Nonobese Diabetic/severe Combined Immunodeficient Research Articles

Feasibility analysis of treating severe intrauterine adhesions by transplanting menstrual blood-derived stem cells.

Intrauterine adhesions (IUA) are associated with the loss of stem cells in the endometrium. Menstrual blood‑derived stem cells (MenSCs) can be isolated from the menstrual blood and differentiated into endometrial cells. To check the transplantation feasibility of MenSCs for the treatment of severe IUA, MenSCs were isolated from menstrual blood, cultured in Dulbecco's modified Eagle's medium (DMEM), identified by immunocytochemistry and flow cytometry, differentiated into endometrial cells invitro, and finally transplanted into the axillary subcutaneous tissue of non‑obese diabetic/severe combined immunodeficiency (NOD‑SCID) mice to create endometrial tissue. Additionally, the cloning efficiency and POU domain class5 transcription factor1 (OCT‑4) positivity of MenSCs from patients with severe IUA were compared with those from healthy women. Immunocytochemistry and flow cytometry results showed that 95.1±0.8% cells were OCT‑4‑positive, 0.9±0.4% were cluster of differentiation (CD)45‑positive, 1.8±0.9% were STRO‑1‑positive and 1.0±0.4% were human leukocyte antigen‑antigen D related‑positive. Following differentiation invitro, the results of immunocytochemistry, reverse transcription‑polymerase chain reaction and western blot analysis showed that the expression of cytokeratin(CK) and vimentin (VIM) was increased in MenSCs compared with that in control subjects. Subsequent to transplantation in mice administered with sequential 17β‑estradiol and progesterone, CK, VIM, estrogen receptor and progesterone receptor were expressed in the transplantation regions, suggesting that MenSCs could differentiate into endometrial tissues invivo. The cloning efficiency and OCT‑4 positivity of MenSCs from patients with severe IUA was significantly decreased. In conclusion, to the best of our knowledge, this is the first study in which MenSCs could differentiate into endometrial cells invitro and create endometrial tissue in NOD‑SCID mice invivo, with impaired cloning efficiency and OCT‑4 expression of MenSCs from patients with IUA. This study will provide a theoretical basis for the treatment of IUA with MenSCs.

PSMA-targeted polyinosine/polycytosine vector induces prostate tumor regression and invokes an antitumor immune response in mice

There is an urgent need for an effective treatment for metastatic prostate cancer (PC). Prostate tumors invariably overexpress prostate surface membrane antigen (PSMA). We designed a nonviral vector, PEI-PEG-DUPA (PPD), comprising polyethylenimine-polyethyleneglycol (PEI-PEG) tethered to the PSMA ligand, 2-[3-(1, 3-dicarboxy propyl)ureido] pentanedioic acid (DUPA), to treat PC. The purpose of PEI is to bind polyinosinic/polycytosinic acid (polyIC) and allow endosomal release, while DUPA targets PC cells. PolyIC activates multiple pathways that lead to tumor cell death and to the activation of bystander effects that harness the immune system against the tumor, attacking nontargeted neighboring tumor cells and reducing the probability of acquired resistance and disease recurrence. Targeting polyIC directly to tumor cells avoids the toxicity associated with systemic delivery. PPD selectively delivered polyIC into PSMA-overexpressing PC cells, inducing apoptosis, cytokine secretion, and the recruitment of human peripheral blood mononuclear cells (PBMCs). PSMA-overexpressing tumors in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice with partially reconstituted immune systems were significantly shrunken following PPD/polyIC treatment, in all cases. Half of the tumors showed complete regression. PPD/polyIC invokes antitumor immunity, but unlike many immunotherapies does not need to be personalized for each patient. The potent antitumor effects of PPD/polyIC should spur its development for clinical use.

A comparative global phosphoproteomics analysis of obinutuzumab (GA101) versus rituximab (RTX) against RTX sensitive and resistant Burkitt lymphoma (BL) demonstrates differential phosphorylation of signaling pathway proteins after treatment.

We recently demonstrated that obinutuzumab (GA101), a novel glycoengineered type II CD20 Ab compared to rituximab (RTX) mediates significantly enhanced antibody-dependent cell cytotoxicity (ADCC) in vitro and increased overall survival in a Burkitt lymphoma (BL) xenograft non-obese diabetic severe combined immunodeficiency gamma (NSG) model. In this study we compared the phosphoproteomic changes by pathway analysis following obinutuzumab vs RTX against RTX-sensitive (Raji) and -resistant BL (Raji4RH). Phosphoproteomic analyses were performed by mass-spectrometry (MS)-based label-free quantitative phosphoproteomic profiling. We demonstrated that 418 proteins in Raji and 377 proteins in Raji 4RH, were differentially phosphorylated (>1.5-fold) after obinutuzumab vs. RTX. Proteins that were significantly differentially phosphorylated included the B cell antigen receptor (BCR) (PLCG2, BTK and GSK3B), Fc gamma phagocytosis (FCRG2B, MAPK1, PLCG2 and RAF1), and natural killer cell-mediated cytotoxicity (MAPK1, RAF1, PLCG2 and MAPK3) signaling pathways. Differential phosphorylation of BCR or cytotoxicity pathway proteins revealed significant up-regulation of BTK, PLCY2 and ERK1/RAF1 after obinutuzumab compared to RTX. Silencing of PLCG2 in the BCR and MAPK1 in the cytotoxicity pathway significantly increased BL proliferation and decreased BL cytotoxicity after obinutuzumab compared to RTX. These results in combination with our previous results demonstrating a significant improvement in in vitro BL cytotoxicity and in vivo BL survival by obinutuzumab compared to RTX may in part be due to differential effects on selected BL protein signaling pathways.

Carbon Monoxide Inhibits Islet Apoptosis via Induction of Autophagy.

Carbon monoxide (CO) functions as a therapeutic molecule in various disease models because of its anti-inflammatory and antiapoptotic properties. We investigated the capacity of CO to reduce hypoxia-induced islet cell death and dysfunction in human and mouse models. Culturing islets in CO-saturated medium protected them from hypoxia-induced apoptosis and preserved β cell function by suppressing expression of proapoptotic (Bim, PARP, Cas-3), proinflammatory (TNF-α), and endoplasmic reticulum (ER) stress (glucose-regulated protein 94, grp94, CHOP) proteins. The prosurvival effects of CO on islets were attenuated when autophagy was blocked by specific inhibitors or when either ATG7 or ATG16L1, two essential factors for autophagy, was downregulated by siRNA. In vivo, CO exposure reduced both inflammation and cell death in grafts immediately after transplantation, and enhanced long-term graft survival of CO-treated human and mouse islet grafts in streptozotocin-induced diabetic non-obese diabetic severe combined immunodeficiency (NOD-SCID) or C57BL/6 recipients. These findings underline that pretreatment with CO protects islets from hypoxia and stress-induced cell death via upregulation of ATG16L1-mediated autophagy. Our results suggested that CO exposure may provide an effective means to enhance survival of grafts in clinical islet cell transplantation, and may be beneficial in other diseases in which inflammation and cell death pose impediments to achieving optimal therapeutic effects. Antioxid. Redox Signal. 28, 1309-1322.

Effects of Klf4 and c-Myc Knockdown on Pluripotency Maintenance in Porcine Induced Pluripotent Stem Cell.

Objective The importance of Oct4 and Sox2 in maintaining pluripotency and self-renewal is well-understood, but the functions of Klf4 and c-Myc has not been fully investigated. In the present study, we attempted to determine the roles of Klf4 and c-Myc on pluripotency maintenance of porcine induced pluripotent stem (piPS) cells. Materials and Methods In this experimental study, we performed short hairpin RNA (shRNA) to knock down the Klf4 and c-Myc functions of piPS cells and examined pluripotency markers and teratoma formation to evaluate piPS cell pluripotency. The shRNA-Klf4 and shRNA-c-Myc vectors containing a reporter gene, TagFP635, were transfected into piPS cells by lentivirus infection. The piPS cells fully expressing infrared fluorescence were selected to confirm gene knockdown of Klf4 and c-Myc reverse transcription-polymerase chain reaction (RT-PCR). Next, for pluripotency evaluation, expression of pluripotency markers was detected by immunocytochemical staining, and capability of teratoma formation was investigated by piPS cell transplantation into nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice. Results Our findings indicated that Klf4 and c-Myc functions of piPS cells were knocked down by shRNA transfection, and knockdown of Klf4 and c-Myc functions impaired expression of pluripotency markers such as Oct4, AP, SSEA-3, SSEA-4, TRA-1-6, and TRA-1-81. Furthermore, piPS cells without Klf4 and c-Myc expression failed to form teratomas. ConclusionThe pluripotency of piPS cells are crucially dependent upon Klf4 and c-Myc expression. These findings, suggesting potential mechanisms of Klf4 and c-Myc contribution to piPS cell formation, have important implications for application, regulation, and tumorigenesis of piPS cells.

Inhibitory effects of HNF4\u03b1 on migration/maltransformation of hepatic progenitors: HNF4\u03b1-overexpressing hepatic progenitors for liver repopulation

BackgroundAlthough they are expandable in vitro, hepatic progenitors are immature cells and share many immunomarkers with hepatocellular carcinoma, raising potential concerns regarding maltransformation after transplantation. This study investigated the effects of hepatic nuclear factor (HNF) 4α on the proliferation, migration, and maltransformation of hepatic progenitors and determined the feasibility of using these manipulated cells for transplantation.MethodsThe effects of HNF4α on rat hepatic progenitors (i.e. hepatic oval cells) were analyzed by HNF4α overexpression and HNF4α shRNA. Nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice injured by carbon chloride (CCl4) were then transplanted with control, HNF4α-overexpressing or HNF4α-suppressing hepatic oval cells. Finally, the engraftment of these cells in the recipient liver was analyzed.ResultsRat hepatic progenitors (i.e. hepatic oval cells) expressed HNF4α, although less than that in hepatocytes. When HNF4α was overexpressed in these cells, the proliferation and migration of hepatic oval cells were reduced; but when HNF4α was suppressed by shRNA, the proliferation and migration, and even anchorage-independent growth, of these cells were accelerated. RNA microarray and gene functional analysis revealed that suppressing HNF4α not only impaired many biosynthesis and metabolism pathways of hepatocytes but also increased pathways for cancer. When transplanted into CCl4-injured NOD/SCID mice, few HNF4α-suppressing hepatic oval cells localized into the liver, while control cells and HNF4α-overexpressing cells engrafted into the liver and differentiated into albumin-positive hepatocytes. Interestingly, the hepatocytes derived from HNF4α-overexpressing cells were less migrative and expressed less c-Myc than the cells derived from control cells.ConclusionHNF4α constrains proliferation, migration, and maltransformation of hepatic progenitors, and HNF4α-overexpressing hepatic progenitors serve as an optimal candidate for cell transplantation.

Amotosalen/ultraviolet A pathogen inactivation technology reduces platelet activatability, induces apoptosis and accelerates clearance.

Amotosalen and ultraviolet A (UVA) photochemical-based pathogen reduction using the Intercept™ Blood System (IBS) is an effective and established technology for platelet and plasma components, which is adopted in more than 40 countries worldwide. Several reports point towards a reduced platelet function after Amotosalen/UVA exposure. The study herein was undertaken to identify the mechanisms responsible for the early impairment of platelet function by the IBS. Twenty-five platelet apheresis units were collected from healthy volunteers following standard procedures and split into 2 components, 1 untreated and the other treated with Amotosalen/UVA. Platelet impedance aggregation in response to collagen and thrombin was reduced by 80% and 60%, respectively, in IBS-treated units at day 1 of storage. Glycoprotein Ib (GpIb) levels were significantly lower in IBS samples and soluble glycocalicin correspondingly augmented; furthermore, GpIbα was significantly more desialylated as shown by Erythrina Cristagalli Lectin (ECL) binding. The pro-apoptotic Bak protein was significantly increased, as well as the MAPK p38 phosphorylation and caspase-3 cleavage. Stored IBS-treated platelets injected into immune-deficient nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice showed a faster clearance. We conclude that the IBS induces platelet p38 activation, GpIb shedding and platelet apoptosis through a caspase-dependent mechanism, thus reducing platelet function and survival. These mechanisms are of relevance in transfusion medicine, where the IBS increases patient safety at the expense of platelet function and survival.

Short-Term Alcohol Abstinence Improves Antibacterial Defenses of Chronic Alcohol-Consuming Mice against Gut Bacteria–Associated Sepsis Caused by Enterococcus faecalis Oral Infection

The effects of short-term alcohol abstinence on host antibacterial resistance against Enterococcus faecalis oral infection was investigated in chronic alcohol-consuming mice [mice with 0.1 g/day of 20% ethanol consumption for 12 or 16 weeks (CAC-mice)]. These mice were highly susceptible to the infection; however, after 7 days of alcohol abstinence (aaCAC-mice), their antibacterial resistances were completely restored to the normal mouse level. Normal mice inoculated with CAC-mouse hepatic macrophages were shown to be susceptible to the infection, whereas the same macrophage preparation from aaCAC-mice did not impair the antibacterial resistance of normal mice. aaCAC-mouse liver macrophages protected nonobese diabetic-severe combined immunodeficiency IL-2Rγnull mice exposed to E. faecalis, whereas those from CAC-mice did not. Monocyte-derived (MD) M2b macrophages were predominantly isolated from CAC-mouse livers, but these cells were not significantly isolated from aaCAC-mouse livers. Hepatic MD macrophages from aaCAC-mice switched to M1 macrophages in response to bacterial antigen, whereas the same macrophage preparation from CAC-mice did not. M1 Kupffer cells, M2a Kupffer cells, and MD M2b macrophages were shown to be not bactericidal, whereas E. faecalis was killed effectively by M1 macrophages derived from aaCAC-mouse hepatic MD macrophages. These results indicate that MD M2b macrophages predominantly distributed in the liver are responsible for the impaired resistance of CAC-mice to E. faecalis oral infection, and aaCAC-mice without MD M2b macrophages in the livers are resistant to the infection.

Abstract 910: Phloridzin docosahexaenoate (PZ-DHA) inhibits breast cancer cell invasion and angiogenesis

Abstract In our previous studies, we showed the selective cytotoxicity, in vitro anti-proliferative and anti-metastatic activities of phloridzin docosahexaenoate (PZ-DHA). PZ-DHA combines phloridzin (PZ), a dihydrochalcone found in apple peels, and docosahexaenoic acid (DHA), an ω-3 fatty acid found in fish oil, through an acylation reaction. Significant suppression of tumor growth resulted from the intratumoral administration of PZ-DHA to non-obese diabetic severe combined immunodeficient (NOD-SCID) female mice xenografted with MDA-MB-231 breast cancer cells. Examination of haematoxylin and eosin-stained tumor sections confirmed that PZ-DHA caused tumor cell death. PZ-DHA also suppressed the invasion of MDA-MB-231 cells through membranes coated with extra cellular matrix proteins, fibronectin and gelatin, in a Boyden chamber assay. The effect of PZ-DHA on the mRNA transcription of the gelatinase, matrix metalloproteinase-2 (MMP-2,) was measured in RT-qPCR analysis, which showed that MMP-2 mRNA expression was inhibited at 12 h and 24 h following PZ-DHA treatments. Furthermore, suppression of MMP-2 protein expression was shown by western blot analysis of cell lysates of PZ-DHA-treated MDA-MB-231 cells. Intraperitoneal administration of PZ-DHA suppressed the growth of primary tumors and metastasis of 4T1 and green fluorescence protein (GFP)-tagged-MDA-MB-231 cells to lungs of BALB/c and NOD-SCID female mice, respectively. Anti-angiogenic activity of PZ-DHA was investigated using human umbilical vein endothelial cells (HUVECs). PZ-DHA suppressed the metabolic activity of HUVECs in MTS assays and the cell death was confirmed using flow cytometric analysis of 7-AAD and Annexin-V-FLUOS/propidium iodide-stained HUVECs. Anti-proliferative activity of PZ-DHA was shown using Oregon Green 488 stained-HUVECs by flow cytometry. PZ-DHA treatment arrested the HUVEC cell cycle at S-phase. PZ-DHA also suppressed the migration of HUVECs in a gap closure assay and inhibited HUVEC tubule formation on a matrigel matrix. For comparison purposes, PZ and DHA were included in all experiments, but in all cases were less effective than PZ-DHA. Taken together, these findings reveal the potential of PZ-DHA to inhibit triple-negative breast cancer progression. We are currently working on identifying potential phase I and II metabolites of PZ-DHA and understanding its pharmacokinetic parameters. Citation Format: Wasundara Fernando, Emma MacLean, Krysta Coyle, Paola Marcato, David W. Hoskin, H. P. Vasantha Rupasinghe. Phloridzin docosahexaenoate (PZ-DHA) inhibits breast cancer cell invasion and angiogenesis [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 910. doi:10.1158/1538-7445.AM2017-910

Abstract 3653: Combining ERY974, a novel T cell-redirecting bispecific antibody targeting glypican-3, with chemotherapy profoundly improved antitumor efficacy over its monotherapy in xenograft model

Abstract Background: ERY974 is a humanized IgG4 bispecific T cell-redirecting antibody (TRAB) currently in Phase 1 clinical trial (NCT02748837). ERY974 consists of a common light chain and two different heavy chains that respectively recognize glypican-3 (GPC3) and CD3. The Fc portion of ERY974 is modified to lose FcγR binding to prevent GPC3-independent Fc-mediated effector function. However, binding activity to FcRn, an important factor in the PK profile of IgG, is maintained. ERY974 simultaneously binds to GPC3 on the cancer cell surface and to CD3 on the T cell surface, and induces TRAB-dependent cellular cytotoxicity mediated by the potent effector function of T cells. ERY974 shows strong antitumor activity against gastric, lung, ovarian, head & neck, and esophageal cancer-derived xenograft tumors in a non-obese diabetic/severe combined immunodeficiency (NOD-SCID) mouse model injected with human T cells. Cancer immunotherapy, as represented by immune checkpoint inhibitors such as PD-1, PD-L1, and CTLA-4 antibodies, has recently been demonstrating remarkable clinical benefit in various tumor types. However, the number of patients who have survival benefit is limited, and combining cancer immunotherapy with other agents is required to improve the efficacy. Although ERY974 monotherapy is expected to show clinical activity based on the preclinical data, we examined whether further improvement of ERY974-induced efficacy is attained by combination with chemotherapy. Method & Results: We evaluated the combination effect of ERY974 with chemotherapy against xenograft tumors of MKN45 (gastric cancer) or NCI-H446 (lung cancer) either in a NOD-SCID mouse model injected with human T cells or in a humanized non-obese diabetic/shi-scid/IL-2Rγnull model in which differentiated human T cells are constitutively supplied. Although ERY974 monotherapy shows only minor antitumor effect against MKN45 and NCI-H446, combination therapy remarkably enhanced efficacy. In particular, ERY974 in combination with paclitaxel or cisplatin in NCI-H446 tumors caused a tumor disappearance without regrowth for a long period. Conclusion: These preclinical data suggest the possibility that the strategy of combining ERY974 with chemotherapy may succeed in increasing the clinical benefit. Now the combination effect is being further investigated to clarify the mechanism. Citation Format: Yuji Sano, Yumiko Azuma, Toshiaki Tsunenari, Yasuko Kinoshita, Yoko Kayukawa, Hironori Mutoh, Yoko Miyazaki, Takahiro Ishiguro, Shohei Kishishita, Yoshiki Kawabe, Mika Endo. Combining ERY974, a novel T cell-redirecting bispecific antibody targeting glypican-3, with chemotherapy profoundly improved antitumor efficacy over its monotherapy in xenograft model [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 3653. doi:10.1158/1538-7445.AM2017-3653

SOX17 Regulates Conversion of Human Fibroblasts Into Endothelial Cells and Erythroblasts by Dedifferentiation Into CD34+ Progenitor Cells.

The mechanisms underlying the dedifferentiation and lineage conversion of adult human fibroblasts into functional endothelial cells have not yet been fully defined. Furthermore, it is not known whether fibroblast dedifferentiation recapitulates the generation of multipotent progenitors during embryonic development, which give rise to endothelial and hematopoietic cell lineages. Here we established the role of the developmental transcription factor SOX17 in regulating the bilineage conversion of fibroblasts by the generation of intermediate progenitors. CD34+ progenitors were generated after the dedifferentiation of human adult dermal fibroblasts by overexpression of pluripotency transcription factors. Sorted CD34+ cells were transdifferentiated into induced endothelial cells and induced erythroblasts using lineage-specific growth factors. The therapeutic potential of the generated cells was assessed in an experimental model of myocardial infarction. Induced endothelial cells expressed specific endothelial cell surface markers and also exhibited the capacity for cell proliferation and neovascularization. Induced erythroblasts expressed erythroid surface markers and formed erythroid colonies. Endothelial lineage conversion was dependent on the upregulation of the developmental transcription factor SOX17, whereas suppression of SOX17 instead directed the cells toward an erythroid fate. Implantation of these human bipotential CD34+ progenitors into nonobese diabetic/severe combined immunodeficiency (NOD-SCID) mice resulted in the formation of microvessels derived from human fibroblasts perfused with mouse and human erythrocytes. Endothelial cells generated from human fibroblasts also showed upregulation of telomerase. Cell implantation markedly improved vascularity and cardiac function after myocardial infarction without any evidence of teratoma formation. Dedifferentiation of fibroblasts to intermediate CD34+ progenitors gives rise to endothelial cells and erythroblasts in a SOX17-dependent manner. These findings identify the intermediate CD34+ progenitor state as a critical bifurcation point, which can be tuned to generate functional blood vessels or erythrocytes and salvage ischemic tissue.

Use of p53-Silenced Endothelial Progenitor Cells to Treat Ischemia in Diabetic Peripheral Vascular Disease.

BackgroundPeripheral vascular disease is a major diabetes mellitus‐related complication. In this study, we noted that expressions of proapoptotic p53 gene and its downstream cascade gene such as p21 are upregulated in hyperglycemia. Therefore, we investigated whether p53‐ and p21‐silenced endothelial progenitor cells (EPCs) were able to survive in hyperglycemic milieu, and whether transplantation of either p53 knockout (KO) or p21KO or p53‐ and p21‐silenced EPCs could improve collateral vessel formation and blood flow in diabetic vaso‐occlusive peripheral vascular disease mouse models.Methods and ResultsWe transplanted p53 and p21KO mouse EPCs (mEPCs) into streptozotocin–induced diabetic (type 1 diabetes mellitus model) C57BL/6J and db/db (B6.BKS(D)‐Leprdb/J) (type 2 model) post–femoral artery occlusion. Similarly, Ad‐p53–silenced and Ad‐p21–silenced human EPCs (CD34+) cells were transplanted into streptozotocin‐induced diabetic NOD.CB17‐Prkdcscid/J mice. We measured blood flow at 3, 7, and 10 days and hindlimb muscles were obtained postsacrifice for mRNA estimation and CD31 staining. Enhanced blood flow was noted with delivery of p53 and p21KO mEPCs in streptozotocin‐induced diabetic C57BL/6J mice. Similar results were obtained when human Ad‐p53shEPCs(CD34+) and Ad‐p21shEPCs(CD34+) were transplanted into streptozotocin‐induced nonobese diabetic severe combined immunodeficiency mice. Gene expression analysis of p53 and p21KO EPCs transplanted hindlimb muscles showed increased expression of endothelial markers such as endothelial nitric oxide synthase, vascular endothelial growth factor A, and platelet endothelial cell adhesion molecule 1. Similarly, quantitative reverse transcriptase polymerase chain reaction of human Ad‐p53shEPCs (CD34+)– and Ad‐p21shEPCs (CD34+)–transplanted hindlimb muscles also showed increased expression of endothelial markers such as vascular endothelial growth factor A, noted primarily in the p53‐silenced EPCs group. However, such beneficial effect was not noted in the db/db type 2 diabetic mouse models.ConclusionsTransient silencing of p53 using adenoviral vector in EPCs may have a therapeutic role in diabetic peripheral vascular disease.

Cuprous oxide nanoparticle-inhibited melanoma progress by targeting melanoma stem cells.

Recent studies have shown that metal and metal oxide have a potential function in antitumor therapy. Our previous studies demonstrated that cuprous oxide nanoparticles (CONPs) not only selectively induce apoptosis of tumor cells in vitro but also inhibit the growth and metastasis of melanoma by targeting mitochondria with little hepatic and renal toxicities in mice. As a further study, our current research revealed that CONPs induced apoptosis of human melanoma stem cells (CD271+/high cells) in A375 and WM266-4 melanoma cell lines and could significantly suppress the expression of MITF, SOX10 and CD271 involved in the stemness maintenance and tumorigenesis of melanoma stem cells. CD271+/high cells could accumulate more CONPs than CD271−/low through clathrin-mediated endocytosis. In addition, lower dosage of CONPs exhibited good anti-melanoma effect by decreasing the cell viability, stemness and tumorigenesis of A375 and WM266-4 cells through reducing the expression of SOX10, MITF, CD271 and genes in MAPK pathway involved in tumor progression. Finally, CONPs obviously suppressed the growth of human melanoma in tumor-bearing nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice, accompanied with tumors structural necrosis and fibrosis remarkably and decreased expression of CD271, SOX10 and MITF. These results above proved the effectiveness of CONPs in inhibiting melanoma progress through multiple pathways, especially through targeting melanoma stem cells.

The distribution of the apparent diffusion coefficient as an indicator of the response to chemotherapeutics in ovarian tumour xenografts

Diffusion-weighted magnetic resonance imaging (DW-MRI) was used to evaluate the effects of single-agent and combination treatment regimens in a spheroid-based animal model of ovarian cancer. Ovarian tumour xenografts grown in non-obese diabetic/severe-combined-immunodeficiency (NOD/SCID) mice were treated with carboplatin or paclitaxel, or combination carboplatin/paclitaxel chemotherapy regimens. After 4 weeks of treatment, tumours were extracted and underwent DW-MRI, mechanical testing, immunohistochemical and gene expression analyses. The distribution of the apparent diffusion coefficient (ADC) exhibited an upward shift as a result of each treatment regimen. The 99-th percentile of the ADC distribution (“maximum ADC”) exhibited a strong correlation with the tumour size (r2 = 0.90) and with the inverse of the elastic modulus (r2 = 0.96). Single-agent paclitaxel (n = 5) and combination carboplatin/paclitaxel (n = 2) treatment regimens were more effective in inducing changes in regions of higher cell density than single-agent carboplatin (n = 3) or the no-treatment control (n = 5). The maximum ADC was a good indicator of treatment-induced cell death and changes in the extracellular matrix (ECM). Comparative analysis of the tumours’ ADC distribution, mechanical properties and ECM constituents provides insights into the molecular and cellular response of the ovarian tumour xenografts to chemotherapy. Increased sample sizes are recommended for future studies. We propose experimental approaches to evaluation of the timeline of the tumour’s response to treatment.

Translating Myocardial Remuscularization.

Myocardial remuscularization can be achieved by the implantation of cardiomyocyte-containing grafts. Critical steps toward clinical translation of the first generation cardiac remuscularization therapies include the choice of animal models with high predictive validity for therapeutic outcome in clinical trials. Clinically relevant remuscularization of the failing human heart remains unachieved to date. Proof-of-concept studies in small and large animal models provide compelling evidence as to the principle feasibility of myocardial remuscularization with human cardiomyocyte grafts.1,2 Clinical trials suggest myocardial protection and preservation by cell therapeutics.3,4 The mechanisms underlying the reported therapeutic effects remain under debate, but likely include the release of protective factors (growth factors, noncoding RNA) and modulation of the disease-related inflammatory response. Further studies are needed to define the paracrine milieu hypothesis and unleash its full therapeutic potential, aiming at delaying or preventing disease progression in patients with acute and subacute myocardial injury. The implantation of cardiomyocyte grafts follows a different strategy, namely remuscularization by integration of exogenously produced cardiomyocytes. Patients with chronically scarred myocardium presenting clinically with end-stage heart failure are the primary target. The suggested plug-and-play mechanism of direct cardiac remuscularization seems straight forward, but similar as for the paracrine milieu hypothesis there is a clear need for a better understanding of how, when, and where to integrate exogenous cardiomyocytes into the failing heart to achieve optimal results. Moreover, the path toward clinical application as off-the-shelf cardiomyocyte allograft therapeutics is less defined as for point-of-care autograft formulations and small chemical or biological compounds. A challenge to direct cardiac remuscularization was for many years the limited availability of bona fide cardiomyocytes. Cardiac biopsies can be used to harvest proliferative mesenchymal cells with progenitor cell properties, but without the capacity to spontaneously convert/transdifferentiate into terminally differentiated cardiomyocytes at a therapeutically relevant scale. Induced …

Teratoma Formation Assay for Assessing Pluripotency and Tumorigenicity of Pluripotent Stem Cells.

Pluripotent stem cells such as induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) form teratomas when transplanted into immunodeficient mice. As teratomas contain all three germ layers (endoderm, mesoderm, ectoderm), teratoma formation assay is widely used as an index of pluripotency (Evans and Kaufman, 1981; Hentze et al., 2009 ; Gropp et al., 2012 ). On the other hand, teratoma-forming tumorigenicity also represents a major risk factor impeding potential clinical applications of pluripotent stem cells ( Miura et al., 2009 ; Okano et al., 2013 ). Recently, we reported that iPSCs derived from naked mole-rat lack teratoma-forming tumorigenicity when engrafted into the testes of non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice due to an ES cell-expressed Ras (ERAS) and Alternative reading frame (ARF)-dependent tumor-suppression mechanism specific to this species ( Miyawaki et al., 2016 ). Here, we describe a method for transplanting pluripotent stem cells into the testes of NOD/SCID mice to generate teratomas for assessing the pluripotency and tumorigenicity.

Estrogen receptor β ligation inhibits Hodgkin lymphoma growth by inducing autophagy.

Although Hodgkin lymphoma (HL) is curable with current therapy, at least 20% of patients relapse or fail to make complete remission. In addition, patients who achieve long-term disease-free survival frequently undergo infertility, secondary malignancies, and cardiac failure, which are related to chemotherapeutic agents and radiation therapies. Hence, new therapeutic strategies able to counteract the HL disease in this important patient population are still a matter of study. Estrogens, in particular 17β-estradiol (E2), have been suggested to play a role in lymphoma cell homeostasis by estrogen receptors (ER) β activation. On these bases, we investigated whether the ligation of ERβ by a selective agonist, the 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN), could impact HL tumor growth. We found that DPN-mediated ERβ activation led to a reduction of in vitro cell proliferation and cell cycle progression by inducing autophagy. In nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice engrafted with HL cells, ERβ activation by DPN was able to reduce lymphoma growth up to 60% and this associated with the induction of tumor cell autophagy. Molecular characterization of ERβ-induced autophagy revealed an overexpression of damage-regulated autophagy modulator 2 (DRAM2) molecule, whose role in autophagy modulation is still debated. After ERβ activation, both DRAM2 and protein 1 light chain 3 (LC3), a key actor in the autophagosome formation, strictly interacted each other and localized at mitochondrial level.Altogether these results suggest that targeting ERβ with selective agonists might affect HL cell proliferation and tumor growth via a mechanism that brings into play DRAM2-dependent autophagic cascade.

Abstract B59: Black raspberries inhibit pancreatic carcinogenesis by suppressing Raf/MEK/ERK/STAT3 signaling pathways and promoting apoptosis

Abstract Background: Despite improved understanding of the pathophysiology of pancreatic ductal adenocarcinoma (PDAC) in the past two decades, PDAC has an extremely low 5-year survival rate (7%). However, diets rich in fruits and vegetables have been reported to reduce the risk of cancer development. We previously demonstrated that black raspberries (BRBs) have chemopreventive effects in human colorectal cancer patients and mouse models of colorectal cancer. Our current study aimed to investigate the potential effects of BRBs against PDAC in mice. Methods: Mice that carry a constitutively activated KrasLSL.G12D/+ oncogene and loss-of-function Trp53LSL.R172H/+ tumor-suppressor gene in the pancreas (KrasLSL.G12D/+-Trp53LSL.R172H/+-Pdx-1-Cre) spontaneously develop PDAC that recapitulates the human PDAC. Four-week-old KrasLSL.G12D/+-Trp53LSL.R172H/+-Pdx-1-Cre mice were given a control diet or a control diet supplemented with 5% BRBs. In addition, luciferase-transfected human Panc-1 (Panc-1-Luc) cells were orthotopically injected into the pancreas of nonobese, diabetic, severe combined immunodeficient (NOD.SCID) mice. Four weeks after the inoculation of Panc-1-Luc cells, the NOD.SCID mice were given a control diet or a control diet supplemented with 5% BRBs for 6 weeks. Gross tumor volume of orthotopic pancreatic tumors was measured. Results: The 4-week-old KrasLSL.G12D/+-Trp53LSL.R172H/+-Pdx-1-Cre mice developed pancreatic intraepithelial neoplasia (PanIN) lesions, which are the precancerous lesions of PDAC, and they were fed control or 5% BRB diet. Kaplan-Meier survival analysis showed that 5% BRBs significantly prolonged the survival of those PDAC mice with a median survival of 189 days versus 154 days for the mice fed the control diet. Molecular studies suggested that 5% BRBs suppress the Raf/MEK/ERK/STAT3 pathway, which is the downstream of Kras activation, and inhibit cancer cell proliferation in pancreatic tumor tissues. In addition, 5% BRBs significantly decreased the gross tumor volume of orthotopic tumors produced by injecting Panc-1-Luc cells into the pancreas. Orthotopic pancreatic tumors in BRB-treated NOD.SCID mice had a higher rate of apoptosis than tumors from mice fed the control diet. Conclusions: These results support the hypothesis that BRBs have the clinical potential to delay pancreatic cancer progression by suppressing cancer cell proliferation and/or promoting apoptosis. Citation Format: Pan Pan, Chad W. Skaer, Hsin-Tzu Wang, Susan Tsai, Kiyoko Oshima, Yi-Wen Huang, Li-Shu Wang.{Authors}. Black raspberries inhibit pancreatic carcinogenesis by suppressing Raf/MEK/ERK/STAT3 signaling pathways and promoting apoptosis. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B59.

In vivo Evaluation of Human Embryonic Stem Cells Isolated by 57-C11 Monoclonal Antibody.

BackgroundThe normal cells derived from human embryonic stem cells (hESCs) are regarded as substitutes for damaged or dysfunctional adult cells. However, tumorigenicity of hESCs remains a major challenge in clinical application of hESC-derived cell transplantation. Previously, we generated monoclonal antibody (MAb) 57-C11 specific to the surface molecule on undifferentiated hESCs. The aim of this study is to prove whether 57-C11-positive hESCs are pluripotent and tumorigenic in immunodeficient mice.MethodsUndifferentiated hESCs were mixed with retinoic acid (RA)-differentiated hESCs at different ratios prior to 57-C11-mediated separation. To isolate 57-C11-positive hESCs from the mixture, biotinylated 57-C11 and streptavidin-coated magnetic beads were added to the mixture. Unbound 57-C11-negative hESCs were first isolated after applying magnet to the cell mixture, and 57-C11-bound hESCs were then released from the magnetic beads. In order to measure the efficiency of separation, 57-C11-positive or -negative hESCs were counted after isolation. To evaluate the efficiency of teratoma formation in vivo, 57-C11-positive or negative cells were further injected into left and right, respectively, testes of nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice.ResultsApproximately 77~100% of undifferentiated hESCs were isolated after applying 57-C11-coated magnetic beads to the mixed cell populations. Importantly, teratomas were not observed in NOD/SCID mice after the injection of isolated 57-C11-negative hESCs, whereas teratomas were observed with 57-C11-positive hESCs.Conclusion57-C11-positive hESCs are pluripotent and tumorigenic. The combination of 57-C11 and magnetic beads will be useful to eliminate remaining undifferentiated hESCs for the safe cell transplantation.

SOX12: a novel potential target for acute myeloid leukaemia.

The role of SRY-related high-mobility-group box (SOX) 12 in leukaemia progression and haematopoiesis remains elusive. This study aimed to examine the expression and function of SOX12 in acute myeloid leukaemia (AML) using human myeloid leukaemia samples and the acute myeloid cell line THP1. Mononuclear cells were isolated from the bone marrow of AML patients and healthy donors. SOX12 expression in haematopoietic cells was evaluated by reverse transcription polymerase chain reaction (RT-PCR). SOX12 short hairpin RNAs (shRNAs) were transduced into THP1 cells, and gene knockdown was confirmed by quantitative RT-PCR and Western blot analysis. SOX12 was preferentially expressed in CD34+ cells in AML patients. The THP1 cells transduced with SOX12 shRNAs exhibited significantly reduced SOX12 expression and cell proliferation. SOX12 knockdown had no effect on apoptosis, but it induced cell cycle arrest at G1 phase and reduced the number of colonies. The transduced THP1 and primary AML cells were reconstituted in non-obese diabetic-severe combined immunodeficient (NOD/SCID) mice, and their numbers were significantly reduced 6-12weeks after transplantation. The mRNA and protein levels of β-catenin were significantly diminished following SOX12 knockdown, accompanied by a decrease in TCF/Wnt activity. SOX12 may be involved in leukaemia progression by regulating the expression of β-catenin and then interfering with TCF/Wnt pathway, which may be a target for AML.