Xenotransplantation Model Research Articles

Repurposing NAD Salvage Inhibitors for GCB Diffuse Large-B Cell Lymphoma

Diffuse large B-cell lymphomas (DLBCLs) are a heterogeneous group of diseases in terms of cell of origin, genetics and clinical outcome. About 30% of all DLBCL patients represent an unmet clinical need as they either do not respond to the standard first line chemo-immunotherapy or recur after initial remission. DLBCL classifications based on the NMF (Non-Negative Matrix Factorization) and LymphGen algorithms have led to the identification of genetic subtypes based on the co-occurrence of specific lesions. Repositioning drugs that are approved or in active clinical development for other indications can be a powerful strategy to match these newly uncovered DLBCL subtypes to targeted therapies.We have previously shown that the screening of large panels of DLBCL cell lines representative of the genetics of the disease can facilitate the repositioning of approved drugs for biomarker-selected populations of DLBCL patients. Repositioned drugs can then be rapidly translated to the clinical use, as they have already been extensively characterized for their safety profile. As a proof of concept, we have demonstrated that Dasatinib, a Src/Abl inhibitor approved for B-cell Acute Lymphoblastic Leukemia and Chronic Myelogenous Leukemia, is highly effective in PTEN-positive DLBCLs, irrespective of their COO class (Scuoppo et al., PNAS 2019).Here we present the results of a new screening that was performed on a panel of eight cell lines (4 ABC- and 4 GCB-DLBCLs) to test the activity of 212 drugs, either approved or in advanced clinical development, for repositioning in DLBCL, followed by validation in a larger panel of 34 genetically characterized cell lines. Our results point to inhibitors of the Nicotinamide Phosphoribosyl Transferase (NAMPT) as potently active in 63% of GCB-DLBCLs. NAMPT catalyzes the conversion of Nicotinamide (NAM) to Beta-Nicotinamide Mononucleotide (Beta-NMN). This reaction is the rate-limiting step of the Nicotinamide Adenine Dinucleotide (NAD) salvage pathway, the major metabolic route of NAD regeneration in mammalian cells. We validated the on-target activity of NAMPT inhibitors by multiple genetic and pharmacological approaches. First, we found that the activities of three chemically distinct drugs (FK-866, STF-118804 and KPT-9274) were highly correlated across the 34 lines DLBCL panel. Second, we were able to abolish the activity of all three NAMPT inhibitors by supplementing cells with Beta-NMN. Third, we showed that transduction of the drug-resistant mutant NAMPT H191R offsets the activity of the drugs.Dose-response assays on the full DLBCL cell line panel confirmed ABC-DLBCL resistance and also highlighted the presence of sensitive (GCB-S) and resistant (GCB-R) GCB subsets that can be separated by a subnanomolar IC50 threshold. To generate biomarkers capable of predicting GCB-S patients, we examined the RNA-seq profiles and genetic make-ups of the DLBCL cell lines collection and observed that the GCB-S subset was associated to the LymphGen EZB subtype, characterized by the presence of EZH2 mutations and BCL2 translocations. Conversely, the GCB-R subtype was linked to a 5-gene expression signature for the Kynurenine De Novo pathway, an alternative route for NAD synthesis. Accordingly, expression of each of the five De Novo Kynurenine pathway genes induced resistance to NAMPT inhibitors. These results were validated in xenotransplants of luciferized DLBCL lines and Patient Derived Xenotransplant models (PDXs) that were transcriptionally classified for the status of the Kynurenine De Novo signature. Together, these data support the repositioning of NAMPT inhibitors as a therapeutically relevant strategy for EZB-type GCB-DLBCLs. DisclosuresPasqualucci: Astra Zeneca: Research Funding; Sanofi: Research Funding.

Combination Therapy of FLT3 Tyrosine Kinase Inhibitors and BH3 Mimetics Targeting Antiapoptotic MCL-1 Synergistically Eliminates FLT3-ITD Acute Myeloid Leukemia Cells in Vitro and In Vivo

Combination Therapy of FLT3 Tyrosine Kinase Inhibitors and BH3 Mimetics Targeting Antiapoptotic MCL-1 Synergistically Eliminates FLT3-ITD Acute Myeloid Leukemia Cells in Vitro and In Vivo

Genetic Dissection of Dyskeratosis Congenita-Causing Mutations in TINF2 Using Human Pluripotent and Hematopoietic Stem Cell Models

The length of telomeres, which cap the ends of linear chromosomes and provide genomic stability, is tightly regulated in adult stem cells. The telomere reserve in the stem cell population sets the replicative potential of its differentiated progeny. For this reason, abnormally short telomeres in stem cells restrict the number of cell divisions that their differentiated progenies can undergo, eventually resulting in stem cell depletion and tissue failure syndromes. Telomere biology disorders (TBDs) display a broad range of clinical features, age of onset, and severity, which are all correlated with the extent of abnormal telomere shortening. One such early-onset TBD is dyskeratosis congenita (DC), which is a bone marrow predisposition syndrome characterized by a mucocutaneous triad (oral leukoplakia, nail dystrophy, and abnormal skin pigmentation) as well as other conditions driven by premature tissue aging. The leading cause of death in DC patients is bone marrow failure and hematopoietic stem cell transplantation is the only definite intervention to restore hematopoiesis.TINF2, which encodes the TIN2 protein, is mutated in 12% of patients and thereby the second most frequently altered gene in DC cases. TIN2 is a member of the shelterin protein complex bridging the double-strand binding shelterin proteins TRF1/TRF2, and the TPP1/POT1 heterodimer. Such interactions implicate a complex role of TIN2 in telomere length regulation: First, TIN2 stabilizes TRF1, which is a negative regulator of telomere length. Secondly, TPP1, which recruits telomerase, strictly requires TIN2 for telomere elongation and maintenance. TINF2-DC mutations are uniformly heterozygous and localize to a 30 amino acid coding stretch in exon 6 called the ‘DC cluster’. TINF2-DC mutations usually arise de novo and result in an earlier disease onset, shorter telomeres, and a more severe manifestation compared to other heterozygous DC-causing mutations in genes such as TR and TERT, which encode the components of the telomerase enzyme.How TINF2-DC mutations cause telomere shortening is unknown. Specifically, whether telomere shortening is caused by reduced telomerase action at telomeres or by degradation of telomeric DNA remains unresolved as studies using different model systems report contrasting results. The discrepancy could be attributed to differences in the model systems used in the studies, highlighting the need for a genetically trackable, primary preclinical human model system.Here, we report the development of two novel endogenous, isogenic model systems to study TINF2-DC mutations. First, we generated human embryonic stem cells (hESCs) engineered to express the TINF2-DC T284R mutation from the endogenous locus, which recapitulated the short telomere phenotype observed in DC patients. Using this model, we identified a gene editing strategy that elongates telomeres in the mutant stem cells and eventually restores replicative potential of the differentiated cells. Next, we used a xenotransplantation model of donor-derived human hematopoietic stem cells (hHSCs) to test the effects of target gene modifications on telomere length and proliferative capacity in vivo. We demonstrate that our models robustly complement each other and offer direct insights into the disease mechanism as well as avenues to potential therapeutic approaches. [Display omitted] DisclosuresBertuch: Elixirgen Therapeutics: Consultancy; ImmunityBio: Current equity holder in publicly-traded company; NIH/NCI,: Research Funding; DOD: Research Funding; Hyundai Hope on Wheels: Research Funding.

Anti-thymoglobulin induction improves neonatal porcine xenoislet engraftment and survival.

Porcine islet xenotransplantation is a viable strategy to treat diabetes. Its translation has been limited by the pre-clinical development of a clinically available immunosuppressive regimen. We tested two clinically relevant induction agents in a non-human primate (NHP) islet xenotransplantation model to compare depletional versus nondepletional induction immunosuppression. Neonatal porcine islets were isolated from GKO or hCD46/GKO transgenic piglets and transplanted via portal vein infusion in diabetic rhesus macaques. Induction therapy consisted of either basiliximab (n=6) or rhesus-specific anti-thymocyte globulin (rhATG, n=6), combined with a maintenance regimen using B7 costimulation blockade, tacrolimus with a delayed transition to sirolimus, and mycophenolate mofetil. Xenografts were monitored by blood glucose levels and porcine C-peptide measurements. Of the six receiving basiliximab induction, engraftment was achieved in 4 with median graft survival of 14 days. All six receiving rhATG induction engrafted with significantly longer xenograft survival at 40.5 days (P=0.03). These data suggest that depletional induction provides superior xenograft survival to nondepletional induction, in the setting of a costimulation blockade-based maintenance regimen.

Humanized von Willebrand factor reduces platelet sequestration in ex vivo and in vivo xenotransplant models.

The transplantation of organs across species offers the potential to solve the shortage of human organs. While activation of human platelets by human von Willebrand factor (vWF) requires vWF activation by shear stress, contact between human platelets and porcine vWF (pvWF) leads to spontaneous platelet adhesion and activation. This non-physiologic interaction may contribute to the thrombocytopenia and coagulation pathway dysregulation often associated with xenotransplantation of pig organs in nonhuman primates. Pigs genetically modified to decrease antibody and complement-dependent rejection (GTKO.hCD46) were engineered to express humanized pvWF (h*pvWF) by replacing a pvWF gene region that encodes the glycoprotein Ib-binding site with human cDNA orthologs. This modification corrected for non-physiologic human platelet aggregation on exposure to pig plasma, while preserving in vitro platelet activation by collagen. Organs from pigs with h*pvWF demonstrated reduced platelet sequestration during lung (p ≤ .01) and liver (p ≤ .038 within 4h) perfusion ex vivo with human blood and after pig-to-baboon lung transplantation (p ≤ .007). Residual platelet sequestration and activation were not prevented by the blockade of canonical platelet adhesion pathways. The h*pvWF modification prevents physiologically inappropriate activation of human or baboon platelets by porcine vWF, addressing one cause of the thrombocytopenia and platelet activation observed with xenotransplantation.

MCPIP1 inhibits Wnt/\u03b2-catenin signaling pathway activity and modulates epithelial-mesenchymal transition during clear cell renal cell carcinoma progression by targeting miRNAs

Epithelial-mesenchymal transition (EMT) refers to the acquisition of mesenchymal properties in cells participating in tumor progression. One hallmark of EMT is the increased level of active β-catenin, which can trigger the transcription of Wnt-specific genes responsible for the control of cell fate. We investigated how Monocyte Chemotactic Protein-1-Induced Protein-1 (MCPIP1), a negative regulator of inflammatory processes, affects EMT in a clear cell renal cell carcinoma (ccRCC) cell line, patient tumor tissues and a xenotransplant model. We showed that MCPIP1 degrades miRNAs via its RNase activity and thus protects the mRNA transcripts of negative regulators of the Wnt/β-catenin pathway from degradation, which in turn prevents EMT. Mechanistically, the loss of MCPIP1 RNase activity led to the upregulation of miRNA-519a-3p, miRNA-519b-3p, and miRNA-520c-3p, which inhibited the expression of Wnt pathway inhibitors (SFRP4, KREMEN1, CXXC4, CSNK1A1 and ZNFR3). Thus, the level of active nuclear β-catenin was increased, leading to increased levels of EMT inducers (SNAI1, SNAI2, ZEB1 and TWIST) and, consequently, decreased expression of E-cadherin, increased expression of mesenchymal markers, and acquisition of the mesenchymal phenotype. This study revealed that MCPIP1 may act as a tumor suppressor that prevents EMT by stabilizing Wnt inhibitors and decreasing the levels of active β-catenin and EMT inducers.

MicroRNA-574-3p Regulates HIF-α Isoforms Promoting Gastric Cancer Epithelial-Mesenchymal Transition via Targeting CUL2

Gastric cancer (GC) is the third leading cause of cancer-related deaths worldwide. MicroRNAs (miRNAs) have been widely validated as potential biomarkers for cancer treatment and diagnosis. This paper intends to study the effect and specific mechanism of miR-574-3p/CUL2 axis in GC. The miR-574-3p expression in GC tissues and cell lines was analyzed by reverse transcription polymerase chain reaction (RT-PCR). GC cell (N87) proliferation, migration and invasion were determined by the Brdu assay and Transwell assay, respectively. The tumor xenotransplantation model was established in vivo to test the effect of miR-574-3p or Cullin 2 (CUL2) on tumor growth. The relationship between miR-574-3p and CUL2 was predicated by bioinformatic analysis and verified by dual-luciferase reporter assay and RIP experiment. The expression of CUL2, hypoxia-induced transcription factor-1α (HIF-1α) as well as E-cadherin, Snail and Vimentin was monitored by western blot and immunohistochemistry. miR-574-3p was overexpressed in GC tissues and cells. Forced upregulation of miR-574-3p enhanced proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) of GC cells (N87), while downregulation of miR-574-3p resulted in reverse effects. Additionally, miR-574-3p promoted N87 cells growth and EMT in vivo. CUL2 was negatively regulated by miR-574-3p in N87 cells, and upregulation of CUL2 repressed the malignant behaviors of N87 cells. Moreover, CUL2 directly interacted with HIF-1α and suppressed HIF-1α expression both in vitro and in vivo. miR-574-3p targeted CUL2 to upregulate HIF-1α, thus facilitating the progression of GC.

Structure-based design, synthesis, and evaluation of inhibitors with high selectivity for PARP-1 over PARP-2

The poly (ADP-ribose) polymerase (PARP) inhibitors play a crucial role in cancer therapy. However, most approved PARP inhibitors have lower selectivity to PARP-1 than to PARP-2, so they will inevitably have side effects. Based on the different catalytic domains of PARP-1 and PARP-2, we developed a strategy to design and synthesize highly selective PARP-1 inhibitors. Compounds Y17, Y29, Y31 and Y49 showed excellent PARP-1 inhibition, and their IC50 values were 0.61, 0.66, 0.41 and 0.96 nM, respectively. Then, Y49 (PARP-1 IC50 = 0.96 nM, PARP-2 IC50 = 61.90 nM, selectivity PARP-2/PARP-1 = 64.5) was proved to be the most selective inhibitor of PARP-1. Compounds Y29 and Y49 showed stronger inhibitory effect on proliferation in BRCA1 mutant MX-1 cells than in other cancer cells. In the MDA-MB-436 xenotransplantation model, Y49 was well tolerated and showed remarkable single dose activity. The design strategy proposed in this paper is of far-reaching significance for the further construction of the next generation of selective PARP-1 inhibitors.

ENGRAFTMENT POTENTIAL OF CD34+ CELLS FROM SEVERE APLASTIC ANEMIA PATIENTS EXPANDED WITH UM171

Immune aplastic anemia (AA) is a hematological disease characterized by pancytopenia and bone marrow (BM) hypocellularity. It is caused by an immune attack against hematopoietic stem and progenitor cells (HSPC). Its treatment is based on allogeneic BM transplantation and immunosuppressive therapy. More recently, the addition of eltrombopag showed that the expansion of hematopoietic precursors is a therapeutic alternative. Fares et al. demonstrated that the small molecule UM171 is capable of expanding the most primitive CD34 + cell compartment of umbilical cord blood (UCB) ex vivo. Our research group showed that UM171 is also capable of effectively expanding immature subpopulations of CD34 + cells from patients with immune AA. The aim of this study was to evaluate the engraftment capacity of CD34 + cells from patients with AA expanded with UM171 in a murine xenotransplantation model. CD34 + cells were purified from either bone marrow of one patient or one UCB unit using immunomagnetic labeling with human CD34 MicroBeads, MS columns, and a magnetic separator. Cells were cultured for seven days in medium supplemented with cytokines and 35 nM UM171 or 0.1% DMSO (control) and then transplanted via retro-orbital injection into pre-conditioned 8-to-12-week-old female NSG-SGM3 (NSGS) mice. The progeny of 3×10 4 UCB CD34 + cells or 8.250 AA patient CD34 + cells was transplanted per mouse (four mice per experimental condition). The conditioning regimen consisted of two intraperitoneal injections of 12.5 mg/kg busulfan administered 48 and 24 hours before transplantation. Human hematopoietic cells were monitored in the murine BM by flow cytometry at week 20 post-transplantation. BM cells were collected from the two femurs and the two tibias of each animal and treated twice with red cell lysis, labeled with APC-Cy7 anti-mouse and PerCP-Cy5.5 anti-human CD45 antibodies, then analyzed on a flow cytometer using the FlowJo V.10 software. We found that both progenies of UCB and AA BM CD34 + cells cultured with UM171 showed an improved reconstitution potential in mice BM 20 weeks after transplant compared to DMSO, which was demonstrated by the detection of higher percentages of human CD45 + cells in the mice BM (UCB: UM171 = 10,07% ±2,29 vs DMSO = 3,73% ±1,99; AA BM: UM171 = 0,095% ±0,075 vs DMSO = 0,042% ±0,002; data represent mean ±Standard Error). Differences were not significant. We wondered whether the UM171 treatment would impact the engraftment capacity of the in vitro expanded CD34 + cells. The enhanced engraftment capacity enabled by UM171 could allow for immune AA patients to be treated with a novel strategy, autologous transplantation of their own CD34 + expanded cells. The present study indicates that UM171 could be a promising candidate drug for treating patients with immune AA. More replicates (patients and controls) are required to firmly establish the benefit of UM171 in conferring repopulation potential to ex vivo expanded CD34 + cells from AA patients. In initial experiments, in vitro treatment with UM171 appears to increase the engraftment of CD34 + cells isolated from UCB and from the BM of immune AA patients. These results need to be confirmed in additional experiments.

Corosolic acid inhibits cancer progression by decreasing the level of CDK19-mediated O-GlcNAcylation in liver cancer cells

Diabetes is an important risk factor for liver cancer, but its mechanism is unknown. Corosolic acid (CA) has been proven to have both hypoglycemic and antitumor effects, so revealing the function of CA can help us understand the relationship between diabetes and liver cancer. In previous studies, we confirmed that CA can effectively inhibit the expression of YAP, an important oncoprotein in HCC cells, and the proliferation of HCC cells. In addition, we also found that O-GlcNAcylation plays an indispensable role in HCC tumorigenesis. However, it is not clear whether CA can inhibit the effect of O-GlcNAcylation on HCC cells. In this study, the antitumor ability of CA was investigated by inhibiting the O-GlcNAcylation level and its corresponding mechanism. The results showed that HG (high glucose) could promote the proliferation of liver cancer cells, while CA could inhibit cell growth under HG conditions and tumor growth in a xenotransplantation model. CA can inhibit the activation of the HBP pathway and reduce the expression of YAP and OGT under HG conditions. Importantly, we found that CA can reduce YAP expression and O-GlcNAcylation by inhibiting the activity of CDK19. Overexpression of CDK19 partially reversed the CA-induced decrease in YAP and O-GlcNAcylation. This is the first evidence that CA can reduce the proliferative capacity of cells with high glucose levels and further inhibit tumor growth by inactivating the CDK19/YAP/O-GlcNAcylation pathway, suggesting that CA is a candidate drug for the development of treatments against diabetes-associated liver cancer.

Ursolic Acid Inhibits Breast Cancer Metastasis by Suppressing Glycolytic Metabolism via Activating SP1/Caveolin-1 Signaling.

Breast cancer remains the most common malignancy and the leading causality of cancer-associated mortality among women worldwide. With proven efficacy, Oldenlandia diffusa has been extensively applied in breast cancer treatment in Traditional Chinese Medicine (TCM) for thousands of years. However, the bioactive compounds of Oldenlandia diffusa accounting for its anti-breast cancer activity and the underlying biological mechanisms remain to be uncovered. Herein, bioactivity-guided fractionation suggested ursolic acid as the strongest anti-breast cancer compound in Oldenlandia diffusa. Ursolic acid treatment dramatically suppressed the proliferation and promoted mitochondrial-mediated apoptosis in breast cancer cells while brought little cytotoxicities in nonmalignant mammary epithelial cells in vitro. Meanwhile, ursolic acid dramatically impaired both the glycolytic metabolism and mitochondrial respiration function of breast cancer cells. Further investigations demonstrated that ursolic acid may impair the glycolytic metabolism of breast cancer cells by activating Caveolin-1 (Cav-1) signaling, as Cav-1 knockdown could partially abrogate the suppressive effect of ursolic acid on that. Mechanistically, ursolic acid could activate SP1-mediated CAV1 transcription by promoting SP1 expression as well as its binding with CAV1 promoter region. More meaningfully, ursolic acid administration could dramatically suppress the growth and metastasis of breast cancer in both the zebrafish and mouse xenotransplantation models of breast cancer in vivo without any detectable hepatotoxicity, nephrotoxicity or hematotoxicity. This study not only provides preclinical evidence supporting the application of ursolic acid as a promising candidate drug for breast cancer treatment but also sheds novel light on Cav-1 as a druggable target for glycolytic modulation of breast cancer.

NOTCH-mediated exvivo expansion of human hematopoietic stem and progenitor cells by culture under hypoxia.

SummaryActivation of NOTCH signaling in human hematopoietic stem/progenitor cells (HSPCs) by treatment with an engineered Delta-like ligand (DELTA1ext-IgG [DXI]) has enabled ex vivo expansion of short-term HSPCs, but the effect on long-term repopulating hematopoietic stem cells (LTR-HSCs) remains uncertain. Here, we demonstrate that ex vivo culture of human adult HSPCs with DXI under low oxygen tension limits ER stress in LTR-HSCs and lineage-committed progenitors compared with normoxic cultures. A distinct HSC gene signature was upregulated in cells cultured with DXI in hypoxia and, after 21 days of culture, the frequency of LTR-HSCs increased 4.9-fold relative to uncultured cells and 4.2-fold compared with the normoxia + DXI group. NOTCH and hypoxia pathways intersected to maintain undifferentiated phenotypes in cultured HSPCs. Our work underscores the importance of mitigating ER stress perturbations to preserve functional LTR-HSCs in extended cultures and offers a clinically feasible platform for the expansion of human HSPCs.

Ascorbic Acid Inhibits Liver Cancer Growth and Metastasis in vitro and in vivo, Independent of Stemness Gene Regulation.

Experimental and clinical evidence has indicated that the natural product ascorbic acid (AA) is effective in preventing and treating various types of cancers. However, the effect of AA on liver cancer metastasis has not yet been reported. Cancer stem cells (CSCs) play pivotal roles in cancer metastasis. Here, we demonstrated that AA selectively inhibited the viability of both liver cancer cells and CSCs, reduced the formation of cancer cell colonies and CSC spheres, and inhibited tumor growth in vivo. Additionally, AA prevented liver cancer metastasis in a xenotransplantation model without suppressing stemness gene expression in liver CSCs. Further study indicated that AA increased the concentration of H2O2 and induced apoptosis in liver CSCs. Catalase attenuated the inhibitory effects of AA on liver CSC viability. In conclusion, AA inhibited the viability of liver CSCs and the growth and metastasis of liver cancer cells in vitro and in vivo by increasing the production of H2O2 and inducing apoptosis. Our findings provide evidence that AA exerts its anti-liver cancer efficacy in vitro and in vivo, in a manner that is independent of stemness gene regulation.

Rhopaloic acid A induces apoptosis, autophagy and MAPK activation through ROS-mediated signaling in bladder cancer

BackgroundBladder cancer (BC) is a very common type of malignant cancer in men and new therapeutic strategies are urgently needed to reduce mortality. Several studies have demonstrated that Rhopaloic acid A (RA), a compound isolated from marine sponges, fights cancer but its potential anti-tumor effect on BC is still unknown. PurposeThe present study was aimed to explore the potential anti-tumor effects of RA against human BC cells and the underlying molecular mechanism. MethodsCell cytotoxicity was determined using the MTT and colony formation assays. Cell cycle distribution, apoptosis induction and generation of mitochondrial reactive oxygen species (ROS) were analyzed by flow cytometry. Mitochondrial membrane potential, acridine orange staining and intracellular ROS levels were observed using fluorescence microscopy. Levels of various signaling proteins were assessed using Western blotting. Furthermore, a zebrafish BC xenotransplantation model was used to confirm the anti-tumor effect of RA in vivo. ResultsTreatment with RA significantly suppressed the proliferation of BC cells that resulted from G2/M cycle arrest. Additionally, RA induced mitochondrial-mediated apoptosis and autophagy in BC cells. The death of BC cells induced by RA was rescued by treatment with inhibitors of apoptosis (Z-VAD-FMA) or autophagy (3-MA). RA activated the MAPK pathway and increased the production of cellular and mitochondrial ROS. Treatment with the ROS scavenger N-acetyl cysteine, effectively reversed the induction of apoptosis, autophagy, JNK activation and DNA damage elicited by RA. Finally, RA significantly inhibited tumor growth in a zebrafish BC xenotransplantation model. ConclusionTaken together, our findings indicate that RA induces apoptosis and autophagy and activates the MAPK pathway through ROS-mediated signaling in human BC cells. This RA-induced pathway offers insights into the molecular mechanism of its antitumor effect and shows that RA is a promising candidate for the treatment of BC.

The m6A Methyltransferase METTL14-Mediated N6-Methyladenosine Modification of PTEN mRNA Inhibits Tumor Growth and Metastasis in Stomach Adenocarcinoma.

BackgroundStomach adenocarcinoma (STAD) is a common reason for tumor-related fatalities globally, as it results in distant metastasis. Methyltransferase-like 14 (METTL14), a notable RNA N6-adenosine methyltransferase (m6A), plays a significant role in the growth of tumor through controlling the RNA working. This study aims to highlight METTL14 in STAD’s biological function and molecular mechanism.MethodsBioinformatics and immunohistochemical (IHC) assays have been utilized for the detection of METTL14 expression in the STAD. METTL14’s biological function has been shown while making use of HGC-27 and AGS cells in vitro experiments. MeRIP-qPCR and luciferase reporter assays were employed for the exploration of METTL14’s mechanism modifying the target of phosphatase and tensin homologue (PTEN). Subcutaneous xeno transplantation model and STAD liver metastasis orthotopic tumor model were used to study METTL14 in STAD in vivo.ResultsMETTL14 expression was substantially downregulated in STAD reflecting contribution to major tumors, progressed TNM stage as well as poor overall survival (OS) in STAD. Moreover, METTL14’s inhibition of STAD cells proliferation, migration and invasion has been verified in vitro assays. Furthermore, an identification of PTEN being METTL14-mediated m6A modification’s substrate has been made. METTL14’s overexpression highly enhanced PTEN mRNA m6A variation, stabilized PTEN mRNA and increased protein expression. Further, it has been found out that METTL14-mediated STAD cells inhibition of proliferation and invasion dependent on PTEN. At last, we demonstrated that METTL14 inhibit STAD growth and metastasis in vivo models.ConclusionsMETTL14 inhibits tumor growth and metastasis of STAD via stabilization of PTEN mRNA expression. Therefore, METTL14 is a potential biomarker of prognosis and therapeutic targets for STAD.

Xenotransplantation of Microencapsulated Parathyroid Cells as a Potential Treatment for Autoimmune-Related Hypoparathyroidism.

Hypoparathyroidism occurs as a part of a complex autoimmune syndrome or iatrogenically after neck surgery. The disease presents many challenges, such as hypocalcemia, hyperphosphatemia, and low/undetectable parathormone levels. Allotransplantation of parathyroid tissue or cells has been reported as a promising option to overcome these effects. Transplantation of microencapsulated parathyroid tissue or cells offers an immune escape, which particularly restores the parathyroid function for autoimmune-related hypoparathyroidism. So far, clinical and in vivo studies have demonstrated limited graft survival and instability for the available biocompatible materials. In addition, the transplant site, proper local isolation, and biocompatibility of materials are directly related to survival rate. A microencapsulated parathyroid xenotransplant model by using high guluronic acid-containing ultrapure alginate transplanted into rat omentum was tested in vivo for 1 year. After stability of empty microcapsules was ensured, parathyroid cells were microencapsulated and transplanted in rats, with results compared versus rats with naked (nonencapsulated) parathyroid cells (both groups followed for 64 weeks). Rats remained normocalcemic, and preinflammatory cytokine levels showed dramatic changes. Despite a delay posttransplant, parathormone levels increased significantly. All retrieved microencapsules elicited pericapsular fibrotic overgrowth; however, the fibrosis area was shown to be well tolerated. The possible role of accumulation/cell infiltration of immune response remains to be elucidated. In conjunction with this, the use of nonencapsulated parathyroid cells was also positively correlated with survival rates. A similar evaluation using ultrapure alginate materials and omental transplantation may enable the future determination for the long-term effects of correction of parathormone insufficiency in patients with severe hypocalcemic responses and other endocrine diseases.

ACBD3 is up-regulated in gastric cancer and promotes cell cycle G1-to-S transition in an AKT-dependent manner

It has been reported that ACBD3 is closely related to the malignant process of cells, but its role in gastric cancer has not been elucidated. This study aims to investigate the expression and function of ACBD3 in human gastric cancer. The Cancer Genome Atlas (TCGA) database were selected to analyze mRNA levels of ACBD3 in gastric cancer tissues and normal gastric epithelial tissues. qPCR and Western blot were conducted to detect the expression of ACBD3 in two normal gastric epithelial cell lines and five gastric cancer cell lines which were cultured in our laboratory. To exclude differences in individual background between different patients, we further detected the expression of ACBD3 in 8 pairs of malignant/non-malignant clinical gastric tissues. Through the establishment of stable cells, in vitro cell experiments and in vivo xenotransplantation models in mice, the role of ACBD3 in the proliferation of gastric cancer cells has been further explored. AKT inhibitors were used to deeply explore the molecular regulation mechanism of ACBD3. The results showed that the elevated ACBD3 in gastric cancer tissue were positively correlated with the clinical grade and prognosis of gastric cancer. In terms of molecular function, we found that ACBD3 can enhance the production and growth of gastric cancer cells. At the same time, the activation of AKT kinase played an important role in ACBD3's promotion of G1-to-S transition. The experiments generally indicate that ACBD3 is expected to become a potential diagnostic molecule or therapeutic target for gastric cancer.

Exogenous insulin-like growth factor 1 accelerates growth and maturation of follicles in human cortical xenografts and increases ovarian output in mice

To measure the influence of exogenous insulin-like growth factor 1 (IGF1) on follicle growth and maturation in human ovarian cortical xenografts. Xenotransplantation model. University-based research laboratory. Ovarian tissue was donated with consent and institutional review board approval by brain-dead organ donors or patients undergoing ovarian tissue cryopreservation for fertility preservation. Cortical fragments were transplanted into immunocompromised mice. Cryopreserved ovarian cortical fragments from four women (aged 19, 25, 33, and 46 years) were transplanted into the gluteus muscle of immunocompromised mice in a fibrin matrix containing endothelial cells that were transduced with lentiviral particles encoding secreted IGF1. Xenografts were recovered after 3, 8, and 14 weeks. In addition, C57/Bl6 mice underwent intraovarian injection of saline or recombinant IGF1 (60 μg), followed by superovulation, analysis of ethynyl-deoxyuridine incorporation, and ribonucleic acid sequencing of the whole ovaries. For xenografts: follicle count and distribution; antral follicle count; and corpora lutea/albicans count. For mice: follicle count and distribution; oocyte yield, ethynyl-deoxyuridine incorporation (granulosa cell proliferation); and ovarian transcriptomic signature. At 3 weeks, xenografts in the IGF1 condition revealed a decreased percentage of primary follicles and increased percentage of secondary follicles that were concentrated in the preantral subtype; at 8 weeks, an increase in secondary follicles was concentrated in the simple subtype; after 14 weeks, primordial follicles were reduced, and while the number of advanced follicles did not power the experiment to demonstrate significance, antral follicles reduced and corpora lutea increased. Supporting experiments in mice revealed an increase in normal oocytes following intraovarian injection of recombinant IGF1 (60 μg) as well as increased proliferative index among follicles of secondary and preantral stages. Ribonucleic acid sequencing analysis of the whole ovaries following injection of recombinant IGF1 (25 μg) revealed an acute (24 hours) upregulation of transcripts related to steroidogenesis and luteinization. Exogenous IGF1 advances the pace of growth among primordial, primary, and secondary stage follicles but results in near absence of antral stage follicles in long-term (14 weeks) xenografts. In mice, acute administration of IGF1 promotes follicle advance and increased oocyte yield. The results suggest that while superphysiological IGF1 alone advances the pace of growth among early/preantral follicles, a sustained and/or later-stage influence undermines antral follicle growth/survival or promotes premature luteinization. These findings provide a temporal framework for interpreting follicle growth/mobilization and may be useful in understanding the clinical application of human growth hormone in the context of assisted reproduction.

Poly(A)-Specific Ribonuclease (PARN) Promotes the Proliferation, Migration and Metastasis of Esophageal Cancer

Background: Esophageal cancer leads to the majority of cancer deaths in the world. Surgery, radiotherapy and chemotherapy are the main treatment methods, but due to its high recurrence and metastasis rate, the overall prognosis is poor. Poly(A)-specific ribonuclease (PARN) is a multifunctional enzyme molecule, which is involved in the occurrence and development of tumors. This study explores the relationship between PARN and the proliferation, metastasis and prognosis of esophageal cancer. Methods: The esophageal cancer tissues and adjacent normal tissues were collected from 91 patients with esophageal cancer after surgery. The expression level of PARN was detected by immunohistochemistry (IHC) using tissue microarray (TMA). PARN expression and its relationships with clinicopathological characteristics and prognosis was detected. In addition, stable PARN knockdown esophageal cancer cell lines TE-1 and Eca-109 were used to study its effect on the phenotype and behavior of tumor cells. Meanwhile, the effect of PARN silencing on tumor growth in vivo was verified by xenotransplantation model in nude mice. Finally, we examined possible downstream signaling mechanisms of PARN in esophageal cancer. Findings: PARN showed high expression in esophageal carcinoma tissues and its expression was significantly correlated with lymphatic metastasis and prognosis of patients. In the culture of PARN knockdown esophageal cancer cell lines, the growth and proliferation were significantly inhibited, and the invasion and migration ability of tumor cells were also significantly decreased. In addition, inhibition of PARN suppressed tumor growth and increased survival time in xenograft murine models. In the PARN knockdown group, BIM, IGFBP-5 and p21 were significantly increased, while the expression levels of anti-apoptotic proteins Survivin and STNF-R1 were significantly down-regulated. In addition, the expression levels of Akt, p-Akt, PIK3CA and CCND1 downstream of the signaling pathway regulating esophageal cancer progression were significantly down-regulated. Interpretation: These results suggest a previously undetected role of PARN as a suppressor of tumor development and its associations of prognosis in esophageal cancer through anti-apoptotic effect. Funding Information: None. Declaration of Interests: The authors declare no conflicts of interests. Ethics Approval Statement: Ethical approval for the study was approved by the Research Ethics Committee of the Second Affiliated Hospital of Guangxi Medical University and Zhujiang Hospital Affiliated to Southern Medical University, and written patient consent was obtained from all patients. Nude mice xenograft model - This study was approved by the Guangxi Medical University Ethics Committee.

Abstract 2976: Establishment of organoids derived from patients with advanced thoracic malignancies

Abstract Background: In the past decade, three-dimensional organoid culture derived from various tumors has been applied to cancer research. Compared to the conventional two-dimensional cell culture, organoid culture recapitulates in vivo system and furthermore, its high establishment rate may hold the potential to help decision-making for precision medicine. However, it remains challenging to establish organoids from advanced thoracic malignancies due to a limited amount of biopsy specimens. Here, we report the attempt to establish organoids from endoscopic biopsy specimens and pleural effusions in patients with advanced thoracic malignancies Methods: Organoid culture was attempted using biopsy specimens or pleural effusions from patients who underwent transbronchial biopsy, thoracoscopic biopsy or thoracentesis. After confirming the presence of malignant cells by rapid on-site evaluation, biopsy specimens were digested with collagenase and dispase at 37°C followed by being harvested and cultured in Matrigel. As for pleural effusions, mononuclear cells were isolated by ficoll density gradient centrifugation and CD45+ cells were depleted using anti-CD45 magnetic beads. Then, the resultant cells were cultured in Matrigel. Next-generation sequencing was performed to confirm cancer-related somatic mutations using Ion PGM system. Cell viability assay was performed by resazurin assay. Results:142 patients were registered in the study between May 2019 and October 2020 at Wakayama Medical University. Fifty-one cases were excluded because of negative diagnosis for malignancy, retraction of the consent or risk of bleeding associated with biopsy. We attempted to culture organoids using 98 samples from 91 patients. The types of malignancies were as follows: non-small cell lung cancers (NSCLC)/SCLC/others, 80/8/3. A total of 33 organoids were successfully cultured with sustainable proliferation and 16 were successfully cryopreserved. Established organoids were confirmed to harbor the same mutations as detected by clinical testing with tissue samples and to form tumors in a xenotransplantation model. HLCO-75, established from pleural effusion at disease progression after osimertinib treatment was genotyped and found to gain ERBB2 V777L mutation in exon 20 in addition to original EGFR L858R mutation. Marked growth-inhibitory effect by poziotinib, a HER2 tyrosine kinase inhibitor with IC50 value 6 nM was observed whereas not by TAS6417, another EGFR/HER2 exon 20 inhibitor, suggesting the potential of organoid culture for decision-making in cancer precision medicine. Conclusion: Our results suggest that organoid culture is feasible from small biopsy specimens and these organoids could be applied for personalized medicine. Citation Format: Jun Oyanagi, Yasuhiro Koh, Koichi Sato, Masanori Tanaka, Katsuyuki Furuta, Takeya Sugimoto, Ryota Shibaki, Shunsuke Teraoka, Daichi Fujimoto, Nahomi Tokudome, Hiroaki Akamatsu, Atsushi Hayata, Atsushi Hayata, Yuichi Ozawa, Hiroki Ueda, Nobuyuki Yamamoto. Establishment of organoids derived from patients with advanced thoracic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2976.