Matsuda, Akiko

AbstractExtracellular vesicles are endogenous biological nanoparticles that have potential for use as therapeutic nanoparticles or as delivery vehicles for therapeutic agents. Milk nanovesicles (MNV) are extracellular vesicles isolated from bovine milk that have been explored for use as delivery vehicles for RNA therapeutics such as small interfering RNA (siRNA). We performed in vivo toxicological studies of MNV or therapeutic MNV (tMNV) loaded with siRNA as a prelude to their clinical use. Development toxicity was assessed in zebrafish embryos. Acute toxicity was assessed in both mice and zebrafish whereas safety, biochemical, histological and immune effects after multiple dosing were assessed in mice. Zebrafish embryo hatching was accelerated with MNV and tMNV. While acute toxicity or effects on mortality were not observed in zebrafish, developmental effects were observed at high concentrations of MNV. There was a lack of discernable toxicity, mortality and systemic inflammatory or immunological responses in mice following administration of either MNVs or tMNVs. The tolerability and lack of discernable developmental or systemic in vivo toxicity support their use as biological nano‐therapeutics. Adoption of a standardized protocol for systematic analysis of in vivo safety and toxicity will facilitate preclinical assessment of EV based formulations for therapeutic use.

Hepatocellular carcinoma (HCC) is a leading cause of cancer‐related death worldwide. Although HCC can respond to immune checkpoint inhibitors, such as monoclonal antibodies against programmed death 1 (PD‐1), many patients fail to respond or develop secondary resistance. Activation of Wnt/β‐catenin signaling can contribute to immune evasion. Mutations in β‐catenin are among the most frequent mutations associated with HCC. Thus, our aim was to directly target β‐catenin to enhance the therapeutic response to immune checkpoint inhibition. A synthetic transgenic mouse model of experimental HCC induced by tyrosine‐protein kinase Met/β‐catenin expression and extracellular vesicles (EVs) as a therapeutic delivery agent was used to evaluate the efficacy of directly targeting β‐catenin on the response to anti‐PD‐1. These studies showed that (1) oncogenic β‐catenin could be therapeutically targeted using a biological nanoparticle‐based delivery approach, (2) targeting β‐catenin using small interfering RNA (siRNA) delivered within EVs can reduce tumor growth, and (3) the therapeutic response to anti‐PD‐1 can be enhanced by concomitantly targeting β‐catenin using therapeutic EVs. These preclinical studies establish the efficacy of the use of biological nanoparticles as an endogenous delivery vehicle for therapeutic RNA delivery and support the use of therapeutic strategies targeting tumor‐intrinsic β‐catenin as an adjunct to anti‐PD‐1‐based therapy.Conclusion:Combination therapy with anti‐PD‐1 and β‐catenin siRNA delivered using biological nanoparticles provides an effective strategy for the treatment of HCC. This strategy could be further exploited into targeted approaches for immune potentiation by countering oncogene‐mediated resistance to immunotherapies.

Aberrant activation of β-catenin signaling is frequently observed in hepatocellular cancer. Although Wnt/β-catenin signaling can be targeted by vitamin D, therapeutic use of vitamin D for this purpose is not currently established. We evaluated the therapeutic use of vitamin D or its analogs using a synthetic transgenic mouse of hepatocarcinogenesis induced by mutant β-catenin, and MET overexpression in which 75% of mice develop well-differentiated HCC within 8 weeks in the absence of fibrosis. Vitamin D receptor expression was similar in both tumoral and nontumoral tissue. There was no significant difference in overall survival, or in tumor progression assessed by imaging, biochemical, or tumor cell burden assessments in mice receiving a vitamin D-supplemented diet containing 12.0 IU VD/g (HVD) compared with a standard diet (SD) containing 2.3 IU VD/g. Furthermore, systemic treatment with calcitriol [vitamin D analog 1α,25(OH) 2 D 3 ] or EB1089 (synthetic vitamin D analog) by intraperitoneal injection for 4 weeks prolonged median survival but did not increase overall survival compared with controls. Although tumor formation was delayed in males compared with that in females, there was no difference in overall survival between males and females. In conclusion, although 1α,25(OH) 2 D 3 is reported to inhibit β-catenin signaling, as well as proliferation, migration, and differentiation in cancer cells, neither dietary supplementation with vitamin D nor treatment with vitamin D analogs altered the formation or growth of HCC associated with β-catenin activation. These results conclusively demonstrate the lack of utility of targeting vitamin D for therapy of HCC in this setting.

Abstract Background: The paucity of effective therapeutic agents for hepatocellular cancer (HCC) underscores the critical need for more effective therapeutic strategies. Recent studies indicate lipid biosynthesis and desaturation is required for HCC survival. Targeting these may prove beneficial because such changes contribute to therapeutic resistance. Stearoyl CoA desaturase (SCD1), a key mediator of fatty acid (FA) biosynthesis and rate-limiting in conversion of saturated fatty acids (SFAs) to mono-unsaturated fatty acids (MUFAs), is upregulated in HCC and many other cancers. As such, we therapeutically targeted a novel lipogenic tumor survival mechanism mediated by SCD1 as a means to combat the chemoresistance associated with HCC. In so doing, we evaluated a novel lead SCD1 inhibitor in HCC. Methods: Paraffin embedded patient HCC tissues were examined for SCD1 expression. Using combined computational and synthetic chemistry approaches, we synthesized four novel specific SCD1 inhibitors with SSI-4 being the lead SCD1 inhibitor. HCC cell lines were examined using proliferation assays for response to SSI-4. IC50 concentrations for blocking SCD1 enzyme activity was determined. Blood half-life and bioavailability of single dose SSI-4 was determined. Mechanisms of action of SCD1 were examined that included Endoplasmic reticulum (ER) stress. In vivo, antitumor activity was determined using HCC patient derived xenograft (PDX) mouse models. Results: We identified elevated SCD1 mRNA and protein in HCCs tissues. SSI-4 dose-dependently inhibits cell proliferation in HCC cell lines with specificity demonstrated by oleic acid (MUFA) co-culture. Single dose oral gavage SSI-4 demonstrated a half-life of ~4 hours and excellent oral bioavailability. SSI-4 was well tolerated with long-term daily dosing. SSI-4 treatment of HCC cells and tumors led to endoplasmic reticulum (ER) stress followed by apoptotic cell death. Single agent SSI-4 demonstrated antitumor activity in HCC PDX mouse models with suppression of ER stress regulated proteins. Conclusions: Targeting a novel lipid metabolic pathway in HCC may provide effective therapy for aggressive HCC. Citation Format: John Alton Copland, Laura A. Marlow, Ilah Bok, James L. Miller, Matsuda Akiko, Yan W. Asmann, Vivekananda Sarangi, Steven R. Alberts, Kabir Mody, Lewis R. Roberts, Mark J. Truty, Tushar C. Patel. Targeting stearoyl CoA desaturase 1 (SCD1) in hepatobilliary carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 192. doi:10.1158/1538-7445.AM2017-192

Hepatic ischemia/reperfusion injury (IRI) and associated inflammation contributes to liver dysfunction and complications after liver surgery and transplantation. Mesenchymal stem cells (MSCs) have been reported to reduce hepatic IRI because of their reparative immunomodulatory effects in injured tissues. Recent studies have highlighted beneficial effects of extracellular vesicles from mesenchymal stem cells (MSC‐EV) on tissue injury. The effects of systemically administered mouse bone marrow–derived MSC‐EV were evaluated in an experimental murine model of hepatic IRI induced by cross‐clamping the hepatic artery and portal vein for 90 minutes followed by reperfusion for periods of up to 6 hours. Compared with controls, intravenous administration of MSC‐EV 30 minutes prior to IRI dramatically reduced the extent of tissue necrosis, decreased caspase 3–positive and apoptotic cells, and reduced serum aminotransferase levels. MSC‐EV increased hepatic messenger RNA (mRNA) expression of NACHT, LRR, and PYD domains‐containing protein 12, and the chemokine (C‐X‐C motif) ligand 1, and reduced mRNA expression of several inflammatory cytokines such as interleukin 6 during IRI. MSC‐EV increased cell viability and suppressed both oxidative injury and nuclear factor kappa B activity in murine hepatocytes in vitro. In conclusion, the administration of extracellular vesicles derived from bone marrow–derived MSCs may ameliorate hepatic IRI by reducing hepatic injury through modulation of the inflammatory response.Liver Transplantation 23 791–803 2017 AASLD.

Abstract Stem cell-based therapies have potential for treatment of liver injury by contributing to regenerative responses, through functional tissue replacement or paracrine effects. The release of extracellular vesicles (EV) from cells has been implicated in intercellular communication, and may contribute to beneficial paracrine effects of stem cell-based therapies. Therapeutic effects of bone-marrow derived mesenchymal stem cells (MSC) and vesicles released by these cells were examined in a lethal murine model of hepatic failure induced by d-galactosamine/tumor necrosis factor-α (TNF-α). Systemically administered EV derived from MSC accumulated within the injured liver following systemic administration, reduced hepatic injury, and modulated cytokine expression. Moreover, survival was dramatically increased by EV derived from either murine or human MSC. Similar results were observed with the use of cryopreserved mMSC-EV after 3 months. Y-RNA-1 was identified as a highly enriched noncoding RNA within hMSC-EV compared to cells of origin. Moreover, siRNA mediated knockdown of Y-RNA-1 reduced the protective effects of MSC-EV on TNF-α/ActD-mediated hepatocyte apoptosis in vitro. These data support a critical role for MSC-derived EV in mediating reparative responses following hepatic injury, and provide compelling evidence to support the therapeutic use of MSC-derived EV in fulminant hepatic failure.

Extracellular vesicles (EVs) are membrane-bound vesicles that are released by cells into their extracellular environment, have selective enrichment of specific proteins and RNA, and can mediate intercellular communication. In this review we highlight recent observations of the role of EVs in liver injury, viral hepatitis, alcoholic or nonalcoholic liver disease, biliary tract disease, and liver cancers. Potential applications as markers of diseases or for therapeutic applications are outlined to emphasize the new opportunities that are arising from the study of EVs.

AbstractExtracellular vesicles (EVs) are membrane‐bound vesicles released from cells into the extracellular environment. There is emerging interest in the use of EVs as potential therapeutic interventions. We sought to evaluate the safety of EVs that may be therapeutically used by performing in vitro toxicological assessments. EVs were obtained from mesenchymal stem cells (MSC‐EV) or from bovine milk (BM‐EV) by differential ultracentrifugation, and quantitated using nanoparticle tracking analysis. Genotoxic effects, hematological effects, immunological effects and endotoxin production were evaluated at two dose levels. Neither MSC‐EVs nor BM‐EVs elicited detectable genotoxic effects using either the alkaline comet assay or micronucleus assay. Hemolysis was observed with BM‐EVs but not with MSC‐EVs. MSC‐EVs did not have any significant effect on either spontaneous or collagen‐induced platelet aggregation. In contrast, BM‐EVs were noted to increase collagen‐induced platelet aggregation, even though no spontaneous increase in platelet aggregation was noted. Both types of EVs induced leukocyte proliferation, which was greater with BM‐EV. Neither MSC‐EVs nor BM‐EVs induced HL‐60 phagocytosis, although BM‐EVs decreased zymosan‐induced phagocytosis. Furthermore, neither MSC‐EVs nor BM‐EVs induced nitric oxide production. Unlike MSC‐EVs, BM‐EVs tested positive for endotoxin and induced complement activation. There are significant differences in toxicological profiles between MSC‐EVs and BM‐EVs that may reflect variations in techniques for EV isolation, EV content or cross‐species differences. The safety of MSC‐EV supports their use for disease therapeutics, whereas detailed safety and toxicological assessment will be necessary before the use of BM‐EVs. Copyright © 2016 John Wiley & Sons, Ltd.