Targeted deletion of CD244 on monocytes promotes differentiation into anti-tumorigenic macrophages and potentiates PD-L1 blockade in melanoma.

BACKGROUND: In the myeloid compartment of the tumor microenvironment, CD244 signaling has been implicated in immunosuppressive phenotype of monocytes. However, the precise molecular mechanism and contribution of CD244 to tumor immunity in monocytes/macrophages remains elusive due to the co-existing lymphoid cells expressing CD244. METHODS: To directly assess the role of CD244 in tumor-associated macrophages, monocyte-lineage-specific CD244-deficient mice were generated using cre-lox recombination and challenged with B16F10 melanoma. The phenotype and function of tumor-infiltrating macrophages along with antigen-specific CD8 T cells were analyzed by flow cytometry and single cell RNA sequencing data analysis, and the molecular mechanism underlying anti-tumorigenic macrophage differentiation, antigen presentation, phagocytosis was investigated ex vivo. Finally, the clinical feasibility of CD244-negative monocytes as a therapeutic modality in melanoma was confirmed by adoptive transfer experiments. RESULTS: CD244fl/flLysMcre mice demonstrated a significant reduction in tumor volume (61% relative to that of the CD244fl/fl control group) 14 days after tumor implantation. Within tumor mass, CD244fl/flLysMcre mice also showed higher percentages of Ly6Clow macrophages, along with elevated gp100+IFN-γ+ CD8 T cells. Flow cytometry and RNA sequencing data demonstrated that ER stress resulted in increased CD244 expression on monocytes. This, in turn, impeded the generation of anti-tumorigenic Ly6Clow macrophages, phagocytosis and MHC-I antigen presentation by suppressing autophagy pathways. Combining anti-PD-L1 antibody with CD244-/- bone marrow-derived macrophages markedly improved tumor rejection compared to the anti-PD-L1 antibody alone or in combination with wild-type macrophages. Consistent with the murine data, transcriptome analysis of human melanoma tissue single-cell RNA-sequencing dataset revealed close association between CD244 and the inhibition of macrophage maturation and function. Furthermore, the presence of CD244-negative monocytes/macrophages significantly increased patient survival in primary and metastatic tumors. CONCLUSION: Our study highlights the novel role of CD244 on monocytes/macrophages in restraining anti-tumorigenic macrophage generation and tumor antigen-specific T cell response in melanoma. Importantly, our findings suggest that CD244-deficient macrophages could potentially be used as a therapeutic agent in combination with immune checkpoint inhibitors. Furthermore, CD244 expression in monocyte-lineage cells serve as a prognostic marker in cancer patients.

Real-Time Live Imaging of Osteoclast Activation via Cathepsin K Activity in Bone Diseases.

Intravital fluorescence imaging of functional osteoclasts within their intact disease context provides valuable insights into the intricate biology at the microscopic level, facilitating the development of therapeutic approaches for osteoclast-associated bone diseases. However, there is a lack of studies investigating osteoclast activity within deep-seated bone lesions using appropriate fluorescent probes, despite the advantages offered by the multi-photon excitation system in enhancing deep tissue imaging resolution. In this study, we report on the intravital tracking of osteoclast activity in three distinct murine bone disease models. We utilized a cathepsin K (CatK)-responsive two-photon fluorogenic probe (CatKP1), which exhibited a notable fluorescence turn-on response in the presence of active CatK. By utilizing CatKP1, we successfully monitored a significant increase in osteoclast activity in hindlimb long bones and its attenuation through pharmacological intervention without sacrificing mice. Thus, our findings highlight the efficacy of CatKP1 as a valuable tool for unraveling pathological osteoclast behavior and exploring novel therapeutic strategies.

Circulating biomarkers for diagnosis and therapeutic monitoring in bone metastasis.

Bone is a frequent site of metastasis for multiple types of solid tumors in organs such as prostate, breast, lung, etc., accounting for significant morbidities and mortalities of afflicted patients. One of the major problems of bone metastasis is lack of biomarkers for early diagnosis and for monitoring therapeutic responses. Medical imaging modalities such as computerized tomography, magnetic resonance imaging, and radioactive isotope-based bone scans are currently standard clinical practices, yet these imaging techniques are limited to detect early lesions or to accurately monitor the metastatic disease progression during standard and/or experimental therapies. Accordingly, development of novel blood biomarkers rationalizes extensive basic research and clinical development. This review article covers the up-to-date information on protein- and cell-based biomarkers of bone metastasis that are currently used in the clinical practices and also are under development.

Unique cartilage matrix-associated protein inhibits the migratory and invasive potential of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) that lacks expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) is a breast cancer subtype with very aggressive metastasis and poor prognosis. Unique cartilage matrix-associated protein (UCMA) is a vitamin K-dependent protein (VKDP) with a high-density γ-carboxyglutamic acid (Gla) domain due to the action of vitamin K. UCMA promotes osteoblast differentiation and mineral deposition in bone and suppresses calcification in vessels. However, correlation between UCMA and TNBC is unknown. This study investigated the inhibitory effect of UCMA on TNBC cell in vitro migration, invasion, and colony formation in addition to in vivo tumorigenesis. Cell migration and invasion significantly decreased in Ucma-overexpressing MDA-MB-231 and 4T1 cells compared to the mock control cells. Also, colony formation and the number of colonies significantly decreased in Ucma-overexpressing MDA-MB-231 and 4T1 cells. These results indicate that UCMA significantly inhibits the migration, invasion, and colony formation of TNBC cells. In an in vivo xenograft mouse model, tumor growth significantly decreased in mice bearing Ucma-overexpressing TNBC cells compared to the mock control cells, indicating that UCMA reduced in vivo tumor growth, similar to the inhibitory role of UCMA in vitro. Survival analysis using publicly available database showed that high UCMA expression significantly correlated with favorable relapse-free survival in TNBC patients compared to those with the other VKDPs, matrix Gla protein (MGP) and osteocalcin (OCN). Collectively, this study suggests that UCMA is a promising new therapeutic agent for TNBC.

The p38-activated ER stress-ATF6α axis mediates cellular senescence.

The importance of proteostasis in preventing cellular senescence has been well recognized. However, the exact mechanism by which the loss of proteostasis or endoplasmic reticulum (ER) stress induces cellular senescence remains unclear. We report that ER stress mediates cellular senescence through the activating transcription factor (ATF)6α branch of the unfolded protein response (UPR). Cellular senescence was induced by the abrogation of neighbor of breast cancer (BRCA)1 gene (NBR1). NBR1 abrogation-induced senescence was p53 dependent and observed in both transformed and nontransformed human cell lines: MCF-7, Caki-1, and MRC-5. NBR1 bound to p38 MAPK, preferentially to an active form, and upon NBR1 abrogation, the activity of p38 increased. NADPH oxidase was activated in turn by p38, and the resulting oxidative stress triggered ER stress. It was found that ER stress mediated cellular senescence through the UPR sensor ATF6α. Knockdown of ATF6α prevented senescence, whereas ATF6α overexpression triggered it. The transcriptional activity of ATF6α was important. The ER stress-ATF6α axis also mediated cellular senescence induced by H-RasV12 overexpression and UV irradiation, suggesting a common role of this axis in senescence induction. In summary, we presented an evidence for the novel role of the ER stress-ATF6α axis in cellular senescence.-Kim, H. S., Kim, Y., Lim, M. J., Park, Y.-G., Park, S. I., Sohn, J. The p38-activated ER stress-ATF6α axis mediates cellular senescence.

Depletion of NBR1 in urothelial carcinoma cells enhances rapamycin-induced apoptosis through impaired autophagy and mitochondrial dysfunction.

Rapamycin is well-recognized in the clinical therapeutic intervention for patients with cancer by specifically targeting mammalian target of rapamycin (mTOR) kinase. Rapamycin regulates general autophagy to clear damaged cells. Previously, we identified increased expression of messenger RNA levels of NBR1 (the neighbor of BRCA1 gene; autophagy cargo receptor) in human urothelial cancer (URCa) cells, which were not exhibited in response to rapamycin treatment for cell growth inhibition. Autophagy plays an important role in cellular physiology and offers protection against chemotherapeutic agents as an adaptive response required for maintaining cellular energy. Here, we hypothesized that loss of NBR1 sensitizes human URCa cells to growth inhibition induced by rapamycin treatment, leading to interruption of protective autophagic activation. Also, the potential role of mitochondria in regulating autophagy was tested to clarify the mechanism by which rapamycin induces apoptosis in NBR1-knockdown URCa cells. NBR1-knockdown URCa cells exhibited enhanced sensitivity to rapamycin associated with the suppression of autophagosomal elongation and mitochondrial defects. Loss of NBR1 expression altered the cellular responses to rapamycin treatment, resulting in impaired ATP homeostasis and an increase in reactive oxygen species (ROS). Although rapamycin treatment-induced autophagy by adenosine monophosphate-activated protein kinase (AMPK) phosphorylation in NBR1-knockdown cells, it did not process the conjugated form of LC3B-II after activation by unc-51 like autophagy-activating kinase 1 (ULK1). NBR1-knockdown URCa cells exhibited rather profound mitochondrial dysfunctions in response to rapamycin treatment as evidenced by Δψm collapse, ATP depletion, ROS accumulation, and apoptosis activation. Therefore, our findings provide a rationale for rapamycin treatment of NBR1-knockdown human urothelial cancer through the regulation of autophagy and mitochondrial dysfunction by regulating the AMPK/mTOR signaling pathway, indicating that NBR1 can be a potential therapeutic target of human urothelial cancer.

Targeting the Hepatocyte Growth Factor and c-Met Signaling Axis in Bone Metastases.

Bone metastasis is the terminal stage disease of prostate, breast, renal, and lung cancers, and currently no therapeutic approach effectively cures or prevents its progression to bone metastasis. One of the hurdles to the development of new drugs for bone metastasis is the complexity and heterogeneity of the cellular components in the metastatic bone microenvironment. For example, bone cells, including osteoblasts, osteoclasts, and osteocytes, and the bone marrow cells of diverse hematopoietic lineages interact with each other via numerous cytokines and receptors. c-Met tyrosine kinase receptor and its sole ligand hepatocyte growth factor (HGF) are enriched in the bone microenvironment, and their expression correlates with the progression of bone metastasis. However, no drugs or antibodies targeting the c-Met/HGF signaling axis are currently available in bone metastatic patients. This significant discrepancy should be overcome by further investigation of the roles and regulation of c-Met and HGF in the metastatic bone microenvironment. This review paper summarizes the key findings of c-Met and HGF in the development of novel therapeutic approaches for bone metastasis.

Osteal macrophages support physiologic skeletal remodeling and anabolic actions of parathyroid hormone in bone.

Cellular subpopulations in the bone marrow play distinct and unexplored functions in skeletal homeostasis. This study delineated a unique role of osteal macrophages in bone and parathyroid hormone (PTH)-dependent bone anabolism using murine models of targeted myeloid-lineage cell ablation. Depletion of c-fms(+) myeloid lineage cells [via administration of AP20187 in the macrophage Fas-induced apoptosis (MAFIA) mouse model] reduced cortical and trabecular bone mass and attenuated PTH-induced trabecular bone anabolism, supporting the positive function of macrophages in bone homeostasis. Interestingly, using a clodronate liposome model with targeted depletion of mature phagocytic macrophages an opposite effect was found with increased trabecular bone mass and increased PTH-induced anabolism. Apoptotic cells were more numerous in MAFIA versus clodronate-treated mice and flow cytometric analyses of myeloid lineage cells in the bone marrow showed that MAFIA mice had reduced CD68(+) cells, whereas clodronate liposome-treated mice had increased CD68(+) and CD163(+) cells. Clodronate liposomes increased efferocytosis (clearance of apoptotic cells) and gene expression associated with alternatively activated M2 macrophages as well as expression of genes associated with bone formation including Wnt3a, Wnt10b, and Tgfb1. Taken together, depletion of early lineage macrophages resulted in osteopenia with blunted effects of PTH anabolic actions, whereas depletion of differentiated macrophages promoted apoptotic cell clearance and transformed the bone marrow to an osteogenic environment with enhanced PTH anabolism. These data highlight a unique function for osteal macrophages in skeletal homeostasis.

Inhibition of cyclic AMP response element-directed transcription by decoy oligonucleotides enhances tumor-specific radiosensitivity.

The radiation stress induces cytotoxic responses of cell death as well as cytoprotective responses of cell survival. Understanding exact cellular mechanism and signal transduction pathways is important in improving cancer radiotherapy. Increasing evidence suggests that cyclic AMP response element binding protein (CREB)/activating transcription factor (ATF) family proteins act as a survival factor and a signaling molecule in response to stress. We postulated that CREB inhibition via CRE decoy oligonucleotide increases tumor cell sensitization to γ-irradiation-induced cytotoxic stress. In the present study, we demonstrate that CREB phosphorylation and CREB DNA-protein complex formation increased in time- and radiation dose-dependent manners, while there was no significant change in total protein level of CREB. In addition, CREB was phosphorylated in response to γ-irradiation through p38 MAPK pathway. Further investigation revealed that CREB blockade by decoy oligonucleotides functionally inhibited transactivation of CREB, and significantly increased radiosensitivity of multiple human cancer cell lines including TP53- and/or RB-mutated cells with minimal effects on normal cells. We also demonstrate that tumor cells ectopically expressing dominant negative mutant CREB (KCREB) and the cells treated with p38 MAPK inhibitors were more sensitive to γ-irradiation than wild type parental cells or control-treated cells. Taken together, we conclude that CREB protects tumor cells from γ-irradiation, and combination of CREB inhibition plus ionizing radiation will be a promising radiotherapeutic approach.

Signaling between transforming growth factor ß (TGF-ß) and transcription factor SNAI2 represses expression of microRNA miR-203 to promote epithelial-mesenchymal transition and tumor metastasis.

TGF-ß promotes tumor invasion and metastasis by inducing an epithelial-mesenchymal transition (EMT). Understanding the molecular and epigenetic mechanisms by which TGF-ß induces EMT may facilitate the development of new therapeutic strategies for metastasis. Here, we report that TGF-ß induced SNAI2 to promote EMT by repressing miR-203. Although miR-203 targeted SNAI2, SNAI2 induced by TGF-ß could directly bind to the miR-203 promoter to inhibit its transcription. SNAI2 and miR-203 formed a double negative feedback loop to inhibit each other's expression, thereby controlling EMT. Moreover, we found that miR-203 was significantly down-regulated in highly metastatic breast cancer cells. The restoration of miR-203 in highly metastatic breast cancer cells inhibited tumor cell invasion in vitro and lung metastatic colonization in vivo by repressing SNAI2. Taken together, our results suggest that the SNAI2 and miR-203 regulatory loop plays important roles in EMT and tumor metastasis.

Mobilization of monocytic myeloid-derived suppressor cells is regulated by PTH1R activation in bone marrow stromal cells.

Myeloid-derived suppressor cells (MDSCs) are bone marrow (BM)-derived immunosuppressive cells in the tumor microenvironment, but the mechanism of MDSC mobilization from the BM remains unclear. We investigated how BM stromal cell activation by PTH1R contributes to MDSC mobilization. PTH1R activation by parathyroid hormone (PTH) or PTH-related peptide (PTHrP), a tumor-derived counterpart, mobilized monocytic (M-) MDSCs from murine BM without increasing immunosuppressive activity. In vitro cell-binding assays demonstrated that α4ß1 integrin and vascular cell adhesion molecule (VCAM)-1, expressed on M-MDSCs and osteoblasts, respectively, are key to M-MDSC binding to osteoblasts. Upon PTH1R activation, osteoblasts express VEGF-A and IL6, leading to Src family kinase phosphorylation in M-MDSCs. Src inhibitors suppressed PTHrP-induced MDSC mobilization, and Src activation in M-MDSCs upregulated two proteases, ADAM-17 and MMP7, leading to VCAM1 shedding and subsequent disruption of M-MDSC tethering to osteoblasts. Collectively, our data provide the molecular mechanism of M-MDSC mobilization in the bones of tumor hosts.

The multifaceted actions of PTHrP in skeletal metastasis.

PTHrP, identified during the elucidation of mediators of malignancy-induced hypercalcemia, plays numerous roles in normal physiology as well as pathological conditions. Recent data support direct functions of PTHrP in metastasis, particularly from tumors with strong bone tropism. Bone provides a unique metastatic environment because of mineralization and the diverse cell populations in the bone marrow. PTHrP is a key regulator of tumor-bone interactions and regulates cells in the bone microenvironment through proliferative and prosurvival activities that prime the 'seed' and the 'soil' of the metastatic lesion. This review highlights recent findings regarding the role of PTHrP in skeletal metastasis, including direct actions in tumor cells, as well as alterations in the bone microenvironment and future perspectives involving the potential roles of PTHrP in the premetastatic niche, and tumor dormancy.

Flow cytometry-based immunophenotyping of myeloid-derived suppressor cells in human breast cancer patient blood samples.

Multi-color flow cytometry is the standard approach for immunophenotyping clinical samples. With the recent advances in cancer immunotherapy, myeloid-derived suppressor cells (MDSC), immature myeloid-lineage cells in cancer patient blood and the tumor microenvironment, are highlighted as an important immune cell population that correlates with prognosis and therapeutic efficacy. In contrast to their clear functions and existence, immunophenotyping of MDSC is not consistent among investigators due to surface antigens overlapping with many normal hematopoietic lineage cell populations. We performed a clinical study and analyzed more than 1000 breast cancer patients blood samples to quantitate MDSC during breast cancer progression. In this methodology manuscript, we described detailed procedures for study design, sample logistics and handling, staining and flow cytometric analysis. This protocol used a 7-color fluorochrome-conjugated antibody panel to analyze polymorphonuclear (PMN)- and monocytic (M)-MDSC subsets simultaneously. The interim analysis results of this study showed that both PMN and M-MDSC populations are increased in patients with bone metastasis compared with patients with visceral organ metastasis. In conclusion, this work provides a versatile, comprehensive, and practical protocol to measure MDSC in patient blood samples.

Nuclear transport of STAT6 determines the matrix rigidity dependent M2 activation of macrophages.

Alternatively activated or M2 macrophages, as opposed to the well characterized pro-inflammatory or M1 macrophages, vitally regulate anti-inflammation, wound healing, and tissue repair to maintain tissue homeostasis. Although ubiquitous presence of macrophages in diverse tissues, exposed to different physical environments, infers distinct immune responses of M2 macrophages with high phenotypic heterogeneity, the underlying mechanism of how the varying extracellular mechanical conditions alter their immunological activation remains unclear. Here, we demonstrate that M2 activation requires a threshold mechanical cue from the extracellular microenvironment, and matrix rigidity-dependent macrophage spreading is mediated by the F-actin formation that is essential to regulate mechanosensitive M2 activation of macrophages. We identified a new mechanosensing function of STAT6 (signal transducer and activator of transcription 6), a key transcription factor for M2 activation, whose intranuclear transportation is promoted by the rigid matrix that facilitates the F-actin formation. Our findings further highlight the critical role of mechanosensitive M2 activation of macrophages in long-term adaptation to the extracellular microenvironment by bridging nuclear mechanosensation and immune responses.

AFAP-110 is overexpressed in prostate cancer and contributes to tumorigenic growth by regulating focal contacts.

The actin filament-associated protein AFAP-110 is an actin cross-linking protein first identified as a substrate of the viral oncogene v-Src. AFAP-110 regulates actin cytoskeleton integrity but also functions as an adaptor protein that affects crosstalk between Src and PKC. Here we investigated the roles of AFAP-110 in the tumorigenic process of prostate carcinoma. Using immunohistochemistry of human tissue arrays, we found that AFAP-110 was absent or expressed at very low levels in normal prostatic epithelium and benign prostatic hyperplasia but significantly increased in prostate carcinomas. The level of AFAP-110 in carcinomas correlated with the Gleason scores. Downregulation of AFAP-110 in PC3 prostate cancer cells inhibited cell proliferation in vitro and tumorigenicity and growth in orthotopic nude mouse models. Furthermore, downmodulation of AFAP-110 resulted in decreased cell-matrix adhesion and cell migration, defective focal adhesions, and reduced integrin beta1 expression. Reintroduction of avian AFAP-110 or a mutant disabling its interaction with Src restored these properties. However, expression of an AFAP-110 lacking the PKC-interacting domain failed to restore properties of parental cells. Thus, increased expression of AFAP-110 is associated with progressive stages of prostate cancer and is critical for tumorigenic growth, in part by regulating focal contacts in a PKC-dependent mechanism.

Liposome-Encapsulated Bacillus Calmette-Guérin Cell Wall Skeleton Enhances Antitumor Efficiency for Bladder Cancer In Vitro and In Vivo via Induction of AMP-Activated Protein Kinase.

The Mycobacterium Bacillus Calmette-Guérin cell wall skeleton (BCG-CWS), the main immune active center of BCG, is a potent candidate non-infectious immunotherapeutic drug and an alternative to live BCG for use against urothelial carcinoma. However, its application in anticancer therapy is limited, as BCG-CWS tends to aggregate in both aqueous and non-aqueous solvents. To improve the internalization of BCG-CWS into bladder cancer cells without aggregation, BCG-CWS was nanoparticulated at a 180 nm size in methylene chloride and subsequently encapsulated with conventional liposomes (CWS-Nano-CL) using an emulsified lipid (LEEL) method. In vitro cell proliferation assays showed that CWS-Nano-CL was more effective at suppressing bladder cancer cell growth compared to nonenveloped BCG-CWS. In an orthotopic implantation model of luciferase-tagged MBT2 bladder cancer cells, encapsulated BCG-CWS nanoparticles could enhance the delivery of BCG-CWS into the bladder and suppress tumor growth. Treatment with CWS-Nano-CL induced the inhibition of the mammalian target of rapamycin (mTOR) pathway and the activation of AMP-activated protein kinase (AMPK) phosphorylation, leading to apoptosis, both in vitro and in vivo. Furthermore, the antitumor activity of CWS-Nano-CL was mediated predominantly by reactive oxygen species (ROS) generation and AMPK activation, which induced endoplasmic reticulum (ER) stress, followed by c-Jun N-terminal kinase (JNK) signaling-mediated apoptosis. Therefore, our data suggest that the intravesical instillation of liposome-encapsulated BCG-CWS nanoparticles can facilitate BCG-CW cellular endocytosis and provide a promising drug-delivery system as a therapeutic strategy for BCG-mediated bladder cancer treatment.

Circulating Osteocalcin-Positive Cells as a Novel Diagnostic Biomarker for Bone Metastasis in Breast Cancer Patients.

Current diagnosis of bone metastasis (BM) in breast cancer relies on structural changes of bone that occur only in the advanced stage. A sensitive biomarker for detecting early progression of bone metastasis is urgently required. We performed clinical and preclinical studies to investigate diagnostic value of circulating osteocalcin-positive cells (cOC) in breast cancer bone metastasis. Metastatic breast cancer patients (n = 92) with or without bone metastasis (ie, BM+ or BM- ) were enrolled, and cOC were measured at enrollment. Patients were followed up for bone metastasis progression for 18 months. BM+ patients (n = 59) were divided into progressive (PD) or stable disease (SD) groups, based on imaging studies at the end of the 18-month study. The PD group had higher baseline cOC compared with the SD group. Furthermore, higher cOC resulted in reduced BM progression-free survival. Three patients in the BM- group (n = 33) developed new BM during the 18-month study, and these patients had a higher level of baseline cOC compared with the remaining BM- patients. In murine preclinical studies, cOC increased at early time points when micro-metastases were evident only by histology but undetectable by bioluminescence imaging. Also, cOC levels predicted the progression of BM and correlated significantly with BM tumor burden. cOC increased in the early phase of breast cancer BM and can predict BM progression, supporting cOC as a potential novel biomarker. © 2020 American Society for Bone and Mineral Research.

Comparison of Various Implant Provisional Resin Materials for Cytotoxicity and Attachment to Human Gingival Fibroblasts.

PURPOSE: The aim of this study was to evaluate the responses of human gingival fibroblast (HGF-1) in contact with provisional materials with various chemical compositions and fabricated using different methods. MATERIALS AND METHODS: A total of 210 specimens in eight experimental groups were used. Groups were divided by chemical compositions (poly[ethyl methacrylate], poly[methyl methacrylate], bis-acryl, and hybrid ceramic) and fabricating methods (direct, indirect, and computer-aided design/computer-aided manufacturing [CAD/CAM]). To evaluate the surface characteristics of each group, roughness, water contact angle, and degree of conversion were measured. The responses of HGF-1 to provisional materials were evaluated with cytotoxicity and cell attachment assay. The roughness, surface energy, degree of conversion, level of cytotoxicity, and cell attachment were compared between groups using one-way analysis of variance (ANOVA) and Tukey's multiple comparison (α = .05). RESULTS: The poly(ethyl methacrylate)-direct/indirect and poly(methyl methacrylate)-direct/indirect groups showed higher roughness than the bis-acryl-direct/indirect, poly(methyl methacrylate)-CAD/CAM, and hybrid ceramic-CAD/CAM groups with statistical significance (P < .05). The poly(ethyl methacrylate)-direct group showed the significantly highest water contact angle, and the hybrid ceramic-CAD/CAM group showed the lowest water contact angle (P < .05). The groups that used indirect fabrication methods showed a higher degree of conversion than those that used direct fabrication methods, regardless of chemical composition (P < .05). The poly(ethyl methacrylate) groups showed significantly lower cell viability than the other groups regardless of fabricating methods (P < .05). The poly(ethyl methacrylate)-direct method group showed the lowest cell attachment, and the hybrid ceramic-CAD/CAM method group showed the highest cell attachment (P < .05). CONCLUSION: Poly(methyl methacrylate) and bis-acryl have lower cytotoxicity to HGF-1 than poly(ethyl methacrylate). Indirect fabrication and CAD/CAM are recommended to prevent residual monomer and achieve high cell attachment. To use direct fabrication methods, the auto-mix system is beneficial for the favorable cell response, as it derives a smooth surface.

A Selective FGFR inhibitor AZD4547 suppresses RANKL/M-CSF/OPG-dependent ostoclastogenesis and breast cancer growth in the metastatic bone microenvironment.

Aberrant activation of fibroblast growth factor receptor (FGFR) signalling contributes to progression and metastasis of many types of cancers including breast cancer. Accordingly, FGFR targeted tyrosine kinase inhibitors (TKIs) are currently under development. However, the efficacy of FGFR TKIs in the bone microenvironment where breast cancer cells most frequently metastasize and also where FGFR is biologically active, has not been clearly investigated. We investigated the FGFR-mediated interactions among cancer and the bone microenvironment stromal cells (osteoblasts and osteoclasts), and also the effects of FGFR inhibition in bone metastasis. We showed that addition of culture supernatant from the MDA-MB-134-VI FGFR-amplified breast cancer cells-activated FGFR siganalling in osteoblasts, including increased expression of RANKL, M-CSF, and osteoprotegerin (OPG). Further in vitro analyses showed that AZD4547, an FGFR TKI currently in clinical trials for breast cancer, decreased RANKL and M-CSF, and subsequently RANKL and M-CSF-dependent osteoclastogenesis of murine bone marrow monocytes. Moreover, AZD4547 suppressed osteoclastogenesis and tumor-induced osteolysis in an orthotopic breast cancer bone metastasis mouse model using FGFR non-amplified MDA-MB-231 cells. Collectively, our results support that FGFR inhibitors inhibit the bone microenvironment stromal cells including osteoblasts and osteoclasts, and effectively suppress both tumor and stromal compartments of bone metastasis.

Interleukin-7 Contributes to the Invasiveness of Prostate Cancer Cells by Promoting Epithelial-Mesenchymal Transition.

Precise mechanisms underlying interleukin-7 (IL-7)-mediated tumor invasion remain unclear. Thus, we investigated the role of IL-7 in tumor invasiveness using metastatic prostate cancer PC-3 cell line derivatives, and assessed the potential of IL-7 as a clinical target using a Janus kinase (JAK) inhibitor and an IL-7-blocking antibody. We found that IL-7 stimulated wound-healing migration and invasion of PC-3 cells, increased phosphorylation of signal transducer and activator of transcription 5, Akt, and extracellular signal-regulated kinase. On the other hand, a JAK inhibitor and an IL-7-blocking antibody decreased the invasiveness of PC-3 cells. IL-7 increased tumor sphere formation and expression of epithelial-mesenchymal transition (EMT) markers. Importantly, lentiviral delivery of IL-7Rα to PC-3 cells significantly increased bone metastasis in an experimental murine metastasis model compared to controls. The gene expression profile of human prostate cancer cells from The Cancer Genome Atlas revealed that EMT pathways are strongly associated with prostate cancers that highly express both IL-7 and IL-7Rα. Collectively, these data suggest that IL-7 and/or IL-7Rα are promising targets of inhibiting tumor metastasis.