Diabetic Severe Combined Immunodeficient Mice Research Articles

Bright ferritin for long-term MR imaging of human embryonic stem cells

BackgroundA non-invasive imaging technology that can monitor cell viability, retention, distribution, and interaction with host tissue after transplantation is needed for optimizing and translating stem cell-based therapies. Current cell imaging approaches are limited in sensitivity or specificity, or both, for in vivo cell tracking. The objective of this study was to apply a novel ferritin-based magnetic resonance imaging (MRI) platform to longitudinal tracking of human embryonic stem cells (hESCs) in vivo.MethodsHuman embryonic stem cells (hESCs) were genetically modified to stably overexpress ferritin using the CRISPR-Cas9 system. Cellular toxicity associated with ferritin overexpression and manganese (Mn) supplementation were assessed based on cell viability, proliferation, and metabolic activity. Ferritin-overexpressing hESCs were characterized based on stem cell pluripotency and cardiac-lineage differentiation capability. Cells were supplemented with Mn and imaged in vitro as cell pellets on a preclinical 3 T MR scanner. T1-weighted images and T1 relaxation times were analyzed to assess contrast. For in vivo study, three million cells were injected into the leg muscle of non-obese diabetic severe combined immunodeficiency (NOD SCID) mice. Mn was administrated subcutaneously. T1-weighted sequences and T1 mapping were used to image the animals for longitudinal in vivo cell tracking. Cell survival, proliferation, and teratoma formation were non-invasively monitored by MRI. Histological analysis was used to validate MRI results.ResultsFerritin-overexpressing hESCs labeled with 0.1 mM MnCl2 provided significant T1-induced bright contrast on in vitro MRI, with no adverse effect on cell viability, proliferation, pluripotency, and differentiation into cardiomyocytes. Transplanted hESCs displayed significant bright contrast on MRI 24 h after Mn administration, with contrast persisting for 5 days. Bright contrast was recalled at 4–6 weeks with early teratoma outgrowth.ConclusionsThe bright-ferritin platform provides the first demonstration of longitudinal cell tracking with signal recall, opening a window on the massive cell death that hESCs undergo in the weeks following transplantation before the surviving cell fraction proliferates to form teratomas.

Plasma cell–derived mtDAMPs activate the macrophage STING pathway, promoting myeloma progression

AbstractMitochondrial damage–associated molecular patterns (mtDAMPs) include proteins, lipids, metabolites, and DNA and have various context-specific immunoregulatory functions. Cell-free mitochondrial DNA (mtDNA) is recognized via pattern recognition receptors and is a potent activator of the innate immune system. Cell-free mtDNA is elevated in the circulation of trauma patients and patients with cancer; however, the functional consequences of elevated mtDNA are largely undefined. Multiple myeloma (MM) relies upon cellular interactions within the bone marrow (BM) microenvironment for survival and progression. Here, using in vivo models, we describe the role of MM cell–derived mtDAMPs in the protumoral BM microenvironment and the mechanism and functional consequence of mtDAMPs in myeloma disease progression. Initially, we identified elevated levels of mtDNA in the peripheral blood serum of patients with MM compared with those of healthy controls. Using the MM1S cells engrafted into nonobese diabetic severe combined immunodeficient gamma mice, we established that elevated mtDNA was derived from MM cells. We further show that BM macrophages sense and respond to mtDAMPs through the stimulator of interferon genes (STING) pathway, and inhibition of this pathway reduces MM tumor burden in the KaLwRij-5TGM1 mouse model. Moreover, we found that MM-derived mtDAMPs induced upregulation of chemokine signatures in BM macrophages, and inhibition of this signature resulted in egress of MM cells from the BM. Here, we demonstrate that malignant plasma cells release mtDNA, a form of mtDAMPs, into the myeloma BM microenvironment, which in turn activates macrophages via STING signaling. We establish the functional role of these mtDAMP-activated macrophages in promoting disease progression and retaining MM cells in the protumoral BM microenvironment.

MAP4K2 Inhibition Reinforces the Iberdomide Sensitivity in MM Cells By Inducing IKZF1 Degradation through a CRBN Independent Mechanism

MAP4K2 is predominantly and highly expressed in the germinal center of B cells, also called Germinal Center Kinase (GCK). Recently we have shown that MAP4K2 knockdown in K- or N-RAS mutated MM cells induces MM cell growth inhibition, associated with the downregulation of critical transcriptional factors including IKZF1/3, BCL-6, and c-MYC proteins. IKZF1 is a critical transcriptional factor regulating IRF4 and c-MYC transcription in MM, therefore we expect that IKZF1 degradation is one of the major mechanisms that contribute to the inhibition of MAP4K2 induced MM cell death. To address the role of IKZF1 degradation in MAP4K2 inhibition induced cytotoxicity in MM, we generated a mutant IKZF1 by substituting Glutamine Q146 to Histidine, which abrogates IKZF1 ubiquitination induced by CRBN. MM.1S cells were transduced with lentiviral constructs to overexpress flag-IKZF1WT or flag-IKZF1Q146H. Lenalidomide failed to induce a complete IKZF1-degradation in IKZF1Q146H MM.1S cells, indicating IMiDs induced IKZF1-degradation is mediated by CRBN binding and subsequent ubiquitination. In contrast, IKZF1 mutation did not rescue MM.1S from MAP4K2 inhibitor (TL4-12) induced IKZF1 downregulation. Proliferation assays further confirmed that IKZF1Q146H MM.1S cells were resistant to LEN induced cell growth inhibition, but not MAP4K2 inhibitor, demonstrating that MAP4K2 inhibition causes the degradation of IKZF1 and cell growth inhibition through a mechanism different from that of lenalidomide in myeloma cells. We further evaluated the effects of MAP4K2 silencing in IKZF1Q146H MM.1S cells. MAP4K2 knockdown resulted in significant inhibition of cell proliferation and increase of apoptotic cells in both cell lines (flag-IKZF1WT or flag-IKZF1Q146H MM.1S). Flag-IKZF1WT or flag-IKZF1Q146H were also overexpressed in IMiDs resistant (RPMI-8266) MM cell line by lentiviral infection, and no differences of MAP4K2 knockdown induced change of IKZF1 levels, cell proliferation, or apoptosis were observed between two cell lines. These data suggest that MAP4K2 inhibition induces anti-MM effects by targeting IKZF1 via an CRBN independent mechanism and subsequently overcomes IMiDs resistance. Our recent work also showed that the combination of iberdomide and MAP4K2 silencing led to synergetic anti-MM effects in vitro. In previous biochemical and structural studies, it was demonstrated that iberdomide binds to cereblon with a higher affinity than lenalidomide or pomalidomide. To investigate the combined effects of Iberdomide with MAP4K2 silencing on MM tumor growth, we generated subcutaneous MM xenografts in non-obese diabetic severe combined immunodeficient (NOD/SCID) mice using the inducible MAP4K2 shRNA MM.1S cells. Combination of Iberdomide with MAP4K2 silencing further enhanced tumor inhibition compared to the single treatment. Furthermore, we found that combination treatment significantly prolonged mice survival, compared to the single treatment. Taken together, our findings demonstrate that MAP4K2 is a novel therapeutic target to bypass IMiDs resistance in RAS mutated MM. Combination of MAP4K2 inhibition with Iberdomide results in synergetic anti-cancer effects in MM, therefore will provide a novel strategy to manage the relapsed or refractory patients with multi-drug resistance.

Breast Cancer Xenograft Murine Models.

Mice are used as model organisms to understand the pathological basis of a variety of human diseases, including breast cancer. Both immunocompetent and immunocompromised mouse models are used depending on the scope of the study. Immunocompetent models allow the study of the impact of the immune system in murine models of mammary cancer, while immunodeficient mice serve as ideal host organisms to understand the behavior of human breast cancers within a biological system. Xenografting of human breast cancer cells into immunocompromised mouse models continues to be the most used fundamental animal model in preclinical breast cancer research. These in vivo models allow critical understanding of tumor biology and assessment of novel treatments, a necessary prelude to testing new drugs in the clinic. In this chapter, we provide detailed methodology for the use of non-obese diabetic (NOD) severe combined immunodeficient (SCID) mice in several breast cancer xenografting procedures, including established cell lines and patient-derived xenografts (PDXs).

Chandipura virus induces cell death in cancer cell lines of human origin and promotes tumor regression in vivo

Chandipura virus (CHPV) is an emerging human pathogen of great clinical significance. In this study, we have investigated the susceptibility pattern of both normal and cancer cell lines of human origin to wild-type (wt) CHPV in order to explore the possibility of developing CHPV as an oncolytic vector (OV). Marked cytopathic effect along with enhanced virus output was observed in cancer cell lines (HeLa, A549, U-138, PC-3, and HepG2) in comparison to normal human adult dermal fibroblast (HADF) cells. At an MOI of 0.1, cancer cell lines were differentially susceptible to CHPV, with cells like HeLa and U-138 having pronounced cell death, while the PC-3 were comparatively resistant. All cell lines used in the study except U-138 restricted CHPV infection to varying degrees with IFN-β pre-treatment and supplementation of interferon (IFN) could neither activate the IFN signaling pathway in U-138 cells. Finally, U-138 tumor xenografts established in non-obese diabetic severe combined immunodeficiency (NOD/SCID) mice showed significant delay in tumor growth in the CHPV-challenged animals. Thus, targeted cytopathic effect in cancer cells at a very low dose with restricted replication in normal cells offers a rationale to exploit CHPV as an oncolytic vector in the future.

Cystathionine β-synthase mediated PRRX2/IL-6/STAT3 inactivation suppresses Tregs infiltration and induces apoptosis to inhibit HCC carcinogenesis

BackgroundHepatocellular carcinoma (HCC) is characterized by inflammation and immunopathogenesis. Accumulating evidence has shown that the cystathionine β-synthase/hydrogen sulfide (CBS/H2S) axis is involved in the regulation of inflammation. However, roles of...

Xenografts derived from patients with head and neck cancer recapitulate patient tumour properties.

Rodent models mimic the heterogeneity of head and neck cancer (HNC) malignancies and are used to investigate HNC-associated biomarkers and evaluate drug responses. To assess the utility of patient-derived xenografts (PDXs) as an HNC model, 18 tumour samples were obtained from surgical specimens of patients with HNC and implanted into non-obese diabetic severe combined immunodeficient mice. The histological features of PDXs and corresponding patient samples were compared. Furthermore, the present study investigated how PDX responses to anticancer drugs mimic patient clinical responses, as well as the expression of adenosine triphosphate-binding cassette transporters through chemotherapy in an HNC-PDX model. A total of five PDXs from patients with HNC exhibiting high correspondence with histopathological features of the original patient samples were established (establishment rate, 28%). The responses of three PDXs to cisplatin were associated with clinical responses of the patients. ABC transporter expression was augmented in one PDX model after anticancer drug treatment, but not in PBS-treated passaged PDXs. PDX models exhibited similar biological and chemosensitive characteristics to those of the primary tumours. PDXs could be a useful preclinical tool to test novel therapeutic agents and identify novel targets and biomarkers in HNC.

Sex difference feeding behaviour of NOD SCID mice in a pharmacological model of type 1 diabetes.

This study aimed to assess the sex differences in the feeding behaviour of non-obese diabetic severe combined immunodeficient (NOD SCID) mice in a pharmacological model of type 1 diabetes mellitus (T1Dm). In our study, we chose NOD SCID mice of both sexes and assessed their feeding behaviour, body weight, body fat and water content under identical experimental conditions and diets. After 1month of diabetes mellitus in mice in the experimental group, males and females did not show any increase in body weight, and they weighed significantly less than the control group. However, compared with the control group, in females with a background of T1Dm, there was a significant decrease in body fat. The amount of water consumed in the experimental groups was higher than that in the control groups. The amount of food consumed by males increased when they increased their water consumption, whereas food consumption in females decreased significantly with an increase in water consumption. Thus, we discovered sex differences in the feeding behaviour, body weight and body fat and water content in the pharmacological model of T1Dm after 1month in NOD SCID mice.

High Oncolytic Activity of a Double-Deleted Vaccinia Virus Copenhagen Strain against Malignant Pleural Mesothelioma

Malignant pleural mesothelioma (MPM) is a cancer of the pleura that lacks efficient treatment. Oncolytic immunotherapy using oncolytic vaccinia virus (VV) may represent an alternative therapeutic approach for the treatment of this malignancy. Here, we studied the oncolytic activity of VV thymidine kinase (TK)-ribonucleotide reductase (RR)-/green fluorescent protein (GFP) against MPM. This virus is a VV from the Copenhagen strain that is deleted of two genes encoding the TK (J2R) and the RR (I4L) and that express the GFP. First, we show in vitro that VVTK-RR-/GFP efficiently infects and kills the twenty-two human MPM cell lines used in this study. We also show that the virus replicates in all eight tested MPM cell lines, however, with approximately a 10-fold difference in the amplification level from one cell line to another. Then, we studied the therapeutic efficiency of VVTK-RR-/GFP in non-obese diabetic (NOD) severe combined immunodeficient (SCID) mice that bear peritoneal human MPM tumors. One intraperitoneal infection of VVTK-RR-/GFP reduces the tumor burden and significantly increases mice survival compared to untreated animals. Thus, VVTK-RR- may be a promising oncolytic virus (OV) for the oncolytic immunotherapy of MPM.

Fyn tyrosine kinase, a downstream target of receptor tyrosine kinases, modulates antiglioma immune responses

High-grade gliomas are aggressive and immunosuppressive brain tumors. Molecular mechanisms that regulate the inhibitory immune tumor microenvironment (TME) and glioma progression remain poorly understood. Fyn tyrosine kinase is a downstream target of the oncogenic receptor tyrosine kinase pathway and is overexpressed in human gliomas. Fyn's role in vivo in glioma growth remains unknown. We investigated whether Fyn regulates glioma initiation, growth and invasion. We evaluated the role of Fyn using genetically engineered mouse glioma models (GEMMs). We also generated Fyn knockdown stem cells to induce gliomas in immune-competent and immune-deficient mice (nonobese diabetic severe combined immunodeficient gamma mice [NSG], CD8-/-, CD4-/-). We analyzed molecular mechanism by RNA sequencing and bioinformatics analysis. Flow cytometry was used to characterize immune cellular infiltrates in the Fyn knockdown glioma TME. We demonstrate that Fyn knockdown in diverse immune-competent GEMMs of glioma reduced tumor progression and significantly increased survival. Gene ontology (GO) analysis of differentially expressed genes in wild-type versus Fyn knockdown gliomas showed enrichment of GOs related to immune reactivity. However, in NSG and CD8-/- and CD4-/- immune-deficient mice, Fyn knockdown gliomas failed to show differences in survival. These data suggest that the expression of Fyn in glioma cells reduces antiglioma immune activation. Examination of glioma immune infiltrates by flow cytometry displayed reduction in the amount and activity of immune suppressive myeloid derived cells in the Fyn glioma TME. Gliomas employ Fyn mediated mechanisms to enhance immune suppression and promote tumor progression. We propose that Fyn inhibition within glioma cells could improve the efficacy of antiglioma immunotherapies.

Immunodeficiency Promotes Adaptive Alterations of Host Gut Microbiome: An Observational Metagenomic Study in Mice.

The crosstalk between the gut microbiota and immune state of the host is an essential focus in academia and clinics. To explore the dynamic role of the microbiota in response to immune deficiency, we comprehensively assessed the microbiome of 90 mouse fecal samples, across three time points including two immunodeficiency models, namely severe combined immunodeficient (SCID) mice and non-obese diabetic SCID (NOD/SCID) mice, with BALB/cA as a control strain. Metagenomic analysis revealed a decrease in alpha diversity and the existence of a clear structural separation in the microbiota of immunodeficient mice. Although nuances exist between SCID and NOD/SCID mice, an increase in the protective microbiota, in particular Lactobacillus, contributed the most to the discrimination of immunodeficient and control mice. Further data regarding the red blood cell (RBC) concentration and serum IgA level during different stages of development support the concept of the microbiota alleviating the advancement of immune deficiency, which is called microbial compensation. Taken together, these results demonstrate the dynamic impact of immunodeficiency on the gut microbiota and the adaptive alteration of the microbiota that may influence the host state.

Apple Peel Flavonoid Fraction 4 Suppresses Breast Cancer Cell Growth by Cytostatic and Cytotoxic Mechanisms.

Many dietary flavonoids possess anti-cancer activities. Here, the effect of apple peel flavonoid fraction 4 (AF4) on the growth of triple-negative (MDA-MB-231, MDA-MB-468), estrogen receptor-positive (MCF-7), and HER2-positive (SKBR3) breast cancer cells was determined and compared with the effect of AF4 on normal mammary epithelial cells and dermal fibroblasts. AF4 inhibited breast cancer cell growth in monolayer cultures, as well as the growth of MCF-7 spheroids, without substantially affecting the viability of non-malignant cells. A sub-cytotoxic concentration of AF4 suppressed the proliferation of MDA-MB-231 cells by inhibiting passage through the G0/G1 phase of the cell cycle. AF4-treated MDA-MB-231 cells also exhibited reduced in vitro migration and invasion, and decreased Akt (protein kinase B) signaling. Higher concentrations of AF4 were selectively cytotoxic for MDA-MB-231 cells. AF4 cytotoxicity was associated with the intracellular accumulation of reactive oxygen species. Importantly, intratumoral administration of AF4 suppressed the growth of MDA-MB-231 xenografts in non-obese diabetic severe combined immunodeficient (NOD-SCID) female mice. The selective cytotoxicity of AF4 for breast cancer cells, combined with the capacity of sub-cytotoxic AF4 to inhibit breast cancer cell proliferation, migration, and invasion suggests that flavonoid-rich AF4 (and its constituents) has potential as a natural therapeutic agent for breast cancer treatment.

Safety and Biodistribution of Human Bone Marrow-Derived Mesenchymal Stromal Cells Injected Intrathecally in Non-Obese Diabetic Severe Combined Immunodeficiency Mice: Preclinical Study.

Mesenchymal stromal cells (MSCs) have potent immunomodulatory and neuroprotective properties, and have been tested in neurodegenerative diseases resulting in meaningful clinical improvements. Regulatory guidelines specify the need to perform preclinical studies prior any clinical trial, including biodistribution assays and tumourigenesis exclusion. We conducted a preclinical study of human bone marrow MSCs (hBM-MSCs) injected by intrathecal route in Non-Obese Diabetic Severe Combined Immunodeficiency mice, to explore cellular biodistribution and toxicity as a privileged administration method for cell therapy in Friedreich's Ataxia. For this purpose, 3 × 105 cells were injected by intrathecal route in 12 animals (experimental group) and the same volume of culture media in 6 animals (control group). Blood samples were collected at 24h (n = 9) or 4months (n = 9) to assess toxicity, and nineorgans were harvested for histology and safety studies. Genomic DNA was isolated from all tissues, and mouse GAPDH and human β2M and β-actin genes were amplified by qPCR to analyze hBM-MSCs biodistribution. There were no deaths nor acute or chronic toxicity. Hematology, biochemistry and body weight were in the range of normal values in all groups. At 24h hBM-MSCs were detected in 4/6 spinal cords and 1/6 hearts, and at 4months in 3/6 hearts and 1/6 brains of transplanted mice. No tumours were found. This study demonstrated that intrathecal injection of hBM-MSCs is safe, non toxic and do not produce tumors. These results provide further evidence that hBM-MSCs might be used in a clinical trial in patients with FRDA.

Preclinical verification of the efficacy by targeting peptide-linked liposomal nanoparticles for hepatocellular carcinoma therapy.

The purpose of this study was to investigate the efficacy of targeting peptides chemotherapy to overcome adverse event in the conventional chemotherapy for human hepatocellular carcinoma. Previously we reported several cancer-targeting peptides that bind specifically to cancer cells and their vascular endothelia: L-peptide (anti-cancer cell membrane), RLLDTNRPLLPY; SP-94-peptide (anti-hepatoma cell membrane), SFSHHTPILP; PC5-52-peptide (anti-tumor endothelia), SVSVGMKPSPRP; and control peptide, RLLDTNRGGGGG. In this study, these peptides were linked to liposomal iron oxide nanoparticles to localize the targeted tumor cells and endothelia, and to dextran-coated liposomal doxorubicin (L-D) to treat nonobese diabetic severe combined immunodeficient mice bearing hepatoma xenografts. Our results showed that L-peptide-linked liposomal doxorubicin could inhibit tumor growth with very mild adverse events. Use of the control peptide led to a decrease in the xenograft size but also led to marked apoptotic change in the visceral organ. In conclusion, L-peptide-linked liposomal doxorubicin, SP-94-peptide, and PC5-52-peptide can be used for the treatment of hepatoma xenografts in nonobese diabetic severe combined immunodeficient mice with minimal adverse events.

Embryonic Stem Cell Models of Human Brain Tumors.

Utilization of human embryonic stem cells (hESCs) as a model system to study highly malignant pediatric cancers has led to significant insight into the molecular mechanisms governing tumor progression and has revealed novel therapeutic targets for these devastating diseases. Here, we describe a method for generating heterogeneous populations of neural precursors from both normal and neoplastic hESCs and the subsequent injection of neoplastic human embryonic neural cells (hENs) into intracerebellar or intracranial xenograft models. Histopathologically, neural tumors derived from neoplastic hENs exhibit features similar to more aggressive medulloblastoma, the most common malignant primary pediatric brain tumor. In this chapter, we will outline the detailed methods for culturing normal and neoplastic neural precursor cells in both adherent and tumorsphere format and the full characterization of the brain tumors generated from these cells in non-obese diabetic severe combined immunodeficiency (NOD SCID) mice.

Therapeutic vulnerability of multiple myeloma to MIR17PTi, a first-in-class inhibitor of pri-miR-17-92

AbstractThe microRNA (miRNA) cluster miR-17-92 is oncogenic and represents a valuable therapeutic target in c-MYC (MYC)–driven malignancies. Here, we developed novel LNA gapmeR antisense oligonucleotides (ASOs) to induce ribonuclease H–mediated degradation of MIR17HG primary transcripts and consequently prevent biogenesis of miR-17-92 miRNAs (miR-17-92s). The leading LNA ASO, MIR17PTi, impaired proliferation of several cancer cell lines (n = 48) established from both solid and hematologic tumors by on-target antisense activity, more effectively as compared with miR-17-92 inhibitors. By focusing on multiple myeloma (MM), we found that MIR17PTi triggers apoptosis via impairment of homeostatic MYC/miR-17-92 feed-forward loops (FFLs) in patient-derived MM cells and induces MYC-dependent synthetic lethality. We show that alteration of a BIM-centered FFL is instrumental for MIR17PTi to induce cytotoxicity in MM cells. MIR17PTi exerts strong in vivo antitumor activity in nonobese diabetic severe combined immunodeficient mice bearing clinically relevant models of MM, with advantageous safety and pharmacokinetic profiles in nonhuman primates. Altogether, MIR17PTi is a novel pharmacological tool to be tested in early-phase clinical trials against MM and other MYC-driven malignancies.

Identification of CD24 as a marker for tumorigenesis of melanoma.

ObjectiveCutaneous melanoma (CM) is a common skin cancer. Surgery is still the primary treatment for CM, as melanoma is resistant to chemotherapy. In the recent years, it has been found that cancer stem-like cells (CSCs) are responsible for this drug resistance. CD24 is a widely used marker to isolate CSCs. In this study, we aimed to analyze the properties of CD24+ and CD24− subpopulation of melanoma cells.Materials and methodsWe isolated CD24+ cells CSCs using magnetic-activated cell sorting system. We extracted total RNA and carried out reverse transcription polymerase chain reaction analysis. We counted the cell colonies using soft agar assay and assessed the cell invasion using cell migration assay. We implanted CD24+ or CD24− cells into the flank of non-obese diabetic severe combined immunodeficiency mice, and measured the tumor volumes every 5 days until the end of the experiment. We carried out immunohistochemical analysis to study the tissue sections.ResultsWe demonstrated that the CD24+ subpopulation has self-renewal properties in vitro and in vivo by using soft agar assay and xenograft tumor model. Furthermore, we confirmed that CD24 expression is accompanied by activation of Notch1 signaling pathway.ConclusionThis study provides new knowledge on the role of CD24 in the tumorigenic ability of melanoma.

Effectiveness of bioengineered islet cell sheets for the treatment of diabetes mellitus

BackgroundThe present study aimed to evaluate whether bioengineered mouse islet cell sheets can be used for the treatment of diabetes mellitus. MethodsIsolated mouse pancreatic islets were dispersed, and cells were plated on temperature-responsive culture plates coated with iMatrix-551. On day 3 of culture, the sheets were detached from the plates and used for further analysis or transplantation. The following parameters were assessed: (1) morphology, (2) expression of β-cell–specific transcription factors and other islet-related proteins, (3) methylation level of the pancreatic duodenal homeobox-1 (Pdx-1) promoter, as determined by bisulfite sequencing, and (4) levels of serum glucose after transplantation of one or two islet cell sheets into the abdominal cavity of streptozotocin-induced diabetic severe combined immunodeficiency mice. ResultsFrom each mouse, we recovered approximately 233.3 ± 12.5 islets and 1.4 ± 0.1 × 105 cells after dispersion. We estimate that approximately 68.2% of the cells were lost during dispersion. The viability of recovered single cells was 91.3 ± 0.9%. The engineered islet cell sheets were stable, but the messenger RNA levels of various β-cell-specific transcription factors were significantly lower than those of primary islets, whereas Pdx-1 promoter methylation and the expression of NeuroD, Pdx-1, and glucagon proteins were similar between sheets and islets. Moreover, transplantation of islet cell sheets did not revert serum hyperglycemia in any of the recipient mice. ConclusionsEngineering effective islet cell sheets require further research efforts, as the currently produced sheets remain functionally inferior compared with primary islets.

Safety and Efficacy of Epigenetically Converted Human Fibroblasts Into Insulin-Secreting Cells: A Preclinical Study.

Type 1 Diabetes Mellitus (T1DM) is a chronic disease that leads to loss of insulin secreting β-cells, causing high levels of blood glucose. Exogenous insulin administration is not sufficient to mimic the normal function of β-cells and, consequently, diabetes mellitus often progresses and can lead to major chronic complications and morbidity. The physiological control of glucose levels can only be restored by replacing the β-cell mass.We recently developed a new strategy that allows for epigenetic conversion of dermal fibroblasts into insulin-secreting cells (EpiCC), using a brief exposure to the demethylating agent 5-aza-cytidine (5-aza-CR), followed by a pancreatic induction protocol. This method has notable advantages compared to the alternative available procedures and may represent a promising tool for clinical translation as a therapy for T1DM. However, a thought evaluation of its therapeutic safety and efficacy is mandatory to support preclinical studies based on EpiCC treatment.We here report the data obtained using human fibroblasts isolated from diabetic and healthy individuals, belonging the two genders. EpiCC were injected into 650 diabetic severe combined immunodeficiency (SCID) mice and demonstrated to be able to restore and maintain glycemic levels within the physiological range. Cells had the ability to self-regulate and not to cause hypoglycemia, when transplanted in healthy animals. Efficacy tests showed that EpiCC successfully re-established normoglycemia in diabetic mice, using a dose range that appeared clinically relevant to the concentration 0.6×106 EpiCC. Necropsy and histopathological investigations demonstrated the absence of malignant transformation and cell migration to organs and lymph nodes.The present preclinical study demonstrates safety and efficacy of human EpiCC in diabetic mice and supports the use of epigenetic converted cells for regenerative medicine of diabetes mellitus.

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