Development of finely tuned liposome nanoplatform for macrophage depletion.

BACKGROUND: Immunotherapy with clodronate-encapsulated liposomes, which induce macrophage depletion, has been studied extensively. However, previously reported liposomal formulation-based drugs (Clodrosome® and m-Clodrosome®) are limited by their inconsistent size and therapeutic efficacy. Thus, we aimed to achieve consistent therapeutic effects by effectively depleting macrophages with uniform-sized liposomes. RESULTS: We developed four types of click chemistry-based liposome nanoplatforms that were uniformly sized and encapsulated with clodronate, for effective macrophage depletion, followed by conjugation with Man-N3 and radiolabeling. Functionalization with Man-N3 improves the specific targeting of M2 macrophages, and radioisotope labeling enables in vivo imaging of the liposome nanoplatforms. The functionalized liposome nanoplatforms are stable under physiological conditions. The difference in the biodistribution of the four liposome nanoplatforms in vivo were recorded using positron emission tomography imaging. Among the four platforms, the clodronate-encapsulated mannosylated liposome effectively depleted M2 macrophages in the normal liver and tumor microenvironment ex vivo compared to that by Clodrosome® and m-Clodrosome®. CONCLUSION: The newly-developed liposome nanoplatform, with finely tuned size control, high in vivo stability, and excellent ex vivo M2 macrophage targeting and depletion effects, is a promising macrophage-depleting agent.

Comprehensive characterization of early-programmed tumor microenvironment by tumor-associated macrophages reveals galectin-1 as an immune modulatory target in breast cancer.

Background: In recent years, there has been considerable interest in the therapeutic targeting of tumor-associated macrophages (TAMs) to modulate the tumor microenvironment (TME), resulting in antitumoral phenotypes. However, key mediators suitable for TAM-mediated remodeling of the TME remain poorly understood. Methods: In this study, we used single-cell RNA sequencing analyses to analyze the landscape of the TME modulated by TAMs in terms of a protumor microenvironment during early tumor development. Results: Our data revealed that the depletion of TAMs leads to a decreased epithelial-to-mesenchymal transition (EMT) signature in cancer cells and a distinct transcriptional state characterized by CD8+ T cell activation. Moreover, notable alterations in gene expression were observed upon the depletion of TAMs, identifying Galectin-1 (Gal-1) as a crucial molecular factor responsible for the observed effect. Gal-1 inhibition reversed immune suppression via the reinvigoration of CD8+ T cells, impairing tumor growth and potentiating immune checkpoint inhibitors in breast tumor models. Conclusion: These results provide comprehensive insights into TAM-mediated early tumor microenvironments and reveal immune evasion mechanisms that can be targeted by Gal-1 to induce antitumor immune responses.

A new era of macrophage-based cell therapy.

Macrophages are essential innate immune cells found throughout the body that have protective and pathogenic functions in many diseases. When activated, macrophages can mediate the phagocytosis of dangerous cells or materials and participate in effective tissue regeneration by providing growth factors and anti-inflammatory molecules. Ex vivo-generated macrophages have thus been used in clinical trials as cell-based therapies, and based on their intrinsic characteristics, they outperformed stem cells within specific target diseases. In addition to the old methods of generating naïve or M2 primed macrophages, the recently developed chimeric antigen receptor-macrophages revealed the potential of genetically engineered macrophages for cell therapy. Here, we review the current developmental status of macrophage-based cell therapy. The findings of important clinical and preclinical trials are updated, and patent status is investigated. Additionally, we discuss the limitations and future directions of macrophage-based cell therapy, which will help broaden the potential utility and clinical applications of macrophages.

Pyruvate dehydrogenase kinase inhibitor dichloroacetate augments autophagy mediated constraining the replication of Mycobacteroides massiliense in macrophages.

Increasing evidence indicates a strong interaction between cellular metabolism and innate macrophage immunity. Here, we show that the intracellular replication of Mycobacteroides massiliense in macrophages depends on host pyruvate dehydrogenase kinase (PDK) activity. Infection with M. massiliense induced a metabolic switch in macrophages by increasing glycolysis and decreasing oxidative phosphorylation. Treatment with dichloroacetate (DCA), a PDK inhibitor, converts this switch in M. massiliense-infected macrophages and restricts intracellular bacterial replication. Mechanistically, DCA resulted in AMPKα1 activation via increased AMP/ATP ratio, consequently inducing autophagy to constrain bacterial proliferation in the phagolysosome. This study suggests that the pharmacological inhibition of PDK could be a strategy for host-directed therapy to control virulent M. massiliense infections.

Mannosylated-serum albumin nanoparticle imaging to monitor tumor-associated macrophages under anti-PD1 treatment.

BACKGROUND: Immune checkpoint inhibitors such as anti-programmed cell death protein 1 (PD1) block tumor growth by reinvigorating the immune system; however, determining their efficacy only by the changes in tumor size may prove inaccurate. As the immune cells including macrophages in the tumor microenvironment (TME) are associated with the response to anti-PD1 therapy, tumor-associated macrophages (TAMs) imaging using nanoparticles can noninvasively provide the immune enrichment status of TME. Herein, the mannosylated-serum albumin (MSA) nanoparticle was labeled with radioactive isotope 68Ga to target the mannose receptors on macrophages for noninvasive monitoring of the TME according to anti-PD1 therapy. RESULTS: B16F10-Luc and MC38-Luc tumor-bearing mice were treated with anti-PD1, and the response to anti-PD1 was determined by the tumor volume. According to the flow cytometry, the responders to anti-PD1 showed an increased proportion of TAMs, as well as lymphocytes, and the most enriched immune cell population in the TME was also TAMs. For noninvasive imaging of TAMs as a surrogate of immune cell augmentation in the TME via anti-PD1, we acquired [68Ga] Ga-MSA positron emission tomography. According to the imaging study, an increased number of TAMs in responders at the early phase of anti-PD1 treatment was observed in both B16F10-Luc and MC38-Luc tumor-bearing mice models. CONCLUSION: As representative immune cells in the TME, non-invasive imaging of TAMs using MSA nanoparticles can reflect the immune cell enrichment status in the TME closely associated with the response to anti-PD1. As non-invasive imaging using MSA nanoparticles, this approach shows a potential to monitor and evaluate anti-tumor response to immune checkpoint inhibitors.

Circulation Time-Optimized Albumin Nanoplatform for Quantitative Visualization of Lung Metastasis via Targeting of Macrophages.

The development of molecular imaging probes to identify key cellular changes within lung metastases may lead to noninvasive detection of metastatic lesions in the lung. In this study, we constructed a macrophage-targeted clickable albumin nanoplatform (CAN) decorated with mannose as the targeting ligand using a click reaction to maintain the intrinsic properties of albumin in vivo. We also modified the number of mannose molecules on the CAN and found that mannosylated serum albumin (MSA) harboring six molecules of mannose displayed favorable pharmacokinetics that allowed high-contrast imaging of the lung, rendering it suitable for in vivo visualization of lung metastases. Due to the optimized control of functionalization and surface modification, MSA enhanced blood circulation time and active/passive targeting abilities and was specifically incorporated by mannose receptor (CD206)-expressing macrophages in the metastatic lung. Moreover, extensive in vivo imaging studies using single-photon emission computed tomography (SPECT)/CT and positron emission tomography (PET) revealed that blood circulation of time-optimized MSA can be used to discern metastatic lesions, with a strong correlation between its signal and metastatic burden in the lung.

Blockade of CD47 enhances the antitumor effect of macrophages in renal cell carcinoma through trogocytosis.

Immune checkpoint inhibitors and vascular endothelial growth factor receptor tyrosine kinase inhibitors (VEGFR TKIs) are mainstream treatments for renal cell carcinoma (RCC). Both T cells and macrophages infiltrate the tumor microenvironment of RCC. CD47, an immune checkpoint of macrophages, transmits the "don't eat me" signal to macrophages. We propose a novel therapeutic strategy that activates the antitumor effect of macrophages. We found that CD47 was expressed in patients with RCC, and high CD47 expression was indicative of worse overall survival in datasets from The Cancer Genome Atlas. We observed that CD47-blocking antibodies enhanced the antitumor effect of macrophages against human RCC cell lines. Trogocytosis, rather than phagocytosis, occurred and was promoted by increased cell-to-cell contact between macrophages and RCC cells. Trogocytosis induced by CD47 blockade occurred in the presence of CD11b integrin signaling in macrophages and was augmented when RCC cells were exposed to VEGFR TKIs, except for sunitinib. In conclusion, this study presents evidence that anti-CD47 blocking antibodies improve the antitumor effect of macrophages in RCC. In combination with VEGFR TKIs, CD47 blockade is a potential therapeutic strategy for patients with RCC.

Liposomal Dexamethasone Reduces A/H1N1 Influenza-Associated Morbidity in Mice.

Re-emerging viral threats have continued to challenge the medical and public health systems. It has become clear that a significant number of severe viral infection cases are due to an overreaction of the immune system, which leads to hyperinflammation. In this study, we aimed to demonstrate the therapeutic efficacy of the dexamethasone nanomedicine in controlling the symptoms of influenza virus infection. We found that the A/Wisconsin/WSLH34939/2009 (H1N1) infection induced severe pneumonia in mice with a death rate of 80%, accompanied by significant epithelial cell damage, infiltration of immune cells, and accumulation of pro-inflammatory cytokines in the airway space. Moreover, the intranasal delivery of liposomal dexamethasone during disease progression reduced the death rate by 20%. It also significantly reduced the protein level of tumor necrosis factor-alpha (TNFα), interleukin-1ß (IL-1ß), IL-6, and the C-X-C motif chemokine ligand 2 (CXCL2) as well as the number of infiltrated immune cells in the bronchoalveolar lavage fluids as compared to the control and free dexamethasone. The liposomal dexamethasone was mainly distributed into the monocyte/macrophages as a major cell population for inducing the cytokine storm in the lungs. Taken together, the intranasal delivery of liposomal dexamethasone may serve as a novel promising therapeutic strategy for the treatment of influenza A-induced pneumonia.

Nicotinamide (niacin) supplement increases lipid metabolism and ROS-induced energy disruption in triple-negative breast cancer: potential for drug repositioning as an anti-tumor agent.

Metabolic dysregulation is an important hallmark of cancer. Nicotinamide (NAM), a water-soluble amide form of niacin (vitamin B3), is currently available as a supplement for maintaining general physiologic functions. NAM is a crucial regulator of mitochondrial metabolism and redox reactions. In this study, we aimed to identify the mechanistic link between NAM-induced metabolic regulation and the therapeutic efficacy of NAM in triple-negative breast cancer (TNBC). The combined analysis using multiomics systems biology showed that NAM decreased mitochondrial membrane potential and ATP production, but increased the activities of reverse electron transport (RET), fatty acid ß-oxidation and glycerophospholipid/sphingolipid metabolic pathways in TNBC, collectively leading to an increase in the levels of reactive oxygen species (ROS). The increased ROS levels triggered apoptosis and suppressed tumour growth and metastasis of TNBC in both human organoids and xenograft mouse models. Our results showed that NAM treatment leads to cancer cell death in TNBC via mitochondrial dysfunction and activation of ROS by bifurcating metabolic pathways (RET and lipid metabolism); this provides insights into the repositioning of NAM supplement as a next-generation anti-metabolic agent for TNBC treatment.

Matrix Stiffening Enhances DNCB-Induced IL-6 Secretion in Keratinocytes Through Activation of ERK and PI3K/Akt Pathway.

Matrix stiffness, a critical physical property of the cellular environment, is implicated in epidermal homeostasis. In particular, matrix stiffening during the pathological progression of skin diseases appears to contribute to cellular responses of keratinocytes. However, it has not yet elucidated the molecular mechanism underlying matrix-stiffness-mediated signaling in coordination with chemical stimuli during inflammation and its effect on proinflammatory cytokine production. In this study, we demonstrated that keratinocytes adapt to matrix stiffening by increasing cell-matrix adhesion via actin cytoskeleton remodeling. Specifically, mechanosensing and signal transduction are coupled with chemical stimuli to regulate cytokine production, and interleukin-6 (IL-6) production is elevated in keratinocytes on stiffer substrates in response to 2,4-dinitrochlorobenzene. We demonstrated that ß1 integrin and focal adhesion kinase (FAK) expression were enhanced with increasing stiffness and activation of ERK and the PI3K/Akt pathway was involved in stiffening-mediated IL-6 production. Collectively, our results reveal the critical role of matrix stiffening in modulating the proinflammatory response of keratinocytes, with important clinical implications for skin diseases accompanied by pathological matrix stiffening.

Prostaglandin E2 receptor PTGER4-expressing macrophages promote intestinal epithelial barrier regeneration upon inflammation.

OBJECTIVE: Dysfunctional resolution of intestinal inflammation and altered mucosal healing are essential features in the pathogenesis of inflammatory bowel disease (IBD). Intestinal macrophages are vital in the process of inflammation resolution, but the mechanisms underlying their mucosal healing capacity remain elusive. DESIGN: We investigated the role of the prostaglandin E2 (PGE2) receptor PTGER4 on the differentiation of intestinal macrophages in patients with IBD and mouse models of intestinal inflammation. We studied mucosal healing and intestinal epithelial barrier regeneration in Csf1r-iCre Ptger4fl/fl mice during dextran sulfate sodium (DSS)-induced colitis. The effect of PTGER4+ macrophage secreted molecules was investigated on epithelial organoid differentiation. RESULTS: Here, we describe a subset of PTGER4-expressing intestinal macrophages with mucosal healing properties both in humans and mice. Csf1r-iCre Ptger4fl/fl mice showed defective mucosal healing and epithelial barrier regeneration in a model of DSS colitis. Mechanistically, an increased mucosal level of PGE2 triggers chemokine (C-X-C motif) ligand 1 (CXCL1) secretion in monocyte-derived PTGER4+ macrophages via mitogen-activated protein kinases (MAPKs). CXCL1 drives epithelial cell differentiation and proliferation from regenerating crypts during colitis. Specific therapeutic targeting of macrophages with liposomes loaded with an MAPK agonist augmented the production of CXCL1 in vivo in conditional macrophage PTGER4-deficient mice, restoring their defective epithelial regeneration and favouring mucosal healing. CONCLUSION: PTGER4+ intestinal macrophages are essential for supporting the intestinal stem cell niche and regeneration of the injured epithelium. Our results pave the way for the development of a new class of therapeutic targets to promote macrophage healing functions and favour remission in patients with IBD.

Histamine causes an imbalance between pro-angiogenic and anti-angiogenic factors in the retinal pigment epithelium of diabetic retina via H4 receptor/p38 MAPK axis.

INTRODUCTION: Systemic histaminergic activity is elevated in patients with diabetes mellitus. There are a few studies suggesting that histamine is implicated in the pathogenesis of diabetes, but the exact role of histamine in the development of diabetic retinopathy is unclear. The aim of this study was to investigate the role of histamine receptor H4 (HRH4) in the regulation of retinal pigment epithelium (RPE)-derived pro-angiogenic and anti-angiogenic factors under diabetic conditions. RESEARCH DESIGN AND METHODS: The levels of vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), histamine and histidine decarboxylase (HDC) in the serum and vitreous samples of patients with diabetes were compared with those of patients without diabetes. The effect of hyperglycemia on expression levels of HRH4, VEGF, IL-6 and pigment epithelium-derived factor (PEDF) in the RPE was determined. The role of HRH4 in high glucose-induced regulation of VEGF, IL-6 and PEDF in ARPE-19 cells and the underlying regulatory mechanism were verified using an RNA interference-mediated knockdown study. RESULTS: The serum and vitreous levels of VEGF, IL-6, histamine and HDC were more increased in patients with diabetic retinopathy than in patients without diabetes. HRH4 was overexpressed in RPE both in vitro and in vivo. Histamine treatment upregulated VEGF and IL-6 and downregulated PEDF expression in ARPE-19 cells cultivated under hyperglycemic conditions. Hyperglycemia-induced phosphorylation of p38 and subsequent upregulation of VEGF and IL-6 and downregulation of PEDF were dampened by small interfering RNA-mediated knockdown of HRH4 in ARPE-19 cells. CONCLUSIONS: Taken together, HRH4 was a critical regulator of VEGF, IL-6 and PEDF in the RPE under hyperglycemic conditions and the p38 mitogen-activated protein kinase pathway mediated this regulatory mechanism.

Human Cytomegalovirus-Induced Interleukin-10 Production Promotes the Proliferation of Mycobacterium massiliense in Macrophages.

Human cytomegalovirus (HCMV) exploits the interleukin-10 (IL-10) pathway as a part of its infection cycle through the manipulation of the host IL-10 signaling cascade. Based on its immunomodulatory nature, HCMV attenuates the host immune response and facilitates the progression of co-infection with other pathogens in an immune-competent host. To investigate the impact of HCMV infection on the burden of non-tuberculous mycobacteria (NTM), whose prevalence is growing rapidly worldwide, macrophages were infected with HCMV and further challenged with Mycobacterium massiliense in vitro. The results showed that HCMV infection significantly increased host IL-10 synthesis and promoted the proliferation of M. massiliense in an IL-10-dependent manner. Transcriptomic analysis revealed that HCMV infection dampened the regulatory pathways of interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin-1 (IL-1), consequently abrogating the immune responses to M. massiliense coinfection in macrophages. These findings provide a mechanistic basis of how HCMV infection may facilitate the development of pathogenic NTM co-infection by upregulating IL-10 expression.

Protein Kinase A Catalytic Subunit Is a Molecular Switch that Promotes the Pro-tumoral Function of Macrophages.

As current therapies benefit only a minority of cancer patients, additional therapeutic targets are needed. Tumor-associated macrophages (TAMs) have attracted attention for improving therapeutic responses, yet regulatory strategies remain elusive. Here, we show that the protein kinase A catalytic subunit (PKA-C) acts as a molecular switch, inducing a pro-tumoral immunosuppressive macrophage phenotype within tumors. In human and murine breast cancer, overactivated PKA in TAMs creates a detrimental microenvironment for cancer progression by inducing vascular endothelial growth factor A (VEGFA), interleukin-10 (IL-10), and macrophage-derived arginase 1 (ARG1) expression. Macrophages with genetic deletion of PKA-C are prone to be pro-inflammatory, suggesting a possible immunotherapeutic target. Delivery of liposomal PKA inhibitor facilitates tumor regression and abrogates pro-tumoral TAM functions in mice. The therapeutic effect of targeting PKA is pronounced when combined with αCTLA-4 antibody, increasing cluster of differentiation 8 (CD8)+GranzymeB+ T cells by about 60-fold. Our findings demonstrate critical roles of TAM PKA-C in tumor progression and suggest that targeting PKA-C efficiently augments cancer treatment responses.

Pyruvate dehydrogenase kinase is a negative regulator of interleukin-10 production in macrophages.

Interleukin-10 (IL-10) is the most potent anti-inflammatory cytokine in the body and plays an essential role in determining outcomes of many inflammatory diseases. Cellular metabolism is a critical determinant of immune cell function; however, it is currently unclear whether metabolic processes are specifically involved in IL-10 production. In this study, we aimed to find the central metabolic molecule regulating IL-10 production of macrophages, which are the main producers of IL-10. Transcriptomic analysis identified that metabolic changes were predominantly enriched in Kupffer cells at the early inflammatory phase of a mouse endotoxemia model. Among them, pyruvate dehydrogenase kinase (PDK)-dependent acute glycolysis was negatively involved in IL-10 production. Inhibition or knockdown of PDK selectively increased macrophage IL-10 expression. Mechanistically, PDK inhibition increased IL-10 production via profound phosphorylation of adenosine monophosphate (AMP)-activated protein kinase alpha 1 (AMPKα1) by restricting glucose uptake in lipopolysaccharide-stimulated macrophages. AMPKα1 consequently activated p38 mitogen-activated protein kinase, c-Jun N-terminal kinase, and cyclic AMP-responsive element-binding protein to regulate IL-10 production. Our study uncovers a previously unknown regulatory mechanism of IL-10 in activated macrophages involving an immunometabolic function of PDK.

Identification of FES as a Novel Radiosensitizing Target in Human Cancers.

PURPOSE: The identification of novel targets for developing synergistic drug-radiation combinations would pave the way to overcome tumor radioresistance. We conducted cell-based screening of a human kinome siRNA library to identify a radiation-specific kinase that has a synergistic toxic effect with radiation upon inhibition and is not essential for cell survival in the absence of radiation. EXPERIMENTAL DESIGN: Unbiased RNAi screening was performed by transfecting A549 cells with a human kinome siRNA library followed by irradiation. Radiosensitizing effects of a target gene and involved mechanisms were examined. RESULTS: We identified the nonreceptor protein tyrosine kinase FES (FEline Sarcoma oncogene) as a radiosensitizing target. The expression of FES was increased in response to irradiation. Cell viability and clonogenic survival after irradiation were significantly decreased by FES knockdown in lung and pancreatic cancer cell lines. In contrast, FES depletion alone did not significantly affect cell proliferation without irradiation. An inducible RNAi mouse xenograft model verified in vivo radiosensitizing effects. FES-depleted cells showed increased apoptosis, DNA damage, G2-M phase arrest, and mitotic catastrophe after irradiation. FES depletion promoted radiation-induced reactive oxygen species formation, which resulted in phosphorylation of S6K and MDM2. The radiosensitizing effect of FES knockdown was partially reversed by inhibition of S6K activity. Consistent with the increase in phosphorylated MDM2, an increase in nuclear p53 levels was observed, which appears to contribute increased radiosensitivity of FES-depleted cells. CONCLUSIONS: We uncovered that inhibition of FES could be a potential strategy for inducing radiosensitization in cancer. Our results provide the basis for developing novel radiosensitizers.

Macrophages in intestinal inflammation and resolution: a potential therapeutic target in IBD.

Macrophages are the gatekeepers of intestinal immune homeostasis as they discriminate between innocuous antigens and potential pathogens to maintain oral tolerance. However, in individuals with a genetic and environmental predisposition, regulation of intestinal immunity is impaired, leading to chronic relapsing immune activation and pathologies of the gastrointestinal tract, such as IBD. As evidence suggests a causal link between defects in the resolution of intestinal inflammation and altered monocyte-macrophage differentiation in patients with IBD, macrophages have been considered as a novel potential target to develop new treatment approaches. This Review discusses the molecular and cellular mechanisms involved in the differentiation and function of intestinal macrophages in homeostasis and inflammation, and their role in resolving the inflammatory process. Understanding the molecular pathways involved in the specification of intestinal macrophages might lead to a new class of targets that promote remission in patients with IBD.

Versatile and Finely Tuned Albumin Nanoplatform based on Click Chemistry.

Albumin is one of the most attractive nanoplatforms for targeted imaging and drug delivery due to its biocompatibility and long circulation half-life. However, previously reported albumin-based nanoplatforms have shown inconsistent blood circulation half-life according to the modified methods, and the affecting factors were not well evaluated, which could hamper the clinical translation of albumin-based nanoplatforms. Herein, we developed a finely tuned click-chemistry based albumin nanoplatform (CAN) with a longer circulation half-life and an efficient tumor targeting ability. Methods: CAN was synthesized in two steps. First, albumin was conjugated with ADIBO-NHS (albumin-ADIBO) by reacting albumin with various molar ratios of ADIBO. The number of attached ADIBO moieties was determined using matrix-assisted laser desorption ionization time of flight (MALDI-TOF). Second, the desired modalities including azide-functionalized chelator, a fluorescence dye, and folate were incorporated into albumin-ADIBO using strain-promoted alkyne-azide cycloaddition reaction (SPAAC reaction). The biodistribution and targeting efficiency of functionalized CANs were demonstrated in mice. Results: The degree of functionalization (DOF) and resulting in vivo biodistribution was controlled precisely using the click chemistry approach. Specifically, the numbers of attached azadibenzocyclooctyne (ADIBO) moieties on albumin, the DOF, were optimized by reacting albumin with varying molar ratios of ADIBO with a high reproducibility. Furthermore, we developed a simple and efficient method to estimate the DOF using UV-visible spectrophotometry (UV-vis), which was further validated by matrix-assisted laser desorption ionization time of flight (MALDI-TOF). The biodistribution of CAN could be controlled by DOF, and CAN with an optimized DOF showed a long circulation half-life (> 18 h). CAN was further functionalized using a simple click chemistry reaction with an azide functionalized chelator, a fluorescence dye, and folate. 64Cu- and folate-labeled CAN (64Cu-CAN-FA) showed effective and specific folate receptor targeting in vivo, with an over two-fold higher uptake than the liver at 24 h post-injection. Conclusions: Our development from the precisely controlled DOF demonstrates that an optimized CAN can be used as a multifunctional nanoplatform to obtain a longer half-life with radioisotopes and ligands, and provides an effective method for the development of albumin-based tumor theranostic agents.

Macrophages-Triggered Sequential Remodeling of Endothelium-Interstitial Matrix to Form Pre-Metastatic Niche in Microfluidic Tumor Microenvironment.

The primed microenvironment of future metastatic sites, called the pre-metastatic niche, is a prerequisite for overt metastasis. However, a mechanistic understanding of the contributions of recruited cells to the niche is hindered by complex in vivo systems. Herein, a microfluidic platform that incorporates endothelial cells and extracellular matrix (ECM) scaffolds is developed, and the distinct role of recruited monocytes and macrophages in establishing pre-metastatic niches is delineated. It is observed that monocyte-derived matrix metalloproteinase 9 facilitates cancer cell extravasation through destruction of endothelial tight junctions. Furthermore, subsequent cancer cell invasiveness is significantly enhanced. Close examination of ECM structures reveals that cancer cells move within characteristic "microtracks" generated by macrophages, suggesting that macrophages could serve as a compensatory mechanism for the reduced migratory capacity of cancer cells. Thus, the first evidence of monocyte/macrophage-induced remodeling is shown, and these findings will open up new horizons for improving characterization of the pre-metastatic niche and corresponding immunotherapies.

Optimizing the cutoff for the identification of skin sensitizers by the HaCaSens assay: Introducing an ROC-analysis-based cutoff approach.

The worldwide restricted use of animal testing makes it challenging to identify the skin sensitizing potentials of newly manufactured products. The HaCaSens assay has shown promise as an in vitro skin sensitizing assay comparable to existing assays, and is currently under pre-validation. However, there is little agreement on how to assess the results of the assay to discriminate sensitizers from non-sensitizers as the stimulation index (SI) cutoff value was arbitrarily chosen without appropriate statistical methods. Here, we investigated the SI cutoff values in identifying sensitizers to obtain the optimal value. Sensitivities and specificities were calculated for a set of 30 test substances, and plotted in receiver operator characteristics (ROC) curves. The SI cutoff values with the highest sum of sensitivity and specificity according to LLNA data were 2.2, 1.8 and 3.0 for interleukin 1α (IL-1α), interleukin 6 (IL-6), and the combination of the two cytokines respectively. Also, the same statistical analysis of human data demonstrated optimal SI cutoff values 2.0, 2.0 and 3.2 for the same respective parameters. When considering the predictive capacity of each possible SI cutoff value determined by ROC curves, the optimal value for HaCaSens is 3.0 for the combination of IL-1α and IL-6 as it had the highest sensitivity (90.9%), specificity (75.0%) and accuracy (86.7%) based on LLNA data. Thus, we recommend the wide use of the SI cutoff value of 3.0 to ensure consistent endpoints.