Non-discriminating engineered masking of immuno-evasive ligands on tumour-derived extracellular vesicles enhances tumour vaccination outcomes.

The success of personalized cancer immunotherapy depends on the initial tumour antigenic presentation to dendritic cells and macrophages. Tumour-derived extracellular vesicles (TEVs) contain abundant tumour antigenic molecules. The presence of anti-phagocytotic signals such as cluster of differentiation 47 (CD47) on the surface of the TEVs, however, leads to evasion of the same dendritic cells and macrophages. Here we show that iron oxide hydroxide nanocomposites can successfully mask TEV surfaces and unblock phagocytosis without affecting extracellular vesicles' elicited immune goals. After internalization, the mask disintegrates in the lysosome, releasing the tumour antigenic cargo. This triggers antigen presentation and promotes dendritic cell activation and maturation and macrophage reprogramming in animal models, leading to a drastic reduction of tumour volume and metastasis, and in human malignant pleural effusion clinical samples. This straightforward masking strategy eliminates the ubiquitous anti-phagocytosis block found in clinical samples and can be applied universally across all patient-specific TEVs as tumour antigenic agents for enhanced immunotherapy.

An image-based Abplex method for high-throughput GPCRs antibody discovery.

As the field of antibody therapeutics advances rapidly, membrane proteins, particularly G protein-coupled receptors (GPCRs), have emerged as highly sought-after drug targets. However, the challenges associated with extracting membrane proteins have created a demand for effective antibody screening systems targeting these proteins. In this study, we propose developing an innovative antibody screening strategy (Abplex) based on high-content imaging. This approach leverages intact cells that express target membrane proteins, facilitating the presentation of proteins in their native conformation. Furthermore, it acquires both specific and non-specific binding signals in a single well, thereby bolstering the robustness of the outcomes. The technique involves just one step and can be completed within 50 min, enabling the analysis of a single sample in just one second. The amalgamation of dependable experimental findings, a simplified workflow, reduced hands-on time, and a swift analytical pace positions our method for superior throughput and precision when juxtaposed with traditional techniques such as CbELISA and FACS. Moreover, we introduce the concept of cell barcoding, wherein cells are labeled with different fluorescence spatial patterns. This feature allows for multiplexed detection to meet the needs of various experiments. The characteristics of Abplex promise to expedite GPCR-targeting antibody discovery, advance therapeutics and enable new disease treatments.

Application of exosome engineering modification in targeted delivery of therapeutic drugs.

Cancer is the leading cause of premature death in humans. Scientists have developed several therapeutic drugs for cancer treatment. However, drug delivery faces many problems. First, traditional drugs do not target tumors and are prone to causing significant toxic side effects. Second, suitable drug carriers are essential for improving drug delivery to tumors or circulating cancer cells. Exosomes are natural extracellular vesicles with low immunogenicity and prolonged blood circulation in vivo. These characteristics render exosomes ideal drug carriers. This review highlights the properties of exosomes and mechanisms of exosome biogenesis. It also summarizes the engineering modification methods for enhancing exosome yield, targeting, and drug-loading capacity.

Gold Nanorods Inhibit Tumor Metastasis by Regulating MMP-9 Activity: Implications for Radiotherapy.

Dysregulation of matrix metalloproteinase (MMP) is strongly implicated in tumor invasion and metastasis. Nanomaterials can interact with proteins and have impacts on protein activity, which provides a potential strategy for inhibiting tumor invasion and metastasis. However, the regulation of MMP activity by nanomaterials has not been fully determined. Herein, we have found that gold nanorods (Au NRs) are able to induce the change of the secondary structure of MMP-9 and thereby inhibit their activity. Interestingly, the inhibition of MMP-9 activity is highly dependent on the aspect ratio of Au NRs, and an aspect ratio of 3.3 shows the maximum inhibition efficiency. Molecular dynamics simulations combined with mathematical statistics algorithm reveal the binding behaviors and interaction modes of MMP-9 with Au NRs in atomic details and disclose the mechanism of aspect ratio-dependent inhibition effect of Au NRs on MMP-9 activity. Au NRs with an aspect ratio of 3.3 successfully suppress the X-ray-activated invasion and metastasis of tumor by inhibiting MMP-9 activity. Our findings provide important guidance for the modulation of MMP-9 activity by tuning key parameters of nanomaterials and demonstrate that gold nanorods could be developed as potential MMP inhibitors.

Gut microbiota mediated the toxicity of high concentration of dietary nitrite in C57BL/6 mice.

Growing evidence indicates that exposure to high levels of nitrite for a prolonged time has adverse health effects. Although gut microbiota is responsible for the transformation of nitrite in the gut, the evidence concerning whether gut microbiota mediates the toxicity of nitrite is still lacking. The present study addressed the long-term effects of dietary nitrite on male C57BL/6 mice and employed fecal microbiota transplantation (FMT) to reveal whether gut microbiota mediated the effects of nitrite. Furthermore, the effect of azoxymethane (AOM) on gut microbiota was detected for mice drinking normal or nitrite-containing water. High nitrite had toxic effects on C57BL/6 mice. Meanwhile, high nitrite induced skin lesions in mice, accompanied with increased serum ALT, colon IL-6, TNF-α, and MDA levels, together with decreased serum Cr, colon sIgA, and T-AOC levels. After fecal microbiota was transplanted into the normal mice, the nitrite-regulated gut microbiota could also induce skin lesions, coupled with reduced serum Cr, and increased colon MDA. The high dose of nitrite caused the upregulations of Alistipes, Prevotella, and Ruminococcus, which could be transplanted into normal mice through FMT. Inversely, gut microbiota from normal mice reduced the effects of nitrite on serum ALT and Cr, together with colon sIgA and MDA. Gut microbiota from normal mice could also upregulate metabolic genes and downregulate stress genes in the nitrite-treated mice. It might due to the upregulation of Akkermansia and Parabacteroides caused by FMT from normal water-treated mice to nitrite-treated mice. In addition, AOM exhibited to be more toxic to the colon in the nitrite-treated mice in comparison with normal water-treated mice, and it might be due to the expression of Hspa1a and Hspa1b in the colon. Interestingly, gut microbiota was more influenced by AOM in the normal water-treated mice than the nitrite-treated mice. Overall, these data demonstrated that gut microbiota mediated the toxicity of a high concentration of dietary nitrite.

Emergence in protein derived nanomedicine as anticancer therapeutics: More than a tour de force.

Cancer has thwarted as a major health problem affecting the global population. With an alarming increase in the patient population suffering from diverse varieties of cancers, the global demographic data predicts sharp escalation in the number of cancer patients. This can be expected to reach 420 million cases by 2025. Among the diverse types of cancers, the most frequently diagnosed cancers are the breast, colorectal, prostate and lung cancer. From years, conventional treatment approaches like surgery, chemotherapy and radiation therapy have been practiced. In the past few years, increasing research on molecular level diagnosis and treatment of cancers have significantly changed the realm of cancer treatment. Lately, uses of advanced chemotherapy and immunotherapy like treatments have gained significant progress in the cancer therapy, but these approaches have several limitations on their safety and toxicity. This has generated lot of momentum for the evolution of new drug delivery approaches for the effective delivery of anticancer therapeutics, which may improve the pharmacokinetic and pharmacodynamic effect of the drugs along with significant reduction in the side effects. In this regard, the protein-based nano-medicines have gained wider attention in the management of cancer. Proteins are organic macromolecules essential, for life and have quite well explored in developing the nano-carriers. Furthermore, it provides passive or active tumour cell targeted delivery, by using protein based nanovesicles or virus like structures, antibody drug conjugates, viral particles, etc. Moreover, by utilizing various formulation strategies, both the animal and plant derived proteins can be converted to produce self-assembled virus like nano-metric structures with high efficiency in targeting the metastatic cancer cells. Therefore, the present review extensively discusses the applications of protein-based nano-medicine with special emphasis on intracellular delivery/drug targeting ability for anticancer drugs.

Differentially Expressed microRNAs in MIA PaCa-2 and PANC-1 Pancreas Ductal Adenocarcinoma Cell Lines are Involved in Cancer Stem Cell Regulation.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies, and thus better understanding of its molecular pathology is crucial for us to devise more effective treatment of this deadly disease. As cancer cell line remains a convenient starting point for discovery and proof-of-concept studies, here we report the miRNA expression characteristics of two cell lines, MIA PaCa-2 and PANC-1, and discovered three miRNAs (miR-7-5p, let-7d, and miR-135b-5p) that are involved in cancer stem cells (CSCs) suppression. After transfection of each miRNA's mimic into PANC-1 cells which exhibits higher stemness feature than MIA-PaCa-2 cells, partial reduction of CSC surface markers and inhibition of tumor sphere formation were observed. These results enlighten us to consider miRNAs as potential therapeutic agents for pancreatic cancer patients via specific and effective inhibition of CSCs.

Cyclic RGD functionalized liposomes encapsulating urokinase for thrombolysis.

Thrombosis, a critical event in blood vessels, not only is associated with myocardial infarction and stroke, but also accounts for considerable morbidity and mortality. Thrombolytic drugs are usually applied to the treatment of acute myocardial infarction, acute cerebral infarction and pulmonary embolism. However, thrombolytic drugs show limited efficacy in clinical practice because of the short half-life in plasma and systemic side effects. In this study, the cyclic RGD (cRGD) functionalized liposomes were prepared to encapsulate urokinase, a cheap and widely used thrombolytic drug in clinic and better thrombolysis efficacy was achieved. The flow cytometry analysis showed that the cRGD liposomes could bind to the activated platelets while not to the resting platelets. In vitro release study revealed that the release percentage reached plateau in about 5 h with 60% urokinase being released from liposomes. Results from the in vitro thrombolysis experiments demonstrated a good thrombolysis potential of the cRGD urokinase liposomes. The in vivo thrombolysis study demonstrated that the cRGD liposomes could significantly reduce the dose of urokinase by 75% while achieving the equivalent thrombolysis effect as the free urokinase in mouse mesenteric thrombosis model. In conclusion, the cRGD liposomes encapsulating urokinase hold great promise in clinic for better thrombolytic efficacy. STATEMENT OF SIGNIFICANCE: In this paper, the cRGD liposomes were prepared to encapsulate urokinase for targeted thrombolysis therapy. The cRGD liposomes could specifically bind to the activated platelets and could stably and continuously release its loaded urokinase. The mouse mesenteric thrombosis model was established to evaluate the thrombolysis effect of the cRGD urokinase liposomes. The results demonstrated that the cRGD liposomes could improve the thrombolytic efficacy by almost 4-fold over free urokinase. In conclusion, the cRGD liposomes encapsulating urokinase had great potential for the clinical treatment of thrombosis.

Peptide Blocking of PD-1/PD-L1 Interaction for Cancer Immunotherapy.

Immunotherapy has become a promising alternative therapeutic approach for cancer patients. Interruption of immune checkpoints, such as CTLA-4 and PD-1, has been verified to be a successful means for cancer therapy in clinical trials. mAb targeting PD-L1 has been approved to treat urothelial carcinoma, non-small cell lung cancer, or Merkel cell carcinoma by the FDA. However, the high cost of the antibody can limit its application. In our study, targeting PD-L1 peptide (TPP-1), which specifically binds to PD-L1 with high affinity, was identified through bacterial surface display methods. Using a T-cell activation assay and mixed lymphocyte reaction, TPP-1 was verified to interfere with the interaction of PD-1/PD-L1. To examine the inhibitory effect of TPP-1 on tumor growth in vivo, a xenograft mouse model using H460 cells was established. The growth rate of tumor masses in TPP-1 or PD-L1 antibody-treated mice was 56% or 71% lower than that in control peptide-treated mice, respectively, indicating that TPP-1 inhibits, or at least retards, tumor growth. IHC of the tumors showed that IFNγ and granzyme B expression increased in the TPP-1 or PD-L1 antibody-treated groups, indicating that TPP-1 attenuates the inhibitory effect of PD-L1 on T cells and that T cells may get reactivated. On the basis of our data, TPP-1 peptide could work as an alternative to antibodies for tumor immunotherapy. Cancer Immunol Res; 6(2); 178-88. ©2017 AACR.

Epithelial cell adhesion molecule independent capture of non-small lung carcinoma cells with peptide modified microfluidic chip.

Circulating tumor cells (CTCs) present in the blood of patients with non-hematological cancers are accessible sources for diagnosis and monitoring of cancers. By the aid of the ability of the anti-EpCAM antibody to recognize the epithelial cells, microsystem-based technologies provide robust means for effectively detecting CTCs in vitro. Considering the EpCAM expression is down-regulated during epithelial-mesenchymal transition (EMT) process, the amount of CTCs detected based on anti-EpCAM antibody is underestimated. In our study, the A549 cells targeting peptide (A-1 peptide), as the substitute of anti-EpCAM antibody, was introduced to microfluidic chip to capture A549 cells. Our results showed that both epithelial-like and mesenchymal-like A549 cells could efficiently be captured by the A-1 peptide modified microfluidic chip, and the capture efficiency for epithelial-like cells is comparable to that captured by the EpCAM antibody. Thus, we concluded that the peptide could be a better supplement to the EpCAM antibody for capturing CTCs in microfluidic system with broader spectrum.

Nanosystem composed with MSNs, gadolinium, liposome and cytotoxic peptides for tumor theranostics.

A dual-functional delivery system, based on mesoporous silica nanoparticles (MSNs) with the integration of Magnetic Resonance (MR) imaging and therapeutic peptide delivery, is reported in this paper. A lipid bilayer is attached onto the surface of the nanoparticles, following the doping of Gadolinium (Gd), a paramagnetic lanthanide ion. The liposome-coated GdMSNs exhibit improved colloidal stability, better biocompatibility and more efficient cellular uptake. The Gd renders the nano carrier a potential T1 contrast agent, confirmed by the MR imaging. A pro-apoptotic peptide, KLA (HGGKLAKLAKKLAKLAK), is encapsulated into the GdMSNs-LP and enters into the cells successfully to induce mitochondrial swelling and apoptosis, while it is nontoxic outside the cells. The synthesis procedure is convenient and free of toxic organic reagents. The nanosystem we construct may contribute to a promising theranostic platform for therapeutic peptide delivery in cancer treatment.

Identification of VEGFR2-binding peptides using high throughput bacterial display methods and functional assessment.

The signal transduction pathway initiated by vascular endothelial growth factor-vascular endothelial growth factor receptor 2 (VEGF-VEGFR2) plays an important role in the angiogenesis of tumors. The effective antagonists of VEGFR2 would behave as potent drugs for the treatment of malignant cancers. In our study, specific binding peptides with high affinity to VEGFR2 were obtained through bacterial display technology. Conserved motif (FF/YEXWGVK) among those peptide sequences was discovered. One of the selected peptides, VRBP1 (YDGNSFYEMWGVKPASES) was identified by screening the biased bacterial peptide library and its physiochemical feature was further characterized. The results of surface plasmon resonance (SPR) assay indicated that the dissociation constant (KD) value of VRBP1 was 228.3 nM and this peptide competed with VEGF binding to VEGFR2. Particles conjugated with VRBP1 could recognize the human umbilical vein endothelial cells (HUVEC) which express VEGFR2 on the surface. Further therapeutic effect of VRBP1 was examined by in vivo experiments. VRBP1 could result in a significant decrease in tumor size of H460 xenografts. The results from the immunohistochemical assay showed that CD31 positive signals in VRBP1-treated group were fewer than those in the control ones. These data highlighted the potential of VEGFR2-binding peptides as effective molecules for cancer diagnosis and therapy.

Identification of stem-like cells in non-small cell lung cancer cells with specific peptides.

Recent studies indicate that tumor maintenance, metastasis and drug-resistance are mainly conducted by a small subset of cancer cells which are termed cancer stem cells (CSCs) or cancer stem-like cells (CSLCs). Successful identification of CSCs/CSLCs might lead to discovery of the novel and effective therapeutic targets for cancers. In our study, lung CSCs/CSLCs were enriched by sphere-forming assay. Screening and selection of specific binding peptides for lung CSCs/CSLCs were performed with bacterial surface display method. Selected peptide named HCBP-1 exhibited highest specific binding capability as examined by flow cytometry and fluorescence microscopy. Drug-resistant lung CSCs/CSLCs might be characterized with HCBP-1 peptide and several microRNAs related to the stem-like properties were discriminatively expressed in HCBP-1+ subpopulation. Moreover, at least two distinct subpopulations in H460 tumor sphere cells could be distinguished by HCBP-1 peptide. Thus, a new method was established to identify lung CSCs/CSLCs, which provided robust approaches for the research of CSCs/CSLCs.

Role of surface charge and oxidative stress in cytotoxicity and genotoxicity of graphene oxide towards human lung fibroblast cells.

Recently, attempts have been made to apply graphene oxide (GO) in the field of biology and medicine, such as DNA sensing and drug delivery with some necessary modifications. Therefore, the toxicity of GO must be evaluated before it is applied further in biomedicine. In this paper, the cytotoxicity and genotoxicity of GO to human lung fibroblast (HLF) cells have been assessed with methyl thiazolyl tetrazolium (MTT), sub-G1 measurement and comet assays, and the mechanism of its toxicity has been explored. Various modifications of GO have been made to help us determine the factors which could affect the toxicity of GO. The results indicated that cytotoxicity and genotoxicity of GO to HLF cells were concentration dependent, and the genotoxicity induced by GO was more severe than the cytotoxicity to HLF cells. Oxidative stress mediated by GO might explain the reason of its toxic effect. Furthermore, the electronic charge on the surface of GO would play a very important role in the toxicity of GO to HLF cells.

Peptide-fluorescent bacteria complex as luminescent reagents for cancer diagnosis.

Currently in clinic, people use hematoxylin and eosin stain (H&E stain) and immunohistochemistry methods to identify the generation and genre of cancers for human pathological samples. Since these methods are inaccurate and time consuming, developing a rapid and accurate method to detect cancer is urgently demanded. In our study, binding peptides for lung cancer cell line A549 were identified using bacteria surface display method. With those binding peptides for A549 cells on the surface, the fluorescent bacteria (Escherichia coli with stably expressed green fluorescent protein) were served as specific detecting reagents for the diagnosis of cancers. The binding activity of peptide-fluorescent bacteria complex was confirmed by detached cancer cells, attached cancer cells and mice tumor xenograft samples. A unique fixation method was developed for peptide-bacteria complex in order to make this complex more feasible for the clinic use. This peptide-fluorescent bacteria complex has great potential to become a new diagnostic tool for clinical application.

A new prospect in cancer therapy: targeting cancer stem cells to eradicate cancer.

According to the cancer stem cell theory, cancers can be initiated by cancer stem cells. This makes cancer stem cells prime targets for therapeutic intervention. Eradicating cancer stem cells by efficient targeting agents may have the potential to cure cancer. In this review, we summarize recent breakthroughs that have improved our understanding of cancer stem cells, and we discuss the therapeutic strategy of targeting cancer stem cells, a promising future direction for cancer stem cell research.