Transient CSF1R inhibition ameliorates behavioral deficits in Cntnap2 knockout and valproic acid-exposed mouse models of autism.

Microglial abnormality and heterogeneity are observed in autism spectrum disorder (ASD) patients and animal models of ASD. Microglial depletion by colony stimulating factor 1-receptor (CSF1R) inhibition has been proved to improve autism-like behaviors in maternal immune activation mouse offspring. However, it is unclear whether CSF1R inhibition has extensive effectiveness and pharmacological heterogeneity in treating autism models caused by genetic and environmental risk factors. Here, we report pharmacological functions and cellular mechanisms of PLX5622, a small-molecule CSF1R inhibitor, in treating Cntnap2 knockout and valproic acid (VPA)-exposed autism model mice. For the Cntnap2 knockout mice, PLX5622 can improve their social ability and reciprocal social behavior, slow down their hyperactivity in open field and repetitive grooming behavior, and enhance their nesting ability. For the VPA model mice, PLX5622 can enhance their social ability and social novelty, and alleviate their anxiety behavior, repetitive and stereotyped autism-like behaviors such as grooming and marble burying. At the cellular level, PLX5622 restores the morphology and/or number of microglia in the somatosensory cortex, striatum, and hippocampal CA1 regions of the two models. Specially, PLX5622 corrects neurophysiological abnormalities in the striatum of the Cntnap2 knockout mice, and in the somatosensory cortex, striatum, and hippocampal CA1 regions of the VPA model mice. Incidentally, microglial dynamic changes in the VPA model mice are also reported. Our study demonstrates that microglial depletion and repopulation by transient CSF1R inhibition is effective, and however, has differential pharmacological functions and cellular mechanisms in rescuing behavioral deficits in the two autism models.

Brain volume and microglial density changes are correlated in a juvenile mouse model of cranial radiation and CSF1R inhibitor treatment.

Microglia have been shown to proliferate and become activated following cranial radiotherapy (CRT), resulting in a chronic inflammatory response. We investigated the role of microglia in contributing to widespread volume losses observed in the brain following CRT in juvenile mice. To manipulate microglia, we used low-dose treatment with a highly selective CSF1R inhibitor called PLX5622 (PLX). We hypothesized that alteration of the post-CRT microglia population would lead to changes in brain development outcomes, as evaluated by structural MRI. Wild-type C57BL/6J mice were provided with daily intraperitoneal injections of PLX (25 mg/kg) or vehicle from postnatal day (P)14 to P19. Mice also received whole-brain irradiation (7 Gy) or sham irradiation (0 Gy) at 16 days of age. In one cohort of mice, immunohistochemical assessment in tissue sections was conducted to assess the impact of the selected PLX and CRT doses as well as their combination. In a separate cohort, mice were imaged using MRI at P14 (pretreatment), P19, P23, P42 and P63 in order to assess induced volume changes, which were measured based on structures from a predefined atlas. We observed that PLX and radiation treatments led to sex-specific changes in the microglial cell population. Across treatment groups, MRI-detected anatomical volumes at P19 and P63 were associated with microglia and proliferating microglia densities, respectively. Overall, our study demonstrates that low-dose PLX treatment produces a sex-dependent response in juvenile mice, that manipulation of microglia alters CRT-induced volume changes and that microglia density and MRI-derived volume changes are correlated in this model.

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.

Pexidartinib and Immune Checkpoint Inhibitors Combine to Activate Tumor Immunity in a Murine Colorectal Cancer Model by Depleting M2 Macrophages Differentiated by Cancer-Associated Fibroblasts.

Tumor-associated macrophages (TAMs) and cancer-associated fibroblasts (CAFs) are known to play supportive roles in tumor development and progression, but their interactions in colorectal cancer (CRC) remain unclear. Here, we investigated the effects of colon-cancer-derived CAFs on TAM differentiation, migration, and tumor immunity, both in vitro and in vivo. When co-cultured with monocytes, CAFs attracted monocytes and induced their differentiation into M2 macrophages. Immunohistology of surgically resected human CRC specimens and orthotopically transplanted mouse tumors revealed a correlation between numbers of CAFs and numbers of M2 macrophages. In a mouse model of CRC orthotopic transplantation, treatment with an inhibitor of the colony-stimulating factor-1 receptor (PLX3397) depleted M2 macrophages and increased CD8-positive T cells infiltrating the tumor nest. While this treatment had a minor effect on tumor growth, combining PLX3397 with anti-PD-1 antibody significantly reduced tumor growth. RNA-seq following combination therapy showed activation of tumor immunity. In summary, CAFs are involved in the induction and mobilization of M2 macrophage differentiation in the CRC tumor immune microenvironment, and the combination of cancer immunotherapy and PLX3397 may represent a novel therapeutic option for CRC.

Cytokine enrichment in deep cerebellar nuclei is contributed by multiple glial populations and linked to reduced amyloid plaque pathology.

Alzheimer's disease (AD) pathology and amyloid-beta (Aß) plaque deposition progress slowly in the cerebellum compared to other brain regions, while the entorhinal cortex (EC) is one of the most vulnerable regions. Using a knock-in AD mouse model (App KI), we show that within the cerebellum, the deep cerebellar nuclei (DCN) has particularly low accumulation of Aß plaques. To identify factors that might underlie differences in the progression of AD-associated neuropathology across regions, we profiled gene expression in single nuclei (snRNAseq) across all cell types in the DCN and EC of wild-type (WT) and App KI male mice at age 7 months. We found differences in expression of genes associated with inflammatory activation, PI3K-AKT signalling, and neuron support functions between both regions and genotypes. In WT mice, the expression of interferon-response genes in microglia is higher in the DCN than the EC and this enrichment is confirmed by RNA in situ hybridisation, and measurement of inflammatory cytokines by protein array. Our analyses also revealed that multiple glial populations are responsible for establishing this cytokine-enriched niche. Furthermore, homogenates derived from the DCN induced inflammatory gene expression in BV2 microglia. We also assessed the relationship between the DCN microenvironment and Aß pathology by depleting microglia using a CSF1R inhibitor PLX5622 and saw that, surprisingly, the expression of a subset of inflammatory cytokines was increased while plaque abundance in the DCN was further reduced. Overall, our study revealed the presence of a cytokine-enriched microenvironment unique to the DCN that when modulated, can alter plaque deposition.

Distinguishing the effects of systemic CSF1R inhibition by PLX3397 on microglia and peripheral immune cells.

Microglia, the primary immune cells of the central nervous system (CNS), are derived from the yolk sac and populate the brain during development. Once microglia migrate to the CNS, they are self-renewing and require CSF1R signaling for their maintenance. Pexidartinib (PLX3397, PLX), a small molecule inhibitor of the CSF1R, has been shown to effectively deplete microglia since microglial maintenance is CSF1R-dependent. There have, however, been several conflicting reports that have shown the potential off-target effects of PLX on peripheral immune cells particularly those of lymphoid origin. Given this controversy in the use of the PLX family of drugs, it has become important to ascertain to what extent PLX affects the peripheral immune profile in lymphoid (spleen, and bone marrow) and non-lymphoid (kidney, lungs, and heart) organs. PLX3397 chow treatment at 660 mg/kg for 7 days significantly reduced CD45+ macrophages, CX3CR1-GFP cells, CD11b+CD45intermediate cells, and P2RY12 expression in the brain. However, there were minimal effects on peripheral immune cells from both lymphoid and non-lymphoid organs except in the heart where there was a significant decrease in CD3+ cells, inflammatory and patrolling monocytes, and CD11b+Ly6G+ neutrophils. We then stimulated the immune system with 1 mg/kg of LPS which resulted in a significant reduction in the number of innate immune cells. In this context, PLX did not alter the cytokine profile in the serum and the brain of naïve mice but did so in the LPS-stimulated group resulting in a significant reduction in TNFα, IL-1α, IFN-γ and IL-1ß. Furthermore, PLX did not alter locomotor activity in the open field test suggesting that microglia do not contribute to LPS-induced sickness behavior. Our results provide an assessment of immune cell populations with PLX3397 treatment on brain, lymphoid and non-lymphoid organs without and during LPS treatment that can serve as a resource for understanding consequences of such approaches.

Microglia preserve visual function loss in the aging retina by supporting retinal pigment epithelial health.

BACKGROUND: Increased age is a risk factor for the development and progression of retinal diseases including age-related macular degeneration (AMD). Understanding the changes that occur in the eye due to aging is important in enhancing our understanding of AMD pathogenesis and the development of novel AMD therapies. Microglia, the resident brain and retinal immune cells are associated with both maintaining homeostasis and protection of neurons and loss of microglia homeostasis could be a significant player in age related neurodegeneration. One important characteristic of retinal aging is the migration of microglia from the inner to outer retina where they reside in the subretinal space (SRS) in contact with the retinal pigment epithelial (RPE) cells. The role of aged subretinal microglia is unknown. Here, we depleted microglia in aged C57/BL6 mice fed for 6 weeks with a chow containing PLX5622, a small molecule inhibitor of colony-stimulating factor-1 receptor (Csf1r) required for microglial survival. RESULTS: The subretinal P2RY12 + microglia in aged mice displayed a highly amoeboid and activated morphology and were filled with autofluorescence droplets reminiscent of lipofuscin. TEM indicates that subretinal microglia actively phagocytize shed photoreceptor outer segments, one of the main functions of retinal pigmented epithelial cells. PLX5622 treatment depleted up to 90% of the retinal microglia and was associated with significant loss in visual function. Mice on the microglia depletion diet showed reduced contrast sensitivity and significantly lower electroretinogram for the c-wave, a measurement of RPE functionality, compared to age-matched controls. The loss of c-wave coincided with a loss of RPE cells and increased RPE swelling in the absence of microglia. CONCLUSIONS: We conclude that microglia preserve visual function in aged mice and support RPE cell function, by phagocytosing shed photoreceptor outer segments and lipids, therefore compensating for the known age-related decline of RPE phagocytosis.

In vitro 2D and 3D cancer models to evaluate compounds that modulate macrophage polarization.

In vitro cancer models that can identify novel immunomodulating compounds are essential. Using a 3D multicellular tumor spheroid (MCTS) model comprising cancer cells, fibroblasts, and macrophages, we tested tumor-associated macrophage (TAM)-inhibiting compounds (CCL2 Ab, CSF1R inhibitor, CSF1R Ab) and TAM-reprograming compounds (poly I:C, CD40 Ab, CD40 ligand) for their effects on monocyte infiltration and polarization in tumor spheroids. For characterization of macrophage polarization, we measured the expression of CD206, CD163, CD86, MHC II, CD40, and CD14 and measured 43 soluble factors in the 3D MCTS cultures. 2D macrophage models were evaluated for comparison. A CSF1R inhibitor prevented infiltration of monocytes into pancreatic cancer spheroids, and macrophages treated with the inhibitor showed decreased expression of M2 markers. Treatment with a CD40 ligand and poly I:C induced M1 macrophage polarization in our models. We propose that these models can be used to improve the drug screening process of anti-cancer immunotherapies targeting macrophages.

Potential Stereoselective Binding of Trans-(±)-Kusunokinin and Cis-(±)-Kusunokinin Isomers to CSF1R.

Breast cancer cell proliferation and migration are inhibited by naturally extracted trans-(-)-kusunokinin. However, three additional enantiomers of kusunokinin have yet to be investigated: trans-(+)-kusunokinin, cis-(-)-isomer and cis-(+)-isomer. According to the results of molecular docking studies of kusunokinin isomers on 60 breast cancer-related proteins, trans-(-)-kusunokinin was the most preferable and active component of the trans-racemic mixture. Trans-(-)-kusunokinin targeted proteins involved in cell growth and proliferation, whereas the cis-(+)-isomer targeted proteins involved in metastasis. Trans-(-)-kusunokinin targeted CSF1R specifically, whereas trans-(+)-kusunokinin and both cis-isomers may have bound AKR1B1. Interestingly, the compound's stereoisomeric effect may influence protein selectivity. CSF1R preferred trans-(-)-kusunokinin over trans-(+)-kusunokinin because the binding pocket required a ligand planar arrangement to form a π-π interaction with a selective Trp550. Because of its large binding pocket, EGFR exhibited no stereoselectivity. MD simulation revealed that trans-(-)-kusunokinin, trans-(+)-kusunokinin and pexidartinib bound CSF1R differently. Pexidartinib had the highest binding affinity, followed by trans-(-)-kusunokinin and trans-(+)-kusunokinin, respectively. The trans-(-)-kusunokinin-CSF1R complex was found to be stable, whereas trans-(+)-kusunokinin was not. Trans-(±)-kusunokinin, a potential racemic compound, could be developed as a selective CSF1R inhibitor when combined.

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.

Functional role of brain-engrafted macrophages against brain injuries.

BACKGROUND: Brain-resident microglia have a distinct origin compared to macrophages in other organs. Under physiological conditions, microglia are maintained by self-renewal from the local pool, independent of hematopoietic progenitors. Pharmacological depletion of microglia during whole-brain radiotherapy prevents synaptic loss and long-term recognition memory deficits. However, the origin or repopulated cells and the mechanisms behind these protective effects are unknown. METHODS: CD45low/int/CD11b+ cells from naïve brains, irradiated brains, PLX5622-treated brains and PLX5622 + whole-brain radiotherapy-treated brains were FACS sorted and sequenced for transcriptomic comparisons. Bone marrow chimeras were used to trace the origin and long-term morphology of repopulated cells after PLX5622 and whole-brain radiotherapy. FACS analyses of intrinsic and exotic synaptic compartments were used to measure phagocytic activities of microglia and repopulated cells. In addition, concussive brain injuries were given to PLX5622 and brain-irradiated mice to study the potential protective functions of repopulated cells after PLX5622 + whole-brain radiotherapy. RESULTS: After a combination of whole-brain radiotherapy and microglia depletion, repopulated cells are brain-engrafted macrophages that originate from circulating monocytes. Comparisons of transcriptomes reveal that brain-engrafted macrophages have an intermediate phenotype that resembles both monocytes and embryonic microglia. In addition, brain-engrafted macrophages display reduced phagocytic activity for synaptic compartments compared to microglia from normal brains in response to a secondary concussive brain injury. Importantly, replacement of microglia by brain-engrafted macrophages spare mice from whole-brain radiotherapy-induced long-term cognitive deficits, and prevent concussive injury-induced memory loss. CONCLUSIONS: Brain-engrafted macrophages prevent radiation- and concussion-induced brain injuries and cognitive deficits.

A phase 1a/1b trial of CSF-1R inhibitor LY3022855 in combination with durvalumab or tremelimumab in patients with advanced solid tumors.

Background LY3022855 is a recombinant, immunoglobulin, human monoclonal antibody targeting the colony-stimulating factor-1 receptor. This phase 1 trial determined the safety, pharmacokinetics, and antitumor activity of LY3022855 in combination with durvalumab or tremelimumab in patients with advanced solid cancers who had received standard anti-cancer treatments. Methods In Part A (dose-escalation), patients received intravenous (IV) LY3022855 25/50/75/100 mg once weekly (QW) combined with durvalumab 750 mg once every two weeks (Q2W) IV or LY3022855 50 or 100 mg QW IV with tremelimumab 75/225/750 mg once every four weeks. In Part B (dose-expansion), patients with non-small cell lung cancer (NSCLC) or ovarian cancer (OC) received recommended phase 2 dose (RP2D) of LY3022855 from Part A and durvalumab 750 mg Q2W. Results Seventy-two patients were enrolled (median age 61 years): Part A = 33, Part B = 39. In Part A, maximum tolerated dose was not reached, and LY3022855 100 mg QW and durvalumab 750 mg Q2W was the RP2D. Four dose-limiting equivalent toxicities occurred in two patients from OC cohort. In Part A, maximum concentration, area under the concentration-time curve, and serum concentration showed dose-dependent increase over two cycles of therapy. Overall rates of complete response, partial response, and disease control were 1.4%, 2.8%, and 33.3%. Treatment-emergent anti-drug antibodies were observed in 21.2% of patients. Conclusions LY3022855 combined with durvalumab or tremelimumab in patients with advanced NSCLC or OC had limited clinical activity, was well tolerated. The RP2D was LY3022855 100 mg QW with durvalumab 750 mg Q2W. ClinicalTrials.gov ID: NCT02718911 (Registration Date: May 3, 2011).

LY3022855, an anti-colony stimulating factor-1 receptor (CSF-1R) monoclonal antibody, in patients with advanced solid tumors refractory to standard therapy: phase 1 dose-escalation trial.

Background Tumor-associated macrophages (TAMs) promote tumor growth, metastasis, and therapeutic resistance via colony-stimulating factor-1 (CSF-1), acting through CSF-1 receptor (CSF-1R) signaling. This phase 1 study determined the safety, tolerability, pharmacokinetics-pharmacodynamics, immunogenicity, and efficacy of the anti-CSF-1R antibody LY3022855 in solid tumors. Methods Patients with advanced solid tumors refractory to standard therapy were enrolled and treated in 2 dosing cohorts: weight-based (part A) and non-weight-based (part B). Part A patients were assigned to intravenous (IV) dose-escalation cohorts: 2.5 mg/kg once per week (QW), 0.3 mg/kg QW, 0.6 mg/kg QW, 1.25 mg/kg once every 2 weeks (Q2W) and 1.25 mg/kg QW doses of LY3022855. Non-weight-based doses in part B were 100 mg and 150 mg IV QW. Results Fifty-two patients (mean age 58.6 ± 10.4 years) were treated with ≥1 dose of LY3022855 (range: 4-6). Five dose-limiting toxicities (left ventricular dysfunction, anemia, pancreatitis, rhabdomyolysis, and acute kidney injury) occurred in 4 patients. The non-weight-based 100 mg QW dose was established as the RP2D. The most common treatment-emergent adverse events were increase in liver function variables, fatigue, nausea, vomiting, diarrhea, anorexia, pyrexia, increased lipase, amylase, and lactate dehydrogenase. Clearance decreased with increasing dose and weight-based dosing had minimal effect on pharmacokinetics. Serum CSF-1, and IL-34 levels increased at higher doses and more frequent dosing, whereas TAMs and CD14dimCD16bright levels decreased. Three patients achieved stable disease. No responses were seen. Conclusions LY3022855 was well tolerated and showed dose-dependent pharmacokinetics-pharmacodynamics and limited clinical activity in a heterogenous solid tumor population. ClinicalTrials.gov ID NCT01346358 (Registration Date: May 3, 2011).

Genetically induced brain inflammation by Cnp deletion transiently benefits from microglia depletion.

Reduced expression of 2'-3'-cyclic nucleotide 3'-phosphodiesterase (Cnp) in humans and mice causes white matter inflammation and catatonic signs. These consequences are experimentally alleviated by microglia ablation via colony-stimulating factor 1 receptor (CSF1R) inhibition using PLX5622. Here we address for the first time preclinical topics crucial for translation, most importantly 1) the comparison of 2 long-term PLX5622 applications (prevention and treatment) vs. 1 treatment alone, 2) the correlation of catatonic signs and executive dysfunction, 3) the phenotype of leftover microglia evading depletion, and 4) the role of intercellular interactions for efficient CSF1R inhibition. Based on our Cnp-/- mouse model and in vitro time-lapse imaging, we report the unexpected discovery that microglia surviving under PLX5622 display a highly inflammatory phenotype including aggressive premortal phagocytosis of oligodendrocyte precursor cells. Interestingly, ablating microglia in vitro requires mixed glial cultures, whereas cultured pure microglia withstand PLX5622 application. Importantly, 2 extended rounds of CSF1R inhibition are not superior to 1 treatment regarding any readout investigated (magnetic resonance imaging and magnetic resonance spectroscopy, behavior, immunohistochemistry). Catatonia-related executive dysfunction and brain atrophy of Cnp-/- mice fail to improve under PLX5622. To conclude, even though microglia depletion is temporarily beneficial and worth pursuing, complementary treatment strategies are needed for full and lasting recovery.-Fernandez Garcia-Agudo, L., Janova, H., Sendler, L. E., Arinrad, S., Steixner, A. A., Hassouna, I., Balmuth, E., Ronnenberg, A., Schopf, N., van der Flier, F. J., Begemann, M., Martens, H., Weber, M. S., Boretius, S., Nave, K.-A., Ehrenreich, H. Genetically induced brain inflammation by Cnp deletion transiently benefits from microglia depletion.

Pexidartinib: first approved systemic therapy for patients with tenosynovial giant cell tumor.

Pexidartinib is an orally administered small molecule tyrosine kinase inhibitor. Phase III ENLIVEN study results provided clinical evidence for US FDA approval for treatment of adult patients with symptomatic tenosynovial giant cell tumor associated with severe morbidity or functional limitations and not amenable to improvement with surgery. Recommended dosage is 400 mg orally twice daily on an empty stomach. Long-term follow-up in pooled analyses showed increased response rates compared with those observed in ENLIVEN. Patients on pexidartinib also experience meaningful improvements in range of motion. Side effects associated with pexidartinib are generally manageable; however, there is a risk of potentially life-threatening mixed or cholestatic hepatotoxicity and pexidartinib has a Risk Evaluation and Mitigation Strategy (REMS) program to ensure appropriate monitoring.

Modulating the Tumor Microenvironment via Oncolytic Viruses and CSF-1R Inhibition Synergistically Enhances Anti-PD-1 Immunotherapy.

Immunotherapy based on the immune checkpoint blockade has emerged as the most promising approach for cancer therapy. However, the proportion of colorectal cancer patients who benefit from immunotherapy is small due to the immunosuppressive tumor microenvironment. Hence, combination immunotherapy is an ideal strategy to overcome this limitation. In this study, we developed a novel combination of CSF-1R (colony-stimulating factor 1 receptor) inhibitor (PLX3397), oncolytic viruses, and anti-PD-1 antibody. Our results demonstrated that the triple treatment synergistically conferred significant tumor control and prolonged the survival of mouse models of colon cancer. Approximately 43% and 82% of mice bearing the CT26 and MC38 tumor, respectively, survived long term following the triple treatment. This combination therapy reprogrammed the immunosuppressive tumor microenvironment toward a CD8+ T cell-biased anti-tumor immunity by increasing T cell infiltration in the tumor and augmenting anti-tumor CD8+ T cell function. Our results provide a robust strategy for clinical combination therapy.

CSF-1R Inhibitor Development: Current Clinical Status.

PURPOSE OF REVIEW: Colony-stimulating factor 1 receptor (CSF-1R) and its ligands, CSF-1 and interleukin 34 (IL-34), regulate the function and survival of tumor-associated macrophages, which are involved in tumorigenesis and in the suppression of antitumor immunity. Moreover, the CSF-1R/CSF-1 axis has been implicated in the pathogenesis of pigmented villonodular synovitis (PVNS), a benign tumor of the synovium. As advanced or metastatic malignant solid tumors and relapsed/refractory PVNS remain unresolved therapeutic problems, new approaches are needed to improve the outcome of patients with these conditions. RECENT FINDINGS: In solid tumors, targeting CSF-1R via either small molecules or antibodies has shown interesting results in vitro but limited antitumor activity in vivo. Concerning PVNS, clinical trials assessing CSF-1R inhibitors have revealed promising initial outcomes. Blocking CSF-1/CSF-1R signaling represents a promising immunotherapy approach and several new potential combination therapies for future clinical testing.

CNS resident macrophages enhance dysfunctional angiogenesis and circulating monocytes infiltration in brain arteriovenous malformation.

Myeloid immune cells present abundantly in both ruptured and unruptured brain arteriovenous malformations (bAVMs). The role of central nervous system (CNS) resident and circulating monocyte-derived macrophages in bAVM pathogenesis has not been fully understood. RNA sequencing using cultured cells and bAVM samples revealed that downregulation of activin-like kinase 1 (ALK1) or endoglin (two bAVM causative genes) increased pro-angiogenic, endothelial inflammation and innate immune signaling, which provided endogenous underpinnings of the active inflammation in bAVM. To further understand the role of CNS resident macrophages in bAVM development and hemorrhage, we administrated a colony-stimulating factor 1 receptor (CSF1R) inhibitor to bAVM mice with endothelial Alk1 deletion. Transient depletion of CNS resident macrophages at early stage of bAVM development remarkably mitigated the subsequent phenotype severity of bAVM. This therapeutic effect exhibited a prolonged inhibition of angiogenesis, dysplastic vasculature formation, and infiltration of CNS resident and circulating monocyte-derived macrophages during bAVM development. Transient depletion of CNS resident macrophages also reduced the dysplasia vessels and improved the integrity of endothelial tight junctions in established bAVMs. Administration of CSF1R inhibitor also prevented severe hemorrhage of bAVMs. Thus, endothelial AVM causative gene mutation can activate CNS resident macrophages promoting bAVM progression. CNS resident macrophages could be specific targets to mitigate the development and severity of bAVMs.

Microglial depletion exacerbates motor impairment and dopaminergic neuron loss in a 6-OHDA model of Parkinson's disease.

6-hydroxydopamine (6-OHDA) is a common neurotoxin used to induce Parkinson's disease (PD) in mice, exerting neurotoxic effects through the production of reactive oxygen species and microglial activation. However, the role of microglia in PD is still not clear, with contradictory reports showing neuroprotection or exacerbation of neuronal death. Microglial depletion aggravates motor coordination impairments and reduces tyrosine hydroxylase positive neurons in the substantia nigra pars compacta. Moreover, MeCP2 and Adora1 genes expression were downregulated, suggesting they may be involved in the neurodegenerative process. This study highlights that microglia plays a protective role in dopaminergic neuron survival during the initial phase of PD, and the investigation of the mechanisms of this effect in future studies will help elucidate the pathophysiology of PD.

Dual CSF1R inhibition and CD40 activation demonstrates anti-tumor activity in a 3D macrophage- HER2+ breast cancer spheroid model.

The complex interaction between tumor-associated macrophages (TAMs) and tumor cells through soluble factors provides essential cues for breast cancer progression. TAMs-targeted therapies have shown promising clinical therapeutical potential against cancer progression. The molecular mechanisms underlying the response to TAMs-targeted therapies depends on complex dynamics of immune cross-talk and its understanding is still incomplete. In vitro models are helpful to decipher complex responses to combined immunotherapies. In this study, we established and characterized a 3D human macrophage-ER+ PR+ HER2+ breast cancer model, referred to as macrophage-tumor spheroid (MTS). Macrophages integrated within the MTS had a mixed M2/M1 phenotype, abrogated the anti-proliferative effect of trastuzumab on tumor cells, and responded to IFNγ with increased M1-like polarization. The targeted treatment of MTS with a combined CSF1R kinase inhibitor and an activating anti-CD40 antibody increased M2 over M1 phenotype (CD163+/CD86+ and CD206+/CD86+ ratio) in time, abrogated G2/M cell cycle phase transition of cancer cells, promoted the secretion of TNF-α and reduced cancer cell viability. In comparison, combined treatment in a 2D macrophage-cancer cell co-culture model reduced M2 over M1 phenotype and decreased cancer cell viability. Our work shows that this MTS model is responsive to TAMs-targeted therapies, and may be used to study the response of ER+ PR+ HER2+ breast cancer lines to novel TAM-targeting therapies.