Colony-stimulating Factor-1 Receptor Signaling Research Articles

CSF1R antagonism results in increased supraspinal infiltration in EAE.

Colony stimulating factor 1 receptor (CSF1R) signaling is crucial for the maintenance and function of various myeloid subsets. CSF1R antagonism was previously shown to mitigate clinical severity in experimental autoimmune encephalomyelitis (EAE). The associated mechanisms are still not well delineated. To assess the effect of CSF1R signaling, we employed the CSF1R antagonist PLX5622 formulated in chow (PLX5622 diet, PD) and its control chow (control diet, CD). We examined the effect of PD in steady state and EAE by analyzing cells isolated from peripheral immune organs and from the CNS via flow cytometry. We determined CNS infiltration sites and assessed the extent of demyelination using immunohistochemistry of cerebella and spinal cords. Transcripts of genes associated with neuroinflammation were also analyzed in these tissues. In addition to microglial depletion, PD treatment reduced dendritic cells and macrophages in peripheral immune organs, both during steady state and during EAE. Furthermore, CSF1R antagonism modulated numbers and relative frequencies of T effector cells both in the periphery and in the CNS during the early stages of the disease. Classical neurological symptoms were milder in PD compared to CD mice. Interestingly, a subset of PD mice developed atypical EAE symptoms. Unlike previous studies, we observed that the CNS of PD mice was infiltrated by increased numbers of peripheral immune cells compared to that of CD mice. Immunohistochemical analysis showed that CNS infiltrates in PD mice were mainly localized in the cerebellum while in CD mice infiltrates were primarily localized in the spinal cords during the onset of neurological deficits. Accordingly, during the same timepoint, cerebella of PD but not of CD mice had extensive demyelinating lesions, while spinal cords of CD but not of PD mice were heavily demyelinated. Our findings suggest that CSF1R activity modulates the cellular composition of immune cells both in the periphery and within the CNS, and affects lesion localization during the early EAE stages.

Differentiated mesenchymal stem cells-derived exosomes immobilized in decellularized sciatic nerve hydrogels for peripheral nerve repair

Peripheral nerve injury (PNI) and the limitations of current treatments often result in incomplete sensory and motor function recovery, which significantly impact the patient's quality of life. While exosomes (Exo) derived from stem cells and Schwann cells have shown promise on promoting PNI repair following systemic administration or intraneural injection, achieving effective local and sustained Exo delivery holds promise to treat local PNI and remains challenging. In this study, we developed Exo-loaded decellularized porcine nerve hydrogels (DNH) for PNI repair. We successfully isolated Exo from differentiated human adipose-derived mesenchymal stem cells (hADMSC) with a Schwann cell-like phenotype (denoted as dExo). These dExo were further combined with polyethylenimine (PEI), and DNH to create polyplex hydrogels (dExo-loaded pDNH). At a PEI content of 0.1%, pDNH showed cytocompatibility for hADMSCs and supported neurite outgrowth of dorsal root ganglions. The sustained release of dExos from dExo-loaded pDNH persisted for at least 21 days both in vitro and in vivo. When applied around injured nerves in a mouse sciatic nerve crush injury model, the dExo-loaded pDNH group significantly improved sensory and motor function recovery and enhanced remyelination compared to dExo and pDNH only groups, highlighting the synergistic regenerative effects. Interestingly, we observed a negative correlation between the number of colony-stimulating factor-1 receptor (CSF-1R) positive cells and the extent of PNI regeneration at the 21-day post-surgery stage. Subsequent in vitro experiments demonstrated the potential involvement of the CSF-1/CSF-1R axis in Schwann cells and macrophage interaction, with dExo effectively downregulating CSF-1/CSF-1R signaling.

Tumor-Microenvironment-Activatable Nanoparticle Mediating Immunogene Therapy and M2 Macrophage-Targeted Inhibitor for Synergistic Cancer Immunotherapy.

Immunotherapy has achieved prominent clinical efficacy in combating cancer and has recently become a mainstream treatment strategy. However, achieving broad efficacy with a single modality is challenging, and the heterogeneity of the tumor microenvironment (TME) restricts the accuracy and effectiveness of immunotherapy strategies for tumors. Herein, a TME-responsive targeted nanoparticle to enhance antitumor immunity and reverse immune escape by codelivering interleukin-12 (IL-12) expressing gene and colony-stimulating factor-1 receptor (CSF-1R) inhibitor PLX3397 (PLX) is presented. The introduction of disulfide bonds and cyclo(Arg-Gly-Asp-d-Phe-Lys) (cRGD) peptides conferred reduction reactivity and tumor targeting to the nanoparticles, respectively. It is hypothesized that activating host immunity by the local expression of IL-12, while modulating the tumor-associated macrophages (TAM) function through blocking CSF-1/CSF-1R signaling, could constitute a feasible approach for cancer immunotherapy. The fabricated functional nanoparticle successfully ameliorated the TME by stimulating the proliferation and activation of T lymphocytes, promoting the repolarization of TAMs, reducing myeloid-derived suppressor cells (MDSCs), and promoting the maturation of dendritic cells (DC) as well as the secretion of antitumor cytokines, which efficiently suppressed tumor growth and metastasis. Finally, substantial changes in the TME were deciphered by single-cell analysis including infiltration of different cells, transcriptional states, secretory signaling and cell-cell communications. These findings provide a promising combinatorial immunotherapy strategy through immunomodulatory nanoparticles.

CSF1R inhibition promotes neuroinflammation and behavioral deficits during graft-versus-host disease in mice

AbstractChronic graft-versus-host disease (cGVHD) remains a significant complication of allogeneic hematopoietic stem cell transplantation. Central nervous system (CNS) involvement is becoming increasingly recognized, in which brain-infiltrating donor major histocompatibility complex (MHC) class II+ bone marrow–derived macrophages (BMDM) drive pathology. BMDM are also mediators of cutaneous and pulmonary cGVHD, and clinical trials assessing the efficacy of antibody blockade of colony-stimulating factor 1 receptor (CSF1R) to deplete macrophages are promising. We hypothesized that CSF1R antibody blockade may also be a useful strategy to prevent/treat CNS cGVHD. Increased blood-brain barrier permeability during acute GVHD (aGVHD) facilitated CNS antibody access and microglia depletion by anti-CSF1R treatment. However, CSF1R blockade early after transplant unexpectedly exacerbated aGVHD neuroinflammation. In established cGVHD, vascular changes and anti-CSF1R efficacy were more limited. Anti-CSF1R–treated mice retained donor BMDM, activated microglia, CD8+ and CD4+ T cells, and local cytokine expression in the brain. These findings were recapitulated in GVHD recipients, in which CSF1R was conditionally depleted in donor CX3CR1+ BMDM. Notably, inhibition of CSF1R signaling after transplant failed to reverse GVHD-induced behavioral changes. Moreover, we observed aberrant behavior in non-GVHD control recipients administered anti-CSF1R blocking antibody and naïve mice lacking CSF1R in CX3CR1+ cells, revealing a novel role for homeostatic microglia and indicating that ongoing clinical trials of CSF1R inhibition should assess neurological adverse events in patients. In contrast, transfer of Ifngr–/– grafts could reduce MHC class II+ BMDM infiltration, resulting in improved neurocognitive function. Our findings highlight unexpected neurological immune toxicity during CSF1R blockade and provide alternative targets for the treatment of cGVHD within the CNS.

CSF1R Inhibition Promotes Neuroinflammation and Behavioural Deficits during Graft-Versus-Host Disease in Mice

Chronic graft-versus-host disease (cGVHD) remains a significant complication of allogeneic hematopoietic stem cell transplantation. Central nervous system (CNS) involvement is becoming increasingly recognised, where brain-infiltrating donor MHC class II + bone marrow-derived macrophages (BMDM) are implicated mediators of pathology. Colony-stimulating factor 1 receptor (CSF1R)-dependent BMDM are established mediators of cutaneous and pulmonary cGVHD, and clinical trials assessing the efficacy of CSF1R antibody blockade to deplete macrophages are promising. We hypothesized that CSF1R antibody blockade may also be a useful strategy to prevent/treat CNS cGVHD. We leveraged studies in acute GVHD (aGVHD) to determine the feasibility of an antibody-based approach for depleting brain microglia and macrophages. Moreover, we queried whether CSF1R blockade may be a viable therapy for CNS aGVHD, which has been shown to be mediated by host microglia. Increased blood-brain barrier permeability during aGVHD facilitated CNS antibody access. However, CSF1R blockade early post-transplant induced region-specific microglia depletion and was associated with unexpected exacerbation of aGVHD neuroinflammation, including increased T cell and inflammatory monocyte infiltration, and whole brain cytokine levels. In established cGVHD, vascular changes and anti-CSF1R efficacy was more limited. Anti-CSF1R-treated mice retained donor BMDM, activated microglia, CD8 + and CD4 + T cells, and local cytokine expression in the brain. These findings were recapitulated in GVHD recipients where CSF1R was conditionally depleted in donor CX3CR1 + BMDM. Notably, inhibition of CSF1R signalling post-transplant failed to reverse either acute or chronic GVHD-induced behavioral changes. Taken together, these data suggest that ongoing clinical trials of CSF1R inhibition should assess neurological adverse events in patients. In contrast to the lack of efficacy with CSF1R inhibition, transfer of Ifngr -/- grafts reduced MHC class II + BMDM infiltration, resulting in improved behaviour. Furthermore, single nuclei RNA sequencing of brains from mice receiving wild-type or Ifngr -/- allografts revealed a disease-associated interferon-inducible mature oligodendrocyte population in the cGVHD brain. This was markedly reduced in recipients of Ifngr -/- grafts, indicating that targeting this axis can promote brain repair beyond the myeloid compartment during cGVHD. In summary, our findings reveal unexpected neurological immune toxicity during CSF1R blockade and highlight the IFNGR pathway as an alternative target for the treatment of cGVHD within the CNS.

Axatilimab Ameliorates Inflammation and Fibrosis By Targeting Macrophages in a Preclinical Model of Chronic GVHD

Introduction: Chronic graft-versus-host disease (cGVHD) is an immune-mediated serious and life-threatening complication after allogeneic hematopoietic stem cell transplantation, occurring in 30%-70% of patients. The pathophysiology of cGVHD may involve inflammation, cell-mediated immunity, humoral immunity, and fibrosis. Axatilimab is a high-affinity humanized IgG4 (kappa light chain) monoclonal antibody targeting colony-stimulating factor 1 receptor (CSF-1R) that is under investigation for the treatment of cGVHD. CSF-1R is a member of the receptor protein tyrosine kinase family of growth factor receptors and is expressed on monocyte precursors, monocytes, and macrophages. Signaling by colony-stimulating factor 1 (CSF-1) or interleukin-34 (IL-34) through CSF-1R has been shown to be the primary regulatory pathway for determining development and differentiation of the mononuclear phagocytic cell lineage. Recent work has established a role for monocyte-derived macrophages as the pathogenic population of cells that establish the fibrotic niche and drive the fibrotic process. Upon entry of monocytes into tissues, the tissue microenvironment regulates their differentiation, resulting in a heterogeneous pool of cells ranging from pro-inflammatory (often referred to as M1 macrophages) to immune suppressive/wound healing (M2 macrophages). An imbalance in the number and activity of these macrophage populations is believed to result in the excess production of fibrotic growth factors, such as transforming growth factor-β and platelet-derived growth factor, leading to uncontrolled fibroblast activation and subsequent fibrosis. Blocking CSF-1R signaling with axatilimab is expected to reduce the circulating levels of pathogenic monocyte-derived macrophage precursors and inhibit their activation in tissues, thereby providing an opportunity to therapeutically intervene in diseases such as cGVHD. The results described herein support this assertion by demonstrating the direct impact of axatilimab exposure to skewing differentiating monocytes away from a pro-inflammatory and pro-fibrotic phenotype. Methods: Primary human monocytes were differentiated in the presence or absence of ascending doses of axatilimab (0.07-7 μM) into distinct phenotypes: activated (M0), pro-inflammatory (M1-like), and suppressive (M2-like) macrophages. Supernatants and cell pellets were then assessed for secreted protein (Luminex Corporation) and gene expression (NanoString Technologies) profiles, respectively. Data were analyzed in R (version 4.1.1). Results: Gene expression profiles of pro-inflammatory cytokines and chemokines (eg, IL-1β, chemokine CC motif ligand 2 [CCL2], CCL7, CCL24, and CCL22) from primary differentiated macrophage subsets were dramatically reduced in the presence of ascending concentrations of axatilimab. Further, macrophages exposed to axatilimab while differentiated under pro-inflammatory conditions showed increased levels of c-MYC expression compared with control cultures. c-MYC is a transcription factor associated with suppressive M2-like macrophages, which suggests that axatilimab interferes with pro-inflammatory macrophage differentiation. Consistent with this, expression of M2-associated cytokines, including IL-10, IL-5, and IL-4, was enhanced in axatilimab-treated macrophage cultures. Conclusions: Blocking CSF-1R signaling in primary, differentiated macrophages dramatically alters the secretomes and gene expression profiles of these cells. Macrophages are key inducers of fibroblast growth factors, which in turn promote fibrotic disease. These data confirm that production of inflammatory and pro-fibrotic factors by pro-inflammatory macrophages is inhibited in the presence of axatilimab, thereby supporting the notion that axatilimab provides a unique opportunity to therapeutically intervene or prevent fibrosis in cGVHD.

Dynamic changes in microglia in the mouse hippocampus during administration and withdrawal of the CSF1R inhibitor PLX3397.

Pexidartinib (PLX3397), a colony-stimulating factor-1 receptor (CSF1R) inhibitor, is currently in phase 1-3 clinical trials as a treatment for a variety of tumours. CSF1R signalling regulates the development, survival and maintenance of microglia, the resident brain innate immune cells. In this study, we examined the effects of PLX3397 in the drinking water of mice on microglia in the hippocampus using ionized calcium-binding adapter molecule 1 (Iba1, a microglial marker) immunocytochemistry. A high concentration of PLX3397 (1 mg/mL) significantly decreased the density of Iba1-immunoreactive cells after 7 days of exposure, but a low concentration of PLX3397 (0.5 mg/mL) did not. In addition, both low and high concentrations of PLX3397 significantly increased the intersection number, total length and maximum length of microglial processes in male mice. PLX3397 administered for 21 days eliminated microglia with 78% efficiency in males and 84% efficiency in females. Significant increases in microglial processes were found after both seven and 21 days of PLX3397 exposure in males, whereas decreases in microglial processes were observed after both 14 and 21 days of exposure in females. After PLX3397 withdrawal following its administration for 14 days in males, the soma size quickly returned to normal levels within a week. However, the microglial density, intersection number and total length of microglial processes after 3 days of recovery stabilized to untreated levels. In summary, these findings provide detailed insight into the dynamic changes in microglial number and morphology in the hippocampus in a dose- and time-dependent manner after PLX3397 treatment and withdrawal.

Effects of regorafenib on the mononuclear/phagocyte system and how these contribute to the inhibition of colorectal tumors in mice

BackgroundRegorafenib was previously shown to reduce tumor-associated macrophages and potently inhibit colony-stimulating factor 1 receptor (CSF1R), also known as CD115, in biochemical assays. The CSF1R signaling pathway is essential in the biology of the mononuclear/phagocyte system, which can promote the development of cancer.MethodsA deeper investigation of regorafenib’s effects on CSF1R signaling was performed using preclinical in vitro and in vivo studies with syngeneic CT26 and MC38 mouse models of colorectal cancer. Peripheral blood and tumor tissue were analyzed mechanistically by flow cytometry using antibodies against CD115/CSF1R and F4/80 and by ELISA for chemokine (C–C motif) ligand 2 (CCL2) levels. These read-outs were correlated with drug levels for the detection of pharmacokinetic/pharmacodynamic relationships.ResultsPotent inhibition of CSF1R by regorafenib and its metabolites M-2, M-4, and M-5 was confirmed in vitro in RAW264.7 macrophages. The dose-dependent growth inhibition of subcutaneous CT26 tumors by regorafenib was associated with a significant reduction in both the number of CD115hi monocytes in peripheral blood and the number of selective subpopulations of intratumoral F4/80hi tumor-associated macrophages. CCL2 levels were not affected by regorafenib in blood but increased in tumor tissue, which may contribute to drug resistance and prevent complete tumor remission. An inverse relationship between regorafenib concentration and the number of CD115hi monocytes and CCL2 levels was observed in peripheral blood, supporting the mechanistic involvement of regorafenib.ConclusionsThese findings may be clinically useful in optimizing drug dosing using blood-based pharmacodynamic markers and in identifying resistance mechanisms and ways to overcome them by appropriate drug combinations.

Putting PLX5622 into perspective: microglia in central nervous system viral infection.

Putting PLX5622 into perspective: microglia in central nervous system viral infection.

Microglia depletion as a therapeutic strategy: friend or foe in multiple sclerosis models?

Multiple sclerosis is a chronic central nervous system demyelinating disease whose onset and progression are driven by a combination of immune dysregulation, genetic predisposition, and environmental factors. The activation of microglia and astrocytes is a key player in multiple sclerosis immunopathology, playing specific roles associated with anatomical location and phase of the disease and controlling demyelination and neurodegeneration. Even though reactive microglia can damage tissue and heighten deleterious effects and neurodegeneration, activated microglia also perform neuroprotective functions such as debris phagocytosis and growth factor secretion. Astrocytes can be activated into pro-inflammatory phenotype A1 through a mechanism mediated by activated neuroinflammatory microglia, which could also mediate neurodegeneration. This A1 phenotype inhibits oligodendrocyte proliferation and differentiation and is toxic to both oligodendrocytes and neurons. However, astroglial activation into phenotype A2 may also take place in response to neurodegeneration and as a protective mechanism. A variety of animal models mimicking specific multiple sclerosis features and the associated pathophysiological processes have helped establish the cascades of events that lead to the initiation, progression, and resolution of the disease. The colony-stimulating factor-1 receptor is expressed by myeloid lineage cells such as peripheral monocytes and macrophages and central nervous system microglia. Importantly, as microglia development and survival critically rely on colony-stimulating factor-1 receptor signaling, colony-stimulating factor-1 receptor inhibition can almost completely eliminate microglia from the brain. In this context, the present review discusses the impact of microglial depletion through colony-stimulating factor-1 receptor inhibition on demyelination, neurodegeneration, astroglial activation, and behavior in different multiple sclerosis models, highlighting the diversity of microglial effects on the progression of demyelinating diseases and the strengths and weaknesses of microglial modulation in therapy design.

Haloperidol Instigates Endometrial Carcinogenesis and Cancer Progression by the NF-κB/CSF-1 Signaling Cascade.

Simple SummaryHaloperidol, a typical antipsychotic, is widely used in schizophrenia and palliative care of cancer; however, the role and impact of chronic haloperidol treatment in endometrial cancer (EC) development are unclear. Here, we showed that haloperidol is a carcinogenic compound capable of inducing endometrial hyperplasia and promoting EC progression in rodents. Mechanistically, haloperidol stimulates the production of colony-stimulating factor 1 (CSF-1) on tumor cells by activating nuclear factor kappa B (NF-κB), and its downstream autocrine oncogenic CSF-1 receptor signaling contributes to this carcinogenesis. Furthermore, we demonstrated that the use of haloperidol is associated with increased EC-specific mortality in EC patients. Overall, these findings highlighted that physicians should be cautious about the use of haloperidol in female patients.Haloperidol is a routine drug for schizophrenia and palliative care of cancer; it also has antitumor effects in several types of cancer. However, the role of haloperidol in endometrial cancer (EC) development is still unclear. Here, we show that chronic haloperidol treatment in clinically relevant doses induced endometrial hyperplasia in normal mice and promoted tumor growth and malignancy in mice with orthotopic EC. The pharmacokinetic study indicated that haloperidol highly accumulated in the uterus of mice. In vitro studies revealed that haloperidol stimulated the cellular transformation of human endometrial epithelial cells (HECCs) and promoted the proliferation, migration, and invasion of human endometrial carcinoma cells (HECCs) by activating nuclear factor kappa B (NF-κB) and its downstream signaling target, colony-stimulating factor 1 (CSF-1). Gain of function of CSF-1 promotes the cellular transformation of HEECs and the malignant progression of HECCs. Moreover, blockade of CSF-1 inhibited haloperidol-promoted EC progression in vitro and in vivo. A population-based cohort study of EC patients further demonstrated that the use of haloperidol was associated with increased EC-specific mortality. Collectively, these findings indicate that clinical use of haloperidol could potentially be harmful to female patients with EC.

PD16-04 THE PROTECTIVE EFFECTS OF CSF-1R INHIBITION ON BLADDER OUTLET OBSTRUCTION (BOO) INDUCED REMODELING

You have accessJournal of UrologyCME1 May 2022PD16-04 THE PROTECTIVE EFFECTS OF CSF-1R INHIBITION ON BLADDER OUTLET OBSTRUCTION (BOO) INDUCED REMODELING Wei Wang, Xiaoshuai Gao, Liao Peng, Lede Lin, Kaiwen Xiao, and Liang Zhou Wei WangWei Wang More articles by this author , Xiaoshuai GaoXiaoshuai Gao More articles by this author , Liao PengLiao Peng More articles by this author , Lede LinLede Lin More articles by this author , Kaiwen XiaoKaiwen Xiao More articles by this author , and Liang ZhouLiang Zhou More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002548.04AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Bladder outlet obstruction (BOO) is a common problem that can affect bladder structure and function. Currently, there is no effective treatment options for BOO-induced remodeling. Previous reports have demonstrated that the pathogenesis of BOO is associated with macrophage infiltration and polarization, which is physiologically dependent on colony-stimulating factor 1 receptor (CSF-1R) activation. Here, we utilized a highly selective CSF-1R inhibitor, GW2580, to determine its therapeutic effects on BOO-induced remodeling. METHODS: Sprague-Dawley rats were randomly divided into sham, BOO + vehicle and BOO + GW2580 group. GW2580 or vehicle control was administrated by oral gavage at daily doses of 40 mg/kg for 6 weeks. Bladder samples were harvested for histopathology, immunohistochemistry, immunofluorescence, western blot and flow cytometry analysis. RESULTS: Our results demonstrated that bladder fibrosis was ameliorated by GW250 compared with vehicle group. Furthermore, treatment with GW2580 induced a significant inhibition of macrophage infiltration and M2 macrophage polarization. Pro-fibrotic F4/80+αSMA+ macrophage to myofibroblast transition and CD163+TGF-β1+ cells were also statistically significant decreased in GW2580 group when compared to vehicle group. CONCLUSIONS: In summary, our findings supported that targeting macrophage through inhibiting CSF-1R signaling might be a promising strategy for the treatment of BOO-induced remodeling. Source of Funding: This study was supported by the National Natural Science Foundation of China (Grant No. 81800667) and the Innovation Spark Project of Sichuan University (Grant No. 2018SCUH0061) © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e270 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Wei Wang More articles by this author Xiaoshuai Gao More articles by this author Liao Peng More articles by this author Lede Lin More articles by this author Kaiwen Xiao More articles by this author Liang Zhou More articles by this author Expand All Advertisement PDF DownloadLoading ...

Neuronal apoptosis drives remodeling states of microglia and shifts in survival pathway dependence.

Microglia serve critical remodeling roles that shape the developing nervous system, responding to the changing neural environment with phagocytosis or soluble factor secretion. Recent single-cell sequencing (scRNAseq) studies have revealed the context-dependent diversity in microglial properties and gene expression, but the cues promoting this diversity are not well defined. Here, we ask how interactions with apoptotic neurons shape microglial state, including lysosomal and lipid metabolism gene expression and dependence on Colony-stimulating factor 1 receptor (CSF1R) for survival. Using early postnatal mouse retina, a CNS region undergoing significant developmental remodeling, we performed scRNAseq on microglia from mice that are wild-type, lack neuronal apoptosis (Bax KO), or are treated with CSF1R inhibitor (PLX3397). We find that interactions with apoptotic neurons drive multiple microglial remodeling states, subsets of which are resistant to CSF1R inhibition. We find that TAM receptor Mer and complement receptor 3 are required for clearance of apoptotic neurons, but that Mer does not drive expression of remodeling genes. We show TAM receptor Axl is negligible for phagocytosis or remodeling gene expression but is consequential for microglial survival in the absence of CSF1R signaling. Thus, interactions with apoptotic neurons shift microglia toward distinct remodeling states and through Axl, alter microglial dependence on survival pathway, CSF1R.

Colony-stimulating factor 1 receptor signaling in the central nervous system and the potential of its pharmacological inhibitors to halt the progression of neurological disorders.

Colony Stimulating Factor-1 (CSF-1)/Colony Stimulating Factor-1 Receptor (CSF-1R) signaling axis plays an essential role in the development, maintenance, and proliferation of macrophage lineage cells. Within the central nervous system, CSF-1R signaling primarily maintains microglial homeostasis. Microglia, being the resident macrophage and first responder to any neurological insults, plays critical importance in overall health of the human brain. Aberrant and sustained activation of microglia along with continued proliferation and release of neurotoxic proinflammatory cytokines have been reported in various neurological and neurodegenerative diseases. Therefore, halting the neuroinflammatory pathway via targeting microglial proliferation, which depends on CSF-1R signaling, has emerged as a potential therapeutic target for neurological disorders. However, apart from regulating the microglial function, recently it has been discovered that CSF-1R has much broader role in central nervous system. These findings limit the therapeutic utility of CSF-1R inhibitors but also highlight the need for a complete understanding of CSF-1R function within the central nervous system. Moreover, it has been found that selective inhibitors of CSF-1R may be more efficient in avoiding non-specific targeting and associated side effects. Short-term depletion of microglial population in diseased conditions have also been found to be beneficial; however, the dose and therapeutic window for optimum effects may need to be standardized further.This review summarizes the present understanding of CSF-1R function within the central nervous system. We discuss the CSF-1R signaling in the context of microglia function, crosstalk between microglia and astroglia, and regulation of neuronal cell function. We also discuss a few of the neurological disorders with a focus on the utility of CSF-1R inhibitors as potential therapeutic strategy for halting the progression of neurological diseases.

Decreased CSF1R Signaling and the Accumulation of Reticular Pseudo-Drusen?

Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) is caused by dominant-acting mutations in the gene colony-stimulating factor 1 receptor (CSF1R). It is an ultra-rare leukoencephalopathy that involves demyelination of white matter and early-onset dementia. It has been well validated that mutations in the kinase region of the gene cause decreased signaling of the receptor via its two cognate ligands interleukin-34 (IL-34) and colony-stimulating factor-1 (CSF-1). In this article, we report a thorough analysis of retinal integrity in a 48-year-old genetically diagnosed ALSP patient. We show that although the optic nerve, optic chiasm, and optic tracts are relatively preserved, the patient has visual field deficits likely due to optic radiation and/or cortical atrophy. Intriguingly, we report the appearance of inner retinal vascular leakage and the appearance of reticular pseudo-drusen (RPD)-like deposits. We propose that the early stages of RPD accumulation may be associated with an attenuated CSF-1 receptor signaling axis. [Ophthalmic Surg Lasers Imaging Retina. 2021;52:666-671.].

Macrophage-Targeted Therapy Unlocks Antitumoral Cross-talk between IFNγ-Secreting Lymphocytes and IL12-Producing Dendritic Cells.

Macrophages often abound within tumors, express colony-stimulating factor 1 receptor (CSF1R), and are linked to adverse patient survival. Drugs blocking CSF1R signaling have been used to suppress tumor-promoting macrophage responses; however, their mechanisms of action remain incompletely understood. Here, we assessed the lung tumor immune microenvironment in mice treated with BLZ945, a prototypical small-molecule CSF1R inhibitor, using single-cell RNA sequencing and mechanistic validation approaches. We showed that tumor control was not caused by CSF1R+ cell depletion; instead, CSF1R targeting reshaped the CSF1R+ cell landscape, which unlocked cross-talk between antitumoral CSF1R- cells. These cells included IFNγ-producing natural killer and T cells, and an IL12-producing dendritic cell subset, denoted as DC3, which were all necessary for CSF1R inhibitor-mediated lung tumor control. These data indicate that CSF1R targeting can activate a cardinal cross-talk between cells that are not macrophages and that are essential to mediate the effects of T cell-targeted immunotherapies and promote antitumor immunity.See related Spotlight by Burrello and de Visser, p. 4.

Abstract PO-106: The extrinsic and modulatory effects of CSF-1/CSF-1R signaling in generating an immunosuppressive pancreatic cancer tumor microenvironment and promoting metastasis

Abstract Introduction: The multistep process of the tumor cell invasion-metastasis cascade, which involves the spread of cancer cells from primary tumor sites to colonization into distant organs, is a major barrier to effective therapy. Both intrinsic factors such as genomic instability and epigenetic alterations and extrinsic factors, such as microenvironmental cues, have been implicated in contributing to the metastatic proclivity of cancer cells. The crosstalk between tumor cells and cells within the heterogenous tumor microenvironment (TME) is a critical driver of tumorigenesis. This can nurture tumor cell migration and invasion into the stroma, providing a foundation for eventual metastasis. Using our mouse models of PDAC, we have identified Colony stimulating factor 1 (Csf-1; also known as M-Csf) to be differentially and significantly upregulated. CSF-1 is secreted by tumor cells to recruit and polarize macrophages into M2-like tumor associated macrophage (TAM) phenotype through binding to its cognate tyrosine kinase receptor colony-stimulating factor 1 receptor (CSF-1R). However, the role and mechanisms of CSF-1/CSF-1R pathway in modulating other elements of the PDAC TME that contributes to invasion and metastasis has yet to be investigated. Methods: We use Pdx1-Cre; LSL-KrasG12D/+; LSL-Trp53R172H; Rosa26LSL-YFP (KPCY) mice and 2D/3D cell culture systems. We overexpressed Csf-1 and established a CRISPR system to knockout Csf-1 in our cells using ribonucleoprotein (RNP) Cas9/gRNA complex via nucleofection. These engineered cells are used to model syngeneic primary and metastatic tumor formation. We have established a novel quantitative multiplex immunofluorescence (qmIF) staining approach to characterize changes in the TME upon modulating Csf-1 expression, specifically focusing on macrophages, myeloid-derived suppressor cell (MDSC), T-cell, B-cell, fibroblast and nerve fiber markers. Results: Our data from in vivo studies suggested that Csf-1 overexpression leads to an increase in primary tumor growth and metastasis, while the depletion of Csf-1 reduces metastatic burden. The automated quantification and analysis of our unbiased IF approaches has yielded the following: CSF-1 overexpression leads to increased tumor infiltration of F4/80+CD163+CD206+ M2-polarized macrophages and decreased number of CD3+CD8+ cytotoxic T-cells, generating an immunosuppressive TME. Furthermore, our preliminary data demonstrated that Csf-1 is also upregulated in cancer associated fibroblasts (CAFs), which potentially synergizes with the epithelial compartment to attract immunosuppressive immune cells and promote immune evasion. Finally, the TCGA data reveals that metastatic PDACs have increased Csf-1 expression compared to primary tumors and overexpression of Csf-1 is associated with reduced survival. Conclusion: We have demonstrated a novel role of CSF-1 upregulation in reprogramming the TME in PDAC and fostering increased metastatic capacity. We believe this can be exploited therapeutically. Citation Format: Gizem Efe, Kensuke Suzuki, Jason R. Pitarresi, Anna M. Chiarella, Alina L. Li, Anil K. Rustgi. The extrinsic and modulatory effects of CSF-1/CSF-1R signaling in generating an immunosuppressive pancreatic cancer tumor microenvironment and promoting metastasis [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-106.

Atorvastatin-Induced Absorption of Chronic Subdural Hematoma Is Partially Attributed to the Polarization of Macrophages.

As one of the main types of secondary craniocerebral injury, the onset, progression, and prognosis of chronic subdural hematoma (CSDH) are closely related to the local inflammation of intracranial hematoma. Atorvastatin is reported to be effective in the conservative treatment of CSDH. This study aimed to clarify whether atorvastatin regulated the inflammatory responses in CSDH by interfering with the function of macrophages. The rat CSDH model was prepared by repeated intracranial blood injection with velocity gradient, and MRI was applied to calculate the intracranial hematoma volume. Changes in rat nerve functions were evaluated by foot-fault and Morris water maze tests. Flow cytometry was applied to detect the number of total macrophages and the percentage of M1 or M2 macrophages. The expression of inflammatory factors was examined by ELISA and western blot. Western bolt was applied to detect the expression of proteins involved in the colony-stimulating factor 1 receptor (CSF-1R) signaling pathway. Our results showed that atorvastatin significantly accelerated the absorption of hematoma and improved the nerve functions of CSDH rats. In addition, atorvastatin treatment effectively suppressed the expression of TNF-α, IL-6, and IL-8 and promoted the expression of IL-10. The total number of macrophages was decreased, and the percentage of M2 macrophages was increased in the intracranial hematoma following atorvastatin treatment. Furthermore, atorvastatin increased the levels of M2-related genes and surface markers in BMDMs stimulated by lipopolysaccharides and IFNγ, and activated the CSF-1R signaling pathway. In conclusion, our study shows that atorvastatin could alleviate the symptoms of CSDH and promote hematoma ablation by polarizing macrophages to M2 type and regulating the inflammatory responses.

Abstract 1444: TPX-0022, a potent MET/SRC/CSF1R inhibitor that modulates the tumor immune microenvironment in preclinical models

Abstract Within the tumor microenvironment (TME), CSF1R (colony stimulating factor 1 receptor) signaling plays a pivotal role in recruitment and enrichment of M2 tumor-associated macrophages (TAM) that dampen the anti-tumor immune response and promote tumor progression. Although CSF1R inhibitors have shown promise in the clinic, tumor cells and TAMs can bypass CSF1R inhibition and expand M2 TAMs through other growth factor receptors, such as MET (hepatocyte growth factor receptor) to confer resistance to targeted therapies and immune checkpoint inhibitors. Through autocrine signaling pathways, CSF1R and MET activation in tumors contribute to tumor invasiveness and metastasis. Therefore, inhibitors that target both CSF1R and MET, such as TPX-0022 (a compact macrocyclic inhibitor of MET/SRC/CSF1R), may more effectively suppress CSF1R mediated signaling in tumor cells and the stroma. In the current study, the role of TPX-0022 in the anti-tumor immune response is evaluated in the context of both normal immune cells and immunocompetent syngeneic murine models. TPX-0022 blocks both HGF and CSF1 mediated signaling and proliferation of MET and CSF1R dependent cell lines. In the MC-38 syngeneic model, TPX-0022 treatment repolarized TAMs towards M1 anti-tumorigenic macrophages, increased CD8 T cells, and inhibited tumor growth. In a mixed lymphocyte reaction assay, we demonstrated that pretreatment of macrophages with TPX-0022 enhances macrophage function in driving the activation and proliferation of CD8 T cells. Furthermore, TPX-0022 treatment of anti-C3/CD28 and or SIINFEKL peptide stimulated murine T cells had no deleterious effects on T cell activation and proliferation. Additionally, TPX-0022 enhanced T cell cytotoxic function towards cancer cells in a dose-dependent manner. Detailed analysis of the TME in CT-26 animals treated with TPX-0022 showed a significant reduction in TAMs, rebalancing both immunosuppressive Treg and M2 macrophages towards anti-tumorigenic CTL (CD8+ CD69+ GnzB+) and M1 macrophages. The combination of TPX-0022 and a PD-1 antibody resulted in significant downregulation of pro-tumorigenic cytokines IL-10, IP-10, IL-1β, and HGF, without impacting the anti-tumor cytokine IFN-γ. In the MC-38 model, TPX-0022 demonstrated single-agent activity and a trend to enhanced efficacy in combination with a PD-1 antibody. Taken together, the preclinical data demonstrates that the MET/SRC/CSF1R inhibitor TPX-0022 can reprogram tumor associated macrophages as well as support antigen presentation and activation of T cells within the TME. A TPX-0022 combination with a PD-1 therapy yielded more anti-tumor activity than single agent treatments. Together, the in vitro and in vivo preclinical data provide a mechanistic rationale for the potential utility of TPX-0022 in combination with immune checkpoint inhibitors. Citation Format: Vikas K. Goel, Wei Deng, Dayong zhai, Nathan V. Lee, Ana Parra, Brion W. Murray. TPX-0022, a potent MET/SRC/CSF1R inhibitor that modulates the tumor immune microenvironment in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1444.

MEOX2 serves as a novel biomarker associated with macrophage infiltration in oesophageal squamous cell carcinoma and other digestive system carcinomas

Background Oesophageal squamous cell carcinoma (ESCC) is a malignant tumour of the digestive system that is associated with high morbidity and mortality rates worldwide. With the increased use of immunotherapy in cancer treatment, there is an urgent need to elucidate the immune-related mechanisms in ESCC and other digestive system carcinomas. Methods In our study, single-sample gene set enrichment analysis (ssGSEA) was first performed to analyse the expression profile downloaded from the NCBI Gene Expression Omnibus (GEO) database. Then, via a series of bioinformatic analyses, including the Mann-Whitney test, weighted gene co-expression network analysis (WGCNA), functional enrichment analysis and differentially expressed genes (DEGs) analysis, we identified target immunocytes and related genes. Finally, we validated the results with the TIMER database. Results Our analyses showed that the numbers of infiltrating macrophages were obviously higher in advanced stages in ESCC compared with other types of immunocytes. MEOX2 was identified as a biomarker correlated with macrophage infiltration in ESCC and other types of digestive system carcinomas. And MEOX2 expression was strongly associated with the mRNA expression of colony-stimulating factor 1 (CSF-1) and CSF-1 receptor (CSF-1R) in these kinds of carcinomas. Conclusion We speculated that MEOX2 could facilitate macrophage infiltration via CSF-1/CSF-1R signalling in ESCC and other kinds of digestive system carcinomas, and MEOX2 might serve as a novel target in prospective tumour immunotherapies.