Stem cell therapy has emerged as a promising approach for treatment of a number of diseases, including delayed and non-healing wounds. However, targeted systemic delivery of therapeutic cells to the dysfunctional tissues remains one formidable challenge. Herein, we present a targeted nanocarrier-mediated cell delivery method by coating the surface of the cell to be delivered with dendrimer nanocarriers modified with adhesion molecules. Infused nanocarrier-coated cells reach to destination via recognition and association with the counterpart adhesion molecules highly or selectively expressed on the activated endothelium in diseased tissues. Once anchored on the activated endothelium, nanocarriers-coated transporting cells undergo transendothelial migration, extravasation and homing to the targeted tissues to execute their therapeutic role. We now demonstrate feasibility, efficacy and safety of our targeted nanocarrier for delivery of bone marrow cells (BMC) to cutaneous wound tissues and grafted corneas and its advantages over conventional BMC transplantation in mouse models for wound healing and neovascularization. This versatile platform is suited for targeted systemic delivery of virtually any type of therapeutic cell.

Notch signaling, which is driven by the Notch1 receptor, plays an essential role in the pathogenesis and stroma-mediated drug resistance of T-cell acute lymphoblastic leukemia (T-ALL). However, little is known about the roles of Notch ligands in the survival or drug resistance of T-ALL cells. In the present study, isolated mesenchymal stem cells (MSCs) from human umbilical cord (hUC) samples, termed hUC-MSCs, were used as stromal cells for the Jurkat T-ALL cell line. The role of the Notch ligand, Jagged1, was assessed in the survival of Jurkat T-ALL cells using this co-culture system. hUC-MSCs and Jurkat cells were observed to express Jagged1. Furthermore, co-culture with hUC-MSCs led to a significant upregulation of Jagged1 and a more significant overexpression of its receptor, Notch1, in the Jurkat cells, indicating that the receptor and ligand pair may play a role in the reciprocal or autonomous activation of the Notch pathway. In addition, a higher level of CD28 expression was observed in the Jurkat cells that were co-cultured with hUC-MSCs. Blocking Jagged1 expression using neutralizing antibodies restored drug-induced apoptosis in the Jurkat cells that were co-cultured with hUC-MSCs, and also increased the drug sensitivity of the Jurkat cells that were cultured alone. By contrast, direct incubation with exogenously recombinant Jagged1 produced the same protective effects in Jurkat cells as those induced by hUC-MSCs. These results indicate a significant role for Jagged1 in hUC-MSC-induced survival and the self-maintenance of the Jurkat T-ALL cell line, making it a potential target for the treatment of human T-ALL.

Growth hormone-releasing hormone (GHRH) is a potent stimulator of growth hormone (GH) secretion from the pituitary gland. Although GHRH is essential for the growth of immune cells, the regulatory effects of its antagonist in granulomatous disease remain unknown.

Primary malignant bone tumors can be life-threatening. Surgical resection of tumor plus chemotherapy is the standard clinical treatment. However, postoperative recovery is hindered due to tumor recurrence caused by residual tumor cells and bone defect caused by resection of tumor tissue. Herein, a multifunctional mussel-inspired film was fabricated on Mg alloy, that is, an inner hydrothermal-treated layer, a middle layer of polydopamine, and an outer layer of doxorubicin. The modified Mg alloy showed excellent photothermal effect and thermal/pH-controlled release of doxorubicin. The synergistic effect of chemotherapy and photothermal therapy enabled the modified Mg alloy to kill bone tumor in vitro and inhibit tumor growth in nude mice. Moreover, because of the controlled release of Mg ions and biocompatibility of polydopamine, the modified Mg alloy supported extracellular matrix mineralization, alkaline phosphatase activity, and bone-related gene expression in C3H10T1/2. Bone implantation model in rats verified that the modified Mg showed excellent osteointegration. These findings prove that the use of mussel-inspired multifunction film on Mg alloy offers a promising strategy for the therapy of primary malignant bone tumor.

Cancer-associated fibroblasts (CAFs) play a crucial role in cancer progression, drug resistance and tumor recurrence. We have recently shown that the Notch pathway determines the tumor-regulatory role of experimentally created 'CAFs'. Here, we examined the status of Notch signaling in human melanoma-associated fibroblasts (MAFs) versus their normal counterparts and tested whether manipulation of the Notch pathway activity in MAFs alters their tumor-regulatory function. Using tissue microarrays, we found that MAFs exhibit decreased Notch pathway activity compared with normal fibroblasts in adjacent and non-adjacent skin. Consistently, MAFs isolated from human metastatic melanoma exhibited lower Notch activity than did normal human fibroblasts, demonstrating that Notch pathway activity is low in MAFs. We then investigated the effect of increasing Notch pathway activity in MAF on melanoma growth in co-cultures and in a mouse co-graft model. We found that activation of the Notch pathway in MAFs significantly restricted melanoma cell growth in vitro and suppressed melanoma skin growth and tumor angiogenesis in vivo. Our study demonstrates that the Notch signaling is inhibited in MAFs. Increase of Notch pathway activity can confer tumor-suppressive function on MAFs. Thus, targeting melanoma by activating Notch signaling in MAF may represent a novel therapeutic approach.

Here, we report a new method to increase the therapeutic potential of mesenchymal stem/stromal cells (MSCs) for ischemic wound healing. We tested biological effects of MSCs modified with E-selectin, a cell adhesion molecule capable of inducing postnatal neovascularization, on a translational murine model.

Hepatoma-derived growth factor-related protein-3 (Hdgfrp3 or HRP-3) was recently reported as a neurotrophic factor and is upregulated in hepatocellular carcinoma to promote cancer cell survival. Here we identified HRP-3 as a new endothelial ligand and characterized its in vitro and in vivo functional roles and molecular signaling. We combined open reading frame phage display with multi-round in vivo binding selection to enrich retinal endothelial ligands, which were systematically identified by next generation DNA sequencing. One of the identified endothelial ligands was HRP-3. HRP-3 expression in the retina and brain was characterized by Western blot and immunohistochemistry. Cell proliferation assay showed that HRP-3 stimulated the growth of human umbilical vein endothelial cells (HUVECs). HRP-3 induced tube formation of HUVECs in culture. Wound healing assay indicated that HRP-3 promoted endothelial cell migration. HRP-3 was further confirmed for its in vitro angiogenic activity by spheroid sprouting assay. HRP-3 extrinsically activated the extracellular-signal-regulated kinase ½ (ERK1/2) pathway in endothelial cells. The angiogenic activity of HRP-3 was independently verified by mouse cornea pocket assay. Furthermore, in vivo Matrigel plug assay corroborated HRP-3 activity to promote new blood vessel formation. These results demonstrated that HRP-3 is a novel angiogenic factor.

P-glycoprotein (P-gp) is encoded by the multidrug resistance (MDR1) gene and is well studied as a multi-drug resistance transporter. Peritoneal adhesion formation following abdominal surgery remains an important clinical problem. Here, we found that P-gp was highly expressed in human adhesion fibroblasts and promoted peritoneal adhesion formation in a rodent model. Knockdown of P-gp expression by intraperitoneal injection of MDR1-targeted siRNA significantly reduced both the peritoneal adhesion development rate and adhesion grades. Additionally, we found that operative injury up-regulated P-gp expression in peritoneal fibroblasts through the TGF-β1/Smad signaling pathway and histone H3 acetylation. The overexpression of P-gp accelerated migration and proliferation of fibroblasts via volume-activated Cl(-) current and cell volume regulation by enhancing phosphorylation of the chloride channel-3. Therefore, P-gp plays a critical role in postoperative peritoneal adhesion formation and may be a valuable therapeutic target for preventing the formation of peritoneal adhesions.

Fibroblasts are an integral component of stroma and important source of growth factors and extracellular matrix (ECM). They play a prominent role in maintaining tissue homeostasis and in wound healing and tumor growth. Notch signaling regulates biological function in a variety of cells. To elucidate the physiological function of Notch signaling in fibroblasts, we ablated Notch1 in mouse (Notch1(Flox/Flox)) embryonic fibroblasts (MEFs). Notch1-deficient (Notch1(-/-)) MEFs displayed faster growth and motility rate compared to Notch1(Flox/Flox) MEFs. Such phenotypic changes, however, were reversible by reconstitution of Notch1 activation via overexpression of the intracellular domain of Notch1 (NICD1) in Notch1-deficient MEFs. In contrast, constitutive activation of Notch1 signaling by introducing NICD1 into primary human dermal fibroblasts (FF2441), which caused pan-Notch activation, inhibited cell growth and motility, whereas cellular inhibition was relievable when the Notch activation was countered with dominant-negative mutant of Master-mind like 1 (DN-MAML-1). Functionally, "Notch-activated" stromal fibroblasts could inhibit tumor cell growth/invasion. Moreover, Notch activation induced expression of Wnt-induced secreted proteins-1 (WISP-1/CCN4) in FF2441 cells while deletion of Notch1 in MEFs resulted in an opposite effect. Notably, WISP-1 suppressed fibroblast proliferation, and was responsible for mediating Notch1's inhibitory effect since siRNA-mediated blockade of WISP-1 expression could relieve cell growth inhibition. Notch1-induced WISP-1 expression appeared to be Wnt11-dependent, but Wnt1-independent. Blockade of Wnt11 expression resulted in decreased WISP-1 expression and liberated Notch-induced cell growth inhibition. These findings indicated that inhibition of fibroblast proliferation by Notch pathway activation is mediated, at least in part, through regulating Wnt1-independent, but Wnt11-dependent WISP-1 expression.

Substantial effort in recent years has been devoted to analyzing data based large-scale biological networks, which provide valuable insight into the topologies of complex biological networks but are rarely context specific and cannot be used to predict the responses of cell signaling proteins to specific ligands or compounds. In this work, we proposed a novel strategy to investigate kinase inhibitor induced pathway signatures by integrating multiplex data in Library of Integrated Network-based Cellular Signatures (LINCS), e.g. KINOMEscan data and cell proliferation/mitosis imaging data. Using this strategy, we first established a PC9 cell line specific pathway model to investigate the pathway signatures in PC9 cell line when perturbed by a small molecule kinase inhibitor GW843682. This specific pathway revealed the role of PI3K/AKT in modulating the cell proliferation process and the absence of two anti-proliferation links, which indicated a potential mechanism of abnormal expansion in PC9 cell number. Incorporating the pathway model for side effects on primary human hepatocytes, it was used to screen 27 kinase inhibitors in LINCS database and PF02341066, known as Crizotinib, was finally suggested with an optimal concentration 4.6 uM to suppress PC9 cancer cell expansion while avoiding severe damage to primary human hepatocytes. Drug combination analysis revealed that the synergistic effect region can be predicted straightforwardly based on a threshold which is an inherent property of each kinase inhibitor. Furthermore, this integration strategy can be easily extended to other specific cell lines to be a powerful tool for drug screen before clinical trials.

The chemokine stromal cell-derived factor-1 (SDF-1) plays a critical role in mobilizing precursor cells in the bone marrow and is essential for efficient vascular regeneration and repair. We recently reported that calcium augments the expression of chemokine receptor CXCR4 and enhances the angiogenic potential of bone marrow derived cells (BMCs). Neovascularization is impaired by aging therefore we suggested that aging may cause defects of CXCR4 expression and cellular responses to calcium. Indeed we found that both the basal and calcium-induced surface expression of CXCR4 on BMCs was significantly reduced in 25-month-old mice compared with 2-month-old mice. Reduced Ca-induced CXCR4 expression in BMC from aged mice was associated with defective calcium influx. Diminished CXCR4 surface expression in BMC from aged mice correlated with diminished neovascularization in an ischemic hindlimb model with less accumulation of CD34(+) progenitor cells in the ischemic muscle with or without local overexpression of SDF-1. Intravenous injection of BMCs from old mice homed less efficiently to ischemic muscle and stimulated significantly less neovascularization compared with the BMCs from young mice. Transplantation of old BMCs into young mice did not reconstitute CXCR4 functions suggesting that the defects were not reversible by changing the environment. We conclude that defects of basal and calcium-regulated functions of the CXCR4/SDF-1 axis in BMCs contribute significantly to the age-related loss of vasculogenic responses.

Cancer stem cells (CSCs) play critical roles in cancer initiation, metastasis, recurrence, and drug resistance. Recent studies have revealed involvement of cancer-associated fibroblasts (CAFs) in regulating CSCs. However, the intracellular molecular mechanisms that determine the regulatory role of CAFs in modulating the plasticity of CSCs remain unknown. Here, we uncovered that intracellular Notch1 signaling in CAFs serves as a molecular switch, which modulates tumor heterogeneity and aggressiveness by inversely controlling stromal regulation of the plasticity and stemness of CSCs. Using mesenchymal stem cell-derived fibroblasts (MSC-DF) harboring reciprocal loss-of-function and gain-of-function Notch1 signaling, we found that MSC-DFNotch1-/- prompted cocultured melanoma cells to form more spheroids and acquire the phenotype (CD271+ and Nestin+ ) of melanoma stem/initiating cells (MICs), whereas MSC-DFN1IC+/+ suppressed melanoma cell sphere formation and mitigated properties of MICs. MSC-DFNotch1-/- increased stemness of CD271+ MIC, which resultantly exhibited stronger aggressiveness in vitro and in vivo, by upregulating Sox2/Oct4/Nanog expression. Consistently, when cografted with melanoma cells into NOD scid gamma (NSG) mice, MSC-DFNotch1-/- increased, but MSC-DFN1IC+/+ decreased, the amounts of CD271+ MIC in melanoma tissue. The amounts of CD271+ MIC regulated by MSC-DF carrying high or low Notch1 pathway activity is well correlated with capability of melanoma metastasis, supporting that melanoma metastasis is MIC-mediated. Our data demonstrate that intracellular Notch1 signaling in CAFs is a molecular switch dictating the plasticity and stemness of MICs, thereby regulating melanoma aggressiveness, and therefore that targeting the intracellular Notch1 signaling pathway in CAFs may present a new therapeutic strategy for melanoma. Stem Cells 2019;37:865-875.

Homing of endothelial progenitor cells (EPC) to the ischemic tissues is a key event in neovascularization and tissue regeneration. In response to ischemic insult, injured tissues secrete several chemo-cytokines, including stromal cell-derived factor-1α (SDF-1α), which triggers mobilization and homing of bone marrow-derived EPC (BMD-EPC). We previously reported that SDF-1α-induced EPC homing is mediated by a panel of adhesion molecules highly or selectively expressed on the activated endothelium in ischemic tissues, including E-selectin. Elevated E-selectin on wound vasculature serve as docking sites for circulating EPC, which express counterpart E-selectin ligands. Here, we show that SDF-1α presented in wound tissue and released into circulation can act both locally and remotely to induce ischemic tissue endothelium and BMD-EPC to express both E-selectin and its ligands. By performing BM transplantation using E-selectin-/- and E-selectin+/+ mice as the donors and recipients respectively, we demonstrate that upregulated dual E-selectin/ligand pairs reciprocally expressed on ischemic tissue endothelium and BMD-EPC act as double-locks to secure targeted EPC- endothelium interactions by which to facilitate EPC homing and promote neovascularization and tissue repair. These findings describe a novel mechanism for BMD-EPC homing and indicate that dual E-selectin/ligand pairs may be effective targets/tools for therapeutic neovascularization and targeted cell delivery.

Introduction:Mycobacteria are aerobic non-motile organisms with lipid rich, hydrophobic cell walls that render them resistant to antibiotics. While there are over 150 different species of NTM, Mycobacterium avium complex (MAC) and Mycobacterium abscessus (MAB) are two of the most common culprits of pulmonary infection. MAB has been found to be most common in southeastern United States (Florida to Texas) and the third most rapidly growing NTM infection. It is responsible for chronic lung infections. Mycobacterial cell wall components initiate the interaction between bacteria and host. The reaction between bronchial epithelia and components in the envelope of mycobacterial cell wall is poorly understood. Methods: A lung-on-membrane model was developed with normal human bronchial epithelial (NHBE) cells re-differentiated at the air-liquid interface (ALI) and human endothelial cells on a transwell® polyester membrane. Microparticles from MAB cell walls were developed by an inhouse protocol and added to the ALI side of lung model. NHBE cells were harvested at day 3. RNA was isolated and analyzed with RNASeq. NHBE cells were lysed and protein assay was performed with western blot. We tested whether lung INF-alpha expression would increase in mice treated with intratracheal MAB cell wall particles. A paired t-test is used to compare two population means using GraphPad Prism 7 software. Results: RNAseq analysis identified 1759 differentially expressed genes between NHBE cells challenged with and without MAB microparticles with FDR < 0.5. 410 genes had a 2.5-fold change (FC) or greater. NHBE cells exposure to MAB microparticles significantly enriched the IFN I signaling pathway. Protein overexpression of IFN I family (2'-5'-Oligoadenylate Synthetase 1, Interferon-induced GTP-binding protein Mx1, Interferon-stimulated gene 15) was found in bronchial epithelial cells following exposure to MAB cell wall microparticles. IFN-α protein and gene expressions were significantly increased in mice lung challenged with microparticles in comparison with controls. Conclusion: These data strongly support the role of Type I IFN in cross-talk between NHBE cells and MAB. They also suggest that initiating immune response by NHBE cells may play a central role in innate immunity. Furthermore, this study underscores the importance of mycobacterial cell wall in initiating innate immune response.

CCL17 is an important chemokine that plays a vital immunomodulatory role in the tumor microenvironment (TME). Analysis of lung adenocarcinoma (LUAD) data in Kaplan-Meier plotter databases found that the overall survival of patients in the CCL17 high-expression group was higher than that of the low-expression group, especially for patients with early (stages I and II) LUAD, which has a more positive prognostic value. Expression of CCL17 in LUAD was positively correlated with the proportion of tumor-infiltrating lymphocytes, immunostimulators, and major histocompatibility complexes using the TISIDB databases. Based on the RNA-seq and clinical data of 491 LUAD patients obtained from the TCGA database, 1,455 differential genes were found between the CCL17 high- and low-expression groups. Using WGCNA analysis confirmed that the expression of differential genes in the blue module is negatively correlated with poor survival and clinical stages of LUAD patients, and CCL17 and CCR4 genes belong to the hub genes in the blue module. Further analysis by the ESTIMATE and CIBERSORT algorithm found that the naive B cells and CD8+ T cells in the CCL17 high-expression group have a higher distribution ratio in the early LUAD patients, and the high immune score has a positive relationship with the overall survival rate. Using somatic mutation data of TCGA-LUAD, we found that 1) the tumor mutation burden values of the CCL17 high-expression group were significantly lower than those of the CCL17 low-expression group and 2) the expression levels of CCL17 and the tumor mutation burden values were negatively correlated. Transwell chemotaxis and cytotoxicity assays confirmed that CCL17 contributes to the migration of CCR4-positive lymphocytes into the H1993 LUAD TME and enhances the specific lysis of LUAD cells. In summary, high expression of CCL17 in the LUAD TME promotes local immune cell infiltration and antitumor immune response, which may contribute to the better survival and prognosis of patients with early LUAD.

The aim of this study was to evaluate the association between Ferredoxin 1 (FDX1) expression and the prognostic survival of tumor patients and predict the efficacy of immunotherapy response to antitumor drug sensitivity. FDX1 plays an oncogenic role in thirty-three types of tumors, based on TCGA and GEO databases, and further experimental validation in vitro was provided through multiple cell lines. FDX1 was expressed highly in multiple types of cancer and differently linked to the survival prognosis of tumorous patients. A high phosphorylation level was correlated with the FDX1 site of S177 in lung cancer. FDX1 exhibited a significant association with infiltrated cancer-associated fibroblasts and CD8+ T cells. Moreover, FDX1 demonstrated correlations with immune and molecular subtypes, as well as functional enrichments in GO/KEGG pathways. Additionally, FDX1 displayed relationships with the tumor mutational burden (TMB), microsatellite instability (MSI), DNA methylation, and RNA and DNA synthesis (RNAss/DNAss) within the tumor microenvironment. Notably, FDX1 exhibited a strong connection with immune checkpoint genes in the co-expression network. The validity of these findings was further confirmed through Western blotting, RT-qPCR, and flow cytometry experiments conducted on WM115 and A375 tumor cells. Elevated FDX1 expression has been linked to the enhanced effectiveness of PD-L1 blockade immunotherapy in melanoma, as observed in the GSE22155 and GSE172320 cohorts. Autodocking simulations have suggested that FDX1 may influence drug resistance by affecting the binding sites of antitumor drugs. Collectively, these findings propose that FDX1 could serve as a novel and valuable biomarker and represent an immunotherapeutic target for augmenting immune responses in various human cancers when used in combination with immune checkpoint inhibitors.

The response to ischemia in peripheral artery disease (PAD) depends on compensatory neovascularization and coordination of tissue regeneration. Identifying novel mechanisms regulating these processes is critical to the development of nonsurgical treatments for PAD. E-selectin is an adhesion molecule that mediates cell recruitment during neovascularization. Therapeutic priming of ischemic limb tissues with intramuscular E-selectin gene therapy promotes angiogenesis and reduces tissue loss in a murine hindlimb gangrene model. In this study, we evaluated the effects of E-selectin gene therapy on skeletal muscle recovery, specifically focusing on exercise performance and myofiber regeneration. C57BL/6J mice were treated with intramuscular E-selectin/adeno-associated virus serotype 2/2 gene therapy (E-sel/AAV) or LacZ/AAV2/2 (LacZ/AAV) as control and then subjected to femoral artery coagulation. Recovery of hindlimb perfusion was assessed by laser Doppler perfusion imaging and muscle function by treadmill exhaustion and grip strength testing. After three postoperative weeks, hindlimb muscle was harvested for immunofluorescence analysis. At all postoperative time points, mice treated with E-sel/AAV had improved hindlimb perfusion and exercise capacity. E-sel/AAV gene therapy also increased the coexpression of MyoD and Ki-67 in skeletal muscle progenitors and the proportion of Myh7+ myofibers. Altogether, our findings demonstrate that in addition to improving reperfusion, intramuscular E-sel/AAV gene therapy enhances the regeneration of ischemic skeletal muscle with a corresponding benefit on exercise performance. These results suggest a potential role for E-sel/AAV gene therapy as a nonsurgical adjunct in patients with life-limiting PAD.

Persistent inflammation on histology after successful hepatitis C (HCV) treatment has been reported. However, data regarding the long-term impact in liver transplant recipients is limited, particularly after using direct-acting antiviral (DAA) therapies.

Novel therapeutic angiogenic concepts for critical limb ischemia are still needed for limb salvage. E-selectin, a cell-adhesion molecule, is vital for recruitment of the stem/progenitor cells necessary for neovascularization in ischemic tissues. We hypothesized that priming ischemic limb tissue with E-selectin/adeno-associated virus (AAV) gene therapy, in a murine hindlimb ischemia and gangrene model, would increase therapeutic angiogenesis and improve gangrene.

Cancer-associated fibroblasts (CAF) play a crucial role in regulating cancer progression, yet the molecular determinant that governs the tumor regulatory role of CAF remains unknown. Using a mouse melanoma model in which exogenous melanoma cells were grafted on the skin of two lines of mice where the genetic activation or inactivation of Notch1 signaling specifically occurs in natural host stromal fibroblasts, we demonstrated that Notch1 pathway activity could determine the tumor-promoting or tumor-suppressing phenotype in CAF. CAF carrying elevated Notch1 activity significantly inhibited melanoma growth and invasion, while those with a null Notch1 promoted melanoma invasion. These findings identify the Notch1 pathway as a molecular determinant that controls the regulatory role of CAF in melanoma skin growth and invasion, unveiling Notch1 signaling as a potential therapeutic target for melanoma and potentially other solid tumors.