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Folate uptake is largely mediated by folate receptor (FR)β, encoded by FOLR2 gene, in myeloid immune cells such as granulocytes, monocytes, and especially in macrophages that constitute the reticuloendothelial system (RES) and infiltrate the tumor microenvironment. Since the myeloid immune compartment dynamically changes during tumorigenesis, it is critical to assess the infiltration status of the tumors by FRβ-expressing myeloid cells to better define the targeting efficacy of folate-functionalized drug delivery systems. On the other hand, clearance by RES is a major limitation for the targeting efficacy of nanoparticles decorated with folate. Therefore, the aims of this study are (i) to determine the amount and subtypes of FRβ + myeloid cells infiltrating the tumors at different stages, (ii) to compare the amount and subtype of FRβ + myeloid cells in distinct organs of tumor-bearing and healthy animals, (iii) to test if the cancer-targeting efficacy and biodistribution of a prototypic folate-functionalized nanoparticle associates with the density of FRβ + myeloid cells. Here, we report that myeloid cell infiltration was enhanced and FRβ was upregulated at distinct stages of tumorigenesis in a mouse breast cancer model. The CD206 + subset of macrophages highly expressed FRβ, prominently both in tumor-bearing and healthy mice. In tumor-bearing mice, the amount of all myeloid cells, but particularly granulocytes, was remarkably increased in the tumor, liver, lungs, spleen, kidneys, lymph nodes, peritoneal cavity, bone marrow, heart, and brain. Compared with macrophages, the level of FRβ was moderate in granulocytes and monocytes. The density of FRβ + immune cells in the tumor microenvironment was not directly associated with the tumor-targeting efficacy of the folate-functionalized cyclodextrin nanoparticles. The lung was determined as a preferential site of accumulation for folate-functionalized nanoparticles, wherein FRβ + CD206 + macrophages significantly engulfed cyclodextrin nanoparticles. In conclusion, our results demonstrate that the tumor formation augments the FR levels and alters the infiltration and distribution of myeloid immune cells in all organs which should be considered as a major factor influencing the targeting efficacy of nanoparticles for drug delivery.

Chronic kidney disease (CKD) is a public health problem driven by myofibroblast accumulation, leading to interstitial fibrosis. Heterogeneity is a recently recognized characteristic in kidney fibroblasts in CKD, but the role of different populations is still unclear. Here, we characterize a proinflammatory fibroblast population (named CXCL-iFibro), which corresponds to an early state of myofibroblast differentiation in CKD. We demonstrate that CXCL-iFibro co-localize with macrophages in the kidney and participate in their attraction, accumulation, and switch into FOLR2+ macrophages from early CKD stages on. In vitro, macrophages promote the switch of CXCL-iFibro into ECM-secreting myofibroblasts through a WNT/β-catenin-dependent pathway, thereby suggesting a reciprocal crosstalk between these populations of fibroblasts and macrophages. Finally, the detection of CXCL-iFibro at early stages of CKD is predictive of poor patient prognosis, which shows that the CXCL-iFibro population is an early player in CKD progression and demonstrates the clinical relevance of our findings., (© 2024. The Author(s).)

Folate receptor (FR) alpha (FOLR1) and beta (FOLR2) are membrane-anchored folate transporters that are expressed at low levels in normal tissues, while their expression is strongly increased in several cancers. Intriguingly, although the function of these receptors in, for example, development and cancer has been studied intensively, their role in aging is still unknown. To address this, we utilized Caenorhabditis elegans, in which FOLR-1 is the sole ortholog of folate receptors. We found that the loss of FOLR-1 does not affect reproduction, physical condition, proteostasis or lifespan, indicating that it is not required for folate transport to maintain health. Interestingly, we found that FOLR-1 is detectably expressed only in uterine-vulval cells, and that the histone-binding protein LIN-53 inhibits its expression in other tissues. Furthermore, whereas knockdown of lin-53 is known to shorten lifespan, we found that the loss of FOLR-1 partially rescues this phenotype, suggesting that elevated folr-1 expression is detrimental for health. Indeed, our data demonstrate that overexpression of folr-1 is toxic, and that this phenotype is dependent on diet. Altogether, this work could serve as a basis for further studies to elucidate the organismal effects of abnormal FR expression in diseases such as cancer., (© 2024. The Author(s).)

Background: Epigenetic alterations contribute greatly to the development and progression of colorectal cancer, and effect of aberrant miR-622 expression is still controversial. This study aimed to discover miR-622 regulation in CRC proliferation., Methods: miR-622 expression and prognosis were analyzed in clinical CRC samples from Nanfang Hospital. miR-622 regulation on cell cycle and tumor proliferation was discovered, and FOLR2 was screened as functional target of miR-622 using bioinformatics analysis, which was validated via dual luciferase assay and gain-of-function and loss-of-function experiments both in vitro and in vivo., Results: miR-622 overexpression in CRC indicated unfavorable prognosis and it regulated cell cycle to promote tumor growth both in vitro and in vivo. FOLR2 is a specific, functional target of miR-622, which negatively correlates with signature genes in cell cycle process to promote CRC proliferation., Conclusions: miR-622 upregulates cell cycle process by targeting FOLR2 to promote CRC proliferation, proposing a novel mechanism and treatment target in CRC epigenetic regulation of miR-622., (© 2024. The Author(s).)

Though firstly identified in cerebral folate deficiency, autoantibodies against folate receptors (FRAbs) have been implicated in pregnancy complications such as miscarriage; however, the underlying mechanism needs to be further elaborated. FRAbs can be produced via sensitization mediated by folate-binding protein as well as gene mutation, aberrant modulation, or degradation of folate receptors (FRs). FRAbs may interfere with folate internalization and metabolism through blocking or binding with FRs. Interestingly, different types of FRs are expressed on trophoblast cells, decidual epithelium or stroma, and macrophages at the maternal-fetal interface, implying FRAbs may be involved in the critical events necessary for a successful pregnancy. Thus, we propose that FRAbs may disturb pregnancy establishment and maintenance by modulating trophoblastic biofunctions, placental development, decidualization, and decidua homeostasis as well as the functions of FOLR2 + macrophages. In light of these findings, FRAbs may be a critical factor in pathological pregnancy, and deserve careful consideration in therapies involving folic acid supplementation for pregnancy complications., Competing Interests: The authors declare no conflicts of interest.

Objectives: Roux-en-Y gastric bypass (RYGB) promotes sustained weight loss, and the resulting new gastrointestinal anatomy can contribute to nutritional depletions. Folate deficiency is one of the most frequently observed nutritional deficiencies after RYGB. The aim of this study was to assess whether RYGB affects the expression of genes related to the intestinal folate metabolism pathway as an additional molecular mechanism contributing to its postoperative deficiency., Methods: Biopsies from the duodenum, jejunum, and ileum of 20 obese women were collected before and 3 mo after RYGB. The expression of genes involved in intestinal folate metabolism was assessed by microarray and reverse transcriptase polymerase chain reaction (RT-qPCR). Folate intake (7-d food record) and plasma levels (electrochemiluminescence) also were measured., Results: Compared with the preoperative phase, transcriptomic alterations were observed in all intestinal segments studied after RYBG, mainly marked by decreased expression of genes encoding folate transporters/receptors and increased expression of genes involved in folate biosynthesis (P < 0.05). Reduced folate intake and plasma folate levels were also observed simultaneously (P < 0.05). Plasma folate concentrations correlated inversely with intestinal FOLR2 and SHMT2 genes (P < 0.001)., Conclusion: The present findings suggested that impaired expression of genes related to intestinal folate metabolism may contribute to the early systemic deficiency after RYGB and highlight a potential transcriptomic reprogramming of the intestine in response to RYGB to compensate for folate depletion induced by this surgical technique., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Interstitial macrophages (IMs) reside in the lung tissue surrounding key structures including airways, vessels, and alveoli. Recent work has described IM heterogeneity during homeostasis, however, there are limited data on IMs during inflammation. We sought to characterize IM origin, subsets, and transcriptomic profiles during homeostasis and lipopolysaccharide (LPS) induced acute lung inflammation. During homeostasis, we used three complementary methods, spectral flow cytometry, single-cell RNA-sequencing, and gene regulatory network enrichment, to demonstrate that IMs can be divided into two core subsets distinguished by surface and transcriptional expression of folate receptor β ( Folr2 /FRβ). These subsets inhabited distinct niches within the lung interstitium. Within FRβ + IMs we identified a subpopulation marked by coexpression of LYVE1. During acute LPS-induced inflammation, lung IM numbers expand. Lineage tracing revealed IM expansion was due to recruitment of monocyte-derived IMs. At the peak of inflammation, recruited IMs were comprised two unique subsets defined by expression of genes associated with interferon signaling and glycolytic pathways. As recruited IMs matured, they adopted the overall transcriptional state of FRβ - resident IMs but retained expression in several origin-specific genes, such as IL-1β. FRβ + IMs were of near-pure resident origin. Taken together our data show that during LPS-induced inflammation, there are distinct populations of IMs that likely have unique functions. FRΒ + IMs comprise a stable, resident population, whereas FRβ - ΙΜs represent a mixed population of resident and recruited IMs.

Knowledge of the transcriptional programs underpinning the functions of human kidney cell populations at homeostasis is limited. We present a single-cell perspective of healthy human kidney from 19 living donors, with equal contribution from males and females, profiling the transcriptome of 27677 cells to map human kidney at high resolution. Sex-based differences in gene expression within proximal tubular cells were observed, specifically, increased anti-oxidant metallothionein genes in females and aerobic metabolism-related genes in males. Functional differences in metabolism were confirmed in proximal tubular cells, with male cells exhibiting higher oxidative phosphorylation and higher levels of energy precursor metabolites. We identified kidney-specific lymphocyte populations with unique transcriptional profiles indicative of kidney-adapted functions. Significant heterogeneity in myeloid cells was observed, with a MRC1 + LYVE1 + FOLR2 + C1QC + population representing a predominant population in healthy kidney. This study provides a detailed cellular map of healthy human kidney, and explores the complexity of parenchymal and kidney-resident immune cells., (© 2022. The Author(s).)

TIM4 has previously been associated with antitumor immunity, yet the pattern of expression and the function of this receptor across human cancer tissues remain poorly explored. Here we combined extensive immunolabeling of human tissues with in silico analysis of pan-cancer transcriptomic data sets to explore the clinical significance of TIM4 expression. Our results unveil that TIM4 is expressed on a fraction of cavity macrophages (CATIM4+MΦ) of carcinoma patients. Moreover, we uncover a high expression of TIM4 on macrophages of the T-cell zone of the carcinoma-associated tertiary lymphoid structures (TLSTIM4+MΦ). In silico analysis of a pan-cancer data set revealed a positive correlation between TIM4 expression and markers of B cells, effector CD8+ T cells, and a 12-chemokine signature defining tertiary lymphoid structure. In addition, TLSTIM4+MΦ were enriched in cancers displaying microsatellite instability and high CD8+ T-cell infiltration, confirming their association with immune-reactive tumors. Both CATIM4+MΦ and TLSTIM4+MΦ express FOLR2, a marker of tissue-resident MΦ. However, CATIM4+MΦ had a higher expression of the immunosuppressive molecules TREM2, IL10, and TGFβ as compared with TLSTIM4+MΦ. By analyzing a scRNA sequence data set of tumor-associated myeloid cells, we identified two TIM4+FOLR2+ clusters coherent with CATIM4+MΦ and TLSTIM4+MΦ. We defined specific gene signatures for each subset and found that the CATIM4+ MΦ signature was associated with worse patient survival. In contrast, TLSTIM4+MΦ gene signature positively correlates with a better prognosis. Together, these data illustrate that TIM4 marks two distinct macrophage populations with distinct phenotypes and tissue localization and that may have opposing roles in tumor immunity., (©2022 American Association for Cancer Research.)

Background: Hypomethylation of long interspersed nuclear element 1 (LINE-1) is characteristic of various cancer types, including colorectal cancer (CRC). Malfunction of several factors or alteration of methyl-donor molecules' (folic acid and S-adenosylmethionine) availability can contribute to DNA methylation changes. Detection of epigenetic alterations in liquid biopsies can assist in the early recognition of CRC. Following the investigations of a Hungarian colon tissue sample set, our goal was to examine the LINE-1 methylation of blood samples along the colorectal adenoma-carcinoma sequence and in inflammatory bowel disease. Moreover, we aimed to explore the possible underlying mechanisms of global DNA hypomethylation formation on a multi-level aspect., Methods: LINE-1 methylation of colon tissue (n = 183) and plasma (n = 48) samples of healthy controls and patients with colorectal tumours were examined with bisulfite pyrosequencing. To investigate mRNA expression, microarray analysis results were reanalysed in silico (n = 60). Immunohistochemistry staining was used to validate DNA methyltransferases (DNMTs) and folate receptor beta (FOLR2) expression along with the determination of methyl-donor molecules' in situ level (n = 40)., Results: Significantly decreased LINE-1 methylation level was observed in line with cancer progression both in tissue (adenoma: 72.7 ± 4.8%, and CRC: 69.7 ± 7.6% vs. normal: 77.5 ± 1.7%, p ≤ 0.01) and liquid biopsies (adenoma: 80.0 ± 1.7%, and CRC: 79.8 ± 1.3% vs. normal: 82.0 ± 2.0%, p ≤ 0.01). However, no significant changes were recognized in inflammatory bowel disease cases. According to in silico analysis of microarray data, altered mRNA levels of several DNA methylation-related enzymes were detected in tumours vs. healthy biopsies, namely one-carbon metabolism-related genes-which met our analysing criteria-showed upregulation, while FOLR2 was downregulated. Using immunohistochemistry, DNMTs, and FOLR2 expression were confirmed. Moreover, significantly diminished folic acid and S-adenosylmethionine levels were observed in parallel with decreasing 5-methylcytosine staining in tumours compared to normal adjacent to tumour tissues (p ≤ 0.05)., Conclusion: Our results suggest that LINE-1 hypomethylation may have a distinguishing value in precancerous stages compared to healthy samples in liquid biopsies. Furthermore, the reduction of global DNA methylation level could be linked to reduced methyl-donor availability with the contribution of decreased FOLR2 expression., (© 2022. The Author(s).)

Non-invasive imaging modalities constitute an increasingly important tool in diagnostic and therapy response monitoring of patients with autoimmune diseases, including rheumatoid arthritis (RA). In particular, macrophage imaging with positron emission tomography (PET) using novel radiotracers based on differential expression of plasma membrane proteins and functioning of cellular processes may be suited for this. Over the past decade, selective expression of folate receptor β (FRβ), a glycosylphosphatidylinositol-anchored plasma membrane protein, on myeloid cells has emerged as an attractive target for macrophage imaging by exploiting the high binding affinity of folate-based PET tracers. This work discusses molecular, biochemical and functional properties of FRβ, describes the preclinical development of a folate-PET tracer and the evaluation of this tracer in a translational model of arthritis for diagnostics and therapy-response monitoring, and finally the first clinical application of the folate-PET tracer in RA patients with active disease. Consequently, folate-based PET tracers hold great promise for macrophage imaging in a variety of (chronic) inflammatory (autoimmune) diseases beyond RA., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Steinz, Ezdoglian, Khodadust, Molthoff, Srinivasarao, Low, Zwezerijnen, Yaqub, Beaino, Windhorst, Tas, Jansen and van der Laken.)

Objective: Folate receptor beta (FR-β) has been used as a clinical marker and target in multiple inflammatory diseases, including osteoarthritis (OA) and rheumatoid arthritis (RA). However, the conditions under which FR-β + macrophages arise remain unclear and could be affected by corticosteroids. Therefore, we studied FR-β expression in vitro in macrophage subtypes and determined their response to triamcinolone acetonide (TA), a clinically often-used corticosteroid., Design: Human monocyte-derived macrophages were differentiated to the known M0, M1, or M2 macrophage phenotypes. The phenotype and FR-β expression and plasticity of the macrophage subtypes were determined using flow cytometry, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and enzyme-linked immunosorbent assay (ELISA)., Results: FR-β expression was low in granulocyte-macrophage colony-stimulating factor (GM-CSF)-generated (M1-like) macrophages and high in macrophage colony-stimulating factor (M-CSF)-generated (M0 and M2-like) macrophages. FR-β expression remained high once the M0 or M2 macrophages were stimulated with pro-inflammatory stimuli (interferon-γ plus lipopolysaccharide) to induce M1-like macrophages. On the contrary, anti-inflammatory TA treatment skewed GM-CSF macrophage differentiation toward an M2 and FR-β + phenotype., Conclusions: As corticosteroids skewed monocytes toward an FR-β-expressing, anti-inflammatory phenotype, even in an M1 priming GM-CSF environment, FR-β has potential as a biomarker to monitor success of treatment with corticosteroids. Without corticosteroid treatment, M-CSF alone induces high FR-β expression which remains high under pro-inflammatory conditions. This explains why pro-inflammatory FR-β + macrophages (exposed to M-CSF) are observed in arthritis patients and correlate with disease severity.

The placenta is a fetal-derived organ whose function is crucial for both maternal and fetal health. The human placenta contains a population of fetal macrophages termed Hofbauer cells. These macrophages play diverse roles, aiding in placental development, function and defence. The outer layer of the human placenta is formed by syncytiotrophoblast cells, that fuse to form the syncytium. Adhered to the syncytium at sites of damage, on the maternal side of the placenta, is a population of macrophages termed placenta associated maternal macrophages (PAMM1a). Here we discuss recent developments that have led to renewed insight into our understanding of the ontogeny, phenotype and function of placental macrophages. Finally, we discuss how the application of new technologies within placental research are helping us to further understand these cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Thomas, Naidu, Appios and McGovern.)

Activated macrophages overexpress the folate receptor β (FR-β) that can be used for targeted delivery of drugs conjugated to folic acid. FR-expressing macrophages contribute to arthritis progression by secreting prostaglandin E 2 (PGE 2 ). Non-steroidal anti-inflammatory drugs (NSAIDs) block PGs and thromboxane by inhibiting the cyclooxygenase (COX) enzymes and are used for chronic pain and inflammation despite their well-known toxicity. New NSAIDs target an enzyme downstream of COXs, microsomal prostaglandin E synthase-1 (mPGES-1). Inhibition of mPGES-1 in inflammatory macrophages promises to retain NSAID efficacy while limiting toxicity. We conjugated a potent mPGES-1 inhibitor, MK-7285, to folate, but the construct released the drug inefficiently. Folate conjugation to the primary alcohol of MK-7285 improved the construct's stability and the release of free drug. Surprisingly, the drug-folate conjugate potentiated PGE 2 in FR-positive KB cells, and reduced PGE 2 in macrophages independently of the FR. Folate conjugation of NSAIDs is not an optimal strategy for targeting of macrophages., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Since activated macrophages express a functional folate receptor β (FRβ), targeting this macrophage population with folate-linked drugs could increase selectivity to treat inflammatory diseases. Using a macrophage-mediated anti-glomerular basement membrane (anti-GBM) glomerulonephritis (GN) in WKY rats, we investigated the effect of a novel folic acid-aminopterin (AMT) conjugate (EC2319) designed to intracellularly deliver AMT via the FR. We found that treatment with EC2319 significantly attenuated kidney injury and preserved renal function. Kidney protection with EC2319 was blocked by a folate competitor, indicating that its mechanism of action was specifically FRβ-mediated. Notably, treatment with methotrexate (MTX), another folic acid antagonist related to AMT, did not protect from kidney damage. EC2319 reduced glomerular and interstitial macrophage infiltration and decreased M1 macrophage recruitment but not M2 macrophages. The expression of CCL2 and the pro-fibrotic cytokine TGF-β were also reduced in nephritic glomeruli with EC2319 treatment. In EC2319-treated rats, there was a significant decrease in the deposition of collagens. In nephritic kidneys, FRβ was expressed on periglomerular macrophages and macrophages present in the crescents, but its expression was not observed in normal kidneys. These data indicate that selectively targeting the activated macrophage population could represent a novel means for treating anti-GBM GN and other acute crescentic glomerulonephritis.

Activated macrophages contribute prominently to the progression and maintenance of almost all inflammatory and autoimmune diseases. Although non-specific elimination of these phagocytes has been shown to treat animal models of inflammatory disease, the same therapies have been compromised by unacceptable toxicities, because they also kill quiescent macrophages in healthy tissues. In the studies below, we exploit upregulation of folate receptor beta (FRβ) on inflammatory (but not resting) macrophages to target a cytotoxic drug selectively to the inflammatory subset of macrophages. Because many of these activated macrophages are nondividing, we also employ verrucarin A as the cytotoxic payload, since it kills both mitotic and nonmitotic cells by blocking protein synthesis. By inserting a redox-sensitive self-immolative linker between the folate and verrucarin A, we further assure that release of unmodified verrucarin A is triggered primarily after internalization by an FRβ-positive cell. The resulting folate-verrucarin A conjugate is shown to kill FR-expressing cells in vitro in a manner that can be inhibited by competition with 100-fold excess folic acid. The folate-verrucarin A conjugate is also shown to successfully treat a murine model of inflammatory peritonitis by eliminating inflammatory macrophages without killing other cells in the same peritonitis fluid. Based on this high specificity for inflammatory macrophages, we conclude that folate-verrucarin A warrants continued exploration as a potential therapy for inflammatory and autoimmune diseases in humans., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The immunosuppressive tumor microenvironment (TME) represents a major barrier for effective immunotherapy. Tumor-associated macrophages (TAMs) are highly heterogeneous and plastic cell components of the TME which can either promote tumor progression (M2-like) or boost antitumor immunity (M1-like). Here, we demonstrate that a subset of TAMs that express folate receptor β (FRβ) possess an immunosuppressive M2-like profile. In syngeneic tumor mouse models, chimeric antigen receptor (CAR)-T cell-mediated selective elimination of FRβ + TAMs in the TME results in an enrichment of pro-inflammatory monocytes, an influx of endogenous tumor-specific CD8 + T cells, delayed tumor progression, and prolonged survival. Preconditioning of the TME with FRβ-specific CAR-T cells also improves the effectiveness of tumor-directed anti-mesothelin CAR-T cells, while simultaneous co-administration of both CAR products does not. These results highlight the pro-tumor role of FRβ + TAMs in the TME and the therapeutic implications of TAM-depleting agents as preparative adjuncts to conventional immunotherapies that directly target tumor antigens.

Although immunotherapies of tumors have demonstrated promise for altering the progression of malignancies, immunotherapies have been limited by an immunosuppressive tumor microenvironment (TME) that prevents infiltrating immune cells from performing their anticancer functions. Prominent among immunosuppressive cells are myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM) that inhibit T cells via release of immunosuppressive cytokines and engagement of checkpoint receptors. Here, we explore the properties of MDSCs and TAMs from freshly isolated mouse and human tumors and find that an immunosuppressive subset of these cells can be distinguished from the nonimmunosuppressive population by its upregulation of folate receptor beta (FRβ) within the TME and its restriction to the TME. This FRβ + subpopulation could be selectively targeted with folate-linked drugs. Delivery of a folate-targeted TLR7 agonist to these cells (i) reduced their immunosuppressive function, (ii) increased CD8 + T-cell infiltration, (iii) enhanced M1/M2 macrophage ratios, (iv) inhibited tumor growth, (v) blocked tumor metastasis, and (vi) improved overall survival without demonstrable toxicity. These data reveal a broadly applicable strategy across tumor types for reprogramming MDSCs and TAMs into antitumorigenic immune cells using a drug that would otherwise be too toxic to administer systemically. The data also establish FRβ as the first marker that distinguishes immunosuppressive from nonimmunosuppressive subsets of MDSCs and TAMs. Because all solid tumors accumulate MDSCs and TAMs, a general strategy to both identify and reprogram these cells should be broadly applied in the characterization and treatment of multiple tumors. SIGNIFICANCE: FRβ serves as both a means to identify and target MDSCs and TAMs within the tumor, allowing for delivery of immunomodulatory compounds to tumor myeloid cells in a variety of cancers., (©2020 American Association for Cancer Research.)

Background: Activated macrophages in the experimental model of multiple sclerosis (MS) express folate receptor-β (FR-β), representing a promising target for the treatment of MS. Here, we both evaluated the efficacy of a novel folate-aminopterin construct (EC2319) in a rat focal model of multiple sclerosis (MS) and investigated the utility of 68 Ga-labeled 1,4,7-triazacyclononane-1,4,7-triacetic acid-conjugated folate ( 68 Ga-FOL) for assessing inflammatory lesions. In addition, we investigated whether FR-β is expressed in the brain of patients with MS., Methods: Focal delayed-type hypersensitivity experimental autoimmune encephalomyelitis (fDTH-EAE) was induced in 40 Lewis rats; 20 healthy Lewis rats were used as controls. Rats were divided into six groups according to the duration of disease (control, acute, or chronic) and intervention (vehicle versus EC2319). 68 Ga-FOL analyses, histology, and immunofluorescence of the brain were performed to evaluate the efficacy of subcutaneously administered EC2319 on lesion development. Immunofluorescence was used to assess FR-β expression in postmortem brain samples from 5 patients with MS and 5 healthy controls., Results: Immunofluorescence and histological analyses revealed significant reductions in FR-β expression (P < 0.05) and lesion size (P < 0.01), as well as improved inducible nitric oxide synthase/mannose receptor C type 1 ratios (P < 0.01) in macrophages and microglia during the chronic but not acute phase of fDTH-EAE in EC2319-treated rats. The uptake of IV-injected 68 Ga-FOL in the brain was low and did not differ between the groups, but the in vitro binding of 68 Ga-FOL was significantly lower in EC2319-treated rats (P < 0.01). FR-β positivity was observed in chronically active lesions and in normal-appearing white matter in MS brain samples., Conclusions: EC2319 was well tolerated and attenuated inflammation and lesion development in a rat model of a chronic progressive form of MS. Human MS patients have FR-β-positive cells in chronically active plaques, which suggests that these results may have translational relevance.

Currently available imaging techniques have limited specificity for the detection of active myocardial inflammation. Aluminum 18 F-labeled 1,4,7-triazacyclononane- N,N',N″ -triacetic acid conjugated folate ( 18 F-FOL) is a PET tracer targeting folate receptor β (FR-β), which is expressed on activated macrophages at sites of inflammation. We evaluated 18 F-FOL PET for the detection of myocardial inflammation in rats with autoimmune myocarditis and studied the expression of FR-β in human cardiac sarcoidosis specimens. Methods: Myocarditis was induced by immunizing rats ( n = 18) with porcine cardiac myosin in complete Freund adjuvant. Control rats ( n = 6) were injected with Freund adjuvant alone. 18 F-FOL was intravenously injected, followed by imaging with a small-animal PET/CT scanner and autoradiography. Contrast-enhanced high-resolution CT or 18 F-FDG PET images were used for coregistration. Rat tissue sections and myocardial autopsy samples from 6 patients with cardiac sarcoidosis were studied for macrophages and FR-β. Results: The myocardium of 10 of 18 immunized rats showed focal macrophage-rich inflammatory lesions, with FR-β expression occurring mainly in M1-polarized macrophages. PET images showed focal myocardial 18 F-FOL uptake colocalizing with inflammatory lesions (SUV mean , 2.1 ± 1.1), whereas uptake in the remote myocardium of immunized rats and controls was low (SUV mean , 0.4 ± 0.2 and 0.4 ± 0.1, respectively; P < 0.01). Ex vivo autoradiography of tissue sections confirmed uptake of 18 F-FOL in myocardial inflammatory lesions. Uptake of 18 F-FOL in inflamed myocardium was efficiently blocked by a nonlabeled FR-β ligand folate glucosamine in vivo. The myocardium of patients with cardiac sarcoidosis showed many FR-β-positive macrophages in inflammatory lesions. Conclusion: In a rat model of autoimmune myocarditis, 18 F-FOL shows specific uptake in inflamed myocardium containing macrophages expressing FR-β, which were also present in human cardiac sarcoid lesions. Imaging of FR-β expression is a potential approach for the detection of active myocardial inflammation., (© 2020 by the Society of Nuclear Medicine and Molecular Imaging.)

We employed scRNA sequencing to extensively characterize the cellular landscape of human liver from development to disease. Analysis of ∼212,000 cells representing human fetal, hepatocellular carcinoma (HCC), and mouse liver revealed remarkable fetal-like reprogramming of the tumor microenvironment. Specifically, the HCC ecosystem displayed features reminiscent of fetal development, including re-emergence of fetal-associated endothelial cells (PLVAP/VEGFR2) and fetal-like (FOLR2) tumor-associated macrophages. In a cross-species comparative analysis, we discovered remarkable similarity between mouse embryonic, fetal-liver, and tumor macrophages. Spatial transcriptomics further revealed a shared onco-fetal ecosystem between fetal liver and HCC. Furthermore, gene regulatory analysis, spatial transcriptomics, and in vitro functional assays implicated VEGF and NOTCH signaling in maintaining onco-fetal ecosystem. Taken together, we report a shared immunosuppressive onco-fetal ecosystem in fetal liver and HCC. Our results unravel a previously unexplored onco-fetal reprogramming of the tumor ecosystem, provide novel targets for therapeutic interventions in HCC, and open avenues for identifying similar paradigms in other cancers and disease., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

This review surveys soluble Folate Receptors (FOLRs) in humans. FOLR1 and FOLR2 are equipped with cellular glycosylphosphatidylinositol (GPI) anchors. FOLR1 is secreted from epithelia with or without a micelle-encapsulated GPI-anchor into milk and other body fluids/secretions, e.g. semen where its interaction with spermatozoa indicates a role in male fertility. FOLR1 and FOLR2 serve as serum biomarkers of various diseases. FOLR3 possesses no GPI-anchor and originates from secretory granules of neutrophil granulocytes; its concentration in serum correlates to the FOLR3 content in leukocytes and rises with increased leukocyte counts (infection, malignancy and pregnancy). FOLR3 exerts anti-microbial and anti-tumor effects by depriving bacteria and tumor cells of natural folates. Megalin receptors mediate reabsorption of ultrafiltered folate-bound FOLR into cells of proximal kidney tubules and of folate-bound FOLR uptake in growing embryos. Megalin receptors overexpressed in malignant tumors could be suitable therapeutic targets for folate-conjugated cytotoxic agents utilizing soluble FOLRs as vectors., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Folate receptor β (FR-β), a marker expressed on macrophages, is a promising target for imaging of inflammation. Here, we report the radiosynthesis and preclinical evaluation of [ 68 Ga]Ga-NOTA-folate ( 68 Ga-FOL). After determining the affinity of 68 Ga-FOL using cells expressing FR-β, we studied atherosclerotic mice with 68 Ga-FOL and 18 F-FDG PET/CT. In addition, we studied tracer distribution and co-localization with macrophages in aorta cryosections using autoradiography, histology, and immunostaining. The specificity of 68 Ga-FOL was assessed in a blocking study with folate glucosamine. As a final step, human radiation doses were extrapolated from rat PET data. We were able to produce 68 Ga-FOL with high radiochemical purity and moderate molar activity. Cell binding studies revealed that 68 Ga-FOL had 5.1 nM affinity for FR-β. Myocardial uptake of 68 Ga-FOL was 20-fold lower than that of 18 F-FDG. Autoradiography and immunohistochemistry of the aorta revealed that 68 Ga-FOL radioactivity co-localized with Mac-3-positive macrophage-rich atherosclerotic plaques. The plaque-to-healthy vessel wall ratio of 68 Ga-FOL was significantly higher than that of 18 F-FDG. Blocking studies verified that 68 Ga-FOL was specific for FR. Based on estimations from rat data, the human effective dose was 0.0105 mSv/MBq. Together, these findings show that 68 Ga-FOL represents a promising new FR-β-targeted tracer for imaging macrophage-associated inflammation.

Tumor-targeted 6-substituted pyrrolo[2,3-d]pyrimidine benzoyl compounds based on 2 were isosterically modified at the 4-carbon bridge by replacing the vicinal (C11) carbon by heteroatoms N (4), O (5) or S (6), or with an N-substituted formyl (7), trifluoroacetyl (8) or acetyl (9). Replacement with sulfur (6) afforded the most potent KB tumor cell inhibitor, ~6-fold better than the parent 2. In addition, 6 retained tumor transport selectivity via folate receptor (FR) α and -β over the ubiquitous reduced folate carrier (RFC). FRα-mediated cell inhibition for 6 was generally equivalent to 2, while the FRβ-mediated activity was improved by 16-fold over 2. N (4) and O (5) substitutions afforded similar tumor cell inhibitions as 2, with selectivity for FRα and -β over RFC. The N-substituted analogs 7-9 also preserved transport selectivity for FRα and -β over RFC. For FRα-expressing CHO cells, potencies were in the order of 8 > 7 > 9. Whereas 8 and 9 showed similar results with FRβ-expressing CHO cells, 7 was ~16-fold more active than 2. By nucleoside rescue experiments, all the compounds inhibited de novo purine biosynthesis, likely at the step catalyzed by glycinamide ribonucleotide formyltransferase. Thus, heteroatom replacements of the CH 2 in the bridge of 2 afford analogs with increased tumor cell inhibition that could provide advantages over 2, as well as tumor transport selectivity over clinically used antifolates including methotrexate and pemetrexed., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

As macrophages exhibit a huge functional plasticity under homeostasis and pathological conditions, they have become a therapeutic target for chronic inflammatory diseases. Hence, the identification of macrophage subset-specific markers is a requisite for the development of macrophage-directed therapeutic interventions. In this regard, the macrophage-specific Folate Receptor β (FRβ, encoded by the FOLR2 gene) has been already validated as a target for molecular delivery in cancer as well as in macrophage-targeting therapeutic strategies for chronic inflammatory pathologies. We now show that the transcriptome of human macrophages from healthy and inflamed tissues (tumor; rheumatoid arthritis, RA) share a significant over-representation of the "anti-inflammatory gene set", which defines the gene profile of M-CSF-dependent IL-10-producing human macrophages (M-MØ). More specifically, FOLR2 expression has been found to strongly correlate with the expression of M-MØ-specific genes in tissue-resident macrophages, tumor-associated macrophages (TAM) and macrophages from inflamed synovium, and also correlates with the presence of the PU.1 transcription factor. In fact, PU.1-binding elements are found upstream of the first exon of FOLR2 and most M-MØ-specific- and TAM-specific genes. The functional relevance of PU.1 binding was demonstrated through analysis of the proximal regulatory region of the FOLR2 gene, whose activity was dependent on a cluster of PU.1-binding sequences. Further, siRNA-mediated knockdown established the importance of PU.1 for FOLR2 gene expression in myeloid cells. Therefore, we provide evidence that FRβ marks tissue-resident macrophages as well as macrophages within inflamed tissues, and its expression is dependent on PU.1.

Non-invasive imaging of arthritis activity in rheumatoid arthritis (RA) patients using macrophage PET holds promise for early diagnosis and therapeutic response monitoring. Previously obtained results with macrophage tracer (R)-[ 11 C]PK11195 were encouraging, but the imaging signal could be further improved by reduction of background uptake. Recently, the novel macrophage tracer [ 18 F]fluoro-PEG-folate was developed. This tracer showed excellent targeting of the folate receptor β on activated macrophages in synovial tissue in a preclinical arthritic rat model. We performed three substudies to investigate the biodistribution, potential for imaging arthritis and kinetic properties of [18F]fluoro-PEG-folate in RA patients. Firstly, biodistribution demonstrated fast clearance of [ 18 F]fluoro-PEG-folate from heart and blood vessels and no dose limiting uptake in organs. Secondly, [ 18 F]fluoro-PEG-folate showed uptake in arthritic joints with significantly lower background and hence significantly higher target-to-background ratios as compared to reference macrophage tracer (R)-[ 11 C]PK11195. Lastly, dynamic scanning demonstrated fast tracer uptake in affected joints, reaching a plateau after 1 minute, co-existing with a rapid blood clearance. In conclusion, this first in man study demonstrates the potential of [ 18 F]fluoro-PEG-folate to image arthritis activity in RA with favourable imaging characteristics of rapid clearance and low background uptake, that allow for detection of inflammatory activity in the whole body.

Background: Folate receptor-β (FR-β) is a cell surface receptor that is significantly upregulated on activated macrophages during inflammation and provides a potential target for folate-based therapeutic and diagnostic agents. FR-β expression in central nervous system inflammation remains relatively unexplored. Therefore, we used focally induced acute and chronic phases of experimental autoimmune encephalomyelitis (EAE) to study patterns of FR-β expression and evaluated its potential as an in vivo imaging target., Methods: Focal EAE was induced in rats using heat-killed Bacillus Calmette-Guérin followed by activation with complete Freund's adjuvant supplemented with Mycobacterium tuberculosis. The rats were assessed with magnetic resonance imaging and positron emission tomography/computed tomography (PET/CT) at acute (14 days) and chronic (90 days) phases of inflammation. The animals were finally sacrificed for ex vivo autoradiography of their brains. PET studies were performed using FR-β-targeting aluminum [ 18 F]fluoride-labeled 1,4,7-triazacyclononane-1,4,7-triacetic acid conjugated folate ([ 18 F]AlF-NOTA-folate, 18 F-FOL) and 18 kDa translocator protein (TSPO)-targeting N-acetyl-N-(2-[ 11 C]methoxybenzyl)-2-phenoxy-5-pyridinamine ( 11 C-PBR28). Post-mortem immunohistochemistry was performed using anti-FR-β, anti-cluster of differentiation 68 (anti-CD68), anti-inducible nitric oxide synthase (anti-iNOS), and anti-mannose receptor C-type 1 (anti-MRC-1) antibodies. The specificity of 18 F-FOL binding was verified using in vitro brain sections with folate glucosamine used as a blocking agent., Results: Immunohistochemical evaluation of focal EAE lesions demonstrated anti-FR-β positive cells at the lesion border in both acute and chronic phases of inflammation. We found that anti-FR-β correlated with anti-CD68 and anti-MRC-1 immunohistochemistry; for MRC-1, the correlation was most prominent in the chronic phase of inflammation. Both 18 F-FOL and 11 C-PBR28 radiotracers bound to the EAE lesions. Autoradiography studies verified that this binding took place in areas of anti-FR-β positivity. A blocking assay using folate glucosamine further verified the tracer's specificity. In the chronic phase of EAE, the lesion-to-background ratio of 18 F-FOL was significantly higher than that of 11 C-PBR28 (P = 0.016)., Conclusion: Our EAE results imply that FR-β may be a useful target for in vivo imaging of multiple sclerosis-related immunopathology. FR-β-targeted PET imaging with 18 F-FOL may facilitate the monitoring of lesion development and complement the information obtained from TSPO imaging by bringing more specificity to the PET imaging armamentarium for neuroinflammation.

Montmorillonite Clay (MMT) is aimed to develop as an orally administrable drug delivery vehicle with enhanced efficacy. Aiming to enhance the therapeutic index of methotrexate, curcumin is concomitantly used with methotrexate in the present study. Being folate antagonist in nature, methotrexate is internalized into cells by folate receptor (FR); which is over-expressed in certain human cancer cells such as cervical carcinoma cells (HeLa). Firstly, montmorillonite Clay (MMT) is organically modified (OMMT) with cetyl trimethyl ammonium bromide (CTAB) and used to intercalate curcumin and methotrexate separately, designated as OMMT-Cur and OMMT-MTX, respectively. XRD pattern demonstrated successful intercalation of therapeutics and an increase in clay interlayer distance facilitated by CTAB. The dissolution kinetics of methotrexate follows Higuchi model for both Simulated Gastric Fluid (SGF) and Simulated Intestinal Fluid (SIF), while the release kinetics for curcumin fitted into Higuchi model for SGF and Hixson-Crowell model for SIF, respectively. OMMT-MTX are able to discriminate FR-positive HeLa cells from FR-negative breast cancer cells (MCF7); irrespective of alike cellular phenotypes. Further, the pre-treatment of HeLa cells with curcumin improves its sensitivity towards methotrexate causing a greater killing of the Hela cells. Together, the results propose the concomitant use of curcumin and methotrexate for successfully targeting highly invasive FR-positive carcinomas by means of folate receptor using MMTs., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Inflammation prevailing conditions delay healing processes of damaged tissues, leading to a functional impairment. Although anti-inflammatory drugs are clinically available, they often cause unwanted side effects thus being considered suboptimal. Here we report drug-free synthetic nanoparticles that target and internalize pro-inflammatory cells and release ions, ultimately demonstrating profound anti-inflammatory functions. We introduce folate-functionalized bioactive glass nanoparticle BGN(F) that can bind to pro-inflammatory cells to endocytose and release ions. The folate-conjugation significantly enhanced the nanoparticle internalization to LPS-induced pro-inflammatory cells. The direct treatment of BGN(F) at proper doses (80-160 μg/mL) substantially down-regulated pro-inflammatory molecules, including TNF-α, IL-6, iNOS and COX-2, at both gene and protein levels. The phosphorylation of intracellular signaling molecules involved in the inflammatory events, such as p38 MAPK, ERK (1/2), SAPK/JANK, IκBα, and NF-κB, were significantly suppressed by the BGN(F) treatment. Furthermore, BGN(F) was potential to switch the macrophage polarization from M1 to M2. The released ions, not the physical interactions, of nanoparticles were observed to contribute in major part to the anti-inflammatory actions of BGN(F). The BGN(F), when locally administered to a Notexin-induced myoinjury tissue in mice, significantly down-regulated IL-6 and TNF-α, switched the macrophage phenotype from M1 to M2, and accelerated tissue healing. The current findings that demonstrate profound anti-inflammatory actions of BGN(F) in vitro and in vivo support their uses as novel drug-free nanotherapeutic platform for the treatment of inflamed tissues., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Introduction: Nonalcoholic steatohepatitis (NASH) afflicts 20-36% of individuals with nonalcoholic fatty liver disease (NAFLD). A lipotoxic hepatic environment, altered innate immune signaling and inflammation are defining features of progression to NASH. Activated resident liver macrophages express folate receptor beta (FR-β) which may be an indicator of progression from steatosis to NASH. The goals of this study were to characterize FR-β protein expression in human NAFLD and rodent models of NASH, and demonstrate liver targeting of an FR-β imaging agent to the liver of a rodent NASH model using FR-β., Methods: Rat liver lysates from methionine choline deficient (MCD) fed rats, high fat diet (HFD) and methionine choline sufficient (MC+) rat controls were analyzed for hepatic FR-β protein. The FR-β-targeted agent, Etarfolatide was injected into MCD and MC + -fed C57BL/6 mice for efficient FastSPECT hepatic imaging. Additionally, FR-β expression across the stages of human NAFLD from normal to NASH was assessed., Results: FastSPECT images show targeting of Etarfolatide to the liver of mice fed 8 weeks of MCD diet but not control-fed mice. The MCD rat model exhibited significantly increased protein expression of hepatic FR-β in contrast to HFD or normal samples. Similarly human liver samples categorized as NASH Fatty or NASH Not Fatty showed elevated FR-β protein when compared to normal liver. FR-β transcript expression levels were elevated across both NASH Fatty and NASH Not Fatty samples., Conclusion: The findings in this study indicate that FR-β expression in NASH may be harnessed to target agents directly to the liver., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Giant cell arteritis (GCA) is a chronic immune-mediated disease of medium-to-large sized arteries that affects older adults. GCA manifests with arthritis and occlusive symptoms of headaches, stroke or vision loss. Macrophages and T-helper lymphocytes infiltrate the vascular wall and produce a pro-inflammatory response that lead to vessel damage and ischemia. To date, there is no GCA biomarker that can monitor disease activity and guide therapeutic response. Folate receptor beta (FRB) is a glycosylphosphatidylinositol protein that is anchored on cell membranes and normally expressed in the myelomonocytic lineage and in the majority of myeloid leukemia cells as well as in tumor and rheumatoid synovial macrophages, where its expression correlates with disease severity. The ability of FRB to bind folate compounds, folic acid-conjugates and antifolate drugs has made it a druggable target in cancer and inflammatory disease research. This report describes the histopathologic and immunohistochemical methods used to assess expression and distribution of FRB in relation to GCAimmunopathology. Formalin-fixed and paraffin-embedded temporal artery biopsies from GCA and normal controls were stained with Hematoxylin and Eosin to review tissue histology and identify pathognomonic features.Immunohistochemistry was used to detect FRB, CD68 and CD3 expression. A microscopic analysis was performed to quantify the number of positively stained cells on 10 selected high-power-field sections and their respective locations in the arterial wall. Lymphohistiocytic (LH) inflammation accompanied by intimal hyperplasia and disrupted elastic lamina was seen in GCA with none found in controls. The LH infiltrate was composed of approximately 60% lymphocytes and 40% macrophages. FRB expression was restricted to macrophages, comprising 31% of the total CD68+ macrophage population and localized to the media and adventitia. No FRB was seen in controls. This protocol demonstrated a distinct numerical and spatial pattern of the FRB macrophage relative to the vascular immune microenvironment in GCA.

Macrophages play a key role in the pathophysiology of rheumatoid arthritis (RA). Notably, positive correlations have been reported between synovial macrophage infiltration and disease activity as well as therapy outcome in RA patients. Hence, macrophages can serve as an important target for both imaging disease activity and drug delivery in RA. Folate receptor β (FRβ) is a glycosylphosphatidyl (GPI)-anchored plasma membrane protein being expressed on myeloid cells and activated macrophages. FRβ harbors a nanomolar binding affinity for folic acid allowing this receptor to be exploited for RA disease imaging (e.g., folate-conjugated PET tracers) and therapeutic targeting (e.g., folate antagonists and folate-conjugated drugs). This review provides an overview of these emerging applications in RA by summarizing and discussing properties of FRβ, expression of FRβ in relation to macrophage polarization, FRβ-targeted in vivo imaging modalities, and FRβ-directed drug targeting.

Background: We previously developed cabazitaxel (CTX)-loaded human serum albumin nanoparticles (NPs-CTX) via a self-assembly method, and these NPs showed efficacy in prostate cancer therapy. Many studies have shown that the levels of folic acid (FA) receptor on the surface of various tumor cells are high. Therefore, FA-modified NPs-CTX may have enhanced antitumor effects compared with unmodified NPs-CTX., Methods: NPs-CTX were first prepared via self-assembly, and FA was conjugated on the surface of NPs-CTX through the -NH 2 groups of the NPs to produce FA-NPs-CTX. The FA-NPs-CTX were evaluated in tumor cells with high FA receptor (FR) expression in vitro and in vivo., Results: Both NPs-CTX and FA-NPs-CTX exhibited good stability and morphology. Drug release from the NPs was not affected by FA conjugation. Compared with CTX dissolved in a mixture of Tween 80 and 13% ethanol (w/w) at a ratio of 1:4 (v/v) (Tween-CTX), the two nanoformulations had lower lytic activity against normal red blood cells. However, FA-NPs-CTX showed greater inhibition of tumor cells with overexpressed FR, compared with NPs-CTX, in the cytotoxicity experiments. Moreover, the cellular uptake of FA-NPs-CTX was enhanced through FR-mediated endocytosis in HeLa cells in vitro and HeLa xenograft tumors in vivo. Although Tween-CTX exhibited tumor growth inhibition similar to FA-NPs-CTX in vivo, this inhibition also caused adverse side effects; the median lethal dose (LD50) of Tween-CTX to mice was 5.68 mg/kg, while FA-NPs-CTX-treated mice survived at doses exceeding 400 mg/kg., Conclusion: The results showed that FA-NPs-CTX caused inhibition of tumor growth in a manner similar to that of Tween-CTX; however, the safety and tolerability of CTX were greatly improved by FA conjugation compared with those of Tween-CTX. In summary, FA-NPs-CTX have great potential in CTX delivery, and this formulation is a promising candidate for the treatment of cancers with high FR levels., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Receptor-targeted delivery of imaging and therapeutic agents has emerged as an attractive strategy to diagnosis and treat many diseases including cancer. One of the most well-studied receptors for targeted therapies is the folate receptor (FR) family. FR-α and FR-β are present on many cancers with little expression in normal tissues; leading to the testing of at least six folate-targeted drugs in human clinical trials for various cancers. However, the expression of FR in blood cancers has not been fully explored with no reports of FR expression in myelomas. Herein, we report the expression of both FR-α and FR-β on CD138 + plasma cells isolated from patients with multiple myeloma. In addition, all-trans retinoic acid was shown to increase the levels of FR-α and FR-β in two myeloma cell lines. Altogether, this data suggests that folate-targeted therapies for the treatment of multiple myeloma warrants further investigation.

BACKGROUND The FOLR2 gene encodes folate receptor-beta (FR-beta), which is expressed by tumor-associated macrophages. The effects of FOLR2 gene expression in non-small cell lung cancer (NSCLC) remains unknown. This study aimed to investigate the effects of FOLR2 gene expression and gene silencing in human NSCLC cell lines and normal human bronchial epithelial (HBE) cells in vitro. MATERIAL AND METHODS Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were performed to detect the expression of the FOLR2 gene, cell cycle and apoptosis-associated genes in normal HBE cells and the NSCLC cell lines, A549, NCI-H1299, NCI-H1650, and NCI-H460. Using small interfering RNA (siRNA), or silencing RNA, FOLR2 gene silencing was performed for NCI-H1650 cells. Cell counting kit-8 (CCK-8) was used to measure cell viability. Cell cycle and apoptosis were determined using flow cytometry. Western blot evaluated the expression of Akt, mTOR, and S6K1 signaling. RESULTS Expression of the FOLR2 gene was increased in NSCLC cells compared with normal HBE cells. Silencing of the expression of the FOLR2 gene in NCI-H1650 cells reduced cell viability, increased cell apoptosis, and arrested cells in the G1 phase of the cell cycle, decreased the expression of cyclin D1, upregulated expression of cell cycle inhibitors, p21 and p27, upregulated the expression of Bax/Bcl-2, and inhibited phosphorylation of AKT, mTOR, and S6K1. CONCLUSIONS Silencing of the FOLR2 gene inhibited phosphorylation of AKT, mTOR, and S6K1, inhibited cell proliferation and increased apoptosis in the NCI-H1650 human NSCLC cell line.

Background: The transcription factor NF-kB plays an important role in the pathogenesis of rheumatoid arthritis (RA). Effective treatment of RA is hindered due to the lack of specificity of small molecules in the inflamed joints. In this study, we aimed to develop a unique hybrid-nanoparticles system comprised of calcium phosphate/liposome to deliver NF-kB-targeted siRNA and methotrexate (MTX) to diseased site., Results: We have successfully demonstrated that the combination of siRNA and MTX in a calcium phosphate/liposome-based hybrid nanocarrier could effectively treat the RA. We have showed that folate receptor-targeted nanocarrier system significantly suppression the arthritis progression in mice model. Substantial accumulation of F-siRML was observed in LPS-activated macrophages. These kind of activated macrophages are generally present in the RA and osteoarthritis and folate-targeted nanoparticle enables the effective accumulation of therapeutics in the diseased site. The combinational nanoparticles effectively blocked the NF-kB signaling pathways and reduced the expression of pro-inflammatory cytokines. Furthermore, siRML and F-siRML did not show any decrease in the lymphocyte count indicating that it can avoid the adverse effect of MTX., Conclusion: Therefore, siRML and F-siRML provides unique benefits of excellent therapeutic efficacy with excellent safety profile in the arthritic mice and could be an promising approach in the treatment of rheumatoid arthritis.

Activated macrophages play a key role in the development and maintenance of inflammatory diseases such as atherosclerosis, lupus, psoriasis, rheumatoid arthritis, ulcerative colitis, and many others. These activated macrophages, but not resting or quiescent macrophages highly up-regulate folate receptor beta (FR-β). This differential expression of FR-β provides a mechanism to selectively deliver imaging and therapeutic agents utilizing folate as a targeting molecule. In an effort to determine whether inflammatory diseases can be targeted utilizing a folate-linked nanosize carrier, a PEG-coated liposome was prepared that incorporated a folate conjugated PEG that also could transport imaging or therapeutic cargo. We demonstrate that these folate-liposomes specifically bind to folate receptor positive cells and accumulate at sites of inflammation in mouse models of colitis and atherosclerosis. These two animal models show that folate-targeted liposomes could be successfully utilized to deliver fluorescent molecules and an anti-inflammatory drug (betamethasone) for diagnostic and therapeutic applications., (Copyright © 2018 Elsevier Inc. All rights reserved.)

On-target drug delivery remains a challenge in cancer precision medicine; it is difficult to deliver a targeted therapy to cancer cells without incurring toxicity to normal tissues. The SERCA (sarco-endoplasmic reticulum Ca 2+ ATPase) inhibitor thapsigargin inhibits mutant NOTCH1 receptors compared with wild type in T cell acute lymphoblastic leukemia (T-ALL), but its administration is predicted to be toxic in humans. Leveraging the addiction of ALL to folic acid, we conjugated folate to an alcohol derivative of thapsigargin via a cleavable ester linkage. JQ-FT is recognized by folate receptors on the plasma membrane and delivered into leukemia cells as a potent antileukemic agent. In mechanistic and translational models of T-ALL, we demonstrate NOTCH1 inhibition in vitro and in vivo. These proof-of-concept studies support the further optimization of this first-in-class NOTCH1 inhibitor with dual selectivity: leukemia over normal cells and NOTCH1 mutants over wild-type receptors. Furthermore, tumor-specific disruption of Notch signaling may overcome legitimate concerns associated with the tumor suppressor function of nontargeted Notch pathway inhibitors., (© 2018 Roti et al.)

Arsenic trioxide (ATO, As 2 O 3 ) is currently used to treat acute promyelocytic leukemia. However, expanding its use to include high-dose treatment of other cancers is severely hampered by serious side effects on healthy organs. To address these limitations, we loaded ATO onto folate (FA)-labeled human serum albumin (HSA) pretreated with glutathione (GSH) based on the low pH- and GSH-sensitive arsenic-sulfur bond, and we termed the resulting smart nanodrug as FA-HSA-ATO. FA-HSA-ATO could specifically recognize folate receptor-β-positive (FRβ+) chronic myeloid leukemia (CML) cells, resulting in more intracellular accumulation of ATO. Furthermore, the nanodrug could upregulate FRβ expression in CML cancer cells and xenograft tumor model, facilitating even more recruitment and uptake of FRβ-targeting drugs. In vitro and in vivo experiments indicate that the nanodrug significantly alleviates side effects and improves therapeutic efficacy of ATO on CML and xenograft tumor model., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cationic liposomes are efficient vectors for systemic delivery of therapeutic small interfering RNA (siRNA), taking advantage of RNA interference (RNAi), a naturally occurring gene-silencing mechanism in mammalian cells. However, toxicity at high concentrations, short circulating half-lives and lack of specificity restrict their successful application in a wider scale. The purpose of this study was to evaluate the efficiency of neutral liposomes containing polyethylene glycol (PEG) to encapsulate siRNA in their aqueous core. This formulation will reduce drastically the toxicity associated to cationic liposomes by bringing surface charge to almost zero, increasing stealth degree and therefore circulation time. In this study, we evaluate the efficiency of folate-targeted liposomes for specific delivery of siRNA to activated macrophages, key effector cells in rheumatoid arthritis (RA) pathology which specifically express folate receptor β (FRβ). Myeloid cell leukaemia-1 (Mcl-1) is a protein essential for synovial macrophage survival, since Mcl-1 suppression results in the induction of apoptosis. The effect of MCL1 siRNA incorporated in liposomal formulation was assessed in primary human macrophages and successful inhibition of Mcl-1 expression was achieved. Here we show that the neutral liposomal derived from DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) formulation developed is efficient to encapsulate MCL1 siRNA and silencing gene expression in activated human macrophages., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Previously, we determined that heterogeneous nuclear ribonucleoprotein E1 (hnRNP-E1) functions as an intracellular physiologic sensor of folate deficiency. In this model, l-homocysteine, which accumulates intracellularly in proportion to the extent of folate deficiency, covalently binds to and thereby activates homocysteinylated hnRNP-E1 to interact with folate receptor-α mRNA; this high-affinity interaction triggers the translational upregulation of cell surface folate receptors, which enables cells to optimize folate uptake from the external milieu. However, integral to this model is the need for ongoing generation of hnRNP-E1 to replenish homocysteinylated hnRNP-E1 that is degraded. Objective: We searched for an interrelated physiologic mechanism that could also maintain the steady-state concentration of hnRNP-E1 during prolonged folate deficiency. Methods: A novel RNA-protein interaction was functionally characterized by using molecular and biochemical approaches in vitro and in vivo. Results: l-homocysteine triggered a dose-dependent high-affinity interaction between hnRNP-E1 and a 25-nucleotide cis element within the 5'-untranslated region of hnRNP-E1 mRNA; this led to a proportionate increase in these RNA-protein complexes, and translation of hnRNP-E1 both in vitro and within placental cells. Targeted perturbation of this RNA-protein interaction either by specific 25-nucleotide antisense oligonucleotides or mutation within this cis element or by small interfering RNA to hnRNP-E1 mRNA significantly reduced cellular biosynthesis of hnRNP-E1. Conversely, transfection of hnRNP-E1 mutant proteins that mimicked homocysteinylated hnRNP-E1 stimulated both cellular hnRNP-E1 and folate receptor biosynthesis. In addition, ferrous sulfate heptahydrate [iron(II)], which also binds hnRNP-E1, significantly perturbed this l-homocysteine-triggered RNA-protein interaction in a dose-dependent manner. Finally, folate deficiency induced dual upregulation of hnRNP-E1 and folate receptors in cultured human cells and tumor xenografts, and more selectively in various fetal tissues of folate-deficient dams. Conclusions: This novel positive feedback loop amplifies hnRNP-E1 during prolonged folate deficiency and thereby maximizes upregulation of folate receptors in order to restore folate homeostasis toward normalcy in placental cells. It will also functionally impact several other mRNAs of the nutrition-sensitive, folate-responsive posttranscriptional RNA operon that is orchestrated by homocysteinylated hnRNP-E1., (© 2017 American Society for Nutrition.)

New folic acid (FA) coupled three layered micelles (3LM) were designed to encapsulate DNA, and their application as delivery system that specifically targets activated macrophages was investigated for new treatment options in rheumatoid arthritis (RA). FA coupled poly(l-lactide)-b-poly(ethylene glycol) (FA-PEG-PLLA) was synthesized via the NHS-ester activated/amine coupling method. Fluorescein labeled folic acid was used for flow cytometric detection of the expression of functional folic receptor β in LPS-activated and resting macrophages. FA coupled 3LM were formulated in a two-step procedure and characterized regarding hydrodynamic diameters and zeta potentials. The presence of the targeting ligand was shown not to increase the size of the 3LM compared to their non-targeted counterparts. Targeted and non-targeted 3LM were used in vitro to optimize uptake conditions in the RAW 264.7 macrophage cell line. The amount of FA coupled polymer in the final formulation was found to be optimal at 75% FA-PEG-PLLA and 25% PLLA-PEG-PLLA. Subsequently, transgene expression in vitro in RAW 264.7 cells and ex vivo in primary activated and resting mouse macrophages was determined as a function of FR-mediated internalization of 3LM encapsulating GFP expressing plasmid. FR-overexpressing activated cells, as successfully identified by internalization of FA-fluorescein, showed significantly higher GFP expression in vitro and ex vivo than resting macrophages with only a basal level of FR expression. Lastly, injectable hydrogels as depot formulation were formed by stereocomplexation, and their degradation, DNA release profiles, and dissociation into intact 3LM were found to be beneficial for potential in vivo application. Our findings confirm that FA-3LM are taken up by activated macrophages via folate receptor mediated endocytosis and that their hydrogels release intact 3LM for efficient transfection of primary macrophages. Therefore, FA-3LM could become a promising delivery system for receptor-mediated drug or gene delivery and novel therapy for rheumatoid arthritis in an in situ forming gel formulation., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Mucin-1 (MUC1), a transmembrane glycoprotein is aberrantly expressed on ∼90% of breast cancer and is an excellent target for nanoparticulate targeted imaging. In this study, the development of a dye-doped NIR emitting mesoporous silica nanoparticles platform conjugated to tumor-specific MUC1 antibody (ab-tMUC1-NIR-MSN) for in vivo optical detection of breast adenocarcinoma tissue is reported. The structural properties, the in vitro and in vivo performance of this nanoparticle-based probe were evaluated. In vitro studies showed that the MSN-based optical imaging nanoprobe is non-cytotoxic and targets efficiently mammary cancer cells overexpressing human tMUC1 protein. In vivo experiments with female C57BL/6 mice indicated that this platform accumulates mainly in the liver and did not induce short-term toxicity. In addition, we demonstrated that the ab-tMUC1-NIR-MSN nanoprobe specifically detects mammary gland tumors overexpressing human tMUC1 in a human MUC1 transgenic mouse model.

Based on the fluorescence quenching of folic acid-sensitive bovine serum albumin-directed gold nanoclusters (BSA-AuNCs) via folic acid-induced the change of environment around BSA-AuNCs, we have constructed a turn on fluorescence imaging of folate receptor overexpressed tumor cells. In this paper, the primary fluorescence intensity of BSA-AuNCs was quenched via self-assembly of folic acid onto BSA-AuNCs to produce negligible fluorescence background, the linear range of the method was 0.1-100μg/mL with the limit of detection (LOD) of 30ng/mL (S/N=3); In the presence of overexpression of folate receptor on the surface of tumor cells, the primary fluorescence intensity of BSA-AuNCs turned on by folic acid desorbing from BSA-AuNCs, the linear range of method was 0.12-2μg/mL with the LOD of 20ng/mL (S/N=3). Additionally, due to specific and high affinity of folic acid and folate receptor, the probe had high selectivity for folate receptor, other interferences hardly changed the fluorescence intensity of the probe. Moreover, the text for cytotoxicity implied that the probe had no toxicity for tumor cells. Consequently, using the fluorescence probe, satisfactory results for the turn on imaging of folate receptor overexpressed tumor cells were obtained. A novel turn-on and red fluorescent probe for folate receptor overexpressed tumor cells was developed based on the recovery of fluorescence intensity of folic acid-sensitive BSA-AuNCs., (Copyright © 2016. Published by Elsevier B.V.)

Aims: Tumor-specific targeted imaging is rapidly evolving in cancer diagnosis. The folate receptor alpha (FR-α) has already been identified as a suitable target for cancer therapy and imaging. FR-α is present on ~40% of human cancers. FR-β is known to be expressed on several hematologic malignancies and on activated macrophages, but little is known about FR-β expression in solid tumors. Additional or simultaneous expression of FR-β could help extend the indications for folate-based drugs and imaging agents. In this study, the expression pattern of FR-β is evaluated in ovarian, breast and colorectal cancer., Methods: FR-β expression was analyzed by semi-quantitative scoring of immunohistochemical staining on tissue microarrays (TMAs) of 339 ovarian cancer patients, 418 breast cancer patients, on 20 slides of colorectal cancer samples and on 25 samples of diverticulitis., Results: FR-β expression was seen in 21% of ovarian cancer samples, 9% of breast cancer samples, and 55% of colorectal cancer samples. Expression was weak or moderate. Of the diverticulitis samples, 80% were positive for FR-β expression in macrophages. FR-β status neither correlated to known disease-related variables, nor showed association with overall survival and progression free survival in ovarian and breast cancer. In breast cancer, negative axillary status was significantly correlated to FR-β expression (p=0.022)., Conclusions: FR-β expression was low or absent in the majority of ovarian, breast and colorectal tumor samples. From the present study we conclude that the low FR-β expression in ovarian and breast tumor tissue indicates limited practical use of this receptor in diagnostic imaging and therapeutic purposes. Due to weak expression, FR-β is not regarded as a suitable target in colorectal cancer.

Folate receptor (FR)-β has been identified as a promising target for antimacrophage and antiinflammatory therapies. In the present study, we investigated EC0565, a folic acid-derivative of everolimus, as a FR-specific inhibitor of the mammalian target of rapamycin (mTOR). Because of its amphiphilic nature, EC0565 was first evaluated for water solubility, critical micelle formation, stability in culture and FR-binding specificity. Using FR-expressing macrophages, the effect of EC0565 on mTOR signaling and cellular proliferation was studied. The pharmacokinetics, metabolism and bioavailability of EC0565 were studied in normal rats. The in vivo activity of EC0565 was assessed in rats with adjuvant arthritis, a "macrophage-rich" model with close resemblance to rheumatoid arthritis. EC0565 forms micellar aggregates in physiological buffers and demonstrates good water solubility as well as strong multivalent FR-binding capacity. EC0565 inhibited mTOR signaling in rat macrophages at nanomolar concentrations and induced G0/G1 cell cycle arrest in serum-starved RAW264.7 cells. Subcutaneously administered EC0565 in rats displayed good bioavailability and a relatively long half-life (~12 h). When given at 250 nmol/kg, EC0565 selectively inhibited proliferating cell nuclear antigen expression in thioglycollate-stimulated rat peritoneal cells. With limited dosing regimens, the antiarthritic activity of EC0565 was found superior to that of etanercept, everolimus and a nontargeted everolimus analog. The in vivo activity of EC0565 was also comparable to that of a folate-targeted aminopterin. Folate-targeted mTOR inhibition may be an effective way of suppressing activated macrophages in sites of inflammation, especially in nutrient-deprived conditions, such as in the arthritic joints. Further investigation and improvement upon the physical and biochemical properties of EC0565 are warranted.

Characterization of markers that identify activated macrophages could advance understanding of inflammatory lung diseases and facilitate development of novel methodologies for monitoring disease activity. We investigated whether folate receptor β (FRβ) expression could be used to identify and quantify activated macrophages in the lungs during acute inflammation induced by Escherichia coli LPS. We found that FRβ expression was markedly increased in lung macrophages at 48 hours after intratracheal LPS. In vivo molecular imaging with a fluorescent probe (cyanine 5 polyethylene glycol folate) showed that the fluorescence signal over the chest peaked at 48 hours after intratracheal LPS and was markedly attenuated after depletion of macrophages. Using flow cytometry, we identified the cells responsible for uptake of cyanine 5-conjugated folate as FRβ(+) interstitial macrophages and pulmonary monocytes, which coexpressed markers associated with an M1 proinflammatory macrophage phenotype. These findings were confirmed using a second model of acute lung inflammation generated by inducible transgenic expression of an NF-κB activator in airway epithelium. Using CC chemokine receptor 2-deficient mice, we found that FRβ(+) macrophage/monocyte recruitment was dependent on the monocyte chemotactic protein-1/CC chemokine receptor 2 pathway. Together, our results demonstrate that folate-based molecular imaging can be used as a noninvasive approach to detect classically activated monocytes/macrophages recruited to the lungs during acute inflammation.

Over-expression of folate receptor alpha on cancer cells has been frequently exploited for delivery of folate-targeted imaging and therapeutic agents to tumors. Because limited information exists on expression of the beta isoform of the folate receptor in human cancers (FR-β), we have evaluated the immunohistochemical staining pattern of FR-β in 992 tumor sections from 20 different human cancer types using a new anti-human FR-β monoclonal antibody. FR-β expression was shown to be more pronounced in cells within the stroma, primarily macrophages and macrophage-like cells than cancer cells in every cancer type studied. Moreover, FR-β expression in both cancer and stromal cells was found to be statistically more prominent in females than males. A significant positive correlation was also observed between FR-β expression on stromal cells and both the stage of the cancer and the presence of lymph node metastases. Based on these data we conclude FR-β may constitute a good target for specific delivery of therapeutic agents to activated macrophages and that accumulation of FR-β positive macrophages in the stroma could serve as a useful indicator of a tumor's metastatic potential.

Unlabelled: In atherosclerotic plaques, the risk of rupture is increased at sites of macrophage accumulation. Activated macrophages express folate receptor-β (FR-β), which can be targeted by folate coupled to radioactive ligands to visualize vulnerability. The aim of this study was to explore the presence of activated macrophages in human atherosclerotic plaques by (99m)Tc-folate imaging and to evaluate whether this technique can discriminate between an M1-like and M2-like macrophage phenotype., Methods: Carotid endarterectomy specimens of 20 patients were incubated with (99m)Tc-folate, imaged using micro-SPECT, and divided into 3-mm slices. The mean accumulation was calculated per slice, and the distribution of M1-like and M2-like macrophages per slice was quantified by immunohistochemical staining for CD86 as well as inducible nitric oxide synthase (iNOS) for M1 and CD163 and FR-β for M2 macrophages. Monocytes from healthy donors were differentiated toward M1-like or M2-like phenotype by in vitro culturing. Messenger RNA levels of specific M1 and M2 markers were measured by reverse-transcription polymerase chain reaction and expression of FR-β, CD86, and CD163 by flow cytometry., Results: There was a heterogeneous accumulation of (99m)Tc-folate in plaques (median, 2.45 [0.77-6.40] MBq/g). Slices with the highest (99m)Tc-folate accumulation of each plaque showed significantly more expression of FR-β and CD163, compared with slices with the lowest (99m)Tc-folate accumulation, which showed significantly more expression of iNOS. In in vitro polarized macrophages, messenger RNA expression of FR-β, mannose receptor, IL-10, and matrix metalloproteinase-9 was significantly increased in M2-like macrophages, compared with M1-like macrophages. On a receptor level, CD86 was shown to be overexpressed on M1-like macrophages whereas FR-β and CD163 were overexpressed on M2-like macrophages measured by flow cytometry., Conclusion: Higher numbers of M2-like macrophages were present in areas of high (99m)Tc-folate accumulation than areas with low accumulation. It is anticipated that (99m)Tc-folate imaging using SPECT as a marker for M2-like macrophages in atherosclerosis might be a good indicator for plaque vulnerability., (© 2014 by the Society of Nuclear Medicine and Molecular Imaging, Inc.)

Activated macrophages overexpress a receptor for the vitamin folic acid termed the folate receptor β (FR-β). Because conjugation of folate to low molecular weight drugs, genes, liposomes, nanoparticles, and imaging agents has minor effects on FR binding, the vitamin can be exploited to target both therapeutic and imaging agents to activated macrophages without promoting their uptake by other healthy cells. In this paper, we characterize the binding, internalization, and recycling kinetics of FR-β on activated macrophages in inflamed tissues of rats with adjuvant-induced arthritis. Our results demonstrate that saturation of macrophage FR is achieved at injection doses of ∼150-300 nmol/kg, with more rapidly perfused tissues saturating at lower doses than inflamed appendages. After binding, FR-β internalizes and recycles back to the cell surface every ∼10-20 min, providing empty receptors for additional folate conjugate uptake. Because the half-life of low molecular weight folate conjugates in the vasculature is usually <1 h, these data suggest that targeting of folate conjugates to activated macrophages in vivo can be maximized by frequent dosing at conjugate concentrations that barely saturate FR (∼150 nmol/kg), thereby minimizing nonspecific binding to receptor-negative tissues and maximizing the probability that unoccupied cell surface receptors will be exposed to folate-drug conjugate.