Colony-stimulating Factor-1 Receptor Signaling Research Articles

Eliminating microglia in Alzheimer's mice prevents neuronal loss without modulating amyloid-β pathology.

In addition to amyloid-β plaque and tau neurofibrillary tangle deposition, neuroinflammation is considered a key feature of Alzheimer's disease pathology. Inflammation in Alzheimer's disease is characterized by the presence of reactive astrocytes and activated microglia surrounding amyloid plaques, implicating their role in disease pathogenesis. Microglia in the healthy adult mouse depend on colony-stimulating factor 1 receptor (CSF1R) signalling for survival, and pharmacological inhibition of this receptor results in rapid elimination of nearly all of the microglia in the central nervous system. In this study, we set out to determine if chronically activated microglia in the Alzheimer's disease brain are also dependent on CSF1R signalling, and if so, how these cells contribute to disease pathogenesis. Ten-month-old 5xfAD mice were treated with a selective CSF1R inhibitor for 1 month, resulting in the elimination of ∼80% of microglia. Chronic microglial elimination does not alter amyloid-β levels or plaque load; however, it does rescue dendritic spine loss and prevent neuronal loss in 5xfAD mice, as well as reduce overall neuroinflammation. Importantly, behavioural testing revealed improvements in contextual memory. Collectively, these results demonstrate that microglia contribute to neuronal loss, as well as memory impairments in 5xfAD mice, but do not mediate or protect from amyloid pathology.

Src family kinase expression and subcellular localization in macrophages: implications for their role in CSF-1-induced macrophage migration.

A major role of colony-stimulating factor-1 is to stimulate the differentiation of mononuclear phagocytic lineage cells into adherent, motile, mature macrophages. The colony-stimulating factor-1 receptor transduces colony-stimulating factor-1 signaling, and we have shown previously that phosphatidylinositol 3-kinase p110δ is a critical mediator of colony-stimulating factor-1-stimulated motility through the colony-stimulating factor-1 receptor pY721 motif. Src family kinases are also implicated in the regulation of macrophage motility and in colony-stimulating factor-1 receptor signaling, although functional redundancy of the multiple SFKs expressed in macrophages makes it challenging to delineate their specific functions. We report a comprehensive analysis of individual Src family kinase expression in macrophage cell lines and primary macrophages and demonstrate colony-stimulating factor-1-induced changes in Src family kinase subcellular localization, which provides clues to their distinct and redundant functions in macrophages. Moreover, expression of individual Src family kinases is both species specific and dependent on colony-stimulating factor-1-induced macrophage differentiation. Hck associated with the activated colony-stimulating factor-1 receptor, whereas Lyn associated with the receptor in a constitutive manner. Consistent with this, inhibitor studies revealed that Src family kinases were important for both colony-stimulating factor-1 receptor activation and colony-stimulating factor-1-induced macrophage spreading, motility, and invasion. Distinct colony-stimulating factor-1-induced changes in the subcellular localization of individual SFKs suggest specific roles for these Src family kinases in the macrophage response to colony-stimulating factor-1.

Abstract PR06: CSF1 signaling is a potential therapeutic target for glioma

Abstract Current therapies for high-grade gliomas only modestly extend survival. Given that intrinsic characteristics of glioma cancer stem cells (gCSCs) make them hard to effectively target with current therapies, cells in the tumor microenvironment such as microglia/macrophages are being investigated as therapy targets. We previously identified a microglial/macrophage growth factor, Macrophage colony stimulating factor (Csf1), as a candidate glioma oncogene in a mouse somatic mutagenesis screen. CSF1 overexpression, CSF1 receptor (CSF1R) overexpression, and phosphorylated CSF1R have also been detected in human gliomas. We are using genetic and pharmacological strategies to investigate the role of CSF1 signaling in gliomagenesis in immunocompetent autochthonous mouse models to examine the therapeutic utility of targeting the CSF1R signaling axis. To determine the relevant CSF1R ligand(s) in human gliomas, we performed semi-quantitative RT-PCR on patient-derived gCSCs to examine expression of splice variants encoding membrane bound or secreted CSF1 as well as the additional CSF1R ligand IL34. We found that gCSCs primarily express the mRNA splice variant encoding secreted CSF1, but a splice variant encoding a membrane bound form was also detected. Most gCSCs lines did not express detectable levels of IL34 mRNA. We therefore developed a tissue-specific secreted CSF1 overexpressing transgenic model to study the effect of modulating CSF1 levels in the central nervous system. Since CSF1 is a growth factor for microglia, we previously studied the effects of increased CSF1 levels on microglial phenotypes and found that CSF1 overexpression increased microglial numbers and promoted microglial proliferation in vivo, but did not inherently polarize microglia toward a M2-like phenotype. Moreover, CSF1 signaling is required for survival of adult microglia in vivo, since inhibition of CSF1 signaling by the small molecule inhibitor PLX3397 induced microglial apoptosis. We now show that CSF1 overexpression accelerates gliomagenesis in a genetic model of spontaneous gliomagenesis driven by activated Ras. Both high- and low-grade gliomas form in mice with activated Ras, but CSF1 overexpression causes a dramatic increase in the percentage of tumors that are high-grade. Additionally, gliomas do not form in mice expressing activated Ras that are also CSF1-deficient. Therefore our data in this in vivo model indicate that CSF1 signaling plays a fundamental role in glioma development. Additional studies are underway to determine the impact of CSF1 on microglial and glioma phenotypes, and to explore how to best target CSF1 signaling for glioma therapy. Citation Format: Ishani De, Megan D. Steffen, Emily Sokn, Clayton Patros, Suzanne Litscher, Paul A. Clark, John S. Kuo, Fausto J. Rodriguez, Lara S. Collier. CSF1 signaling is a potential therapeutic target for glioma. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr PR06.

CmFPA008, an anti-mouse CSF-1R antibody, combines with multiple immunotherapies to reduce tumor growth in nonclinical models

The colony stimulating factor 1 receptor (CSF1R) signaling pathway drives the recruitment, differentiation, and survival of tumor-associated macrophages (TAMs) in the neoplastic microenvironment, promoting tumor progression and modulation of the immune response. As such, CSF1R represents an intriguing therapeutic target for immuno-oncology. FivePrime has developed FPA008, an IgG4 antibody with high affinity (KD = 0.35 nM) for CSF1R and the ability to block binding of both CSF1 and IL-34 to this receptor. In order to interrogate the impact of CSF1R signaling inhibition on tumor growth and the immune environment, we generated a surrogate antibody, cmFPA008, which targets mouse CSF1R and demonstrates equivalent affinity and ligand-blocking ability as FPA008. Consistent with other reports on molecules targeting this pathway, cmFPA008 as monotherapy results in statistically significant but modest growth inhibition in multiple preclinical tumor models, including MC38 colon adenocarcinoma and B16 melanoma. Utilizing a combination of flow cytometric, immunohistochemical, and gene expression analyses, we show that CSF1R inhibition induces dramatic reduction of TAMs and an increase in the CD8+ T cell to regulatory T cell ratio in syngeneic tumor models. Given that the regulation an anti-tumor immune response is complex, effective cancer therapy may require combining multiple immunotherapy agents. We sought to determine whether inhibition of CSF1R when combined with other immuno-oncology therapeutics enhanced the anti-tumor impact. We observed increased PD-L1 (CD274) expression in the tumor after treatment with cmFPA008 monotherapy, thereby providing a rationale for a combination of FPA008 with a compound targeting the PD-1 pathway. Our results show that cmFPA008 significantly enhances anti-tumor efficacy when combined with an anti-PD1 therapeutic in multiple syngeneic tumor models. In addition, we show that co-administration of cmFPA008 with an agonist anti-CD40 significantly enhances tumor suppression compared to either therapy alone. Changes in tumor-infiltrating lymphocyte (TIL) populations upon treatment provide important insight into the mechanism of action of cmFPA008 monotherapy and in combination with other immunotherapies. These results provide support for ongoing clinical efforts to evaluate FPA008 as an anti-cancer immunotherapy, particularly in combination with other immuno-oncology therapeutics. FivePrime has planned a clinical trial in collaboration with Bristol-Myers Squibb (BMS) to investigate the efficacy of FPA008 in combination with the anti-PD1 therapeutic Opdivo® (nivolumab) in six tumor types.

Colony stimulating factor-1 receptor signaling networks inhibit mouse macrophage inflammatory responses by induction of microRNA-21

AbstractMacrophage polarization between the M2 (repair, protumorigenic) and M1 (inflammatory) phenotypes is seen as a continuum of states. The detailed transcriptional events and signals downstream of colony-stimulating factor 1 receptor (CSF-1R) that contributes to amplification of the M2 phenotype and suppression of the M1 phenotype are largely unknown. Macrophage CSF-1R pTyr-721 signaling promotes cell motility and enhancement of tumor cell invasion in vitro. Combining analysis of cellular systems for CSF-1R gain of function and loss of function with bioinformatic analysis of the macrophage CSF-1R pTyr-721–regulated transcriptome, we uncovered microRNA-21 (miR-21) as a downstream molecular switch controlling macrophage activation and identified extracellular signal-regulated kinase1/2 and nuclear factor-κB as CSF-1R pTyr-721–regulated signaling nodes. We show that CSF-1R pTyr-721 signaling suppresses the inflammatory phenotype, predominantly by induction of miR-21. Profiling of the miR-21–regulated messenger RNAs revealed that 80% of the CSF-1–regulated canonical miR-21 targets are proinflammatory molecules. Additionally, miR-21 positively regulates M2 marker expression. Moreover, miR-21 feeds back to positively regulate its own expression and to limit CSF-1R–mediated activation of extracellular signal-regulated kinase1/2 and nuclear factor-κB. Consistent with an anti-inflammatory role of miRNA-21, intraperitoneal injection of mice with a miRNA-21 inhibitor increases the recruitment of inflammatory monocytes and enhances the peritoneal monocyte/macrophage response to lipopolysaccharide. These results identify the CSF-1R–regulated miR-21 network that modulates macrophage polarization.

The development and maintenance of the mononuclear phagocyte system of the chick is controlled by signals from the macrophage colony-stimulating factor receptor.

BackgroundMacrophages have many functions in development and homeostasis as well as innate immunity. Recent studies in mammals suggest that cells arising in the yolk sac give rise to self-renewing macrophage populations that persist in adult tissues. Macrophage proliferation and differentiation is controlled by macrophage colony-stimulating factor (CSF1) and interleukin 34 (IL34), both agonists of the CSF1 receptor (CSF1R). In the current manuscript we describe the origin, function and regulation of macrophages, and the role of CSF1R signaling during embryonic development, using the chick as a model.ResultsBased upon RNA-sequencing comparison to bone marrow-derived macrophages grown in CSF1, we show that embryonic macrophages contribute around 2% of the total embryo RNA in day 7 chick embryos, and have similar gene expression profiles to bone marrow-derived macrophages. To explore the origins of embryonic and adult macrophages, we injected Hamburger-Hamilton stage 16 to 17 chick embryos with either yolk sac-derived blood cells, or bone marrow cells from EGFP+ donors. In both cases, the transferred cells gave rise to large numbers of EGFP+ tissue macrophages in the embryo. In the case of the yolk sac, these cells were not retained in hatched birds. Conversely, bone marrow EGFP+ cells gave rise to tissue macrophages in all organs of adult birds, and regenerated CSF1-responsive marrow macrophage progenitors. Surprisingly, they did not contribute to any other hematopoietic lineage. To explore the role of CSF1 further, we injected embryonic or hatchling CSF1R-reporter transgenic birds with a novel chicken CSF1-Fc conjugate. In both cases, the treatment produced a large increase in macrophage numbers in all tissues examined. There were no apparent adverse effects of chicken CSF1-Fc on embryonic or post-hatch development, but there was an unexpected increase in bone density in the treated hatchlings.ConclusionsThe data indicate that the yolk sac is not the major source of macrophages in adult birds, and that there is a macrophage-restricted, self-renewing progenitor cell in bone marrow. CSF1R is demonstrated to be limiting for macrophage development during development in ovo and post-hatch. The chicken provides a novel and tractable model to study the development of the mononuclear phagocyte system and CSF1R signaling.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-015-0121-9) contains supplementary material, which is available to authorized users.

Targeting cell-intrinsic and cell-extrinsic mechanisms of intravasation in invasive breast cancer.

The survival of breast cancer patients with metastatic disease has not markedly improved over recent decades, highlighting the need to better understand this process. In this issue of Science Signaling, Pignatelli et al. used freshly obtained invasive ductal carcinoma cells from patients to demonstrate the need for high abundance of the invasive isoform of the Mena protein (Mena(INV)) in cancer cells and colony-stimulating factor 1 (CSF-1)-mediated paracrine signaling in macrophages for efficient transendothelial migration and metastasis formation in all clinical breast cancer subtypes. Furthermore, the triple negative and HER2(+) subtypes, but not the ERPR(+)/HER2(-) subtype, had high CSF-1 receptor (CSF-1R) abundance and also partially used autocrine CSF-1/CSF-1R signaling for invasion. These data establish Mena(INV), CSF-1/CSF-1R, and macrophages as potential therapeutic targets for most human breast cancers.

CSF-1R signaling in health and disease: a focus on the mammary gland.

Colony-stimulating factor-1 (CSF-1), also known as macrophage-colony stimulating factor (M-CSF), is the primary growth factor regulating survival, proliferation and differentiation of macrophages. It is also a potent chemokine for macrophages and monocytes. Signaling via the CSF-1 receptor (CSF-1R) is necessary for the production of almost all tissue resident macrophage populations and these macrophages participate, via trophic mechanisms, in the normal development and homeostasis of tissues and organs in which they reside, including the mammary gland. The drawback of this close interaction between macrophages and parenchymal cells is that dysregulation of macrophage trophic functions assists in the development and progression of many cancers, including breast cancer. Furthermore, tumour cells secrete CSF-1 to attract more macrophages to the tumour microenvironment where CSF-1R signaling frequently drives the behaviour of these tumour-associated macrophages (TAMs) to promote tumour progression and metastasis. Evidence is mounting that treated tumours secrete more CSF-1 and the increased recruitment of TAMs limits treatment efficacy. Thus, therapeutic targeting of the CSF-1R to inhibit TAM function is likely to enhance tumour response and improve patient outcomes in the treatment of cancer, including breast cancer.

CSF-1 receptor signaling in myeloid cells.

The CSF-1 receptor (CSF-1R) is activated by the homodimeric growth factors colony-stimulating factor-1 (CSF-1) and interleukin-34 (IL-34). It plays important roles in development and in innate immunity by regulating the development of most tissue macrophages and osteoclasts, of Langerhans cells of the skin, of Paneth cells of the small intestine, and of brain microglia. It also regulates the differentiation of neural progenitor cells and controls functions of oocytes and trophoblastic cells in the female reproductive tract. Owing to this broad tissue expression pattern, it plays a central role in neoplastic, inflammatory, and neurological diseases. In this review we summarize the evolution, structure, and regulation of expression of the CSF-1R gene. We discuss the structures of CSF-1, IL-34, and the CSF-1R and the mechanism of ligand binding to and activation of the receptor. We further describe the pathways regulating macrophage survival, proliferation, differentiation, and chemotaxis downstream from the CSF-1R.

Colony-stimulating factor 1 receptor signaling is necessary for microglia viability, unmasking a microglia progenitor cell in the adult brain.

The colony-stimulating factor 1 receptor (CSF1R) is a key regulator of myeloid lineage cells. Genetic loss of the CSF1R blocks the normal population of resident microglia in the brain that originates from the yolk sac during early development. However, the role of CSF1R signaling in microglial homeostasis in the adult brain is largely unknown. To this end, we tested the effects of selective CSF1R inhibitors on microglia in adult mice. Surprisingly, extensive treatment results in elimination of ∼99% of all microglia brain-wide, showing that microglia in the adult brain are physiologically dependent upon CSF1R signaling. Mice depleted of microglia show no behavioral or cognitive abnormalities, revealing that microglia are not necessary for these tasks. Finally, we discovered that the microglia-depleted brain completely repopulates with new microglia within 1 week of inhibitor cessation. Microglial repopulation throughout the CNS occurs through proliferation of nestin-positive cells that then differentiate into microglia.

Delivery of CSF-1R to the lumen of macropinosomes promotes its destruction in macrophages.

Activation of the macrophage colony stimulating factor-1 receptor (CSF-1R) by CSF-1 stimulates pronounced macropinocytosis and drives proliferation of macrophages. Although the role of macropinocytosis in CSF-1R signaling remains unknown, we show here that, despite internalizing large quantities of plasma membrane, macropinosomes contribute little to the internalization of the CSF-1-CSF-1R complex. Rather, internalization of the CSF-1R in small endocytic vesicles that are sensitive to clathrin disruption, outcompetes macropinosomes for CSF-1R endocytosis. Following internalization, small vesicles carrying the CSF-1R underwent homotypic fusion and then trafficked to newly formed macropinosomes bearing Rab5. As these macropinosomes matured, acquiring Rab7, the CSF-1R was transported into their lumen and degraded. Inhibition of macropinocytosis delayed receptor degradation despite no disruption to CSF-1R endocytosis. These data indicate that CSF-1-stimulated macropinosomes are sites of multivesicular body formation and accelerate CSF-1R degradation. Furthermore, we demonstrate that macropinocytosis and cell growth have a matching dose dependence on CSF-1, suggesting that macropinosomes might be a central mechanism coupling CSF-1R signaling and macrophage growth.

Haploinsufficiency of CSF-1R and clinicopathologic characterization in patients with HDLS

Objective:To clarify the genetic, clinicopathologic, and neuroimaging characteristics of patients with hereditary diffuse leukoencephalopathy with spheroids (HDLS) with the colony stimulating factor 1 receptor (CSF-1R) mutation.Methods:We performed molecular genetic analysis of CSF-1R in patients with HDLS. Detailed clinical and neuroimaging findings were retrospectively investigated. Five patients were examined neuropathologically.Results:We found 6 different CSF-1R mutations in 7 index patients from unrelated Japanese families. The CSF-1R mutations included 3 novel mutations and 1 known missense mutation at evolutionarily conserved amino acids, and 1 novel splice-site mutation. We identified a novel frameshift mutation. Reverse transcription PCR analysis revealed that the frameshift mutation causes nonsense-mediated mRNA decay by generating a premature stop codon, suggesting that haploinsufficiency of CSF-1R is sufficient to cause HDLS. Western blot analysis revealed that the expression level of CSF-1R in the brain from the patients was lower than from control subjects. The characteristic MRI findings were the involvement of the white matter and thinning of the corpus callosum with signal alteration, and sequential analysis revealed that the white matter lesions and cerebral atrophy relentlessly progressed with disease duration. Spotty calcifications in the white matter were frequently observed by CT. Neuropathologic analysis revealed that microglia in the brains of the patients demonstrated distinct morphology and distribution.Conclusions:These findings suggest that patients with HDLS, irrespective of mutation type in CSF-1R, show characteristic clinical and neuroimaging features, and that perturbation of CSF-1R signaling by haploinsufficiency may play a role in microglial dysfunction leading to the pathogenesis of HDLS.

CSF1R inhibition delays cervical and mammary tumor growth in murine models by attenuating the turnover of tumor-associated macrophages and enhancing infiltration by CD8+ T cells

Increased numbers of tumor-infiltrating macrophages correlate with poor disease outcome in patients affected by several types of cancer, including breast and prostate carcinomas. The colony stimulating factor 1 receptor (CSF1R) signaling pathway drives the recruitment of tumor-associated macrophages (TAMs) to the neoplastic microenvironment and promotes the differentiation of TAMs toward a pro-tumorigenic phenotype. Twelve clinical trials are currently evaluating agents that target the CSF1/CSF1R signaling pathway as a treatment against multiple malignancies, including breast carcinoma, leukemia, and glioblastoma. The blockade of CSF1R signaling has been shown to greatly decrease the number of macrophages in a tissue-specific manner. However, additional mechanistic insights are needed in order to understand how macrophages are depleted and the global effects of CSF1R inhibition on other tumor-infiltrating immune cells. Using BLZ945, a highly selective small molecule inhibitor of CSF1R, we show that CSF1R inhibition attenuates the turnover rate of TAMs while increasing the number of CD8+ T cells that infiltrate cervical and breast carcinomas. Specifically, we find that BLZ945 decreased the growth of malignant cells in the mouse mammary tumor virus-driven polyomavirus middle T antigen (MMTV-PyMT) model of mammary carcinogenesis. Furthermore, we show that BLZ945 prevents tumor progression in the keratin 14-expressing human papillomavirus type 16 (K14-HPV-16) transgenic model of cervical carcinogenesis. Our results demonstrate that TAMs undergo a constant turnover in a CSF1R-dependent manner, and suggest that continuous inhibition of the CSF1R pathway may be essential to maintain efficacious macrophage depletion as an anticancer therapy.

Abstract A252: A phase 1 study of ARRY-382, an oral inhibitor of colony-stimulating factor-1 receptor (CSF1R), in patients with advanced or metastatic cancers.

Abstract Introduction: Within the tumor microenvironment, CSF1R signaling is thought to play an important role in recruitment and differentiation of tumor-associated macrophages and osteoclasts, promoting disease progression through suppression of anti-tumor immune response, promotion of angiogenesis, tumor cell metastasis and tumor-induced osteolysis. ARRY-382 is a potent, highly selective, oral inhibitor of CSF1R. This Phase 1 dose-escalation study was designed to determine the maximum tolerated dose (MTD) and to assess the safety, pharmacokinetics (PK) and pharmacodynamics (PD) of ARRY-382 in patients (pts) with advanced or metastatic cancers refractory to standard treatment. Methods: Pts received ARRY-382 orally once daily (QD) in 28-day cycles. Study design included an initial accelerated titration phase, followed by 3+3 dose escalation and an expansion phase. Safety was assessed by adverse events (AEs), ECGs, clinical laboratory tests, physical exams and vital signs. PK parameters were estimated based on serial plasma PK samples. Levels of pERK in pt monocytes stimulated with CSF1 ex vivo, circulating CD14dim/CD16+ nonclassical monocytes (NCM) and CSF1 were evaluated as markers of CSF1R inhibition. Urinary collagen type 1 cross-linked N[[Unable to Display Character: ‑]]telopeptide (NTX) was evaluated as a marker of bone turnover. Results: Twenty-six pts received ARRY-382 at doses ranging from 25 to 500 mg QD. The MTD of ARRY-382 was 400 mg QD. Dose-limiting toxicities were increased creatine kinase (CK), increased AST and pyrexia (1 pt each, all Grade 3). The most common drug-related AEs were fatigue (42%), increased CK (27%), nausea (23%), decreased appetite (15%) and vomiting (12%). Grade 3/4 AEs in >1 pt were increased CK (23%), pneumonia (15%), anemia (8%) and vomiting (8%). Increases in ARRY-382 exposure were approximately dose proportional. At the MTD, a Cmax of 3.06 µg/mL was achieved with a Ctrough > 1 µg/mL after repeated dosing. Doses ≥ 200 mg QD resulted in > 80% mean reduction from Baseline in monocyte pERK, consistent with exposures above the ARRY-382 IC50 value. The PD activity of ARRY-382 was consistent with CSF1R inhibition. Reductions in NCM were observed at doses ≥ 200 mg QD, with a 96% mean decrease from Baseline observed at the MTD. Reductions in urinary NTX were observed at doses ≥ 50 mg QD. Five of 7 pts with elevated urinary NTX at Baseline experienced reductions to within the normal range in the first cycle, including 3 pts with bone metastases. Increases in CSF1 appeared dose-dependent with ∼28-fold maximal increase from Baseline observed at the MTD in the first cycle. No objective responses were observed, although 4 pts (15%) experienced stable disease which lasted > 3 months for 2 pts. Conclusions: ARRY-382 was generally well tolerated in pts with advanced or metastatic cancers. Exposure and PD activity of ARRY[[Unable to Display Character: ‑]]382 indicate that CSF1R was significantly inhibited at tolerated doses. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A252. Citation Format: Johanna C. Bendell, Anthony W. Tolcher, Suzanne F. Jones, Muralidhar Beeram, Jeffrey R. Infante, Paul Larsen, Kevin Rasor, Jennifer E. Garrus, Jinfang Li, P. Louann Cable, Christine Eberhardt, Jennifer Schreiber, Selena Rush, Kenneth W. Wood, Emma Barrett, Amita Patnaik. A phase 1 study of ARRY-382, an oral inhibitor of colony-stimulating factor-1 receptor (CSF1R), in patients with advanced or metastatic cancers. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A252.

CSF-1 Receptor-Dependent Colon Development, Homeostasis and Inflammatory Stress Response

The colony stimulating factor-1 (CSF-1) receptor (CSF-1R) directly regulates the development of Paneth cells (PC) and influences proliferation and cell fate in the small intestine (SI). In the present study, we have examined the role of CSF-1 and the CSF-1R in the large intestine, which lacks PC, in the steady state and in response to acute inflammation induced by dextran sulfate sodium (DSS). As previously shown in mouse, immunohistochemical (IHC) analysis of CSF-1R expression showed that the receptor is baso-laterally expressed on epithelial cells of human colonic crypts, indicating that this expression pattern is shared between species. Colons from Csf1r null and Csf1op/op mice were isolated and sectioned for IHC identification of enterocytes, enteroendocrine cells, goblet cells and proliferating cells. Both Csf1r−/− and Csf1op/op mice were found to have colon defects in enterocytes and enteroendocrine cell fate, with excessive goblet cell staining and reduced cell proliferation. In addition, the gene expression profiles of the cell cycle genes, cyclinD1, c-myc, c-fos, and c-myb were suppressed in Csf1r−/− colonic crypt, compared with those of WT mice and the expression of the stem cell marker gene Lgr5 was markedly reduced. However, analysis of the proliferative responses of immortalized mouse colon epithelial cells (lines; Immorto-5 and YAMC) indicated that CSF-1R is not a major regulator of colonocyte proliferation and that its effects on proliferation are indirect. In an examination of the acute inflammatory response, Csf1r +/− male mice were protected from the adverse affects of DSS-induced colitis compared with WT mice, while Csf1r +/− female mice were significantly less protected. These data indicate that CSF-1R signaling plays an important role in colon homeostasis and stem cell gene expression but that the receptor exacerbates the response to inflammatory challenge in male mice.

Tumor-infiltrating macrophages, cancer stem cells and therapeutic responses

While the genetic and epigenetic changes that regulate cell proliferation, survival and/or differentiation are known ‘initiators’ of tumor development, these events do not occur in isolation but rather, in the context of a diverse organ specific tumor stroma. Of the stromal components, tumor-infiltrating immune cells are a hallmark of most solid tumors, and the presence of varied immune populations can significantly affect clinical outcomes for cancer patients. Historically, tumor-infiltrating immune cells have been viewed as restraining tumor progression, but in recent years, this view has been expanded to appreciate that chronic immune responses also play critical roles in promoting tumor progression, metastasis, and resistance to cytotoxic therapies[1]. Therefore, understanding the molecular mechanisms by which malignant cells derail antitumor immune responses to favor disease progression is critical to identify potential therapeutic targets. In addition to immune regulation of cancer progression and chemoresistance, tumor cells that acquire stem-like or tumor-initiating properties (often called “Cancer Stem Cells”) exhibit enhanced resistance to cytotoxic therapy and increased propensity for metastatic dissemination[2]. Several lines of evidence suggest that the tumor-initiating capacity of malignant cells is rooted in inflammatory signals [3]. However, the mechanisms by which different populations of leukocytes might regulate cancer stem cells (CSCs) are unknown. Recently, three studies have demonstrated that tumor-infiltrating macrophages (TAMs) enhance tumor-initiating properties in malignant cells and that these regulatory pathways can be therapeutically exploited. Work by Tahara et al. identified Milk Fat Globulin Epidermal growth factor-8 (MFGE-8) as a macrophage derived factor, which potently increase the tumor initiating properties of murine Colon and Lung Carcinoma cell lines[4]. This activity was attributed to both activation of Signal transducer and activator of transcription 3 (STAT3) signaling and enhancement of the Hedgehog signaling. These pathways are major contributors in triggering tumorigenicity and resistance to anti-cancer therapy. They also found that IL-6 co-ordinates with MFGE8 and plays a critical role in increasing tumorigenic activities in subsets of CSCs of primary human tumors. These data suggest that TAMs play a key role in CSC maintenance and/or expansion. Similar work by Yunping Luo et al. found that the crosstalk between TAMs and tumor cells can regulate the induction of pluripotency gene SOX-2 through EGFR mediated activation of STAT3 signaling[5]. Their data also suggests that inhibition of EGF or STAT3 prevents the up regulation of SOX-2 and thus improves chemo-sensitivity in human patients. An alternative approach to targeting these signaling pathways is to block the recruitment or bioactivity of the tumor-infiltrating macrophages directly. Work from several research groups have shown the potential efficacy in targeting macrophages and monocytes through either Colony Stimulating Factor 1 Receptor (CSF1R) or C-C chemokine receptor 2 (CCR2) to block tumor progression, metastasis and/or improve response to chemotherapy [6, 7]. This research has spurred several new clinical trials of agents targeting macrophages through inhibition of these receptors or their ligands. However, the impact of such therapies on CSCs is not well understood. Recent work by Mitchem et al. showed that blockade of CCR2 or CSF1R signaling in established tumors not only results in a significant decrease in tumor infiltration by macrophages, but also reduces the frequency of CD44+ALDH1+ pancreatic CSCs[8]. According to their results, targeting macrophages through CSF1R blockade also improved response to chemotherapy. Additionally, while treatment with single-agent Gemcitabine, CCR2i, or CSF1Ri did not alter T-cell infiltration but the combination therapy significantly improved the T cell immune response, suggesting that both tumor cell destruction and macrophage depletion are necessary to sustain cytotoxic T lymphocyte (CTL) infiltration in pancreatic ductal adenocarcinoma (PDAC). Consistent with the studies in mammary and colon cancer cells, Mitchem et al. found that TAMs can directly induce CSC properties and chemo-resistance in PDAC cells through the activation of STAT3 in-vitro and that targeting CSF1R or CCR2 decreases STAT3 signaling in-vivo. Interestingly, previous studies have demonstrated that inhibition of STAT3 signaling can increase anti-tumor immune responses by T cells, suggesting that there might be a connection between STAT3, CSCs and CTL responses. Supporting this possible connection, Mitchem et al observed that CD44+ALDHBright PDAC CSCs induce immunosuppressive behavior in TAMs in a STAT3 dependent manner. Taken together, with the observation that macrophage depletion in combination with chemo can induce CTL responses[6, 8], these data together suggest that crosstalk between TAMs and CSCs may enhance immune suppression and thus blunt anti-tumor T cell activity during chemotherapy (Figure 1). Thus the results from these three groups suggest that reprograming of the immune microenvironment might be an effective way to target CSCs and improve outcomes for high-risk patients.

Stroma-Derived Interleukin-34 Controls the Development and Maintenance of Langerhans Cells and the Maintenance of Microglia

Colony stimulating factor-1 (Csf-1) receptor and its ligand Csf-1 control macrophage development, maintenance, and function. The development of both Langerhans cells (LCs) and microglia is highly dependent on Csf-1 receptor signaling but independent ofCsf-1. Here we show that in both mice and humans, interleukin-34 (IL-34), an alternative ligand for Csf-1 receptor, is produced by keratinocytes in the epidermis and by neurons in the brain. Mice lacking IL-34 displayed a marked reduction of LCs and a decrease of microglia, whereas monocytes, dermal, and lymphoid tissue macrophages and DCs were unaffected. We identified IL-34 asa nonredundant cytokine for the development of LCs during embryogenesis as well as for their homeostasis in the adult skin. Whereas inflammation-induced repopulation ofLCs appears to be dependent on Csf-1, once inflammation is resolved, LC survival is again IL-34-dependent. In contrast, microglia and their yolk sac precursors develop independently of IL-34 but rely on it for their maintenance in the adult brain.

PSTPIP2 deficiency in mice causes osteopenia and increased differentiation of multipotent myeloid precursors into osteoclasts

AbstractMissense mutations that reduce or abrogate myeloid cell expression of the F-BAR domain protein, proline serine threonine phosphatase-interacting protein 2 (PSTPIP2), lead to autoinflammatory disease involving extramedullary hematopoiesis, skin and bone lesions. However, little is known about how PSTPIP2 regulates osteoclast development. Here we examined how PSTPIP2 deficiency causes osteopenia and bone lesions, using the mouse PSTPIP2 mutations, cmo, which fails to express PSTPIP2 and Lupo, in which PSTPIP2 is dysfunctional. In both models, serum levels of the pro-osteoclastogenic factor, MIP-1α, were elevated and CSF-1 receptor (CSF-1R)–dependent production of MIP-1α by macrophages was increased. Treatment of cmo mice with a dual specificity CSF-1R and c-Kit inhibitor, PLX3397, decreased circulating MIP-1α and ameliorated the extramedullary hematopoiesis, inflammation, and osteopenia, demonstrating that aberrant myelopoiesis drives disease. Purified osteoclast precursors from PSTPIP2-deficient mice exhibit increased osteoclastogenesis in vitro and were used to probe the structural requirements for PSTPIP2 suppression of osteoclast development. PSTPIP2 tyrosine phosphorylation and a functional F-BAR domain were essential for PSTPIP2 inhibition of TRAP expression and osteoclast precursor fusion, whereas interaction with PEST-type phosphatases was only required for suppression of TRAP expression. Thus, PSTPIP2 acts as a negative feedback regulator of CSF-1R signaling to suppress inflammation and osteoclastogenesis.

CSF-1 receptor signalling from endosomes mediates the sustained activation of Erk1/2 and Akt in macrophages

Colony stimulating factor-1 (CSF-1) mediates its pleiotropic effects on macrophages through the CSF-1 receptor (CSF-1R), a receptor tyrosine kinase. Current models of CSF-1 signalling imply that the CSF-1R activates signalling pathways exclusively at the plasma membrane and the subsequent internalisation of the CSF-1R simply facilitates its lysosomal degradation in order to prevent on-going signalling. Here, we sought to establish if the CSF-1R may in fact continue to signal following its internalisation. Erk1/2, Akt and Stat3 activation were abrogated when the internalisation of the CSF-1R was impaired, with the effects on Stat3 distinct from those for Erk1/2 and Akt. Pharmacologic inhibition of the CSF-1R following its internalisation resulted in less sustained Erk1/2 and Akt activity, whereas Stat3 activity was unaffected. Significantly, the suppressive effects of the CSF-1R inhibitor on the up-regulation of gene expression by CSF-1 (e.g. cyclin D1 and Bcl-xL gene expression) were comparable irrespective of whether the inhibitor was added prior to CSF-1 stimulation or following the internalisation of the CSF-1R. Similarly, pharmacologic inhibition of Erk1/2 (or Akt) activity either prior to CSF-1 stimulation or subsequent to CSF-1R internalisation had comparable effects on the regulation of gene expression by CSF-1. Together, our data argue that key signalling responses to CSF-1 depend on the ability of the CSF-1R to signal from endosomes following its internalisation, thus adding an important spatiotemporal aspect to CSF-1R signalling.

Abstract 3539: Anti-CSF-1R antibodies reduce tumor-associated macrophages and inhibit tumor growth in preclinical models

Abstract In cancer, increased infiltration of macrophages within and surrounding the tumor mass correlates with increased tumor invasiveness and growth. In addition, presence of tumor-associated macrophages (TAMs) has been shown to correlate with poor prognosis, particularly in breast, prostate, ovarian and cervical cancer. TAM proliferation, differentiation and survival is dependent on Colony Stimulating Factor - 1 Receptor (CSF-1R) activation, a type III integral membrane tyrosine kinase receptor selectively expressed on cells of the mononuclear phagocyte lineage. Given that TAMs enhance tumor growth and that activation of the CSF-1R pathway is required for TAM function, an antibody against mouse CSF-1R was generated for proof-of-principle studies. CS7, a monoclonal anti-mouse CSF-1R antibody inhibited both CSF-1 and IL-34 binding to mouse CSF-1R, leading to inactivation of the receptor and downstream signaling molecules. In addition, CS7 prevented monocyte proliferation and macrophage differentiation with IC50s of 0.1 nM and 0.75 nM, respectively. In murine models of breast cancer using CSF-1-secreting MDA-MB-231, Hcc1954, 4T1 or EMT6 tumor cells, CS7 treatment led to a marked reduction in TAMs and an associated decrease in tumor growth. In contrast, breast tumor xenografts with CSF-1 non-secreting breast tumor cell lines JimT1 and MCF-7 had limited or no decrease in tumor volume following CS7 treatment. In prostate models, DU145 tumor cell line xenografts (CSF-1-secreting) but not PC3 (CSF-1 non-secreting) tumor growth was inhibited by CS7, recaptitulating the results seen with breast xenografts. Thus, in breast and prostate preclinical models, CSF-1 secretion by tumor cells is a prerequisite for sensitivity to anti-CSF-1R treatment. Taken together, targeting CSF-1R with a monoclonal antibody inhibits CSF-1R signaling via CSF-1 and IL-34, prevents monocytic to macrophage differentiation, and reduces tumor volume in preclinical models, validating CSF-1R as a target for therapeutic application in cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3539. doi:1538-7445.AM2012-3539