Photosensitive small extracellular vesicles regulate the immune microenvironment of triple negative breast cancer.

Currently, the treatment of triple-negative breast cancer (TNBC) is limited by the special pathological characteristics of this disease. In recent years, photodynamic therapy (PDT) has created new hope for the treatment of TNBC. Moreover, PDT can induce immunogenic cell death (ICD) and improve tumor immunogenicity. However, even though PDT can improve the immunogenicity of TNBC, the inhibitory immune microenvironment of TNBC still weakens the antitumor immune response. Therefore, we used the neutral sphingomyelinase inhibitor GW4869 to inhibit the secretion of small extracellular vesicles (sEVs) by TNBC cells to improve the tumor immune microenvironment and enhance antitumor immunity. In addition, bone mesenchymal stem cell (BMSC)-derived sEVs have good biological safety and a strong drug loading capacity, which can effectively improve the efficiency of drug delivery. In this study, we first obtained primary BMSCs and sEVs, and then the photosensitizers Ce6 and GW4869 were loaded into the sEVs by electroporation to produce immunomodulatory photosensitive nanovesicles (Ce6-GW4869/sEVs). When administered to TNBC cells or orthotopic TNBC models, these photosensitive sEVs could specifically target TNBC and improve the tumor immune microenvironment. Moreover, PDT combined with GW4869-based therapy showed a potent synergistic antitumor effect mediated by direct killing of TNBC and activation of antitumor immunity. Here, we designed photosensitive sEVs that could target TNBC and regulate the tumor immune microenvironment, providing a potential approach for improving the effectiveness of TNBC treatment. STATEMENT OF SIGNIFICANCE: We designed an immunomodulatory photosensitive nanovesicle (Ce6-GW4869/sEVs) with the photosensitizer Ce6 to achieve photodynamic therapy and the neutral sphingomyelinase inhibitor GW4869 to inhibit the secretion of small extracellular vesicles (sEVs) by triple-negative breast cancer (TNBC) cells to improve the tumor immune microenvironment and enhance antitumor immunity. In this study, the immunomodulatory photosensitive nanovesicle could target TNBC cells and regulate the tumor immune microenvironment, thus providing a potential approach for improving the treatment effect in TNBC. We found that the reduction in tumor sEVs secretion induced by GW4869 improved the tumor-suppressive immune microenvironment. Moreover, similar therapeutic strategies can also be applied in other kinds of tumors, especially immunosuppressive tumors, which is of great value for the clinical translation of tumor immunotherapy.

Involvement of epidermal growth factor receptor overexpression in the promotion of breast cancer brain metastasis.

BACKGROUND: Brain-metastatic breast cancer (BMBC) is increasing and poses a severe clinical problem because of the lack of effective treatments and because the underlying molecular mechanisms are largely unknown. Recent work has demonstrated that deregulation of epidermal growth factor receptor (EGFR) may correlate with BMBC progression. However, the exact contribution that EGFR makes to BMBC remains unclear. METHODS: The role of EGFR in BMBC was explored by serial analyses in a brain-trophic clone of human MDA-MB-231 breast carcinoma cells (231-BR cells). EGFR expression was inhibited by stable short-hairpin RNA transfection or by the kinase inhibitor erlotinib, and it was activated by heparin-binding epidermal growth factor-like growth factor (HB-EGF). Cell growth and invasion activities also were analyzed in vitro and in vivo. RESULTS: EGFR inhibition or activation strongly affected 231-BR cell migration/invasion activities as assessed by an adhesion assay, a wound-healing assay, a Boyden chamber invasion assay, and cytoskeleton staining. Also, EGFR inhibition significantly decreased brain metastases of 231-BR cells in vivo. Surprisingly, changes to EGFR expression affected cell proliferation activities less significantly as determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, an anchorage-independent growth assay, and cell cycle analysis. Immunoblot analysis suggested that EGFR drives cells' invasiveness capability mainly through phosphoinositide 3-kinase/protein kinase B and phospholipase C γ downstream pathways. In addition, EGFR was involved less in proliferation because of the insensitivity of the downstream mitogen-activated protein kinase pathway. CONCLUSIONS: The current results indicated that EGFR plays more important roles in cell migration and invasion to the brain than in cell proliferation progression on 231-BR cells, providing new evidence of the potential value of EGFR inhibition in treating BMBC.

[Manganese enhanced magnetic resonance imaging for tracing corticospinal tracts in rat brain using 7.0 T MRI].

OBJECTIVE: To investigate the methods of manganese enhanced magnetic resonance imaging in 7.0 T magnetic field for tracing corticospinal tract in rat brain in vivo. METHODS: 0.4 microl volume of 1 mol/L aqueous solution of MnCl(2) was injected into the primary motor cortex of 9 SD rats under stereotaxis. MRI studies were performed for tracing corticospinal tract and other coherent nerve tracts before injection and 24 hours, 48 hours, 72 hours, 7 days after injection respectively using 7.0T Micro-MRI. RESULTS: Corticospinal tract was visualized perfectly from primary motor cortex, thalamus, cerebral peduncle to pons at different time points after Mn(2+) administration, and the best contrast was achieved after 24-48 h. At the same time, a small quantity of Mn(2+) reached the opposite somatosensory cortex through the corpus callosum. CONCLUSION: Manganese enhanced MRI visualizes perfectly the transport of Mn(2+) through axoplasmic flow in corticospinal tracts. This method may be used to investigate the change of corticospinal tract and the functional connectivity between two sides of hemisphere in rat brain.

[Magnetic resonance imaging of mesenchymal stem cells transplanted intravascularly in treatment of acute renal failure:in vivo experiment with rats].

OBJECTIVE: To evaluate the efficacy of in vivo magnetic resonance imaging (MRI) of mesenchymal stem cells (MSCs) injected intravascularly in treatment of acute renal failure (ARF) , and to investigate the changes of renal function and pathology of ARF after MSC transplantation. METHODS: Rat MSCs were isolated and labeled with Fe2O3-PLL in vitro. Thirty SD rats underwent intramuscular injection of glycerol so as to establish ARF models and then randomly divided into 3 equal groups: Group I undergoing injection of labeled MSCs into abdominal aorta via transcatheter, Group II injected with unlabelled MSCs, and Group III injected with normal saline as controls. MRI of kidney was conducted before injection, and 0.5 h, and 1, 2, and 5 days after injection. One and 2 days after the transplantation 3 rats from each group underwent MRI and extraction of blood samples from the abdominal aorta and then killed with their kidneys taken out, and 5 days after the rest rats were all killed after MRI with their kidneys taken out. Serum creatinine (Scr) and blood urea nitrogen (BUN) were examined so as to evaluate the renal function. Microscopy was conducted to observe the pathological changes. Prussian blue + CD68 antibody staining was performed to identify the labeled MSCs. RESULTS: MRI showed decrease of signal intensity in renal cortex on the T2 *-weighted MR images up to 5 days after transplantation. Histological analysis showed that most Prussian blue-positive cells were in the glomerular capillaries, corresponding to the areas where signal intensity decrease was observed by MRI. The Scr and BUN levels 2 and 5 days after the implantation of Group I were both lower than those of the control group, and there were not significant differences in the Scr and BUN levels between Groups I and II. Renal tubular injury scoring showed that the renal tubular injury was significantly lighter than that of the control group. CONCLUSION: 1.5-T MRI seems a good in vivo technique to monitor the magnetically labeled MSCs administered into the abdominal aorta of ARF animals, which are distributed in the glomerular capillaries in the early stage after transplantation. MSCs may promote the recovery of ARF.

Targeting Early Apoptosis in Acute Ischemic Stroke with a Small-Molecule Probe.

Inhibiting apoptotic cells helps ameliorate ischemic injury. Actually, only the apoptotic cells in early stage could be rescued. Molecular imaging of the early apoptosis would make sense in ischemic stroke; however, few of apoptosis molecular probes could specifically target early apoptosis. This study developed a small-molecule early apoptosis targeting probe CYS-F, which was synthesized by cystine with fluorescein isothiocyanate dyes. And the final molecular weight of CYS-F was only 1013 Da, which was much smaller than the traditional apoptosis marker annexin V. CYS-F showed excellent early apoptosis targeting ability both in vitro and in vivo. And CYS-F was cleared rapidly from the circulation with a blood half-life of 1.325 h. A favorable match was obtained between the images in fluorescence imaging and magnetic resonance imaging in stroke models. The target-to-background ratio of the lesions on 0 h was negative, which reflected the decreased blood flow. Multimodal molecular imaging showed the therapeutic effect of cystamine was dose dependence and CYS-F could also predict the outcome of ischemic stroke at an early stage. The versatility of CYS-F provides a comprehensive and convenient route for clinical decision-making in acute ischemic stroke.

Endothelial Progenitor Cells Combined with Cytosine Deaminase-Endostatin for Suppression of Liver Carcinoma.

Transplantation of gene transfected endothelial progenitor cells (EPCs) provides a novel method for treatment of human tumors. To study treatment of hepatocellular carcinoma using cytosine deaminase (CD)- and endostatin (ES)-transfected endothelial progenitor cells (EPCs), mouse bone marrow-derived EPCs were cultured and transfected with Lenti6.3-CD-EGFP and Lenti6.3-ES-Monomer-DsRed labeled with superparamagnetic iron oxide (SPIO) nanoparticles. DiD (lipophilic fluorescent dye)-labeled EPCs were injected into normal mice and mice with liver carcinoma. The EPCs loaded with CD-ES were infused into the mice through caudal veins and tumor volumes were measured. The tumor volumes in the EPC + SPIO + CD/5-Fc + ES group were found to be smaller as a result and grew more slowly than those from the EPC + SPIO + LV (lentivirus, empty vector control) group. Survival times were also measured after infusion of the cells into the mice. The median survival time was found to be longer in the EPC + SPIO + CD/5-Fc + ES group than in the others. In conclusion, the EPCs transfected with CD-ES suppressed the liver carcinoma cells in vitro, migrated primarily to the carcinoma, inhibited tumor growth, and also extended the median survival time for the mice with liver carcinoma.

Genetic immunotherapy for hepatocellular carcinoma by endothelial progenitor cells armed with cytosine deaminase.

Endothelial progenitor cells (EPCs) serve as cellular vehicles for targeting cancer cells and are a powerful tool for delivery of therapeutic genes. Cytosine deaminase (CD), a kind of frequent suicide gene which can kill carcinoma cells by converting a non-poisonous pro-drug 5-flucytosine (5-FC) into a poisonous cytotoxic 5-fluorouracil (5-FU). We combined super-paramagnetic iron oxide (SPIO) nanoparticles labeled EPCs with CD gene to treat grafted liver carcinomas and tracked them with 7.0 T Magnetic resonance imaging (MRI). Results showed that the therapeutic EPCs loaded with CD plus 5-Fc provided stronger carcinoma growth suppression compared with treatment using CD alone. The CD/5-Fc significantly inhibited the growth of endothelial cells and induced carcinoma cells apoptosis. These results indicate that EPCs transfected with anti-carcinoma genes can be used in carcinoma therapy as a novel therapeutic modality.

Preparation of DOX/BSANP and its antitumor effect on bel-7404 liver cancer cells in vitro and in vivo.

This paper aimed to investigate the preparation of doxorubicin-loaded bovine serum albumin nanoparticles (DOX/BSANP) and their effect on killing liver cancer cells in vitro and in vivo. DOX/BSANP was prepared using a desolvation-chemical crosslinking method. Their morphology and particle size were observed using transmission electron microscopy (TEM). The envelopment, drug-loading rates and slow-release characteristics were determined spectrophotometrically. Their ability to kill liver cancer cells in vitro was determined using the methyl thiazolyl tetrazolium (MTT) assay and flow cytometry (FCM). The tumor-suppressing effect of the nanoparticles in experimental animals in vivo was also evaluated. Under TEM, DOX/BSANP appeared spherical and was distributed uniformly, with a diameter of about 120 nm and hydrated particle size of 170 nm determined by dynamic light diffraction. The envelopment rate was 82% and the drug-loading rate was 11.2%. The in vitro drug-release experiment showed that about 50% of the drug in drug-loaded nanoparticles was released continuously and slowly for 7 days. The MTT assay showed that DOX/BSANP significantly inhibited cell proliferation, while FCM showed that it induced tumor cell apoptosis. The in vivo tumor suppression test showed that the therapeutic effect of drug-loaded nanoparticles was superior to that of DOX alone.

Magnetic resonance imaging of post-ischemic blood-brain barrier damage with PEGylated iron oxide nanoparticles.

Blood-brain barrier (BBB) damage during ischemia may induce devastating consequences like cerebral edema and hemorrhagic transformation. This study presents a novel strategy for dynamically imaging of BBB damage with PEGylated supermagnetic iron oxide nanoparticles (SPIONs) as contrast agents. The employment of SPIONs as contrast agents made it possible to dynamically image the BBB permeability alterations and ischemic lesions simultaneously with T2-weighted MRI, and the monitoring could last up to 24 h with a single administration of PEGylated SPIONs in vivo. The ability of the PEGylated SPIONs to highlight BBB damage by MRI was demonstrated by the colocalization of PEGylated SPIONs with Gd-DTPA after intravenous injection of SPION-PEG/Gd-DTPA into a mouse. The immunohistochemical staining also confirmed the leakage of SPION-PEG from cerebral vessels into parenchyma. This study provides a novel and convenient route for imaging BBB alteration in the experimental ischemic stroke model.

Preparation and characterization of realgar nanoparticles and their inhibitory effect on rat glioma cells.

AIM: Our objective was to prepare a new nano-sized realgar particle and characterize its anti-tumor effect on tumor cells. METHODS: Nanoparticles were prepared by coprecipitation and were detected by transmission electron microscopy, scanning electron microscopy, energy dispersive spectrometry (EDS), and dynamic light scattering. An anti-proliferative effect of realgar nanoparticles on rat glioma (C6) cells was determined by the MTT assay. Cell cycle and apoptosis rates were observed by flow cytometry. Apoptosis-related gene expression was detected by immunofluorescence staining. RESULTS: Realgar nanoparticles were successfully prepared. The particles were spherical, with an average diameter of approximately 80 nm, and contained arsenic and sulfur elements. Realgar nanoparticles inhibited C6 cell proliferation and induced apoptosis in a dose- and time-dependent manner. Treatment of C6 cells with realgar nanoparticles significantly increased the proportions of cells in S and G2/M phases, decreased the proportion of cells in G0/G1 phase, downregulated Bcl-2 expression, and substantially upregulated Bax expression. CONCLUSION: Realgar nanoparticles significantly inhibited C6 glioma cell proliferation and promoted cell apoptosis by inducing the upregulation of Bax and downregulation of Bcl-2 expression. Realgar nanoparticles are a promising in vitro anti-cancer strategy and may be applicable for human cancer therapy studies.

Enhancement of p53 gene transfer efficiency in hepatic tumor mediated by transferrin receptor through trans-arterial delivery.

Transferrin-DNA complex mediated by transferrin receptor in combination with interventional trans-arterial injection into a target organ may be a duel-target-oriented delivery means to achieve an efficient gene therapy. In this study, transferrin receptor expression in normal human hepatocyte and two hepatocellular-carcinoma cells (Huh7/SK-Hep1) was determined. p53-LipofectAMINE with different amounts of transferrin was transfected into the cells and the gene transfection efficiency was evaluated. After VX2 rabbit hepatocarcinoma model was established, the transferrin-p53-LipofectAMINE complex was delivered into the hepatic artery via interventional techniques to analyze the therapeutic p53 gene transfer efficiency in vivo by Western blot, immunohistochemical/immunofluorescence staining analysis and survival time. The results were transferrin receptor expression in Huh7 and SK-Hep1 cells was higher than in normal hepatocyte. Transfection efficiency of p53 was increased in vitro in both Huh7 and SK-Hep1 cells with increasing transferrin in a dose-dependent manner. As compared to intravenous administration, interventional injection of p53-gene complex into hepatic tumor mediated by transferrin-receptor, could enhance the gene transfer efficiency in vivo as evaluated by Western blot, immunohistochemical/immunofluorenscence staining analyses and improved animal survival (H = 12.567, p = 0.0019). These findings show the transferrin-transferrin receptor system combined with interventional techniques enhanced p53-gene transfer to hepatic tumor and the duel-target-oriented gene delivery may be an effective approach for gene therapy.