Phosphatidylserine-Exposing Annexin A1-Positive Extracellular Vesicles: Potential Cancer Biomarkers.

Under physiological conditions, phosphatidylserine (PS) predominantly localizes to the cytosolic leaflet of the plasma membrane of cells. During apoptosis, PS is exposed on the cell surface and serves as an "eat-me" signal for macrophages to prevent releasing self-immunogenic cellular components from dying cells which could potentially lead to autoimmunity. However, increasing evidence indicates that viable cells can also expose PS on their surface. Interestingly, tumor cell-derived extracellular vesicles (EVs) externalize PS. Recent studies have proposed PS-exposing EVs as a potential biomarker for the early detection of cancer and other diseases. However, there are confounding results regarding subtypes of PS-positive EVs, and knowledge of PS exposure on the EV surface requires further elucidation. In this study, we enriched small EVs (sEVs) and medium/large EVs (m/lEVs) from conditioned media of breast cancer cells (MDA-MB-231, MDA-MB-468) and non-cancerous cells (keratinocytes, fibroblasts). Since several PS-binding molecules are available to date, we compared recombinant proteins of annexin A5 and the carboxylated glutamic acid domain of Protein S (GlaS), also specific for PS, to detect PS-exposing EVs. Firstly, PS externalization in each EV fraction was analyzed using a bead-based EV assay, which combines EV capture using microbeads and analysis of PS-exposing EVs by flow cytometry. The bulk EV assay showed higher PS externalization in m/lEVs derived from MDA-MB-468 cells but not from MDA-MB-231 cells, while higher binding of GlaS was also observed in m/lEVs from fibroblasts. Second, using single EV flow cytometry, PS externalization was also analyzed on individual sEVs and m/lEVs. Significantly higher PS externalization was detected in m/lEVs (annexin A1+) derived from cancer cells compared to m/lEVs (annexin A1+) from non-cancerous cells. These results emphasize the significance of PS-exposing m/lEVs (annexin A1+) as an undervalued EV subtype for early cancer detection and provide a better understanding of PS externalization in disease-associated EV subtypes.

Evaluation of the Efficacy and Safety of Antiplatelet Therapeutics in Rabbits.

Hemostasis is a multifactorial process that involves vasoconstriction of blood vessels, activation of the coagulation cascade, and platelet aggregation. Inappropriate activation of hemostatic processes can result in thrombosis and tissue ischemia. In patients at risk for thrombotic events, antiplatelet therapeutic agents inhibit platelet activation, thereby reducing the incidence of pathologic clot formation. Platelets are activated by several endogenous chemical mediators, including adenosine diphosphate, thrombin, and thromboxane. These activation pathways serve as attractive drug targets. The protocols described in this article are designed to evaluate the preclinical efficacy and safety of novel antiplatelet therapeutics in rabbits. Here, we provide two protocols for blood collection, two for determining platelet activation, and one for assessing bleeding safety. Together, these protocols can be used to characterize the efficacy and safety of antiplatelet agents for hemostasis. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Blood collection via the central ear artery Alternative Protocol 1: Blood collection via the jugular vein Basic Protocol 2: Platelet aggregation assessment via light transmission aggregometry Alternative Protocol 2: Platelet activation assessment via flow cytometry Basic Protocol 3: Determination of tongue bleeding time.

Small Molecule 20S Proteasome Enhancer Regulates MYC Protein Stability and Exhibits Antitumor Activity in Multiple Myeloma.

Despite the addition of several new agents to the armamentarium for the treatment of multiple myeloma (MM) in the last decade and improvements in outcomes, the refractory and relapsing disease continues to take a great toll, limiting overall survival. Therefore, additional novel approaches are needed to improve outcomes for MM patients. The oncogenic transcription factor MYC drives cell growth, differentiation and tumor development in many cancers. MYC protein levels are tightly regulated by the proteasome and an increase in MYC protein expression is found in more than 70% of all human cancers, including MM. In addition to the ubiquitin-dependent degradation of MYC by the 26S proteasome, MYC levels are also regulated in a ubiquitin-independent manner through the REGγ activation of the 20S proteasome. Here, we demonstrate that a small molecule activator of the 20S proteasome, TCH-165, decreases MYC protein levels, in a manner that parallels REGγ protein-mediated MYC degradation. TCH-165 enhances MYC degradation and reduces cancer cell growth in vitro and in vivo models of multiple myeloma by enhancing apoptotic signaling, as assessed by targeted gene expression analysis of cancer pathways. Furthermore, 20S proteasome enhancement is well tolerated in mice and dogs. These data support the therapeutic potential of small molecule-driven 20S proteasome activation for the treatments of MYC-driven cancers, especially MM.

In Vitro and In Vivo Analysis of Extracellular Vesicle-Mediated Metastasis Using a Bright, Red-Shifted Bioluminescent Reporter Protein.

Cancer cells produce heterogeneous extracellular vesicles (EVs) as mediators of intercellular communication. This study focuses on a novel method to image EV subtypes and their biodistribution in vivo. A red-shifted bioluminescence resonance energy transfer (BRET) EV reporter is developed, called PalmReNL, which allows for highly sensitive EV tracking in vitro and in vivo. PalmReNL enables the authors to study the common surface molecules across EV subtypes that determine EV organotropism and their functional differences in cancer progression. Regardless of injection routes, whether retro-orbital or intraperitoneal, PalmReNL positive EVs, isolated from murine mammary carcinoma cells, localized to the lungs. The early appearance of metastatic foci in the lungs of mammary tumor-bearing mice following multiple intraperitoneal injections of the medium and large EV (m/lEV)-enriched fraction derived from mammary carcinoma cells is demonstrated. In addition, the results presented here show that tumor cell-derived m/lEVs act on distant tissues through upregulating LC3 expression within the lung.

Microvesicle-Mediated Delivery of Minicircle DNA Results in Effective Gene-Directed Enzyme Prodrug Cancer Therapy.

An emerging approach for cancer treatment employs the use of extracellular vesicles, specifically exosomes and microvesicles, as delivery vehicles. We previously demonstrated that microvesicles can functionally deliver plasmid DNA to cells and showed that plasmid size and sequence, in part, determine the delivery efficiency. In this study, delivery vehicles comprised of microvesicles loaded with engineered minicircle (MC) DNA that encodes prodrug converting enzymes developed as a cancer therapy in mammary carcinoma models. We demonstrated that MCs can be loaded into shed microvesicles with greater efficiency than their parental plasmid counterparts and that microvesicle-mediated MC delivery led to significantly higher and more prolonged transgene expression in recipient cells than microvesicles loaded with the parental plasmid. Microvesicles loaded with MCs encoding a thymidine kinase (TK)/nitroreductase (NTR) fusion protein produced prolonged TK-NTR expression in mammary carcinoma cells. In vivo delivery of TK-NTR and administration of prodrugs led to the effective killing of both targeted cells and surrounding tumor cells via TK-NTR-mediated conversion of codelivered prodrugs into active cytotoxic agents. In vivo evaluation of the bystander effect in mouse models demonstrated that for effective therapy, at least 1% of tumor cells need to be delivered with TK-NTR-encoding MCs. These results suggest that MC delivery via microvesicles can mediate gene transfer to an extent that enables effective prodrug conversion and tumor cell death such that it comprises a promising approach to cancer therapy.

DT-678 inhibits platelet activation with lower tendency for bleeding compared to existing P2Y12 antagonists.

The novel clopidogrel conjugate, DT-678, is an effective inhibitor of platelets and thrombosis in preclinical studies. However, a comparison of the bleeding risk with DT-678 and currently approved P2Y12 antagonists has yet to be determined. The objective of this study was to evaluate the bleeding tendency of animals treated with clopidogrel, ticagrelor, and DT-678. Ninety-one New Zealand white rabbits were randomized to one of 13 treatment groups (n = 7). Platelet activation was assessed by flow cytometry and light transmission aggregometry before and after the administration of various doses of DT-678, clopidogrel, and ticagrelor. Tongue template bleeding times were also measured before and after drug treatment. Treatment with P2Y12 receptor antagonists caused a dose-dependent reduction in markers of platelet activation (P-selectin and integrin αIIbß3) and aggregation in response to adenosine diphosphate stimulation. At the same doses required for platelet inhibition, clopidogrel and ticagrelor significantly prolonged bleeding times, while DT-678 did not. DT-678 and the FDA-approved P2Y12 antagonists clopidogrel and ticagrelor are effective inhibitors of platelet activation and aggregation. However, unlike clopidogrel and ticagrelor, DT-678 did not prolong bleeding times at equally effective antiplatelet doses. The results suggest a more favorable benefit/risk ratio for DT-678 and potential utility as part of a dual antiplatelet therapy regimen.

Identifying chemopreventive agents for obesity-associated cancers using an efficient, 3D high-throughput transformation assay.

Obesity is associated with ~40% of cancer diagnoses but there are currently no effective preventive strategies, illustrating a need for chemoprevention. We previously demonstrated that fibroblast growth factor 2 (FGF2) from adipose tissue stimulates malignant transformation, as measured by growth in soft agar, the gold-standard in vitro transformation assay. Because the soft agar assay is unsuitable for high throughput screens (HTS), we developed a novel method using 3D growth in ultra-low attachment conditions as an alternative to growth in agar to discover compounds that inhibit transformation. Treating non-tumorigenic, skin epithelial JB6 P+ cells with FGF2 stimulates growth in ultra-low attachment conditions analogous to growth in the soft agar. This transformation HTS identified picropodophyllin, an insulin growth factor 1 receptor (IGF1R) inhibitor, and fluvastatin, an HMG-CoA reductase inhibitor, as potential chemopreventive agents. These compounds were validated for efficacy using two non-tumorigenic cell lines in soft agar. Another IGF1R inhibitor and other statins were also tested and several were able to inhibit growth in soft agar. This novel 3D HTS platform is fast, robust and has the potential to identify agents for obesity-associated cancer prevention.

Inhibition of cyclooxygenase-2 impairs the expression of essential plasma cell transcription factors and human B-lymphocyte differentiation.

Cyclooxygenase (Cox) inhibitors are among the most widely used and commonly prescribed medications. Relatively little is understood about their influence on human immune responses. Herein, we discovered a novel and important mechanism whereby non-steroidal anti-inflammatory drugs (NSAIDs) blunt human B-cell antibody production. We demonstrate that the Cox-2 selective small molecule inhibitors SC-58125 and NS-398 attenuate the production of human antibody isotypes including immunoglobulin M (IgM), IgG1, IgG2, IgG3 and IgG4. In addition, inhibition of Cox-2 significantly reduced the generation of CD38+ IgM+ and CD38+ IgG+ antibody-secreting cells. Interestingly, we discovered that inhibition of Cox-2 activity in normal human B cells severely reduced the messenger RNA and protein levels of the essential plasma cell transcription factor, Blimp-1. These observations were mirrored in Cox-2-deficient mice, which had reduced CD138+ plasma cells and a near loss of Blimp-1 expression. These new findings demonstrate a critical role for Cox-2 in the terminal differentiation of human B lymphocytes to antibody-secreting plasma cells. The use of NSAIDs may adversely influence the efficacy of vaccines, especially in the immunocompromised, elderly and when vaccines are weakly immunogenic.