Bone Marrow-derived Cell Line Research Articles

AREG Upregulation in Cancer Cells via Direct Interaction with Cancer-Associated Fibroblasts Promotes Esophageal Squamous Cell Carcinoma Progression Through EGFR-Erk/p38 MAPK Signaling.

Cancer-associated fibroblasts (CAFs) are a key component of the tumor microenvironment and significantly contribute to the progression of various cancers, including esophageal squamous cell carcinoma (ESCC). Our previous study established a direct co-culture system of human bone marrow-derived mesenchymal stem cells (progenitors of CAFs) and ESCC cell lines, which facilitates the generation of CAF-like cells and enhances malignancy in ESCC cells. In this study, we further elucidated the mechanism by which CAFs promote ESCC progression using cDNA microarray analysis of monocultured ESCC cells and those co-cultured with CAFs. We observed an increase in the expression and secretion of amphiregulin (AREG) and the expression and phosphorylation of its receptor EGFR in co-cultured ESCC cells. Moreover, AREG treatment of ESCC cells enhanced their survival and migration via the EGFR-Erk/p38 MAPK signaling pathway. Immunohistochemical analysis of human ESCC tissues showed a positive correlation between the intensity of AREG expression at the tumor-invasive front and the expression level of the CAF marker FAP. Bioinformatics analysis confirmed significant upregulation of AREG in ESCC compared with normal tissues. These findings suggest that AREG plays a crucial role in CAF-mediated ESCC progression and could be a novel therapeutic target for ESCC.

Biosynthesized Silver Nanoparticles from Cyperus conglomeratus Root Extract Inhibit Osteogenic Differentiation of Immortalized Mesenchymal Stromal Cells.

Silver nanoparticles (AgNPs) are a focus of huge interest in biological research, including stem cell research. AgNPs synthesized using Cyperus conglomeratus root extract have been previously reported but their effects on mesenchymal stromal cells have yet to be investigated. The aim of this study is to investigate the effects of C. conglomeratus-derived AgNPs on adipogenesis and osteogenesis of mesenchymal stromal cells. AgNPs were synthesized using C. conglomeratus root extract, and the phytochemicals involved in AgNPs synthesis were analyzed using gas chromatography-mass spectrometry (GCMS). The cytotoxicity of the AgNPs was tested on telomerase-transformed immortalized human bone marrow-derived MSCs-hTERT (iMSC3) and human osteosarcoma cell line (MG-63) using MTT and apoptosis assays. The uptake of AgNPs by both cells was confirmed using inductively coupled plasma-optical emission spectrometry (ICP-OES). Furthermore, the effect of AgNPs on iMSC3 adipogenesis and osteogenesis was analyzed using stain quantification and reverse transcription- quantitative polymerase chain reaction (RT-qPCR). The phytochemicals predominately identified in both the AgNPs and C. conglomeratus root extract were carbohydrates. The AgNP concentrations tested using MTT and apoptosis assays (0.5-64 µg/ml and 1,4 and 32 µg/ml, respectively) showed no significant cytotoxicity on iMSC3 and MG-63. The AgNPs were internalized in a concentration-dependent manner in both cell types. Additionally, the AgNPs exhibited a significant negative effect on osteogenesis but not on adipogenesis. C. conglomeratus-derived AgNPs had an impact on the differentiation capacity of iMSC3. Our results indicated that C. conglomeratus AgNPs and the associated phytochemicals could exhibit potential medical applications.

Stimulator of Interferon Gene Agonists Induce an Innate Antiviral Response against Influenza Viruses.

The devastating effects of COVID-19 have highlighted the importance of prophylactic and therapeutic strategies to combat respiratory diseases. Stimulator of interferon gene (STING) is an essential component of the host defense mechanisms against respiratory viral infections. Although the role of the cGAS/STING signaling axis in the innate immune response to DNA viruses has been thoroughly characterized, mounting evidence shows that it also plays a key role in the prevention of RNA virus infections. In this study, we investigated the role of STING activation during Influenza virus (IFV) infection. In both mouse bone marrow-derived macrophages and monocytic cell line THP-1 differentiated with PMA, we found that dimeric amidobenzimidazole (diABZI), a STING agonist, had substantial anti-IFV activity against multiple strains of IFV, including A/H1N1, A/H3N2, B/Yamagata, and B/Victoria. On the other hand, a pharmacological antagonist of STING (H-151) or the loss of STING in human macrophages leads to enhanced viral replication but suppressed IFN expression. Furthermore, diABZI was antiviral against IFV in primary air-liquid interface cultures of nasal epithelial cells. Our data suggest that STING agonists may serve as promising therapeutic antiviral agents to combat IFV.

Discovery of YS-1 as a cell line of gastric inflammatory cancer-associated fibroblasts.

Inflammatory cancer-associated fibroblasts (iCAFs) was first identified by co-culture of pancreatic stellate cells and tumor organoids. The key feature of iCAFs is IL-6high/αSMAlow. We examine this phenomenon in gastric cancer using two cell lines of gastric fibroblasts (HGF and YS-1). HGF or YS-1 were co-cultured with MKN7 (a gastric adenocarcinoma cell line) in Matrigel. IL-6 protein levels in the culture supernatant were measured by ELISA. The increased production of IL-6 was not observed in any of the combinations. Instead, the supernatant of YS-1 exhibited the higher levels of IL-6. YS-1 showed IL-6high/αSMA (ACTA2)low in real-time PCR, mRNA-seq and immunohistochemistry. In mRNA-seq, iCAFs-associated genes and signaling pathways were up-regulated in YS-1. No transition to myofibroblastic phenotype was observed by monolayer culture, or the exposure to sonic hedgehog (SHH) or TGF-β. YS-1 conditioned medium induced changes of morphology and stem-ness/differentiation in NUGC-3 (a human gastric adenocarcinoma cell line) and UBE6T-15 (a human bone marrow-derived mesenchymal stem cell line). YS-1 is a stable cell line of gastric iCAFs. This discovery will promote further research on iCAFs for many researchers.

Targeting HDAC6 improves anti-CD47 immunotherapy

BackgroundCancer cells can overexpress CD47, an innate immune checkpoint that prevents phagocytosis upon interaction with signal regulatory protein alpha (SIRPα) expressed in macrophages and other myeloid cells. Several clinical trials have reported that CD47 blockade reduces tumor growth in hematological malignancies. However, CD47 blockade has shown modest results in solid tumors, including melanoma. Our group has demonstrated that histone deacetylase 6 inhibitors (HDAC6is) have immunomodulatory properties, such as controlling macrophage phenotype and inflammatory properties. However, the molecular and cellular mechanisms controlling these processes are not fully understood. In this study, we evaluated the role of HDAC6 in regulating the CD47/SIRPα axis and phagocytosis in macrophages.MethodsWe tested the role of HDAC6is, especially Nexturastat A, in regulating macrophage phenotype and phagocytic function using bone marrow-derived macrophages and macrophage cell lines. The modulation of the CD47/SIRPα axis and phagocytosis by HDAC6is was investigated using murine and human melanoma cell lines and macrophages. Phagocytosis was evaluated via coculture assays of macrophages and melanoma cells by flow cytometry and immunofluorescence. Lastly, to evaluate the antitumor activity of Nexturastat A in combination with anti-CD47 or anti-SIRPα antibodies, we performed in vivo studies using the SM1 and/or B16F10 melanoma mouse models.ResultsWe observed that HDAC6is enhanced the phenotype of antitumoral M1 macrophages while decreasing the protumoral M2 phenotype. In addition, HDAC6 inhibition diminished the expression of SIRPα, increased the expression of other pro-phagocytic signals in macrophages, and downregulated CD47 expression in mouse and human melanoma cells. This regulatory role on the CD47/SIRPα axis translated into enhanced antitumoral phagocytic capacity of macrophages treated with Nexturastat A and anti-CD47. We also observed that the systemic administration of HDAC6i enhanced the in vivo antitumor activity of anti-CD47 blockade in melanoma by modulating macrophage and natural killer cells in the tumor microenvironment. However, Nexturastat A did not enhance the antitumor activity of anti-SIRPα despite its modulation of macrophage populations in the SM1 tumor microenvironment.ConclusionsOur results demonstrate the critical regulatory role of HDAC6 in phagocytosis and innate immunity for the first time, further underscoring the use of these inhibitors to potentiate CD47 immune checkpoint blockade therapeutic strategies.

Long Noncoding RNA Gpr137b-ps Promotes Advanced Atherosclerosis via the Regulation of Autophagy in Macrophages.

Current therapies cannot completely reverse advanced atherosclerosis. High levels of amino acids, induced by Western diet, stimulate mTORC1 (mammalian target of rapamycin complex 1)-autophagy defects in macrophages, accelerating atherosclerotic plaque progression. In addition, autophagy-lysosomal dysfunction contributes to plaque necrotic core enlargement and lipid accumulation. Therefore, it is essential to investigate the novel mechanism and molecules to reverse amino acid-mTORC1-autophagy signaling dysfunction in macrophages of patients with advanced atherosclerosis. We observed that Gpr137b-ps (G-protein-coupled receptor 137B, pseudogene) was upregulated in advanced atherosclerotic plaques. The effect of Gpr137b-ps on the progression of atherosclerosis was studied by generating advanced plaques in ApoE-/- mice with cardiac-specific knockout of Gpr137b-ps. Bone marrow-derived macrophages and mouse mononuclear macrophage cell line RAW264.7 cells were subjected to starvation or amino acid stimulation to study amino acid-mTORC1-autophagy signaling. Using both gain- and loss-of-function approaches, we explored the mechanism of Gpr137b-ps-regulated autophagy. Our results demonstrated that Gpr137b-ps deficiency led to enhanced autophagy in macrophages and reduced atherosclerotic lesions, characterized by fewer necrotic cores and less lipid accumulation. Knockdown of Gpr137b-ps increased autophagy and prevented amino acid-induced mTORC1 signaling activation. As the downstream binding protein of Gpr137b-ps, HSC70 (heat shock cognate 70) rescued the impaired autophagy induced by Gpr137b-ps. Furthermore, Gpr137b-ps interfered with the HSC70 binding to G3BP (Ras GTPase-activating protein-binding protein), which tethers the TSC (tuberous sclerosis complex) complex to lysosomes and suppresses mTORC1 signaling. In addition to verifying that the NTF2 (nuclear transport factor 2) domain of G3BP binds to HSC70 by in vitro protein synthesis, we further demonstrated that HSC70 binds to the NTF2 domain of G3BP through its W90-F92 motif by using computational modeling. These findings reveal that Gpr137b-ps plays an essential role in the regulation of macrophage autophagy, which is crucial for the progression of advanced atherosclerosis. Gpr137b-ps impairs the interaction of HSC70 with G3BP to regulate amino acid-mTORC1-autophagy signaling, and these results provide a new potential therapeutic direction for the treatment of advanced atherosclerosis.

Synthesis of Functionalized Triblock Copolyesters Derived from Lactic Acid and Macrolactones for Bone Tissue Regeneration.

Synthetic and functional grafts are a great alternative to conventional grafts. They can provide a physical support and the precise signaling for cells to heal damaged tissues. In this study, a novel RGD peptide end-functionalized poly(ethylene glycol)-b-poly(lactic acid)-b-poly(globalide)-b-poly(lactic acid)-b-poly(ethylene glycol) (RGD-PEG-PLA-PGl-PLA-PEG-RGD) is synthetized and used to prepare functional scaffolds. The PGl inner block is obtained by enzymatic ring-opening polymerization of globalide. The outer PLA blocks are obtained by ring-opening polymerization of both, l-lactide or a racemic mixture, initiated by the α-ω-telechelic polymacrolactone. The presence of PGl inner block enhances the toughness of PLA-based scaffolds, with an increase of the elongation at break up to 300% when the longer block of PGl is used. PLA-PGl-PLA copolymer is coupled with α-ω-telechelic PEG diacids by esterification reaction. PEGylation provides hydrophilic scaffolds as the contact angle is reduced from 114° to 74.8°. That difference improves the contact between the scaffolds and the culture media. Moreover, the scaffolds are functionalized with RGD peptides at the surface significantly enhancing the adhesion and proliferation of bone marrow-derived primary mesenchymal stem cells and MC3T3-E1 cell lines in vitro. These results place this multifunctional polymer as a great candidate for the preparation of temporary grafts.

Inhibition of glutaminase 1 activity reverses airway hyperresponsiveness and decreases IL-1β+ M1s and IL-17 producing ILC3s in high-fat diet-fed obese mice.

In obesity, disturbed glutamine metabolism contributes to enhanced inflammation by inducing alterations in immune cells. As macrophages and innate lymphoid cells (ILCs) are known to be involved in the pathogenesis of obesity-related asthma, we tested our hypothesis that altered glutamine metabolism may link obesity to airway hyperresponsivenss (AHR), a cardinal feature of asthma, focusing on these innate immune cells. Four-week-old male C57BL/6 mice were fed a high-fat diet (HFD) for 13 wk in the presence or absence of BPTES [Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide, a selective inhibitor of glutaminase 1 which converts glutamine to glutamate] and their blood, lung, and adipose tissues were analyzed. We then conducted in vitro experiments using bone marrow-derived macrophages (BMDMs) and mouse alveolar macrophage cell line. Furthermore, we investigated plasma glutamine and glutamate levels in obese and nonobese asthmatics. BPTES treatment prevented HFD-induced AHR and significantly decreased IL-1β+ classically activated macrophages (M1s) and type 3 ILCs (ILC3s) which increased in the lungs of HFD-fed obese mice. In in vitro experiments, BPTES treatment or glutamine supplement significantly reduced the proportion of IL-1β+NLRP3+ M1s in lipopolysaccharide-stimulated BMDMs and mouse alveolar macrophage cell line. BPTES treatment also significantly reduced the IL-17 producing ILC3s differentiated from ILCs in naïve mouse lung. In addition, plasma glutamate/glutamine ratios were significantly higher in obese asthmatics compared to nonobese asthmatics. Inhibition of glutaminolysis reverses AHR in HFD-induced obese mice and decreases IL-1β + NLRP3+ M1s and IL-17 producing ILC3s, which suggests altered glutamine metabolism may have a role in the pathogenesis of obesity-related AHR.

Fabrication and assessment of bifunctional electrospun poly(l-lactic acid) scaffolds with bioglass and zinc oxide nanoparticles for bone tissue engineering

Electrospun scaffolds based on poly(l-lactic acid) (PLLA) with bioglass (n-BG) and zinc oxide (n-ZnO), and mixture of both, were developed to design bifunctional biomaterials with enhanced bioactive and biocidal properties. The presence of n-BG increased the fiber diameter of the pure PLA from 1.5 ± 0.3 μm to 3.0 ± 0.8 μm for 20 wt%. ZnO and the mixed nanoparticles did not significantly affect the morphology. The mechanical properties decreased with the presence of nanoparticles. Scaffolds based on PLA/n-BG promoted hydroxyapatite (HA) formation in simulated body fluid (SBF) that was inhibited with the presence of ZnO. Notably, mixed particles produced bioactivity although at longer times. The incorporation of n-ZnO produced a biocidal capacity against S. aureus in the polymeric scaffold, reaching a viability reduction of 60 % after 6 h of exposure. When both types of nanoparticles were combined, the bacterial viability reduction was 30 %. Pure PLA scaffolds and the composites with n-BG showed good ST-2 bone marrow-derived cell line viability, scaffolds with n-BG (pure or mixture) presented lower viability. Results validated the use of both n-BG and n-ZnO fillers for the development of novel bifunctional PLA-based scaffolds with both bioactive and biocidal properties for bone tissue engineering applications.

Stable human cartilage progenitor cell line stimulates healing of meniscal tears and attenuates post-traumatic osteoarthritis.

Meniscal tearing in the knee increases the risk of post-traumatic osteoarthritis (OA) in patients. The therapeutic application of tissue-specific mesenchymal progenitor cells is currently being investigated as an emerging biologic strategy to help improve healing of musculoskeletal tissues like meniscal fibrocartilage and articular hyaline cartilage. However, many of these approaches involve isolating cells from healthy tissues, and the low yield of rare progenitor populations (< 1% of total cells residing in tissues) can make finding a readily available cell source for therapeutic use a significant logistical challenge. In the present study, we investigated the therapeutic efficacy of using expanded cartilage-derived and bone marrow-derived progenitor cell lines, which were stabilized using retroviral SV40, for repair of meniscus injury in a rodent model. Our findings indicate that these cell lines express the same cell surface marker phenotype of primary cells (CD54+, CD90+, CD105+, CD166+), and that they exhibit improved proliferative capacity that is suitable for extensive expansion. Skeletally mature male athymic rats treated with 3.2 million cartilage-derived progenitor cell line exhibited approximately 79% greater meniscal tear reintegration/healing, compared to injured animals that left untreated, and 76% greater compared to animals treated with the same number of marrow-derived stromal cells. Histological analysis of articular surfaces also showed that cartilage-derived progenitor cell line treated animals exhibited reduced post-traumatic OA associated articular cartilage degeneration. Stable cell line treatment did not cause tumor formation or off-target engraftment in animals. Taken together, we present a proof-of-concept study demonstrating, for the first time, that intra-articular injection of a stable human cartilage-derived progenitor cell line stimulates meniscus tear healing and provide chondroprotection in an animal model. These outcomes suggest that the use of stable cell lines may help overcome cell source limitations for cell-based medicine.

Multiomics surface receptor profiling of the NCI-60 tumor cell panel uncovers novel theranostics for cancer immunotherapy

BackgroundImmunotherapy with immune checkpoint inhibitors (ICI) has revolutionized cancer therapy. However, therapeutic targeting of inhibitory T cell receptors such as PD-1 not only initiates a broad immune response against tumors, but also causes severe adverse effects. An ideal future stratified immunotherapy would interfere with cancer-specific cell surface receptors only.MethodsTo identify such candidates, we profiled the surface receptors of the NCI-60 tumor cell panel via flow cytometry. The resulting surface receptor expression data were integrated into proteomic and transcriptomic NCI-60 datasets applying a sophisticated multiomics multiple co-inertia analysis (MCIA). This allowed us to identify surface profiles for skin, brain, colon, kidney, and bone marrow derived cell lines and cancer entity-specific cell surface receptor biomarkers for colon and renal cancer.ResultsFor colon cancer, identified biomarkers are CD15, CD104, CD324, CD326, CD49f, and for renal cancer, CD24, CD26, CD106 (VCAM1), EGFR, SSEA-3 (B3GALT5), SSEA-4 (TMCC1), TIM1 (HAVCR1), and TRA-1-60R (PODXL). Further data mining revealed that CD106 (VCAM1) in particular is a promising novel immunotherapeutic target for the treatment of renal cancer.ConclusionAltogether, our innovative multiomics analysis of the NCI-60 panel represents a highly valuable resource for uncovering surface receptors that could be further exploited for diagnostic and therapeutic purposes in the context of cancer immunotherapy.

Targeting Castration-Resistant Prostate Cancer Using Mesenchymal Stem Cell Exosomes for Therapeutic MicroRNA-let-7c Delivery.

Castration-resistant prostate cancer (PCa; CRPC) has a poor response to androgen deprivation therapy and is considered an incurable disease. MicroRNA (miR)-lethal 7c (let-7c) was implied to be a tumor suppressor in PCa, and treatment with exogenous let-7c targets both cancer cells and their associated mesenchymal stem cells (MSCs) to prevent CRPC progression and metastasis. Exosomes are nanometer-sized membrane-bound vesicles which have an absolute predominance in biocompatibility for drug delivery and gene therapy by mediating cell-to-cell communication. By utilizing the intrinsic tumor-targeting property of MSCs, this study aimed to investigate the feasibility of MSC-derived exosomes as an exogenous miR delivery system to target CRPC, using miR let-7c as an example. Bioinformatics analysis was performed to observe miR-let-7c expression in clinical samples by utilizing the GEO database. MSC-derived exosomes were collected from a human bone marrow-derived MSC cell line after cell transfection with either a pre-miR negative control or pre-miR-let-7c, and further characterized through nanoparticle tracking analysis and Western blotting. miR-let-7c expression was determined using RT-qPCR, and the phenotypic effects of both naked and MSC-exosome-encapsulated let-7c on CRPC cells (PC3 and CWR22Rv1) were determined by WST-1 cell proliferation assay and wound healing migration assay. miR-let-7c was downregulated in metastatic PCa and high grade group patients. miR-let-7c expression was confirmed to be downregulated in PCa cell lines, with massively decreased in most metastatic CRPC-like cells. Exogenous miR-let-7c can be successfully packaged into MSC exosomes. Treatment with either naked or MSC-exosome-encapsulated miR-let-7c resulted in significant reductions in cell proliferation and migration in CRPC-like PC3 and CWR22Rv1 cells. MSC-derived exosomes could serve as a therapeutic let-7c delivery system to target CRPC.

CASTING: A Potent Supramolecular Strategy to Cytosolically Deliver STING Agonist for Cancer Immunotherapy and SARS-CoV-2 Vaccination

CASTING: A Potent Supramolecular Strategy to Cytosolically Deliver STING Agonist for Cancer Immunotherapy and SARS-CoV-2 Vaccination

The Outer Membrane Vesicles of Salmonella enterica Serovar Typhimurium Activate Chicken Immune Cells through Lipopolysaccharides and Membrane Proteins.

Salmonella is a common pathogen which can secrete outer membrane vesicles (OMVs). However, the effect of OMVs from Salmonella enterica Serovar Typhimurium (S. Typhimurium) of poultry origin on cells of the chicken innate immune system is not well known. In this study, S. Typhimurium OMVs were first isolated from three different poultry strains of Salmonella, Salmonella CVCC542, SALA, and SALB. In order to investigate the effect of OMVs on the maturation of monocytes into macrophages, both bone marrow-derived (BMD) monocytes and macrophage cell line HD11 cells were used. OMVs promoted the formation of monocyte dendrites in both types of cells, enabled BMD cells to become larger, and stimulated expression of LPS-induced TNF-αfactor (LITAF), IL-6, and inducible nitric oxide synthase (iNOS) genes in HD11 cells. These results demonstrated the capability of OMVs to promote the development of chicken monocytes into macrophages and the maturation of macrophages. In order to study the effect of OMVs on the phagocytosis of macrophages, chicken spleen-derived monocytes and HD11 cells were used. Phagocytosis of FITC-Salmonella and FITC-dextran by these two types of cells was enhanced after stimulation with OMVs. To determine which components in OMVs were responsible for the above observed results, OMVs were treated with proteinase K(PK) or polymyxin B (PMB). Both treatments reduced the phagocytosis of FITC-Salmonella by HD11 cells and chicken spleen mononuclear cells and reduced the secretion of IL-1β, LITAF, and IL-6 cytokines. These results demonstrated that Salmonella OMVs activated chicken macrophages and spleen mononuclear cells and the activation was achieved mainly through lipopolysaccharides and membrane proteins.

Magnetic 3D scaffolds for tissue engineering applications: bioactive glass (45S5) coated with iron-loaded hydroxyapatite nanoparticles

Magnetic 45S5 bioactive glass (BG) based scaffolds covered with iron-loaded hydroxyapatite (Fe-HA-BG) nanoparticles were obtained and its cytotoxicity investigated. Fe-HA nanoparticles were synthesized by a wet chemical method involving the simultaneous addition of Fe2+/Fe3+ ions. BG based scaffolds were prepared by the foam replica procedure and covered with Fe-HA by dip-coating. Fe-HA-BG magnetic saturation values of 0.049 emu g−1 and a very low remanent magnetization of 0.01 emu g−1 were observed. The mineralization assay in simulated body fluid following Kokubo’s protocol indicated that Fe-HA-BG scaffolds exhibited improved hydroxyapatite formation in comparison to uncoated scaffolds at shorter immersion times. The biocompatibility of the material in vitro was assessed using human osteoblast-like MG-63 cell cultures and mouse bone marrow-derived stroma cell line ST-2. Overall, the results herein discussed suggest that magnetic Fe-HA coatings seem to enhance the biological performance of 45S5 BG based scaffolds. Thus, this magnetic Fe-HA coated scaffold is an interesting system for bone tissue engineering applications and warrant further investigation.

Abstract 1151: Targeting castration-resistant prostate cancer using mesenchymal stem cell exosomes for therapeutic microRNA let-7c delivery

Abstract Introduction: Castration-resistant prostate cancer (CRPC) has poor response to androgen deprivation therapy, which is caused by a sustained androgen receptor (AR) signal and therefore is considered as an incurable disease. MicroRNA let-7c has been implied as a tumor suppressor in prostate cancer by antagonizing AR expression and activity. In addition to cancer cells, our group previously also demonstrated that downregulation of let-7c by cancer-associated mesenchymal stem cells (MSCs) triggering a reactive stromal response to facilitate prostate cancer growth and metastasis (PLoS ONE 8(8): e71637). Accordingly, treatment with exogenous let-7c would target both cancer cells and their associated MSCs and could be an attractive therapeutic approach to effectively inhibit CRPC recurrence and prevent metastasis. Exosomes are nanometer-sized membrane-bound vesicles with functions as mediators of cell-cell communication, which have the absolute predominance in biocompatibility for clinical applications in drug delivery and gene therapy. Objective: By taking the intrinsic tumor-targeting property of MSCs, this study aimed to investigate the feasibility of using MSC-derived exosomes as an exogenous let-7c delivery system to target CRPC. Methods: Relative miRNA-let-7c expression across various prostate cancer cell lines was examined using q-RT PCR. MSC-derived exosomes were collected from a 3A6 human bone marrow-derived MSC cell line after cell transfection with pre-miR negative control and pre-miR-let-7c, and further characterized through transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blot. The effects of either naked or MSC-exosome encapsulated let-7c on CRPC cells was determined by WST-1 cell proliferation assay and wound healing migration assay. Results: MiRNA-let-7c expression was confirmed to be downregulated in most of CRPC-like cell lines compared with androgen-sensitive LNCaP. Overexpression of pre-miR-let-7 inhibited the ability of cell proliferation and migration of prostate cancer cells. Exogenous microRNAs can be successfully packaged into MSC-exosomes by cell transfection, without a disruption of exosome integrity. Treatment of CRPC-like PC3 (AR-negative) and CWR22r-v1 (AR-positive) cells with MSC-exosome encapsulated let-7c effectively delivered let-7c into cells and resulted in a significant reduction in cancer cell growth and metastasis in vitro. Conclusion: Herein, we have provided in vitro evidences to prove the concept that microRNA let-7c is a potential therapeutic intervention for CRPC, as well as MSC-derived exosomes can serve as a therapeutic let-7c delivery system to target the CRPC. Further in vivo validation of distribution and therapeutic efficacy of MSC-exosome-delivered let-7c in CRPC xenograft mouse model is currently being explored. Citation Format: Ida Kurniawati, Chia-Yen Hsueh, Shian-Ying Sung, Chia-Ling Hsieh. Targeting castration-resistant prostate cancer using mesenchymal stem cell exosomes for therapeutic microRNA let-7c delivery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1151.

Chemotactic and Angiogenic Potential of Mineralized Collagen Scaffolds Functionalized with Naturally Occurring Bioactive Factor Mixtures to Stimulate Bone Regeneration.

To develop cost-effective and efficient bone substitutes for improved regeneration of bone defects, heparin-modified mineralized collagen scaffolds were functionalized with concentrated, naturally occurring bioactive factor mixtures derived from adipose tissue, platelet-rich plasma and conditioned medium from a hypoxia-treated human bone marrow-derived mesenchymal stem cell line. Besides the analysis of the release kinetics of functionalized scaffolds, the bioactivity of the released bioactive factors was tested with regard to chemotaxis and angiogenic tube formation. Additionally, functionalized scaffolds were seeded with human bone marrow-derived mesenchymal stromal cells (hBM-MSC) and their osteogenic and angiogenic potential was investigated. The release of bioactive factors from the scaffolds was highest within the first 3 days. Bioactivity of the released factors could be confirmed for all bioactive factor mixtures by successful chemoattraction of hBM-MSC in a transwell assay as well as by the formation of prevascular structures in a 2D co-culture system of hBM-MSC and human umbilical vein endothelial cells. The cells seeded directly onto the functionalized scaffolds were able to express osteogenic markers and form tubular networks. In conclusion, heparin-modified mineralized collagen scaffolds could be successfully functionalized with naturally occurring bioactive factor mixtures promoting cell migration and vascularization.

Erythropoietin Protects against Diffuse Alveolar Hemorrhage in Mice by Regulating Macrophage Polarization through the EPOR/JAK2/STAT3 Axis.

Macrophages play an important role in the pathogenesis of systemic lupus erythematosus-associated diffuse alveolar hemorrhage (DAH). The immunomodulation of macrophage responses might be a potential approach for the prevention and treatment of DAH. Erythropoietin (EPO) could regulate macrophage bioactivities by binding to the EPO receptor expressing on macrophages. This study assessed the effects of EPO on DAH protection using an immune-mediated DAH murine model with macrophages as the major contributor. A DAH murine model was established in female C57BL/6 mice by an i.p. injection of pristane. We found that EPO administration alleviates DAH by reducing pulmonary macrophages recruitment and promoting phenotype switch toward M2 macrophages in vivo. EPO drove macrophages to the anti-inflammatory phenotype in the primary murine bone marrow-derived macrophages and macrophages cell line RAW 264.7 with LPS, IFN-γ, and IL-4 in vitro. Moreover, EPO treatment increases the expression of EPOR and decreases the expression of miR-494-3p, resulting in increased phosphorylation of JAK2 and STAT3. In conclusion, EPO can be a potential therapeutic agent in DAH by reducing cell apoptosis and regulating macrophage polarization through the EPOR/JAK2/STAT3 axis. Further studies are also needed to validate the direct target of miR-494-3p in regulating JAK2/STAT3 signaling transduction.

Molecular Beacon Imaging to Visualize Ki67 mRNA for Cell Proliferation Ability

The objective of this study is to visualize the ability of cell proliferation based on molecular beacons (MB). Two types of MB to detect messenger RNA (mRNA) were used. One is a Ki67 MB of a target for cell proliferation ability. The other one is a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) MB as a control of stable fluorescence in cells. To enhance the MB internalization into cells, the MB were incorporated into cationized gelatin nanospheres (cGNS). There was no difference in the physicochemical properties and the cell internalization between the cGNSKi67 MB and cGNSGAP MB. When basic fibroblast growth factor (bFGF) was added to KUM6 cells of a mouse bone marrow-derived mesenchymal stem cell line, the expression of Ki67 and the cell proliferation increased with the bFGF concentration. After the incubation for the cell internalization of cGNS incorporating MB (cGNSMB), the cells were further incubated for 24 h with or without different concentrations of bFGF. The fluorescence of cGNSKi67 MB significantly increased with the increase of bFGF concentration, whereas that of cGNSGAP MB was constant, irrespective of the bFGF concentration. A time-lapse imaging assay revealed a fast enhancement of cGNSKi67 MB fluorescence after the bFGF addition compared with no bFGF addition. On the other hand, for cGNSGAP MB, a constant fluorescence was observed even at any time point after the bFGF addition. It is concluded that the cGNSMB system is promising for the chronological visualization of proliferation ability in living cells.

Visualization of Ki67 mRNa by Cationized Gelatin Nanospheres Incorporating Molecular Beacon for Cell Proliferation Ability

The objective of this study is to visualize the ability of cell proliferation based on molecular beacons (MB). Two types of MB to detect messenger RNA (mRNA) were used. One is a Ki67 MB of a target for cell proliferation ability. The other one is a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) MB as a control of stable fluorescence in cells. To enhance the MB internalization into cells, the MB were incorporated into cationized gelatin nanospheres (cGNS). There was no difference in the physicochemical properties and the cell internalization between the cGNSKi67 MB and cGNSGAP MB. When basic fibroblast growth factor (bFGF) was added to KUM6 cells of a mouse bone marrow-derived mesenchymal stem cell line, the expression of Ki67 and the cell proliferation increased with the bFGF concentration. After the incubation for the cell internalization of cGNSMB, the cells were further incubated for 24 hr with or without different concentrations of bFGF. The fluorescence of cGNSKi67 MB significantly increased with the increase of bFGF concentration, whereas that of cGNSGAP MB was constant, irrespective of the bFGF concentration. A timelapse imaging assay revealed that a fast enhancement of cGNSKi67 MB fluorescence after the bFGF addition compared with no bFGF addition. On the other hand, for cGNSGAP MB, a constant fluorescence was observed even at any time point after the bFGF addition. It is concluded that the cGNSMB system is a promising for the chronological visualization of proliferation ability in living cells.