kinase. Compounds 8g and 8h showed the most potent activities against FMS kinase with IC50 values of 21.5 and 73.9 nM, respectively. The two compounds were also tested in NanoBRET assay to investigate their ability to inhibit FMS kinase in cells (IC50 = 563 nM (8g) and 1347 nM (8h) vs. IC50 = 1654 nM for sorafenib). Furthermore, compounds 8g and 8h possess potent inhibitory activities against macrophages when investigated in bone marrow-derived macrophages (BMDM) assay (IC50 = 56 nM and 167 nM, respectively, 164 nM for sorafenib). The safety and selectivity of these compounds were confirmed when tested against normal cell lines. Their safety profile was further confirmed using hERG assay. In silico studies were carried out to investigate their binding modes in the active site of FMS kinase, and to develop a QSAR model for these new motifs.

Therapeutic potential of human microglia transplantation in a chimeric model of CSF1R-related leukoencephalopathy.

Microglia replacement strategies are increasingly being considered for the treatment of primary microgliopathies like adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP). However, available mouse models fail to recapitulate the diverse neuropathologies and reduced microglia numbers observed in patients. In this study, we generated a xenotolerant mouse model lacking the fms-intronic regulatory element (FIRE) enhancer within Csf1r, which develops nearly all the hallmark pathologies associated with ALSP. Remarkably, transplantation of human induced pluripotent stem cell (iPSC)-derived microglial (iMG) progenitors restores a homeostatic microglial signature and prevents the development of axonal spheroids, white matter abnormalities, reactive astrocytosis, and brain calcifications. Furthermore, transplantation of CRISPR-corrected ALSP-patient-derived iMG reverses pre-existing spheroids, astrogliosis, and calcification pathologies. Together with the accompanying study by Munro and colleagues, our results demonstrate the utility of FIRE mice to model ALSP and provide compelling evidence that iMG transplantation could offer a promising new therapeutic strategy for ALSP and perhaps other microglia-associated neurological disorders.

Leukoencephalopathy with calcifications, developmental brain abnormalities and skeletal dysplasia due to homozygosity for a hypomorphic CSF1R variant: A report of three siblings.

We report three siblings homozygous for CSF1R variant c.1969 + 115_1969 + 116del to expand the phenotype of "brain abnormalities, neurodegeneration, and dysosteosclerosis" (BANDDOS) and discuss its link with "adult leukoencephalopathy with axonal spheroids and pigmented glia" (ALSP), caused by heterozygous CSF1R variants. We evaluated medical, radiological, and laboratory findings and reviewed the literature. Patients presented with developmental delay, therapy-resistant epilepsy, dysmorphic features, and skeletal abnormalities. Secondary neurological decline occurred from 23 years in sibling one and from 20 years in sibling two. Brain imaging revealed multifocal white matter abnormalities and calcifications during initial disease in siblings two and three. Developmental brain anomalies, seen in all three, were most severe in sibling two. During neurological decline in siblings one and two, the leukoencephalopathy was progressive and had the MRI appearance of ALSP. Skeletal survey revealed osteosclerosis, most severe in sibling three. Blood markers, monocytes, dendritic cell subsets, and T-cell proliferation capacity were normal. Literature review revealed variable initial disease and secondary neurological decline. BANDDOS presents with variable dysmorphic features, skeletal dysplasia, developmental delay, and epilepsy with on neuro-imaging developmental brain anomalies, multifocal white matter abnormalities, and calcifications. Secondary neurological decline occurs with a progressive leukoencephalopathy, in line with early onset ALSP. Despite the role of CSF1R signaling in myeloid development, immune deficiency is absent. Phenotype varies within families; skeletal and neurological manifestations may be disparate.

Unifying considerations and evidence of macrophage activation mosaicism through human CSF1R and M1/M2 genes.

Addressing the mononuclear phagocyte system (MPS) and macrophage M1/M2 activation is important in diagnosing hematological disorders and inflammatory pathologies and designing therapeutic tools. CSF1R is a reliable marker to identify all circulating MPS cells and tissue macrophages in humans using a single surface protein. CSF1R permits the quantification and isolation of monocyte and dendritic cell (DC) subsets in conjunction with CD14, CD16, and CD1c and is stable across the lifespan and sexes in the absence of overt pathology. Beyond cell detection, measuring M1/M2 activation in humans poses challenges due to response heterogeneity, transient signaling, and multiple regulation steps for transcripts and proteins. MPS cells respond in a conserved manner to M1/M2 pathways such as interleukin-4 (IL-4), steroids, interferon-γ (IFNγ), and lipopolysaccharide (LPS), for which we propose an ad hoc modular gene expression tool. Signature analysis highlights macrophage activation mosaicism in experimental samples, an emerging concept that points to mixed macrophage activation states in pathology.

Identification and characterization of a novel intronic splicing mutation in CSF1R-related leukoencephalopathy.

AIMS: Colony stimulating factor 1 receptor (CSF1R)-related leukoencephalopathy is a rapidly progressing neurodegenerative disease caused by CSF1R gene mutations. This study aimed to identify and investigate the effect of a novel intronic mutation (c.1754-3C>G) of CSF1R on splicing. METHODS: A novel intronic mutation was identified using whole-exome sequencing. To investigate the impact of this mutation, we employed various bioinformatics tools to analyze the transcription of the CSF1R gene and the three-dimensional structure of its encoded protein. Furthermore, reverse transcription polymerase chain reaction (RT-PCR) was performed to validate the findings. RESULTS: A novel mutation (c.1754-3C>G) in CSF1R was identified, which results in exon 13 skipping due to the disruption of the 3' splice site consensus sequence NYAG/G. This exon skipping event was further validated in the peripheral blood of the mutation carrier through RT-PCR and Sanger sequencing. Protein structure prediction indicated a disruption in the tyrosine kinase domain, with the truncated protein showing significant structural alterations. CONCLUSIONS: Our findings underscore the importance of intronic mis-splicing mutations in the diagnosis and management of CSF1R-related leukoencephalopathy.

Allogenic microglia replacement: A novel therapeutic strategy for neurological disorders.

Microglia are resident immune cells in the central nervous system (CNS) that play vital roles in CNS development, homeostasis and disease pathogenesis. Genetic defects in microglia lead to microglial dysfunction, which in turn leads to neurological disorders. The correction of the specific genetic defects in microglia in these disorders can lead to therapeutic effects. Traditional genetic defect correction approaches are dependent on viral vector-based genetic defect corrections. However, the viruses used in these approaches, including adeno-associated viruses, lentiviruses and retroviruses, do not primarily target microglia; therefore, viral vector-based genetic defect corrections are ineffective in microglia. Microglia replacement is a novel approach to correct microglial genetic defects via replacing microglia of genetic defects with allogenic healthy microglia. In this paper, we systematically review the history, rationale and therapeutic perspectives of microglia replacement, which would be a novel strategy for treating CNS disorders.

Assessing Chitinases and Neurofilament Light Chain as Biomarkers for Adult-Onset Leukodystrophies.

Leukodystrophies represent a large and complex group of inherited disorders affecting the white matter of the central nervous system. Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) is a rare leukodystrophy which still needs the proper identification of diagnostic, prognostic, and monitoring biomarkers. The aim of this study was to determine the diagnostic and prognostic value of chitinases and neurofilament light chain as biomarkers for ALSP. A cross-sectional study was performed to analyze cerebrospinal fluid levels of chitinases (chitotriosidase and chitinase 3-like 2) and neurofilament light chain in five different groups: (i) normal health individuals; (ii) patients with definitive diagnosis of ALSP and genetic confirmation; (iii) asymptomatic patients with CSF1R variants; (iv) patients with other adult-onset leukodystrophies; and (v) patients with amyotrophic lateral sclerosis (external control group). Chitinase levels showed a statistical correlation with clinical assessment parameters in ALSP patients. Chitinase levels were also distinct between ALSP and the other leukodystrophies. Significant differences were noted in the levels of chitinases and neurofilament light chain comparing symptomatic (ALSP) and asymptomatic individuals with CSF1R variants. This study is the first to establish chitinases as a potential biomarker for ALSP and confirms neurofilament light chain as a good biomarker for primary microgliopathies.

Chlorin e6 and BLZ945 Based Self-Assembly for Photodynamic Immunotherapy Through Immunogenic Tumor Induction and Tumor-Associated Macrophage Depletion.

Immunotherapeutic effect is restricted by the nonimmunogenic tumor phenotype and immunosuppression behaviors of tumor-associated macrophages (TAMs). In this work, a drug self-assembly (designated as CeBLZ) is fabricated based on chlorin e6 (Ce6) and BLZ945 to activate photodynamic immunotherapy through tumor immunogenic induction and tumor-associated macrophage depletion. It is found that Ce6 tends to assemble with BLZ945 without any drug excipients, which can enhance the cellular uptake, tumor penetration, and blood circulation behaviors. The robust photodynamic therapy effect of CeBLZ efficiently suppresses the primary tumor growth and also triggers immunogenic cell death to reverse the nonimmunogenic tumor phenotype. Moreover, CeBLZ can deplete TAMs in tumor tissues to reverse the immunosuppression microenvironment, activating abscopal effect for distant tumor inhibition. In vitro and in vivo results confirm the superior antitumor effect of CeBLZ with negligible side effect, which might promote the development of sophisticated drug combinations for systematic tumor management.

An adapted protocol to derive microglia from stem cells and its application in the study of CSF1R-related disorders.

BACKGROUND: Induced pluripotent stem cell-derived microglia (iMGL) represent an excellent tool in studying microglial function in health and disease. Yet, since differentiation and survival of iMGL are highly reliant on colony-stimulating factor 1 receptor (CSF1R) signaling, it is difficult to use iMGL to study microglial dysfunction associated with pathogenic defects in CSF1R. METHODS: Serial modifications to an existing iMGL protocol were made, including but not limited to changes in growth factor combination to drive microglial differentiation, until successful derivation of microglia-like cells from an adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) patient carrying a c.2350G > A (p.V784M) CSF1R variant. Using healthy control lines, the quality of the new iMGL protocol was validated through cell yield assessment, measurement of microglia marker expression, transcriptomic comparison to primary microglia, and evaluation of inflammatory and phagocytic activities. Similarly, molecular and functional characterization of the ALSP patient-derived iMGL was carried out in comparison to healthy control iMGL. RESULTS: The newly devised protocol allowed the generation of iMGL with enhanced transcriptomic similarity to cultured primary human microglia and with higher scavenging and inflammatory competence at ~ threefold greater yield compared to the original protocol. Using this protocol, decreased CSF1R autophosphorylation and cell surface expression was observed in iMGL derived from the ALSP patient compared to those derived from healthy controls. Additionally, ALSP patient-derived iMGL presented a migratory defect accompanying a temporal reduction in purinergic receptor P2Y12 (P2RY12) expression, a heightened capacity to internalize myelin, as well as heightened inflammatory response to Pam3CSK4. Poor P2RY12 expression was confirmed to be a consequence of CSF1R haploinsufficiency, as this feature was also observed following CSF1R knockdown or inhibition in mature control iMGL, and in CSF1RWT/KO and CSF1RWT/E633K iMGL compared to their respective isogenic controls. CONCLUSIONS: We optimized a pre-existing iMGL protocol, generating a powerful tool to study microglial involvement in human neurological diseases. Using the optimized protocol, we have generated for the first time iMGL from an ALSP patient carrying a pathogenic CSF1R variant, with preliminary characterization pointing toward functional alterations in migratory, phagocytic and inflammatory activities.

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.

The plasma-soluble CSF1R level is a promising prognostic indicator for pediatric Langerhans cell histiocytosis.

Langerhans cell histiocytosis (LCH) is a rare hematologic neoplasm characterized by the clonal proliferation of Langerhans-like cells. Colony-stimulating factor 1 receptor (CSF1R) is a membrane-bound receptor that is highly expressed in LCH cells and tumor-associated macrophages. In this study, a soluble form of CSF1R protein (sCSF1R) was identified by plasma proteome profiling, and its role in evaluating LCH prognosis was explored. We prospectively measured plasma sCSF1R levels in 104 LCH patients and 10 healthy children using ELISA. Plasma sCSF1R levels were greater in LCH patients than in healthy controls (p < .001) and significantly differed among the three disease extents, with the highest level in MS RO+ LCH patients (p < .001). Accordingly, immunofluorescence showed the highest level of membrane-bound CSF1R in MS RO+ patients. Furthermore, the plasma sCSF1R concentration at diagnosis could efficiently predict the prognosis of LCH patients treated with standard first-line treatment (AUC = 0.782, p < .001). Notably, dynamic monitoring of sCSF1R levels could predict relapse early in patients receiving BRAF inhibitor treatment. In vitro drug sensitivity data showed that sCSF1R increased resistance to Ara-C in THP-1 cells expressing ectopic BRAF-V600E. Overall, the plasma sCSF1R level at diagnosis and during follow-up is of great clinical importance in pediatric LCH patients.

Stigmasterol alleviates neuropathic pain by reducing Schwann cell-macrophage cascade in DRG by modulating IL-34/CSF1R.

AIMS: This study aimed to investigate the potential therapeutic applications of stigmasterol for treating neuropathic pain. METHODS: Related mechanisms were investigated by DRG single-cell sequencing analysis and the use of specific inhibitors in cellular experiments. In animal experiments, 32 male Sprague-Dawley rats were randomly divided into the sham operation group, CCI group, ibuprofen group, and stigmasterol group. We performed behavioral tests, ELISA, H&E staining and immunohistochemistry, and western blotting. RESULTS: Cell communication analysis by single-cell sequencing reveals that after peripheral nerve injury, Schwann cells secrete IL-34 to act on CSF1R in macrophages. After peripheral nerve injury, the mRNA expression levels of CSF1R pathway and NLRP3 inflammasome in macrophages were increased in DRG. In vitro studies demonstrated that stigmasterol can reduce the secretion of IL-34 in LPS-induced RSC96 Schwann cells; stigmasterol treatment of LPS-induced Schwann cell-conditioned medium (L-S-CM) does not induce the proliferation and migration of RAW264.7 macrophages; L-S-CM reduces CSF1R signaling pathway (CSF1R, P38MAPK, and NFκB) activation, NLRP3 inflammasome activation, and ROS production. In vivo experiments have verified that stigmasterol can reduce thermal and cold hyperalgesia in rat chronic compressive nerve injury (CCI) model; stigmasterol can reduce IL-1ß, IL-6, TNF-α, CCL2, SP, and PGE2 in serum of CCI rats; immunohistochemistry and western blot confirmed that stigmasterol can reduce the levels of IL-34/CSF1R signaling pathway and NLRP3 inflammasome in DRG of CCI rats. CONCLUSION: Stigmasterol alleviates neuropathic pain by reducing Schwann cell-macrophage cascade in DRG by modulating IL-34/CSF1R axis.

Deplete and repeat: microglial CSF1R inhibition and traumatic brain injury.

Traumatic brain injury (TBI) is a public health burden affecting millions of people. Sustained neuroinflammation after TBI is often associated with poor outcome. As a result, increased attention has been placed on the role of immune cells in post-injury recovery. Microglia are highly dynamic after TBI and play a key role in the post-injury neuroinflammatory response. Therefore, microglia represent a malleable post-injury target that could substantially influence long-term outcome after TBI. This review highlights the cell specific role of microglia in TBI pathophysiology. Microglia have been manipulated via genetic deletion, drug inhibition, and pharmacological depletion in various pre-clinical TBI models. Notably, colony stimulating factor 1 (CSF1) and its receptor (CSF1R) have gained much traction in recent years as a pharmacological target on microglia. CSF1R is a transmembrane tyrosine kinase receptor that is essential for microglia proliferation, differentiation, and survival. Small molecule inhibitors targeting CSF1R result in a swift and effective depletion of microglia in rodents. Moreover, discontinuation of the inhibitors is sufficient for microglia repopulation. Attention is placed on summarizing studies that incorporate CSF1R inhibition of microglia. Indeed, microglia depletion affects multiple aspects of TBI pathophysiology, including neuroinflammation, oxidative stress, and functional recovery with measurable influence on astrocytes, peripheral immune cells, and neurons. Taken together, the data highlight an important role for microglia in sustaining neuroinflammation and increasing risk of oxidative stress, which lends to neuronal damage and behavioral deficits chronically after TBI. Ultimately, the insights gained from CSF1R depletion of microglia are critical for understanding the temporospatial role that microglia develop in mediating TBI pathophysiology and recovery.

Clinical presentation and diagnosis of adult-onset leukoencephalopathy with axonal spheroids and pigmented glia: a literature analysis of case studies.

Introduction: Because adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) is a rare, rapidly progressive, debilitating, and ultimately fatal neurodegenerative disease, a rapid and accurate diagnosis is critical. This analysis examined the frequency of initial misdiagnosis of ALSP via comprehensive review of peer-reviewed published cases. Methods: Data were extracted from a MEDLINE search via PubMed (January 1, 1980, through March 22, 2022) from eligible published case reports/series for patients with an ALSP diagnosis that had been confirmed by testing for the colony-stimulating factor-1 receptor gene (CSF1R) mutation. Patient demographics, clinical symptoms, brain imaging, and initial diagnosis data were summarized descriptively. Categorical data for patient demographics, symptoms, and brain imaging were stratified by initial diagnosis category to test for differences in initial diagnosis based on each variable. Results: Data were extracted from a cohort of 291 patients with ALSP from 93 published case reports and case series. Mean (standard deviation) age of symptom onset was 43.2 (11.6) years. A family history of ALSP was observed in 59.1% of patients. Cognitive impairment (47.1%) and behavioral and psychiatric abnormalities (26.8%) were the most frequently reported initial symptoms. Of 291 total cases, an accurate initial diagnosis of ALSP was made in 72 cases (24.7%) and the most frequent initial misdiagnosis categories were frontotemporal dementia (28 [9.6%]) and multiple sclerosis (21 [7.2%]). Of the 219 cases (75.3%) that were initially mis- or undiagnosed, 206 cases (94.1%) were later confirmed as ALSP by immunohistology, imaging, and/or genetic testing; for the remaining 13 cases, no final diagnosis was reported. Initial diagnosis category varied based on age, family history, geographic region, mode of inheritance, and presenting symptoms of pyramidal or extrapyramidal motor dysfunction, behavioral and psychiatric abnormalities, cognitive impairment, and speech difficulty. Brain imaging abnormalities were common, and initial diagnosis category was significantly associated with white matter hyperintensities, white matter calcifications, and ventricular enlargement. Discussion: In this literature analysis, ALSP was frequently misdiagnosed. Improving awareness of this condition and distinguishing it from other conditions with overlapping presenting symptoms is important for timely management of a rapidly progressive disease such as ALSP.

Relative contributions of osteal macrophages and osteoclasts to postnatal bone development in CSF1R-deficient rats and phenotype rescue following wild-type bone marrow cell transfer.

Macrophage and osteoclast proliferation, differentiation and survival are regulated by colony-stimulating factor-1 receptor (CSF1R) signaling. Osteopetrosis associated with Csf1 and Csf1r mutations has been attributed to the loss of osteoclasts and deficiency in bone resorption. Here we demonstrate that homozygous Csf1r mutation in rat leads to delayed postnatal skeletal ossification associated with substantial loss of osteal macrophages (osteomacs) in addition to osteoclasts. Osteosclerosis and site-specific skeletal abnormalities were reversed by intraperitoneal transfer of wild-type bone marrow cells (BMT) at weaning. Following BMT, IBA1+ macrophages were detected before TRAP+ osteoclasts at sites of ossification restoration. These observations extend evidence that osteomacs independently contribute to bone anabolism and are required for normal postnatal bone growth and morphogenesis.

Identification of Key Molecular Pathways and Associated Genes as Targets to Overcome Radiotherapy Resistance Using a Combination of Radiotherapy and Immunotherapy in Glioma Patients.

Recent mechanistic studies have indicated that combinations of radiotherapy (RT) plus immunotherapy (via CSF-1R inhibition) can serve as a strategy to overcome RT resistance and improve the survival of glioma mice. Given the high mortality rate for glioma, including low-grade glioma (LGG) patients, it is of critical importance to investigate the mechanism of the combination of RT and immunotherapy and further translate the mechanism from mouse studies to improve survival of RT-treated human glioma patients. Using the RNA-seq data from a glioma mouse study, 874 differentially expressed genes (DEGs) between the group of RT-treated mice at glioma recurrence and the group of mice with combination treatment (RT plus CSF-1R inhibition) were translated to the human genome to identify significant molecular pathways using the KEGG enrichment analysis. The enrichment analysis yields statistically significant signaling pathways, including the phosphoinositide 3-kinase (PI3K)/AKT pathway, Hippo pathway, and Notch pathway. Within each pathway, a candidate gene set was selected by Cox regression models as genetic biomarkers for resistance to RT and response to the combination of RT plus immunotherapies. Each Cox model is trained using a cohort of 295 RT-treated LGG patients from The Cancer Genome Atlas (TCGA) database and validated using a cohort of 127 RT-treated LGG patients from the Chinese Glioma Genome Atlas (CGGA) database. A four-DEG signature (ITGB8, COL9A3, TGFB2, JAG1) was identified from the significant genes within the three pathways and yielded the area under time-dependent ROC curve AUC = 0.86 for 5-year survival in the validation set, which indicates that the selected DEGs have strong prognostic value and are potential intervention targets for combination therapies. These findings may facilitate future trial designs for developing combination therapies for glioma patients.

Antiandrogen Flutamide-Induced Restoration of miR-449 Expression Mitigates Functional Biomarkers Associated with Ovarian Cancer Risk.

Background: The involvement of the androgen and androgen receptor (AR) pathway in the development of epithelial ovarian cancer is increasingly recognized. However, the specific mechanisms by which anti-androgen agents, such as flutamide, may prevent ovarian cancer and their efficacy remain unknown. We examined the effects of flutamide on the miRNA expression profile found in women at high risk (HR) for ovarian cancer. Methods: Ovarian and tubal tissues, free from ovarian, tubal, peritoneal cancers, and serous tubal intraepithelial carcinoma (STIC), were collected from untreated and flutamide-treated HR women. Low-risk (LR) women served as controls. Transcriptomic miRNA sequencing was performed on these 3 sample cohorts. The miRNAs that showed the most notable differential expression were subjected to functional assays in primary ovarian epithelial cells and ovarian cancer cells. Results: Flutamide treatment demonstrated a normalization effect on diminished miRNA levels in HR tissues compared to LR tissues. Particularly, the miR-449 family was significantly upregulated in HR ovarian tissues following flutamide treatment, reaching levels comparable to those in LR tissues. MiR-449a and miR-449b-5p, members of the miR-449 family, were computationally predicted to target the mRNAs of AR and colony-stimulating factor 1 receptor (CSF1R, also known as c-fms), both of which are known contributors to ovarian cancer progression, with emerging evidence also supporting their roles in ovarian cancer initiation. These findings were experimentally validated in primary ovarian epithelial cells and ovarian cancer cell lines (SKOV3 and Hey): flutamide treatment resulted in elevated levels of miR-449a and miR-449b-5p, and introducing mimics of these miRNAs reduced the mRNA and protein levels of CSF1R and AR. Furthermore, introducing miR-449a and miR-449b-5p mimics showed inhibitory effects on the migration and proliferation of ovarian cancer cells. Conclusion: Flutamide treatment restored the reduced expression of miR-449a and miR-449b-5p in HR tissues, thereby decreasing the expression of CSF1R and AR, functional biomarkers associated with an increased risk of ovarian cancer. In addition to the known direct binding of flutamide to the AR, we found that flutamide also suppresses AR expression via miR-449a and miR-449b-5p upregulation, revealing a novel dual-inhibitory mechanism on the AR pathway. Taken together, our study highlights mechanisms supporting the chemopreventive potential of flutamide in ovarian cancer, particularly in HR patients with reduced miR-449 expression.

CSF1R inhibition reprograms tumor-associated macrophages to potentiate anti-PD-1 therapy efficacy against colorectal cancer.

PD-1 blockade therapy has made great breakthroughs in treatment of multiple solid tumors. However, patients with microsatellite-stable (MSS) colorectal cancer (CRC) respond poorly to anti-PD-1 immunotherapy. Although CRC patients with microstatellite instability (MSI) or microsatellite instability-high (MSI-H) can benefit from PD-1 blockade therapy, there are still some problems such as tumor recurrence. Tumor-associated macrophages (TAMs), most abundant immune components in tumor microenvironment (TME), largely limit the therapeutic efficacy of anti-PD-1 against CRC. The CSF1/CSF1R pathway plays a key role in regulating macrophage polarization, and blocking CSF1R signaling transduction may be a potential strategy to effectively reprogram macrophages and remodel TME. Here, we found that increasing expression of CSF1R in macrophages predicted poor prognosis in CRC cohort. Furthermore, we discovered a novel potent CSF1R inhibitor, PXB17, which significantly reprogramed M2 macrophages to M1 phenotype. Mechanically, PXB17 significantly blocked activation of PI3K/AKT/mTORC1 signaling, resulting in inhibition of cholesterol biosynthesis. Results from 3D co-culture system suggested that PXB17-repolarized macrophages could induce infiltration of CD8+ T lymphocytes in tumors and improve the immunosuppressive microenvironment. In vivo, PXB17 significantly halted CRC growth, with a stronger effect than PLX3397. In particular, PXB17 potently enhanced therapeutic activity of PD-1 mAb in CT-26 (MSS) model and prevented tumor recurrence in MC-38 (MSI-H) model by promoting formation of long-term memory immunity. Our study opens a new avenue for CSF1R in tumor innate and adaptive anti-tumor immunomodulatory activity and suggests that PXB17 is a promising immunotherapy molecule for enhancing the efficacy of PD-1 mAb or reducing tumor recurrence of CRC.