Combined Immunodeficient Mice Research Articles

BI-907828, a novel potent MDM2 inhibitor, inhibits glioblastoma brain tumor stem cells in vitro and prolongs survival in orthotopic xenograft mouse models.

The tumor suppressor TP53 (p53) is frequently mutated, and its downstream effectors inactivated in many cancers, including glioblastoma (GBM). In tumors with wild-type status, p53 function is frequently attenuated by alternate mechanisms including amplification and overexpression of its key negative regulator, MDM2. We investigated the efficacy of the MDM2 inhibitor, BI-907828, in GBM patient-derived brain tumor stem cells (BTSCs) with different amplification statuses of MDM2, in vitro and in orthotopic xenograft models. In vitro growth inhibition and on-target efficacy of BI-907828 were assessed by cell viability, co-immunoprecipitation assays, and western blotting. In vivo efficacy of BI-907828 treatments was assessed with qPCR, immunohistochemistry, and in intracranial xenograft models. BI-907828 decreases viability and induces cell death at picomolar concentrations in both MDM2 amplified and normal copy number TP53 wild-type BTSC lines. Restoration of p53 activity, including robust p21 expression and apoptosis induction, was observed in TP53 wild-type but not in TP53 mutant BTSCs. shRNA-mediated knock-down of TP53 in wild-type BTSCs abrogated the effect of BI-907828, confirming the specificity of the inhibitor. Pharmacokinetic-pharmacodynamic studies in orthotopic tumor-bearing severe combined immunodeficiency (SCID) mice demonstrated that a single 50mg/kg p.o. dose of BI-907828 resulted in strong activation of p53 target genes p21 and MIC1. Long-term weekly or bi-weekly treatment with BI-907828 in orthotopic BTSC xenograft models was well-tolerated and improved survival both as a single-agent and in combination with temozolomide, with dose-dependent efficacy observed in the MDM2 amplified model. BI-907828 provides a promising new therapeutic option for patients with TP53 wild-type primary brain tumors.

Transplantation of chemically-induced liver progenitor cells ameliorates hepatic fibrosis in mice with diet-induced nonalcoholic steatohepatitis.

Primary mature hepatocytes were isolated from 7-week-old male Wistar rats. To establish CLiPs, isolated hepatocytes were cultured in differentiation medium composed of Y-27632, A-83-01, and CHIR99021 (YAC medium). As an animal model that reproduces NASH pathophysiology, 6-week-old severe combined immunodeficient (SCID) mice were carefully selected and prepared and fed with choline-deficient, L-amino acid-defined, high-fat diet (HFD). After 12 weeks' HFD feeding, the mice were assigned to continue HFD with or without the administration of rat CLiPs (HFD+CLiPs and HFD-CLiPs, respectively). Rat CLiPs were administered from the spleen. Hepatic fibrosis was semi-quantitatively evaluated according to histology. Liver parenchyma and blood samples were collected for biochemical analyses. Rat CLiPs were positive for CK19 and EpCAM were successfully delivered to the liver. At 8 weeks after CLiPs transplantation, the HFD+CLiPs group showed significantly less positive staining than the HFD-CLiPs group. Alanine aminotransferase significantly improved in the HFD+CLiPs group, as demonstrated by Azan staining and αSMA immunostaining. RT qPCR showed that the liver expression of MMP2 and 9 tended to be higher in the HFD+CLiPs group. The anti-fibrotic effect of CLiPs was demonstrated in the immunodeficient NASH animal model and may have therapeutic applications in humans.

Establishment and characterization of urothelial carcinoma cell lines with and without BRAF mutation (V595E) in dogs

Each 5 urothelial carcinoma (UC) cell lines with and without the v-Raf murine sarcoma virus oncogene homolog B (BRAF) gene mutation (V595E) were established and examined V595E-related tumorigenic characteristics in dogs. No typical morphological features were observed in cloned cells with and without V595E. The cell proliferation of both cloned cells showed logarithmic growth curve and those doubling time were 24.9 ± 4.1 h in V595E ( +) and 29.3 ± 11.3 h in V595E ( −). On the growth curve of xenotransplanted tumor in severe combined immunodeficiency mice, 3 out of 5 V595E ( +) and 2 out of 5 V595E ( −) cloned cells revealed gradually and remarkably increasing curve, indicating clearly tumorigenicity. The xenotransplanted tumors with V595E ( +) showed typical features of UC, such as solid proliferation of pleomorphic tumor cells, formation of papillary structure, and glandular structure. Additionally, various vascular formation was observed, probably indicating an advanced growth phase of UC. In mitogen-activated protein kinase (MAPK) signaling pathway, cytoplasmic phosphorylated-BRAF (pBRAF) and cytoplasmic and nuclear phosphorylated-ERK1/2 (pERK1/2) were detected in all 4 tumors with V595E ( +), whereas only cytoplasmic and nuclear pERK1/2 was detected in tumors with V595E ( −). Since V595E can directly activate MAPK signaling pathway, coincidence of V595E with pBRAF (phosphor Thr598/Ser601) indicates acquired resistance to BRAF inhibitors. These established UC cell lines, especially V595E ( +) cell lines, are useful tool for understanding pathophysiological states and controlling therapeutic manners of UC in dogs.

The use of animal models in rheumatoid arthritis research.

The pathological hallmark of rheumatoid arthritis (RA) is a synovial pannus that comprises proliferating and invasive fibroblast-like synoviocytes, infiltrating inflammatory cells, and an associated neoangiogenic response. Animal models have been established to study these pathological features of human RA. Spontaneous and induced animal models of RA primarily reflect inflammatory aspects of the disease. Among various induced animal models, collagen-induced arthritis (CIA) and collagen antibody-induced arthritis (CAIA) models are widely used to study the pathogenesis of RA. Improved transplantation techniques for severe combined immunodeficiency (SCID) mouse models of RA can be used to evaluate the effectiveness of potential therapeutics in human tissues and cells. This review provides basic information on various animal models of RA, including CIA and CAIA. In addition, we describe a SCID mouse coimplantation model that can measure the long-distance migration of human RA synoviocytes and cartilage destruction induced by these cells.

Bioluminescent Monitoring of Graft Survival in an Adoptive Transfer Model of Autoimmune Diabetes in Mice.

Type 1 diabetes is characterized by the autoimmune destruction of the insulin-producing beta cells of the pancreas. A promising treatment for this disease is the transplantation of stem cell-derived beta cells. Genetic modifications, however, may be necessary to protect the transplanted cells from persistent autoimmunity. Diabetic mouse models are a useful tool for the preliminary evaluation of strategies to protect transplanted cells from autoimmune attack. Described here is a minimally invasive method for transplanting and imaging cell grafts in an adoptive transfer model of diabetes in mice. In this protocol, cells from the murine pancreatic beta cell line NIT-1 expressing the firefly luciferase transgene luc2 are transplanted subcutaneously into immunodeficient non-obese diabetic (NOD)-severe combined immunodeficient (scid) mice. These mice are simultaneously injected intravenously with splenocytes from spontaneously diabetic NOD mice to transfer autoimmunity. The grafts are imaged at regular intervals via non-invasive bioluminescent imaging to monitor the cell survival. The survival of mutant cells is compared to that of control cells transplanted into the same mouse.

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.

Visualization of Multiple Myeloma Mouse Model with 89zr-Labeled Anti-CD38 Antibody By a New Imaging System; Whole-Gamma Imaging

Introduction Visualization and measurement of multiple myeloma (MM) cells in vivo by means of positron emission tomography (PET) is expected to determine biopsy sites, personalize treatments, and make an early diagnosis of recurrence. The positron radioisotope 89Zr is one of the promising radionuclides for PET image due to the long radioactive half-life of 3 days and stable labelling of antibodies. 89Zr also emits 909 keV single gamma-rays about 4 times more frequently than positrons, causing just noise to make PET images. To overcome this disadvantage of 89Zr, we propose a new imaging method that detects and uses the single gamma-rays as well as positrons for imaging, called whole gamma imaging (WGI). WGI is composed of PET and Compton imaging methods, equipped with two PET detector rings; both of the inner ring and the outer ring also acts as a scatterer and an absorber, respectively, for Compton imaging method (Fig. 1). In this study, we have firstly validated the affinity and specificity of antibodies recognizing CD38 and SLAMF7 in two types of human MM cell lines: RPMI8226 with CD38 expression and U226 with CD38 and SLAMF7. Then we carried out imaging in xenograft model mice with WGI using an 89Zr-antibody tracer and finally compared the quantitative values by WGI to those by ex-vivo biodistribution method. Methods Specific binding of antibodies was evaluated up to 10 × 106 cells of each MM cell type. An inhibition assay was performed using unlabeled IgG. 89Zr was produced in a cyclotron and neutralized 89Zr-oxalate was added to chelate-conjugated antibodies. Two severe combined immunodeficiency (SICD) mice after 3 Gy gamma-ray irradiation were implanted subcutaneously in the right flank with 3×107 MM cells in 100μL of PBS. On 12 days after the implantation, the tumor volume reached approximately 180 mm3 (mouse 1) and 110 mm3 (mouse 2). Based on the in-vitro study, 89Zr-anti-CD38 antibodies at the dose of 3.3 MBq were injected via a tail-vein. WGI imaging was performed on 4, 6, 8, and 12 days after the tracer injection. After WGI imaging on day 12, mice were euthanized for ex-vivo biodistribution analysis; blood was collected via cardiac puncture into counting tubes. Tumor and normal tissues: lung, liver, spleen, kidney, muscle, heart, bone (tibia), pancreas, stomach, intestine, brain, and lymph node were excised, and their weight and radioactivity were measured, and %ID/g values were calculated. Results High specific binding fractions of anti-CD38 antibodies were observed for RPMI8226 cells, reaching to approximately 50% at 10 × 106 cells; while only 5% for U226 cells. Those of anti-SLAMF7 antibodies were under 1% for both types of tumor cells. The inhibition assay using cold IgG blocked the binding of anti-CD 38 antibodies (Kd = 1.2 nM) and chelate-conjugated anti-CD38 antibodies (Kd = 1.4 nM) to RPMI8226. Then we decided to use 89Zr-anti-CD38 antibody and RPMI8226 cells for the WGI imaging study. WIG imaging showed that 89Zr-anti-CD38 antibody uptake in tumors increased over time, being up to standardized uptake value (SUV) 9.8 on 12 days after tracer injection. The uptake in the healthy tissues gradually decreased with time except for the bone (Fig. 2). Ex-vivo biodistribution revealed that the highest 89Zr-anti-CD38 antibody uptake in tumors was 92.4%ID/g (SUV, 18.5) and 97.5%ID/g (SUV, 19.5) in mice 1 and 2, respectively. That in healthy tissues was under 27.7%ID/g. Considering the specific activity of 89Zr-anti-CD38 antibodies of 4.2 MBq/nmol, 89Zr-anti-CD38 antibodies accumulated in RPMI8226 tumors at approximately 730 fmol/mg. Conclusion We proposed a new imaging method, called WGI, which clearly visualized MM cells and was able to estimate the uptake values of 89Zr-anti-CD38 antibody binding to specific antigen in MM tumors. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Long-term and non-invasive in vivo tracking of DiD dye-labeled human hepatic progenitors in chronic liver disease models.

Chronic liver diseases (CLD) are the major public health burden due to the continuous increasing rate of global morbidity and mortality. The inherent limitations of organ transplantation have led to the development of stem cell-based therapy as a supportive and promising therapeutic option. However, identifying the fate of transplanted cells in vivo represents a crucial obstacle. To evaluate the potential applicability of DiD dye as a cell labeling agent for long-term, and non-invasive in vivo tracking of transplanted cells in the liver. Magnetically sorted, epithelial cell adhesion molecule positive (1 × 106 cells/mL) fetal hepatic progenitor cells were labeled with DiD dye and transplanted into the livers of CLD-severe combined immunodeficiency (SCID) mice. Near-infrared (NIR) imaging was performed for in vivo tracking of the DiD-labeled transplanted cells along with colocalization of hepatic markers for up to 80 d. The existence of human cells within mouse livers was identified using Alu polymerase chain reaction and sequencing. NIR fluorescence imaging of CLD-SCID mice showed a positive fluorescence signal of DiD at days 7, 15, 30, 45, 60, and 80 post-transplantation. Furthermore, positive staining of cytokeratin, c-Met, and albumin colocalizing with DiD fluorescence clearly demonstrated that the fluorescent signal of hepatic markers emerged from the DiD-labeled transplanted cells. Recovery of liver function was also observed with serum levels of glutamic-oxaloacetic transaminase, glutamate-pyruvate transaminase, and bilirubin. The detection of human-specific Alu sequence from the transplanted mouse livers provided evidence for the survival of transplanted cells at day 80. DiD-labeling is promising for long-term and non-invasive in vivo cell tracking, and understanding the regenerative mechanisms incurred by the transplanted cells.

Oral administration of human carbonic anhydrase I suppresses colitis in a murine inflammatory bowel disease model

The incidence of inflammatory bowel disease (IBD) is increasing; hence, effective treatments are warranted. The therapeutic effect of human carbonic anhydrase I (hCA I) in IBD remains unknown. Therefore, we investigated whether oral tolerization to hCA I would induce antigen-specific protection from intestinal inflammation in vivo. Severe combined immunodeficient mice received hCA I, keyhole limpet hemocyanin (KLH), or phosphate-buffered saline (PBS) orally for 7 days. Colons and mesenteric lymph nodes (MLNs) were collected 4 weeks after cell transfer. Additionally, the mechanisms underlying the therapeutic effects were investigated. The comparison between the effects of well-established drugs and hCA I oral administration was investigated. Oral administration of hCA I ameliorated colitis remarkably. hCA I reached the cecum and ameliorated colitis more effectively than mesalazine and similarly to prednisolone. Compared with PBS treatment, hCA I treatment reduced interleukin (IL)-17a, IL-6, and retinoic acid-related orphan receptor gamma t (RORγt) expression in the colon or MLNs; moreover, hCA I markedly reduced IL-6, IL-17, and interferon-gamma (IFN-γ) levels in the MLN. Oral administration of hCA I induced immune tolerance and suppressed colitis in vivo. Thus, hCA I administration could be proposed as a new treatment option for IBD.

Enhancing regulatory T-cell function via inhibition of high mobility group box 1 protein signaling in immune thrombocytopenia.

Primary immune thrombocytopenia (ITP) is the most common acquired autoimmune bleeding disorder. Abnormally increased levels of High Mobility Group Box 1 (HMGB1) protein associate with thrombocytopenia and therapeutic outcome in ITP. Previous studies proposed that a natural inhibitor of HMGB1, 18β-glycyrrhetinic acid (18β-GA), could be used for its anti-inflammatory and immune-modulatory effects, although its ability to correct immune balance in ITP is unclear. In this study, we showed that plasma HMGB1 correlated negatively with platelet counts in ITP patients, and confirmed that 18β-GA stimulated the production of regulatory T cells (Treg), restored the balance of CD4+ T-cell subsets and enhanced the suppressive function of Treg through blocking the effect on HMGB1 in patients with ITP. HMGB1 short hairpin RNA interference masked the effect of 18β-GA in Treg of ITP patients. Furthermore, we found that 18β-GA alleviated thrombocytopenia in mice with ITP. Briefly, anti-CD61 immune-sensitized splenocytes were transferred into severe combined immunodeficient mice to induce a murine model of severe ITP. The proportion of circulating Treg increased significantly, while the level of plasma HMGB1 and serum antiplatelet antibodies decreased significantly in ITP mice along 18β-GA treatment. In addition, 18β-GA reduced phagocytic activity of macrophages towards platelets both in ITP patients and ITP mice. These results indicate that 18β-GA has the potential to restore immune balance in ITP via inhibition of HMGB1 signaling. In short, this study reveals the role of HMGB1 in ITP, which may serve as a potential target for thrombocytopenia therapy.

Individualized therapy based on the combination of mini-PDX and NGS for a patient with metastatic AFP-producing and HER-2 amplified gastric cancer.

Mini-patient-derived xenograft (mini-PDX) is a novel, rapid and accurate method used to assess in vivo drug susceptibility. In the present study, a mini-PDX combined with next-generation sequencing (NGS) was used to guide the individualized treatment of a patient with metastatic a-fetoprotein-producing and human epidermal growth factor receptor 2 (HER-2) amplified gastric cancer (GC). Tumor cells were isolated from the tumor tissue obtained from gastroscopic biopsy, transferred into capsules and implanted into severe combined immunodeficiency mice to determine their sensitivity to various drug regimens. NGS was also performed to assess the mutation spectrum of the cells. The results were analyzed to select the most appropriate treatment regimen for the patient. The mini-PDX model confirmed that the patient's tumor was sensitive to a combination regimen of irinotecan and tegafur-gimeracil-oteracil (S-1). Fluorescence in situ hybridization assay of the tumor tissue confirmed HER-2 amplification. The NGS results indicated ERBB2 amplification, and tumor protein P53 [c.659A>G (p.Y220C)], ataxia-telengiectasia mutated [c.125A>G (p.H42R)] and MutS homolog 6 [c.3254C(8>7) (p.F1088Sfs*2)] mutation, in which UGT1A1*28, TA6/7 (rs8175347) was a mutant heterozygote. After six courses of treatment with a regimen comprising 300 mg irinotecan on day 1 + 40 mg S-1 twice daily on days 2–15 + 350 mg trastuzumab once-every 3 weeks, the patient continued with S-1 treatment for 4 courses and trastuzumab for 1 year. The patient retained progression-free survival status at the 32-month follow-up. Thus, the mini-PDX model combined with the NGS rapidly assessed drug sensitivity in a patient with GC and revealed key genetic mutations. However, the proposed technique requires further research to confirm its potential in the individualized treatment of patients with refractory malignancies.

Human duodenal submucosal glands contain a defined stem/progenitor subpopulation with liver-specific regenerative potential

Common precursors for the liver, biliary tree, and pancreas exist at an early stage of development in the definitive endoderm forming the foregut. We have identified and characterised endodermal stem/progenitor cells with regenerative potential persisting in the adult human duodenum. Human duodena were obtained from organ donors, and duodenal submucosal gland cells were isolated after removal of the mucosa layer. Cells were cultured on plastic or as organoids and were transplanted into severe combined immunodeficient (SCID) mouse livers. In situ studies of submucosal glands in the human duodenum revealed cells expressing stem/progenitor cell markers that had unique phenotypic traits distinguishable from intestinal crypt cells. Genetic signature studies indicated that the cells are closer to biliary tree stem cells and to definitive endodermal cells than to adult hepatocytes, supporting the interpretation that they are endodermal stem/progenitor cells. Invitro, human duodenal submucosal gland cells demonstrated clonal growth, capability to form organoids, and ability to acquire functional hepatocyte traits. Invivo, transplanted cells engrafted into the livers of immunocompromised mice and differentiated to mature liver cells. In an experimental model of fatty liver, human duodenal submucosal gland cells were able to rescue hosts from liver damage by supporting repopulation and regeneration of the liver. A cell population with clonal growth and organoid formation capability, which has liver differentiation potency invitro and invivo in murine experimental models, is present within adult duodenal submucosal glands. These cells can be isolated, do not require reprogramming, and thus could potentially represent a novel cell source for regenerative medicine of the liver. Cell therapies for liver disease could represent an option to support liver function, but the identification of sustainable and viable cell sources is critical. Here, we describe a cell population with organoid formation capability and liver-specific regenerative potential in submucosal glands of the human duodenum. Duodenal submucosal gland cells are isolated from adult organs, do not require reprogramming, and could rescue hepatocellular damage in preclinical models of chronic, but not acute, liver injury. Duodenal submucosal gland cells could represent a potential candidate cell source for regenerative medicine of the liver, but the determination of cell dose and toxicity is needed before clinical testing in humans.

Low-dose decitabine modulates myeloid-derived suppressor cell fitness via LKB1 in immune thrombocytopenia

AbstractMyeloid-derived suppressor cells (MDSCs) are heterogeneous immature cells and natural inhibitors of adaptive immunity. Metabolic fitness of MDSCs is fundamental for its suppressive activity toward effector T cells. Our previous studies showed that the number and inhibitory function of MDSCs were impaired in patients with immune thrombocytopenia (ITP) compared with healthy controls. In this study, we analyzed the effects of decitabine on MDSCs from patients with ITP, both in vitro and in vivo. We found that low-dose decitabine promoted the generation of MDSCs and enhanced their aerobic metabolism and immunosuppressive functions. Lower expression of liver kinase 1 (LKB1) was found in MDSCs from patients with ITP, which was corrected by decitabine therapy. LKB1 short hairpin RNA (shRNA) transfection effectively blocked the function of MDSCs and almost offset the enhanced effect of decitabine on impaired MDSCs. Subsequently, anti-CD61 immune-sensitized splenocytes were transferred into severe combined immunodeficient (SCID) mice to induce ITP in murine models. Passive transfer of decitabine-modulated MDSCs significantly raised platelet counts compared with that of phosphate buffered saline–modulated MDSCs. However, when LKB1 shRNA-transfected MDSCs were transferred into SCID mice, the therapeutic effect of decitabine in alleviating thrombocytopenia was quenched. In conclusion, our study suggests that the impaired aerobic metabolism of MDSCs is involved in the pathogenesis of ITP, and the modulatory effect of decitabine on MDSC metabolism contributes to the improvement of its immunosuppressive function. This provides a possible mechanism for sustained remission elicited by low-dose decitabine in patients with ITP.

Nm23-H1 induces apoptosis in primary effusion lymphoma cells via inhibition of NF-κB signaling through interaction with oncogenic latent protein vFLIP K13 of Kaposi's sarcoma-associated herpes virus.

Primary effusion lymphoma (PEL) is an aggressive form of non-Hodgkin lymphoma of B cells caused by Kaposi's Sarcoma-associated Herpes Virus (KSHV). KSHV encoded latent and lytic antigens promote oncogenic transformation and evade apoptosis through the modulation of various host cellular signaling pathways. Nm23-H1 is a known metastatic suppressor whose expression inversely correlates with the metastatic potential of different cancers. Here, we set out to assess the role of Nm23-H1 in PEL development. Flow cytometry and real-time PCR assays were performed for Nm23-H1 expression analysis. Induction of apoptosis was assessed using Western blotting and flow cytometry-based assays in Nm23-H1 overexpressing cells. Co-immunoprecipitation assays, confocal microscopy and imaging flow cytometry were performed to determine Nm23-H1 and vFLIP K13 protein-protein interaction. A PEL cell-induced xenograft model was established in non-obese diabetic/severely combined immunodeficient (NOD/SCID) mice to validate the effect of Nm23-H1 overexpression. We found that Nm23-H1 expression was significantly downregulated both at transcriptional and protein levels in PEL cell lines and that its overexpression triggered mitochondrial-mediated caspase-dependent apoptosis. We revealed Nm23-H1 interacts with the latent protein vFLIP K13 and that Nm23-H1 overexpression leads to inhibition of vFLIP K13 driven nuclear factor-kappa B (NF-κB) signaling with concurrent inhibition of autocrine and paracrine growth factors and downregulation of latent KSHV antigens without induction of active lytic reactivation. We also confirmed the effects of Nm23-H1 overexpression in a PEL cell-induced xenograft model in NOD/SCID mice. Downregulation of Nm23-H1 expression in KSHV-infected PEL cells and its overexpression trigger apoptosis by impairing vFLIP K13-driven NF-κB signaling, suggesting therapeutic implications of Nm23-H1 for primary effusion lymphomas.

Induction of iPSC-derived Prg4-positive cells with characteristics of superficial zone chondrocytes and fibroblast-like synovial cells

BackgroundLubricin, a proteoglycan encoded by the PRG4 gene, is synthesised by superficial zone (SFZ) chondrocytes and synovial cells. It reduces friction between joints and allows smooth sliding of tendons. Although lubricin has been shown to be effective against osteoarthritis and synovitis in animals, its clinical application remains untested. In this study, we aimed to induce lubricin-expressing cells from pluripotent stem cells (iPSCs) and applied them locally via cell transplantation.MethodsTo generate iPSCs, OCT3/4, SOX2, KLF4, and L-MYC were transduced into fibroblasts derived from Prg4-mRFP1 transgenic mice. We established a protocol for the differentiation of iPSC-derived Prg4-mRFP1-positive cells and characterised their mRNA expression profile. Finally, we injected Prg4-mRFP1-positive cells into the paratenon, surrounding the Achilles tendons and knee joints of severe combined immunodeficient mice and assessed lubricin expression.ResultWnt3a, activin A, TGF-β1, and bFGF were applied to induce the differentiation of iPSC-derived Prg4-mRFP1-positive cells. Markers related to SFZ chondrocytes and fibroblast-like synovial cells (FLSs) were expressed during differentiation. RNA-sequencing indicated that iPSC-derived Prg4-mRFP1-positive cells manifested expression profiles typical of SFZ chondrocytes and FLSs. Transplanted iPSC-derived Prg4-mRFP1-positive cells survived around the Achilles tendons and in knee joints.ConclusionsThe present study describes a protocol for the differentiation of iPSC-derived Prg4-positive cells with characteristics of SFZ chondrocytes and FLSs. Transplantation of lubricin-expressing cells offers promise as a therapy against arthritis and synovitis.

Flavokawain A Reduces Tumor-Initiating Properties and Stemness of Prostate Cancer.

We have previously demonstrated the in vivo chemopreventive efficacy of flavokawain A (FKA), a novel chalcone from the kava plant, in prostate carcinogenesis models. However, the mechanisms of the anticarcinogenic effects of FKA remain largely unknown. We evaluated the effect of FKA on prostate tumor spheroid formation by prostate cancer stem cells, which were sorted out from CD44+/CD133+ prostate cancer cells 22Rv1 and DU145. FKA treatment significantly decreased both the size and numbers of the tumor spheroids over different generations of spheroid passages. In addition, the dietary feeding of FKA-formulated food to Nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice bearing CD44+/CD133+ 22Rv1 xenograft tumors resulted in a significant reduction of tumor growth compared to those fed with vehicle control food–fed mice. Furthermore, the expression of stem cell markers, such as Nanog, Oct4, and CD44, were markedly downregulated in both tumor spheroids and tumor tissues. We also observed that FKA inhibits Ubc12 neddylation, c-Myc, and keratin-8 expression in both CD44+/CD133+ prostate tumor spheroids and xenograft tumors. Our results suggest that FKA can reduce the tumor-initiating properties and stemness of prostate cancer, which provides a new mechanism for the chemoprevention efficacy of FKA.

Ultrasound Imaging of Pancreatic Perfusion Dynamics Predicts Therapeutic Prevention of Diabetes in Preclinical Models of Type 1 Diabetes.

In type 1 diabetes (T1D), immune-cell infiltration into islets of Langerhans (insulitis) and β-cell decline occur years before diabetes presents. There is a lack of validated clinical approaches for detecting insulitis and β-cell decline, to diagnose eventual diabetes and monitor the efficacy of therapeutic interventions. We previously determined that contrast-enhanced ultrasound measurements of pancreas perfusion dynamics predict disease progression in T1D pre-clinical models. Here, we test whether these measurements predict therapeutic prevention of T1D. We performed destruction-reperfusion measurements with size-isolated microbubbles in non-obese diabetic (NOD)-severe combined immunodeficiency (SCID) mice receiving an adoptive transfer of diabetogenic splenocytes. Mice received vehicle control or the following treatments: (i) anti-CD3 to block T-cell activation; (ii) anti-CD4 to deplete CD4+ T cells; (iii) verapamil to reduce β-cell apoptosis; or (iv) tauroursodeoxycholic acid (TUDCA) to reduce β-cell endoplasmic reticulum stress. We compared measurements of pancreas perfusion dynamics with subsequent progression to diabetes. Anti-CD3, anti-CD4, and verapamil delayed diabetes development. Blood flow dynamics was significantly altered in treated mice with delayed/absent diabetes development compared with untreated mice. Conversely, blood flow dynamics in treated mice with unchanged diabetes development was similar to that in untreated mice. Thus, measurement of pancreas perfusion dynamics predicts the successful prevention of diabetes. This strategy may provide a clinically deployable predictive marker for therapeutic prevention in asymptomatic T1D.

O-034 Xenotransplantation of ovarian cortex tissue from young girls with Turner Syndrome in a mice model: is normal follicular development possible?

Abstract Study question Does aneuploidy of granulosa and stromal cells affect folliculogenesis of small follicles in girls with mosaic Turner Syndrome (TS)? Summary answer Our results showed that despite high level aneuploidy in granulosa cells of small follicles and stromal cells folliculogenesis was not affected in mosaic TS patients. What is known already In 2018 the TurnerFertility study was initiated to explore if ovarian tissue cryopreservation (OTC) is a viable option for TS patients to preserve their fertility at an early age. In this study, karyotyping of ovarian cells in 7 mosaic TS patients showed that the majority of oocytes had a normal X chromosomal content, while granulosa and stromal cells were aneuploid. Until now, it remains unclear if follicular development is affected, and consequently the success rate of autotransplantation after OTC in girls with TS. Study design, size, duration A collaboration project was initiated between Radboudumc, The Netherlands and UCLouvain, Belgium to assess folliculogenesis of small follicles in TS patients by using a xenograft mice model. Ovarian cortex tissue was obtained from 18 mosaic TS patients and 13 age-matched controls. Participants/materials, setting, methods After unilateral ovariectomy, one fragment of the ovarian tissue was used for research. One part of this fragment was used for fluorescence in situ hybridization (FISH) to determine the X chromosomal content of ovarian cells in non-grafted tissue. The other part was xenografted into severe combined immunodeficient mice. After 5 months, grafts were retrieved and analysed for aneuploidy by FISH. The expression of 6 proteins essential for folliculogenesis was analysed by immunohistochemistry and immunofluorescence. Main results and the role of chance The mean follicle density in ovarian tissue of TS patients before grafting was significantly lower compared to controls. FISH analysis showed that 97% of the oocytes in non-grafted tissue had a normal X chromosomal content. Follicles of TS patients contained mainly or exclusively 45,X granulosa cells, but different levels of X chromosome mosaicism between TS patients and between follicles of the same patient were observed. In total, 12/18 grafts contained follicles after 5 months xenografting. Follicle density of both TS patients and controls decreased significantly after xenotransplantation. Despite the presence of high level aneuploidy in granulosa and stromal cells in the tissue before grafting, secondary and even antral follicles were observed after xenotransplantation. Remarkably, a shift from high to low percentage of 45,X granulosa cells was observed during folliculogenesis. Immunohistochemistry showed that proliferating cell nuclear antigen (PCNA) positive follicles from TS patients increased during grafting to almost 100%. Secretion of anti-Müllerian hormone by granulosa cells was impaired before grafting in peri/postpubertal TS girls, but recovered after grafting. Expression of c-kit receptor and bone morphogenetic protein 15 (BMP15) in peri/postpubertal TS patients remained abnormal after xenotransplantation, while secretion of growth differentiation factor 9 (GDF9) and kit ligand was similar to controls. Limitations, reasons for caution In this study, only ovarian tissue of mosaic TS patients was examined, because the chances of finding ovarian follicles in this subgroup is considerably higher than in other TS patients. Subtle effects of reduced expression of c-kit receptor and BMP15 on folliculogenesis might have gone unnoticed. Wider implications of the findings Small follicles of mosaic TS patients are able to grow to secondary and antral follicles, despite the presence of aneuploid granulosa and stromal cells before grafting. Therefore, OTC could be a realistic option for young mosaic TS patients to preserve their fertility provided that sufficient numbers of follicles are present. Trial registration number NCT03381300

Development and evaluation of a novel xeno-free culture medium for human-induced pluripotent stem cells

BackgroundHuman-induced pluripotent stem cells (hiPSCs) are considered an ideal resource for regenerative medicine because of their ease of access and infinite expansion ability. To satisfy the sizable requirement for clinical applications of hiPSCs, large-scale, expansion-oriented, xeno-free, and cost-effective media are critical. Although several xeno-free media for hiPSCs have been generated over the past decades, few of them are suitable for scalable expansion of cultured hiPSCs because of their modest potential for proliferation and high cost.MethodsIn this study, we developed a xeno-free ON2/AscleStem PSC medium (ON2) and cultured 253G1 hiPSCs on different matrices, including iMatrix-511 and gelatin nanofiber (GNF) in ON2. Over 20 passages, we evaluated cell proliferation by doubling times; pluripotency by flow cytometry, immunofluorescence staining and qRT-PCR; and differentiation ability by three germ layer differentiation in vitro and teratoma formation in severe combined immunodeficiency mice, followed by histological analysis. In addition, we compared the maintenance effect of ON2 on hiPSCs with StemFit® AK02 (AK02N) and Essential 8™ (E8). Besides 253G1 hiPSCs, we cultivated different hiPSC lines, including Ff-l01 hiPSCs, ATCC® ACS-1020™ hiPSCs, and Down’s syndrome patient-specific ATCC® ACS-1003™ hiPSCs in ON2.ResultsWe found that 253G1 hiPSCs in ON2 demonstrated normal morphology and karyotype and high self-renewal and differentiation abilities on the tested matrices for over 20 passages. Moreover, 253G1 hiPSCs kept on GNF showed higher growth and stemness, as verified by the shorter doubling time and higher expression levels of pluripotent markers. Compared to AK02N and E8 media, 253G1 hiPSCs grown in ON2 showed higher pluripotency, as demonstrated by the increased expression level of pluripotent factors. In addition, all hiPSC lines cultivated in ON2 were able to grow for at least 10 passages with compact clonal morphology and were positive for all detected pluripotent markers.ConclusionsOur xeno-free ON2 was compatible with various matrices and ideal for long-term expansion and maintenance of not only healthy-derived hiPSCs but also patient-specific hiPSCs. This highly efficient medium enabled the rapid expansion of hiPSCs in a reliable and cost-effective manner and could act as a promising tool for disease modeling and large-scale production for regenerative medicine in the future.

Clinical Dose Preparation of [177Lu]Lu-DOTA-Pertuzumab Using Medium Specific Activity [177Lu]LuCl3 for Radioimmunotherapy of Breast and Epithelial Ovarian Cancers, with HER2 Receptor Overexpression.

Background: The overexpression of human epidermal growth factor receptor 2 (HER2) is commonly associated with metastatic breast cancer and epithelial ovarian cancer. The U.S. Food and Drug Administration (FDA) has approved Trastuzumab as an anti-HER2 agent for the metastatic breast and epithelial ovarian cancer. However, Trastuzumab has severe limitations in the treatment of metastatic breast cancer associated with ligand-dependent dimerization of HER2 receptor at the extracellular domain-II (ECD-II) region. The therapeutic approach in combination of pertuzumab and trastuzumab is found to be effective in preventing HER2 dimerization at the ECD-II region. The radioimmunotherapeutic approach, utilizing both these anti-HER2 agents (trastuzumab/pertuzumab), radiolabeled with [177Lu]Lu3+, has proved to be clinically efficacious with promising potential. Toward this, the formulation for clinical doses of [177Lu]Lu-DOTA-pertuzumab has been optimized using medium specific activity (0.81 GBq/μg) [177Lu]LuCl3. Materials and Methods: Preconcentrated pertuzumab was conjugated with p-NCS-benzyl-DOTA. Purified DOTA-benzyl-pertuzumab conjugate was radiolabeled with carrier-added [177Lu]LuCl3. Quality control parameters were evaluated for the [177Lu]Lu-DOTA-pertuzumab. In vivo biodistribution was carried out at different time points postadministration. Specific cell binding, immunoreactivity, and internalization were investigated by using SKOV3 and SKBR3 cells. Results: In this study, the authors reported a consistent and reproducible protocol for clinical dose formulations of [177Lu]Lu-DOTA-pertuzumab, with a radiochemical yield of 86.67% ± 1.03% and radiochemical purity (RCP) of 99.36% ± 0.36% (n = 10). Preclinical cell binding studies of [177Lu]Lu-DOTA-pertuzumab revealed specific binding with SKOV3 and SKBR3 cells up to 24.4% ± 1.4% and 23.2% ± 0.8%, respectively. The uptakes in SKOV3- and SKBR3-xenografted tumor in severe combined immunodeficiency mice were observed to be 25.9% ± 0.8% and 25.2% ± 1.2% ID/g at 48 and 120 h postinjection, respectively. Conclusions: A protocol was optimized for the preparation of ready-to-use clinical dose of [177Lu]Lu-DOTA-pertuzumab, in hospital radiopharmacy settings. The retention of RCP of the radiopharmaceutical, on storage in saline and serum, at -20°C, up to 120 h postradiolabeling, confirmed its in vitro stability.