Detection of Binding of a Synthetic Granzyme B-like Peptide Fluorescent Conjugate within Platelet-like Structures in Cancer-related Peripheral Blood Specimens and Tissue Sections

Platelets and cytotoxic T lymphocytes (CTL) are important whole blood components in peripheral blood. Studies have shown that platelets, from precursor megakaryocytes, are significant factors in cancer prognosis, cancer progression, and metastasis; but a direct platelet-cancer relationship remains unclear. CTL play an essential role in cancer surveillance by inducing cancer cell death with granzyme B. A recent report has shown the presence of binding targets with binding affinity to a synthetic granzyme B-like peptide fluorescent conjugate (GP1R) in different types of cancer cells grown in vitro. It suggests that these binding targets may serve as a “universal-pathologic-biomarker”. It is not known if similar biomarkers may be present in platelets of cancer patients. We show with fluoroscopic images that GP1R can bind to binding targets: 1) within platelets in methanol-fixed whole blood smears of patients with breast cancer and lung cancer, and 2) within platelet-like structures in formalin-fixed-paraffin-embedded (FFPE) nude mouse xenogeneic breast tumor tissues. Samples without cancer-association displayed no discernible GP1R-binding in platelet-like structures. Our data demonstrate for the first time that a similar “universal-pathologic-biomarker” detectable by GP1R-binding is present in circulating platelets of cancer patients. The data depict a co-existence of animal-platelets and human-breast cancer cells, both have a common pathologic biomarker detectable by GP1R, in the tumor growth. The fluoroscopic images indicate a visual direct connection between pathologic platelets and cancer. These preliminary results may lead to developments of novel platelet-based cancer diagnostics and therapeutics and a better understanding of the potential multifunction of GP1R and its relationship to megakaryocytes and PD1.     

Connecting Effective Immune Response,Fluorescent Granzyme B-like Peptide,Specific Peptide Binding Patterns,Patients with Cancer and Viral Infection,in Remission,Clinical Significance,and Liquid Biopsy

Functional cytotoxic-T-lymphocytes (CTL) with granzyme B play an important role in an effective immune response to tumor growth and infection progression. Tumor cells and platelets in peripheral whole blood smears of cancer patients have shown the presence of innate binding targets for GP1R, a fluorescent synthetic Granzyme B-like peptide. It is not known if similar GP1R-binding targets and specific binding patterns are detectable in peripheral blood of patients with viral infection. It is also not known if a specific binding pattern may be associated with an effective immune response to indicate a favorable prognosis. We reviewed the GP1R-binding patterns in the peripheral blood smears of 5 patients in remission at the time of sampling (3 with cancer and 2 with flu-like symptoms) and a negative control. We show with fluoroscopic images that there are: 1) fluorescent GP1R-binding targets mostly in the cytoplasmic areas of nucleated cells in patients with breast and lung cancer who have longer survival, 2) intense fluorescent deposits mostly in the nuclear areas of segmented neutrophils in patients recovered from severe to mild flu-like symptoms, 3) discernible fluorescent deposits in the cytoplasmic areas of small lymphocyte-like elements and overall intense fluorescent stain in large cells in the patient with advanced pancreatic cancer who had shorter survival, 4) GP1R-binding targets in numerous platelet-like elements in all 5 patients. The control sample did not show similar binding patterns. The potential association between specific GP1R-binding patterns in peripheral blood samples and prognostic significance, and its use as liquid biopsy are discussed.     

Membranes,peptides, and disease: Unraveling the mechanisms of viral proteins with solid state nuclear magnetic resonance spectroscopy

The interplay between peptides and lipid bilayers drives crucial biological processes. For example, a critical step in the replication cycle of enveloped viruses is the fusion of the viral membrane and host cell endosomal membrane, and these fusion events are controlled by viral fusion peptides. Thus such membrane-interacting peptides are of considerable interest as potential pharmacological targets. Deeper insight is needed into the mechanisms by which fusion peptides and other viral peptides modulate their surrounding membrane environment, and also how the particular membrane environment modulates the structure and activity of these peptides. An important step toward understanding these processes is to characterize the structure of viral peptides in environments that are as biologically relevant as possible. Solid state nuclear magnetic resonance (ssNMR) is uniquely well suited to provide atomic level information on the structure and dynamics of both membrane-associated peptides as well as the lipid bilayer itself; further ssNMR can delineate the contribution of specific membrane components, such as cholesterol, or changing cellular conditions, such as a decrease in pH on membrane-associating peptides. This paper highlights recent advances in the study of three types of membrane associated viral peptides by ssNMR to illustrate the more general power of ssNMR in addressing important biological questions involving membrane proteins.     

Peptide antagonists of superantigen toxins

Superantigens produced by Staphylococcus aureus and Streptococcus pyogenes are among the most lethal of toxins. Toxins in this large family trigger an excessive cellular immune response leading to toxic shock. Superantigens are secreted by the bacteria as diverse natural mixtures, a complexity that demands development of broad-spectrum countermeasures. We used a rational approach to design short peptides with homology to various domains in a typical superantigen (staphylococcal enterotoxin B) and screened each peptide for its ability to antagonize, in human peripheral blood mononuclear cells, superantigen-mediated induction of the genes encoding T helper 1 cytokines that mediate shock: interleukin-2, interferon-gamma and tumor necrosis factor. A dodecamer peptide proved a potent antagonist against widely different superantigens. This peptide protected mice from killing by superantigens and it was able to rescue mice undergoing toxic shock. The antagonist peptide shows homology to a beta-strand-hinge-alpha-helix domain that is structurally conserved among superantigens, yet currently of unknown function and remote from the binding sites for the known ligands essential for T cell activation, the major histocompatibility complex class II molecule and T cell receptor. The antagonist activity of this peptide thus identifies a novel domain in superantigens that is critical for their toxic action. The antagonist peptide provides a new tool for understanding the mechanism of excessive human immune response activation by superantigens that occurs during toxic shock and for identification of a novel target ligand that may interact with this superantigen domain.     

Study on the relationship between structure and taste activity of the umami peptide of Stropharia rugosoannulata prepared by ultrasound

Through virtual screening, electronic tongue verification, and molecular docking technology, the structure-taste activity relationship of 47 kinds of umami peptides (octapeptide - undecapeptide) from Stropharia rugosoannulata prepared by simultaneous ultrasonic-assisted directional enzymatic hydrolysis was analyzed. The umami peptides of S.rugosoannulata can form hydrogen bond interaction and electrostatic interaction with umami receptors T1R1/T1R3. The amino acid residues at the peptides' N-terminal and C-terminal play a vital role in binding with the receptors to form a stable complex. D, E, and R are the primary amino acids in the peptides that easily bind to T1R1/T1R3. The basic amino acid in the peptides is more easily bound to T1R1, and the acidic amino acid is more easily bound to T1R3. The active amino acid sites of the receptors to which the peptides bind account for 42%−65% of the total active amino acid residues in the receptors. ASP147 and ASP219 are the critical amino acid residues for T1R1 to recognize the umami peptides, and ARG64, GLU45, and GLU48 are the critical amino acid residues for T1R3 to recognize the umami peptides. The increase in the variety and quantity of umami peptides is the main reason for improving the umami taste of the substrate prepared by synchronous ultrasound-assisted directional enzymatic hydrolysis. This study provides a theoretical basis for understanding simultaneous ultrasound-assisted directional enzymatic hydrolysis for preparing umami peptides from S.rugosoannulata, enhancing the flavor of umami, and the relationship between peptide structure and taste activity.     

CTLA-4 blockade during dendritic cell based booster vaccination influences dendritic cell survival and CTL expansion

Dendritic cells (DCs) are potent antigen-presenting cells and critical for the priming of CD8+ T cells. Therefore the use of these cells as adjuvant cells has been tested in a large number of experimental and clinical vaccination studies, in particular cancer vaccine studies. A number of protocols are emerging that combine vaccination with CTL expanding strategies, such as e.g. blockade of CTLA-4 signalling. On the other hand, the lifespan and in vivo survival of therapeutic DCs have only been addressed in a few studies, although this is of importance for the kinetics of CTL induction during vaccination. We have previously reported that DCs loaded with specific antigens are eliminated by antigen specific CTLs in vivo and that this elimination affects the potential for in vivo CTL generation. We now show that CTLA-4 blockade increases the number of DC vaccine induced LCMV gp33 specific CTLs and the lysis of relevant in vivo targets. However, the CTLA-4 blockage dependent expansion of CTLs also affect DC survival during booster DC injections and our data suggest that during a booster DC vaccine, the largest increase in CTL levels is already obtained during the first vaccination.     

Multifunctional Gelatin Nanoparticle Integrated Microchip for Enhanced Capture,Release, and Analysis of Circulating Tumor Cells

Detection of rare circulating tumor cells (CTCs) from patients has an important effect on clinical cancer diagnosis and prognosis. Here, an integrated microfluidic chip for efficient isolation and downstream analysis of CTCs is developed. This new designed platform, gelatin nanoparticle (GNP) integrated microchip, combines a series of curved herringbone structures, which generate enhanced interactions between CTCs and the immunomodified channel surface, with multifunctional GNP based nanostructured surface, which not only provide more binding sites for antibodies and targets and avoid nonspecific absorption of blood cells due to its electronegative surface charge, but also enable viable cell release under a mild enzymatic treatment. The chip allows cell isolation with ≈85% capture yield and ≈90% release efficiency using spiked cell samples. Results demonstrate that the released cancer cells maintain good viability and proliferation ability. Furthermore, the microchip is successfully applied to capture noninvasive release and genetically analyze CTCs from clinical cancer patients. The proposed platform may provide a potential in clinics.     

The feasibility of assessing branched-chain amino acid metabolism in cellular models of prostate cancer with hyperpolarized [1-C]-ketoisocaproate

Recent advancements in the field of hyperpolarized ¹³C magnetic resonance spectroscopy (MRS) have yielded powerful techniques capable of real-time analysis of metabolic pathways. These non-invasive methods have increasingly shown application in impacting disease diagnosis and have further been employed in mechanistic studies of disease onset and progression. Our goals were to investigate branched-chain aminotransferase (BCAT) activity in prostate cancer with a novel molecular probe, hyperpolarized [1-¹³C]-2-ketoisocaproate ([1-¹³C]-KIC), and explore the potential of branched-chain amino acid (BCAA) metabolism to serve as a biomarker. Using traditional spectrophotometric assays, BCAT enzymatic activities were determined in vitro for various sources of prostate cancer (human, transgenic adenocarcinoma of the mouse prostate (TRAMP) mouse and human cell lines). These preliminary studies indicated that low levels of BCAT activity were present in all models of prostate cancer but enzymatic levels are altered significantly in prostate cancer relative to healthy tissue. The MR spectroscopic studies were conducted with two cellular models (PC-3 and DU-145) that exhibited levels of BCAA metabolism comparable to the human disease state. Hyperpolarized [1-¹³C]-KIC was administered to prostate cancer cell lines, and the conversion of [1-¹³C]-KIC to the metabolic product, [1-¹³C]-leucine ([1-¹³C]-Leu), could be monitored via hyperpolarized ¹³C MRS.     

High‐Density Lipoprotein Nanoparticles Deliver RNAi to Endothelial Cells to Inhibit Angiogenesis

Systemic delivery of therapeutic nucleic acids to target cells and tissues outside of the liver remains a major challenge. A biomimetic high‐density lipoprotein nanoparticle (HDL NP) is synthesized for delivery of a cholesteryl‐modified therapeutic nucleic acid to vascular endothelial cells (ECs), a cell type naturally targeted by HDL. HDL NPs adsorb cholesteryl‐modified oligonucleotides and protect them from nuclease degradation. As proof of principle, we deliver RNAi targeting vascular endothelial growth factor receptor 2 (VEGFR2) to ECs to effectively silence target mRNA and protein expression in vitro. In addition, data show that treatment strongly attenuates in vivo neovascularization measured using a standard angiogenesis assay and in hypervascular tumor allografts where a striking reduction in tumor growth is observed. For effective delivery, HDL NPs require the expression of the cell surface protein scavenger receptor type‐B1 (SR‐B1). No toxicity of HDL NPs is measured in vitro or after in vivo administration. Thus, by using a biomimetic approach to nucleic acid delivery, data demonstrate that systemically administered RNAi–HDL NPs target SR‐B1 expressing ECs to deliver functional anti‐angiogenic RNAi as a potential treatment of cancer and other neovascular diseases.     

Exploring the anti-proliferative activity of <Emphasis Type="BoldItalic">Pelargonium sidoides</Emphasis> DC with in silico target identification and network pharmacology

Pelargonium sidoides DC (Geraniaceae) is a medicinal plant indigenous to Southern Africa that has been widely evaluated for its use in the treatment of upper respiratory tract infections. In recent studies, the anti-proliferative potential of P. sidoides was shown, and several phenolic compounds were identified as the bioactive compounds. Little, however, is known regarding their anti-proliferative protein targets. In this study, the anti-proliferative mechanisms of P. sidoides through in silico target identification and network pharmacology methodologies were evaluated. The protein targets of the 12 phenolic compounds were identified using the target identification server PharmMapper and the server for predicting Drug Repositioning and Adverse Reactions via the Chemical–Protein Interactome (DRAR-CPI). Protein–protein and protein–pathway interaction networks were subsequently constructed with Cytoscape 3.4.0 to evaluate potential mechanisms of action. A total of 142 potential human target proteins were identified with the in silico target identification servers, and 90 of these were found to be related to cancer. The protein interaction network was constructed from 86 proteins involved in 209 interactions with each other, and two protein clusters were observed. A pathway enrichment analysis identified over 80 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enriched with the protein targets and included several pathways specifically related to cancer as well as various signaling pathways that have been found to be dysregulated in cancer. These results indicate that the anti-proliferative activity of P. sidoides may be multifactorial and arises from the collective regulation of several interconnected cell signaling pathways.     

Antigenized antibodies expressing Vbeta8.2 TCR peptides immunize against rat experimental allergic encephalomyelitis

BACKGROUND: Immunity against the T cell receptor (TCR) is considered to play a central role in the regulation of experimental allergic encephalomyelitis (EAE), a model system of autoimmune disease characterized by a restricted usage of TCR genes. Methods of specific vaccination against the TCR of pathogenetic T cells have included attenuated T cells and synthetic peptides from the sequence of the TCR. These approaches have led to the concept that anti-idiotypic immunity against antigenic sites of the TCR, which are a key regulatory element in this disease. METHODS: The present study in the Lewis rat used a conventional idiotypic immunization based on antigenized antibodies expressing selected peptide sequences of the Vbeta8.2 TCR (93ASSDSSNTE101 and 39DMGHGLRLIHYSYDVNSTEKG59). RESULTS: The study demonstrates that vaccination with antigenized antibodies markedly attenuates, and in some instances, prevents clinical EAE induced with the encephalitogenic peptide 68GSLPQKSQRSQDENPVVHF88 in complete Freunds' adjuvant (CFA). Antigenized antibodies induced an anti-idiotypic response against the Vbeta8.2 TCR, which was detected by ELISA and flowcytometry. No evidence was obtained of a T cell response against the corresponding Vbeta8.2 TCR peptides. CONCLUSIONS: The results indicate that antigenized antibodies expressing conformationally-constrained TCR peptides are a simple means to induce humoral anti-idiotypic immunity against the TCR and to vaccinate against EAE. The study also suggests the possibility to target idiotypic determinants of TCR borne on pathogenetic T cells to vaccinate against disease.     

Hybrid gold nanoparticles in molecular imaging and radiotherapy

Metallic nanoparticles, because of their size, chemical and physical properties, are particularly attractive as therapeutic probes in treating cancer. Central to any clinical advances in nanoparticulate based therapy will be to produce hybrid nanoparticles that can be targeted to vascular, extracellular or cell surface receptors. Development of hybrid nanoparticles that specifically target cancer vasculature has received considerable attention. Most cancers have leaky vasculature and the defective vascular architecture, created due to the rapid vascularization necessary to serve fast growing cancers, in combination with poor lymphatic drainage allows increased permeation and retention effects. The leaky vasculature, because of higher porosity and permeability, serve as natural high affinity targets to metallic nanoparticles. Another attractive approach toward the application of nanotechnology to nanomedicine is the utility of nanoparticles that display inherent therapeutic properties. For example radioactive gold nanoparticles present attractive prospects in therapy of cancer. The radioactive properties of Au-198 (β_max=0.96 MeV; t_1/2=2.7 d) and Au-199 (β_max=0.46 MeV; t_1/2=3.14 d) make them ideal candidates for use in radiotherapeutic applications. In addition, they both have imageable gamma emissions for dosimetry and pharmacokinetic studies and Au-199 can be made carrier-free by indirect methods. Gold nanoparticles are of interest for treatment of disease as they can deliver agents directly into cells and cellular components with a higher concentration of radioactivity, e.g. higher dose of radioactivity, to cancerous tumor cells.     

Enhancement of Intracellular Delivery of CdTe Quantum Dots (QDs) to Living Cells by Tat Conjugation

Quantum dots (QDs), as novel fluorescence probes, have shown a great potential for bio-molecular labeling and cellular imaging. To stain cellular targets, the sufficient intracellular delivery of QDs is required. In this work the tat, a typical membrane-permeable carrier peptide, was conjugated with thiol-capped CdTe QDs to form CdTe Tat-QDs, and the intracellular deliveries of CdTe QDs or CdTe Tat-QDs were compared in human hepatocellular carcinoma (QGY) cells and human breast cancer (MCF7) cells in vitro by means of confocal laser scanning microscopy. Added into the cell dishes, both QDs and Tat-QDs adhered to the outer leaflet of the plasma membrane of cells within a few minutes, but the binding amount of Tat-QDs was obviously higher than that of QDs. Then both QDs and Tat-QDs can penetrate into cells, and their cellular contents increased with incubation time but both saturated after 3 hours incubation. However the cellular levels of Tat-QDs were higher than those of QDs, with the ratio of 2.1 (±0.3) times in QGY cells and 1.5 (±0.2) times in MCF7 cells, demonstrating the enhancing effect of Tat conjugation on the intracellular delivery of QDs.     

Enzymatic quantification of cell-matrix and cell-cell adhesion

Adhesion assays are powerful tools to investigate the adhesive properties of cells. The quantification of cell adhesion enables determination of the capacity of cells to stick to a target, screening for novel adhesion involved binding molecules, exploration of structure-function relationships of adhesion molecules, evaluation of adhesion targets, and examination of compounds interfering with cell adhesion. Thus, quantification of cell adhesion needs simple and reliable methods that might be applied for both research and diagnostic purposes. This review presents methodological principles of enzymatic approaches for quantification of cell adhesion. In particular, the advantages of exogenous cell labelling with horseradish peroxidase are described.     

Activation of microbubbles by low-intensity pulsed ultrasound enhances the cytotoxicity of curcumin involving apoptosis induction and cell motility inhibition in human breast cancer MDA-MB-231 cells

Ultrasound and microbubbles-mediated drug delivery has become a promising strategy to promote drug delivery and its therapeutic efficacy. The aim of this research was to assess the effects of microbubbles (MBs)-combined low-intensity pulsed ultrasound (LPUS) on the delivery and cytotoxicity of curcumin (Cur) to human breast cancer MDA-MB-231 cells. Under the experimental condition, MBs raised the level of acoustic cavitation and enhanced plasma membrane permeability; and cellular uptake of Cur was notably improved by LPUS–MBs treatment, aggravating Cur-induced MDA-MB-231 cells death. The combined treatment markedly caused more obvious changes of cell morphology, F-actin cytoskeleton damage and cell migration inhibition. Our results demonstrated that combination of MBs and LPUS may be an efficient strategy for improving anti-tumor effect of Cur, suggesting a potential effective method for antineoplastic therapy.     

The use of a combinatorial library method to isolate human tumor cell adhesion peptides

Summary Tumor cell progression is dependent in part on the successful adhesive interactions of the cells with the extracellular matrix. In this study, a new approach is described to isolate linear peptide ligand candidates involved in cellular adhesion. A synthetic combinatorial peptide library based on the one-bead-one-peptide concept was incubated with live human prostate cancer cells for 90 min at 37 °C. The peptide bead coated with a monolayer of cells was then isolated for micro sequencing. The DU145 (DU-H) cells were chosen since they have been previously characterized as containing elevated levels of a laminin receptor for cell adhesion, the ₆₁ integrin on the cell surface. The use of a function-blocking antibody (GoH3) allows for the detection of peptides which are ₆-specific ligand candidates. From two different libraries (linear 9-mer and 11-mer) of a total of 1 500 000 beads, 68 peptide beads containing attached cells were isolated. These positive beads were then retested to determine the ability of the GoH3 antibody to block binding of the cells to the peptide beads. The ₆ integrin candidate peptide beads (five in total) were recovered and two of the beads were microsequenced. These two peptides, RU-1 (LNIVSVNGRHX) and RX-1 (DNRIRLQAKXX), resemble the previously reported active peptide sequences (GD-2 and AG-73) from native laminin. The RU-1, RX-1 and AG-73 peptides were tested for their ability to support cell attachment and to bind the cell surface of DU-H prostate carcinoma cells in suspension using fluorescence-activated cell-sorting (FACS) analysis. Both RU-1 and AG-73 peptides supported cellular attachment within 1 h. In contrast, after 1 h, EHS laminin supported both cellular attachment and spreading. The RX-1 peptide exhibited only weak binding to the DU-H prostate carcinoma cells. FACS analysis indicated that AG-73 peptide attached to tumor cell surfaces over a range of concentrations, whereas the RU-1 peptide showed a homogeneous concentration required for attachment. The described strategy for screening a random peptide library offers three advantages: (i) ligands for conformationally sensitive receptors of adhesion can be isolated using live cells; (ii) specific binding can be selected for using function-blocking antibodies; and (iii) peptides supporting adhesion independent of spreading properties can be distinguished. In principle, specific adhesive peptides without prior knowledge of the sequence could be isolated for any epithelial cell surface receptor for which a function-blocking reagent is available.     

Effective combinatorial strategy to increase affinity of carbohydrate binding by peptides

The Thomsen-Friedenreich antigen, a carcinoma-associated disaccharide involved in carcinoma cell homotypic aggregation and increased metastatic potential, has clinical value as a prognostic indicator and a marker of metastasized cells. Hence, it can reasonably be predicted that antigen-binding macromolecules are valuable clinical in vivo diagnostic/therapeutic targeting agents. Recently, we have selected first-generation antigen-binding peptides from a random peptide bacteriophage display library and have applied combinatorial affinity maturation to select functionally-maturated peptides, which target cultured carcinoma cells and inhibit carcinoma cell aggregation. In the current study we hypothesize that a targeted search of sequence space surrounding the antigen-binding consensus sequence will select unpredictable amino acid sequences in the non-consensus portions of the peptides, leading to increased affinity for the carbohydrate and greater solubility in physiological buffers. This comprehensive in vitro analysis demonstrates that preferential evolution of the amino-terminal sequence of the peptides occurred, which correlated, in structure/function studies, with the acquisition of maturated function. The maturated peptides are more soluble than the earlier peptides. Studies of peptide binding to the disaccharide indicate that two maturated peptides (P-30-1, F03) have higher affinity for the antigen and bind with higher intensity to the surface of cultured human carcinoma cells than the first-generation peptides. The results support our hypothesis that affinity maturation can improve carbohydrate binding by peptides and have theoretical importance as the first report of maturation of carbohydrate-binding affinity in a small, soluble peptide.     

A novel method to identify and characterise peptide mimotopes of heat shock protein 70-associated antigens

The heat shock protein, Hsp70, has been shown to play an important role in tumour immunity. Vaccination with Hsp70-peptide complexes (Hsp70-PCs), isolated from autologous tumour cells, can induce protective immune responses. We have developed a novel method to identify synthetic mimic peptides of Hsp70-PCs and to test their ability to activate T-cells. Peptides (referred to as "recognisers") that bind to Hsp70-PCs from the human breast carcinoma cell line, MDA-MB-231, were identified by bio-panning a random peptide M13 phage display library. Synthetic recogniser peptides were subsequently used as bait in a reverse bio-panning experiment to identify potential Hsp70-PC mimic peptides. The ability of the recogniser and mimic peptides to prime human lymphocyte responses against tumour cell antigens was tested by stimulating lymphocytes with autologous peptide-loaded monocyte-derived dendritic cells (DCs). Priming and subsequent stimulation with either the recogniser or mimic peptide resulted in interferon-γ (IFN-γ) secretion by the lymphocytes. Furthermore, DCs loaded with Hsp70, Hsp70-PC or the recogniser or the mimic peptide primed the lymphocytes to respond to soluble extracts from breast cells. These results highlight the potential application of synthetic peptide-mimics of Hsp70-PCs, as modulators of the immune response against tumours.     

Phase Differential Enhancement of FLIM to Distinguish FRET Components of a Biosensor for Monitoring Molecular Activity of Membrane Type 1 Matrix Metalloproteinase in Live Cells

Fluorescence lifetime-resolved imaging microscopy (FLIM) has been used to monitor the enzymatic activity of a proteolytic enzyme, Membrane Type 1 Matrix Metalloproteinase (MT1-MMP), with a recently developed FRET-based biosensor in vitro and in live HeLa and HT1080 cells. MT1-MMP is a collagenaise that is involved in the destruction of extra-cellular matrix (ECM) proteins, as well as in various cellular functions including migration. The increased expression of MT1-MMP has been positively correlated with the invasive potential of tumor cells. However, the precise spatiotemporal activation patterns of MT1-MMP in live cells are still not well-established. The activity of MT1-MMP was examined with our biosensor in live cells. Imaging of live cells was performed with full-field frequency-domain FLIM. Image analysis was carried out both with polar plots and phase differential enhancement. Phase differential enhancement, which is similar to phase suppression, is shown to facilitate the differentiation between different conformations of the MT1-MMP biosensor in live cells when the lifetime differences are small. FLIM carried out in differential enhancement or phase suppression modes, requires only two acquired phase images, and permits rapid imaging of the activity of MT1-MMP in live cells.     

Use of ultraviolet-light irradiated multiple myeloma cells as immunogens to generate tumor-specific cytolytic T lymphocytes

Background

As the eradication of tumor cells in vivo is most efficiently performed by cytolytic T lymphocytes (CTL), various methods for priming tumor-reactive lymphocytes have been developed. In this study, a method of priming CTLs with ultraviolet (UV)-irradiated tumor cells, which results in termination of tumor cell proliferation, apoptosis, as well as upregulation of heat shock proteins (HSP) expression is described.

Methods

Peripheral blood mononuclear cells (PBMC) were primed weekly with UV-irradiated or mitomycin-treated RPMI 8226 multiple myeloma cells. Following three rounds of stimulation over 21 days, the lymphocytes from the mixed culture conditions were analyzed for anti-MM cell reactivity.

Results

By day 10 of cultures, PBMCs primed using UV-irradiated tumor cells demonstrated a higher percentage of activated CD8+/CD4- T lymphocytes than non-primed PBMCs or PBMCs primed using mitomycin-treated MM cells. Cytotoxicity assays revealed that primed PBMCs were markedly more effective (p < 0.01) than non-primed PBMCs in killing RPMI 8226 MM cells. Surface expression of glucose regulated protein 94 (Grp94/Gp96) and Grp78 were both found to be induced in UV-treated MM cells.

Conclusion

Since, HSP-associated peptides are known to mediate tumor rejection; these data suggest that immune-mediated eradication of MM cells could be elicited via a UV-induced HSP process. The finding that the addition of 17-allylamide-17-demethoxygeldanamycin (17AAG, an inhibitor of HSP 90-peptide interactions) resulted in decreased CTL-induced cytotoxicity supported this hypothesis. Our study, therefore, provides the framework for the development of anti-tumor CTL cellular vaccines for treating MM using UV-irradiated tumor cells as immunogens.