Enhancement of Tumorigenicity, Spheroid Niche, and Drug Resistance of Pancreatic Cancer Cells in Three-Dimensional Culture System.

The three-dimensional (3D) cell culture technique has been applied comprehensively as a variable platform for medical research, biochemical signal pathway analysis, and evaluation of anti-tumor treatment response due to an excellent recapitulation of a tumor microenvironment (TME) in the in vitro cultured cancer cells. Pancreatic cancer (PaC) is one of the toughest malignancies with a complex TME and refractory treatment response. To comprehensively study the TME of PaC, there is an eager need to develop a 3D culture model to decompose the cellular components and their cross interactions. Herein, we establish a 3D PaC culture system with cancer stem cell (CSC) and scalability properties. To validate our model, we tested the individual PaC cell and the combined effects with cancer-associated fibroblasts (CAFs) on cancer tumorigenicity, the cellular interaction through the CXCR3/CXCL10 axis, and cellular responses reflection of anti-cancer treatments. With the help of our 3D technology, a simulated malignant spheroid with important stromal populations and TME physiochemical properties may be successfully recreated. It can be used in a wide range of preclinical research and helpful in advancing basic and translational cancer biology.

Effect of chimeric antigen receptor T cells against protease-activated receptor 1 for treating pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a devastating malignancy with a 5-year survival rate of 6% following a diagnosis, and novel therapeutic modalities are needed. Protease-activated receptor 1 (PAR1) is abundantly overexpressed by both tumor cells and multiple stroma cell subsets in the tumor microenvironment (TME), thereby offering a suitable immunotherapy target. METHODS: A chimeric antigen receptor (CAR) strategy was applied to target PAR1 using a human anti-PAR1 scFv antibody fused to the transmembrane region with two co-stimulatory intracellular signaling domains of cluster of differentiation 28 (CD28) and CD137 (4-1BB), added to CD3ζ in tandem. RESULTS: The engineered PAR1CAR-T cells eliminated PAR1 overexpression and transforming growth factor (TGF)-ß-mediated PAR1-upregulated cancer cells by approximately 80% in vitro. The adoptive transfer of PAR1CAR-T cells was persistently enhanced and induced the specific regression of established MIA PaCa-2 cancer cells by > 80% in xenograft models. Accordingly, proinflammatory cytokines/chemokines increased in CAR-T-cell-treated mouse sera, whereas Ki67 expression in tumors decreased. Furthermore, the targeted elimination of PAR1-expressing tumors reduced matrix metalloproteinase 1 (MMP1) levels, suggesting that the blocking of the PAR1/MMP1 pathway constitutes a new therapeutic option for PDAC treatment. CONCLUSIONS: Third-generation PAR1CAR-T cells have antitumor activity in the TME, providing innovative CAR-T-cell immunotherapy against PDAC.

LncRNA MALAT1 Facilitates Ovarian Cancer Progression through Promoting Chemoresistance and Invasiveness in the Tumor Microenvironment.

Upregulation of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1, also known as nuclear-enriched abundant transcript 2 (NEAT2) or LINC00047) was found in various solid tumors, including epithelial ovarian cancer (EOC). MALAT1 is a long noncoding (lnc)RNA that regulates many functional signaling pathways, including tumorigenesis. Herein, we observed the consistent upregulation of MALAT1 in MYST4-overexpressing cell lines, while MALAT1 was frequently found to be upregulated in various types of clinical carcinoma tissues, especially EOC. To further investigate the lncRNA MALAT1 in EOC progression, the transduced overexpression of MALAT1 in EOC cell lines and cancer-associated fibroblasts (CAFs) was employed. We found that MALAT1 overexpression in EOC cell lines significantly increased drug resistance, cell migration, and invasion. Furthermore, the concomitant overexpression of MALAT1 in EOC cells and CAFs dramatically increased EOC cell invasion. Accordingly, a mechanistic investigation of MALAT1 overexpression in EOC cells showed that expressions of the cytokines interleukin (IL)-1ß and p-P38/p-NFκB/Cox2/prostaglandin E2 (PGE2) signaling were significantly increased, which stimulated inflammatory responses, whereas cell apoptosis was inhibited due to increased Bcl-2 levels and reduced Caspase3 levels. After MALAT1 was overexpressed in EOC cells, and the cyclin D1, p-PI3K, and p-Akt expressions increased, suggesting the promotion of tumor cell proliferation, while increased zinc finger E-box-binding homeobox-2 (ZEB2), yes-associated protein (YAP), and vimentin expression with E-cadherin downregulation indicated the enhancement of the epithelial-to-mesenchymal transition (EMT) in terms of metastasis, thereby triggering EOC progression. Together, our findings demonstrate how MALAT1 overexpression facilitates an oncogenic function through inhibiting tumor cell apoptosis, combined with increasing tumor cell inflammation, proliferation, and invasion in the EOC tumor microenvironment. MALAT1 is thus a potential diagnostic marker and therapeutic for this malignancy.

The Molecular Landscape Influencing Prognoses of Epithelial Ovarian Cancer.

Epithelial ovarian cancer (EOC) is one of the major increasing lethal malignancies of the gynecological tract, mostly due to delayed diagnosis and chemoresistance, as well as its very heterogeneous genetic makeup. Application of high-throughput molecular technologies, gene expression microarrays, and powerful preclinical models has provided a deeper understanding of the molecular characteristics of EOC. Therefore, molecular markers have become a potent tool in EOC management, including prediction of aggressiveness, prognosis, and recurrence, and identification of novel therapeutic targets. In addition, biomarkers derived from genomic/epigenomic alterations (e.g., gene mutations, copy number aberrations, and DNA methylation) enable targeted treatment of affected signaling pathways in advanced EOC, thereby improving the effectiveness of traditional treatments. This review outlines the molecular landscape and discusses the impacts of biomarkers on the detection, diagnosis, surveillance, and therapeutic targets of EOC. These findings focus on the necessity to translate these potential biomarkers into clinical practice.

Alterations of Specific Lymphocytic Subsets with Aging and Age-Related Metabolic and Cardiovascular Diseases.

To investigate the association of immunosenescence with aged-related morbidity in the elderly, a clinical study was conducted to analyze and compare the alterations in peripheral blood (PB) T-cell subsets among young healthy (YH) controls, elderly healthy (EH) controls, and age-matched elderly patients with metabolic diseases (E-MDs), with cardiovascular diseases (E-CVDs) or with both (E-MDs/E-CVDs). The frequencies of CD3T, CD8T and invariant natural killer T (iNKT) cells were decreased in the EH, E-MD and E-CVD cohorts, indicating a decline in defense function. Although CD4T and regulatory T (Treg) cell frequencies tended to increase with aging, they were lower in patients with E-MDs and E-CVDs. Subset analyses of T-cells consistently showed the accumulation of senescent T-cell in aging and in patients with E-MDs and E-CVDs, compared with YH volunteers. These accumulated senescent T-cells were undergoing apoptosis upon stimulation due to the replicative senescence stage of T-cells. In addition, serum levels of cytokines, including interferon (IF)-γ, transforming growth factor (TGF)-ß and growth differentiation factor (GDF)-15, consistently reflected alterations in T-cell subsets. This study demonstrated that T-cell subset changes with paralleled alterations in cytokines were associated with aging and age-related pathogenesis. These altered T-cell subsets and/or cytokines can potentially serve as biomarkers for the prevention, diagnosis and treatment of age-related morbidities.

Characteristics of CD133-Sustained Chemoresistant Cancer Stem-Like Cells in Human Ovarian Carcinoma.

Cancer stem cells (CSCs) are considered to be the origin of ovarian cancer (OC) development, recurrence, and chemoresistance. We investigated changes in expression levels of the CSC biomarker, cluster of differentiation 133 (CD133), from primary OC cell lines to induction of CSC-spheres in an attempt to explore the mechanisms related to modulation of stemness, drug resistance, and tumorigenesis in CSCs, thus facilitating the search for new therapeutics for OC. The effect of CD133 overexpression on the induction of CSC properties was evaluated by sphere-forming assays, RT-qPCR, flow cytometry, cell viability assays, and in vivo xenograft experiments. Moreover, the potential signaling molecules that participate in CD133 maintenance of stemness were screened by RNA-sequencing. CD133 expression was upregulated during OCSC induction and chemotherapeutic drug treatment over time, which increased the expressions of stemness-related markers (SOX2, OCT4, and Nanog). CD133 overexpression also promoted tumorigenesis in NOD/SCID mice. Several signalings were controlled by CD133 spheres, including extracellular matrix receptor interactions, chemokine signaling, and Wnt signaling, all of which promote cell survival and cell cycle progression. Our findings suggest that CD133 possesses the ability to maintain functional stemness and tumorigenesis of OCSCs by promoting cell survival signaling and may serve as a potential target for stem cell-targeted therapy of OC.

CD4+ T cells engineered with FVIII-CAR and murine Foxp3 suppress anti-factor VIII immune responses in hemophilia a mice.

Although protein replacement therapy provides effective treatment for hemophilia A patients, about a third of severe patients develop neutralizing inhibitor antibodies to factor VIII. Adoptive transfer of regulatory T cells (Tregs) has shown promise in treating unwanted immune responses. In previous studies, transferred polyclonal Tregs ameliorated the anti-factor VIII immune responses in hemophilia A mice. In addition, factor VIII-primed Tregs demonstrated increased suppressive function. However, antigen-specific Tregs are a small fraction of the total lymphocyte population. To generate large numbers of factor VIII-specific Tregs, the more abundant murine primary CD4+ T cells were lentivirally transduced ex vivo to express Foxp3 and a chimeric antigen receptor specific to factor VIII (F8CAR). Transduced cells significantly inhibited the proliferation of factor VIII-specific effector T cells in suppression assays. To monitor the suppressive function of the transduced chimeric antigen receptor expressing T cells in vivo, engineered CD4+CD25+Foxp3+F8CAR-Tregs were sorted and adoptively transferred into hemophilia A mice that are treated with hydrodynamically injected factor VIII plasmid. Mice receiving engineered F8CAR-Tregs showed maintenance of factor VIII clotting activity and did not develop anti-factor VIII inhibitors, while control CD4+T cell or PBS recipient mice developed inhibitors and had a sharp decrease in factor VIII activity. These results show that CD4+ cells lentivirally transduced to express Foxp3 and F8CAR can promote factor VIII tolerance in a murine model. With further development and testing, this approach could potentially be applied to human hemophilia patients.

Engineering Chimeric Antigen Receptor T Cells against Immune Checkpoint Inhibitors PD-1/PD-L1 for Treating Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with a 5-year survival rate of 9%. Major obstacles to successful treatment of pancreatic cancer are the immunosuppressive tumor microenvironment (TME) and antigenic complexity or heterogeneity. Programmed death-ligand 1 (PD-L1) is expressed on PDAC and immunosuppressed cells within the TME, providing suitable immunotherapy targets. We applied a chimeric antigen receptor (CAR) strategy to target immune checkpoint programmed death-1 (PD-1)/PD-L1 interactions. Lentiviral vectors were used to express the extracellular domain of human PD-1 (PD-1-CD28-4-1BB activating chimeric receptor [PD1ACR]) or the single-chain variable fragment (scFv) region of anti-PD-L1 (PDL1CAR) that binds to PD-L1, and each was fused to intracellular signaling domains containing CD3 zeta, CD28, and 4-1BB (CD137). Both engineered CAR T cells recognized and eliminated PD-L1-overexpressing CFPAC1 cells efficiently at approximately 80% in vitro. Adoptive transfer of both CAR T cells enhanced T cell persistence and induced specific regression of established CFPAC1 cancer by >80% in both xenograft and orthotopic models. Ki67 expression in tumors decreased, whereas proinflammatory cytokines/chemokines increased in CAR T cell-treated mouse sera. PD1ACR and PDL1CAR obtained a similar therapeutic efficacy. Thus, these armed third-generation PD-L1-targeted CAR T cells confer antitumor activity and the ability to combat T cell exhaustion, providing a potentially new and innovative CAR T cell immunotherapy against pancreatic cancers.

Antifibrotic Effects of a Barbituric Acid Derivative on Liver Fibrosis by Blocking the NF-κB Signaling Pathway in Hepatic Stellate Cells.

Hepatic stellate cells (HSCs) are the major profibrogenic cells that promote the pathogenesis of liver fibrosis. The crosstalk between transforming growth factor-ß1 (TGF-ß1) signaling and lipopolysaccharide (LPS)-induced NF-κB signaling plays a critical role in accelerating liver fibrogenesis. Until now, there have been no FDA-approved drug treatments for liver fibrosis. Barbituric acid derivatives have been used as antiasthmatic drugs in the clinic; however, the effect of barbituric acid derivatives in treating liver fibrosis remains unknown. In this study, we synthesized a series of six barbituric acid (BA) derivatives, and one of the compounds, BA-5, exhibited the best ability to ameliorate TGF-ß1-induced HSC activation without overt cytotoxic effects. Then, we treated HSCs and RAW264.7 macrophages with BA-5 to analyze the cross-talk of anti-fibrotic and anti-inflammatory effects. Carbon tetrachloride (CCl4)-induced liver fibrosis mouse model was used to evaluate the therapeutic effects of BA-5. Treatment with BA-5 inhibited TGF-ß1-induced α-SMA, collagen1a2, and phosphorylated smad2/3 expression in HSCs. Furthermore, BA-5 treatment reversed the LPS-induced reduction in BAMBI protein and decreased IκBα and NF-κB phosphorylation in HSCs. NF-κB nuclear translocation, MCP-1 secretion, and ICAM-1 expression were also inhibited in BA-5-treated HSCs. Conditioned medium collected from BA-5-treated HSCs showed a reduced ability to activate RAW264.7 macrophages by inhibiting the MAPK pathway. In the mouse model, BA-5 administration reduced CCl4-induced liver damage, liver fibrosis, and F4/80 expression without any adverse effects. In conclusion, our study showed that the barbituric acid derivative BA-5 inhibits HSCs activation and liver fibrosis by blocking both the TGF-ß1 and LPS-induced NF-κB signaling pathways and further inhibits macrophages recruitment and activation.

Treatment with a new benzimidazole derivative bearing a pyrrolidine side chain overcomes sorafenib resistance in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a major cause of cancer-related death worldwide. Currently, sorafenib is the standard first-line drug for patients with advanced HCC. However, long-term exposure to sorafenib often results in reduced sensitivity of tumour cells to the drug, leading to acquired resistance. Therefore, developing new compounds to treat sorafenib resistance is urgently needed. Although benzimidazole and its derivatives have been reported to exert antimicrobial and antitumour effects, the anti-drug resistance potential of these molecules is still unknown. In this study, we established sorafenib-resistant (SR) cell lines and an acquired sorafenib resistance xenograft model. We showed that treatment with a benzimidazole derivative bearing a pyrrolidine side chain (compound 9a) inhibited the proliferation of SR cells by blocking the phosphorylation of AKT, p70S6 and the downstream molecule RPS6. In addition, caspase 3/PARP-dependent apoptotic signals were induced in 9a-treated cells. Regarding epithelial-mesenchymal transition (EMT) activities, 9a treatment significantly suppressed the migration of SR cells. In particular, the levels of EMT-related transcription factors (snail, slug and twist) and mesenchymal markers (vimentin and N-cadherin) were downregulated. In the acquired sorafenib resistance xenograft model, compound 9a administration decreased the growth of tumours with acquired sorafenib resistance and the expression of the HCC markers α-fetoprotein, glypican 3 and survivin. In conclusion, treatment with this compound may be a novel therapeutic strategy for patients with sorafenib resistance.

HMGCS2 Mediates Ketone Production and Regulates the Proliferation and Metastasis of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the most common primary malignant tumor worldwide; however, the traditional therapeutic approaches and survival rates are still limited. To improve current therapies, it is necessary to investigate the molecular mechanisms underlying liver cancer and to identify potential therapeutic targets. The aims of this study were to verify the mechanisms and therapeutic potential of the ketogenesis rate-limiting enzyme 3-Hydroxymethylglutaryl-CoA synthase 2 (HMGCS2) in HCC. Immunohistochemical staining of human liver disease tissue arrays showed that HMGCS2 is abundantly expressed in normal liver tissues but is downregulated in cirrhosis and HCC tissues. In HCC patients, lower HMGCS2 expression was correlated with higher pathological grades and clinical stages. In our investigation of the molecular mechanisms of HMGCS2 in HCC, we showed that knockdown of HMGCS2 decreased ketone production, which promoted cell proliferation, cell migration, and xenograft tumorigenesis by enhancing c-Myc/cyclinD1 and EMT signaling and by suppressing the caspase-dependent apoptosis pathway. Ketone body treatment reduced the proliferation- and migration-promoting effects of HMGCS2 knockdown in cells. In contrast, HMGCS2 overexpression increased the intracellular ketone level and inhibited cell proliferation, cell migration, and xenograft tumorigenesis. Finally, ketogenic diet administration significantly inhibited liver cancer cell growth in mice. Our studies highlight the potential therapeutic strategy of targeting HMGCS2-mediated ketogenesis in liver cancer.

SRPX and HMCN1 regulate cancer­associated fibroblasts to promote the invasiveness of ovarian carcinoma.

Cancer­associated fibroblasts (CAFs) are known to be essential in cancer initiation and development. However, the role of CAFs in promoting ovarian cancer (OC) invasion remains to be fully elucidated. To address this in the present study, 49 clinical OC specimens were used to evaluate the roles of CAFs in promoting ovarian tumor migration and invasion and disease progression. It was found that the sushi repeat­containing protein, X­linked (SRPX) and hemicentin 1 (HMCN1) genes were significantly upregulated in CAFs from high­grade serous carcinoma (HGSC) and clear cell carcinoma (CCC) samples, the two major histological types of OC with frequently poor patient survival rates. The short hairpin (sh)RNA­mediated silencing of SRPX and HMCN1 in fibroblasts significantly suppressed the Transwell invasive activities of OC cells. Further experiments showed that SRPX and HMCN1 regulated the invasiveness of OC via the Ras homology family member A (RhoA) signaling pathway in fibroblasts. Therefore, the findings of the present study suggest that targeting the CAF genes, SRPX and HMCN1, can inhibit OC migration and invasion. These data highlight the importance of CAF­OC crosstalk signaling in cancer invasion and demonstrate the potential for improved efficacy of OC treatment by targeting CAF­SRPX/HMCN1.

Ex Vivo Expanded Human Vγ9Vδ2 T-Cells Can Suppress Epithelial Ovarian Cancer Cell Growth.

γδ-T-cells have attracted attention because of their potent cytotoxicity towards tumors. Most γδ-T-cells become activated via a major histocompatibility complex (MHC)-independent pathway by the interaction of their receptor, Natural Killer Group 2 Member D (NKG2D) with the tumor-specific NKG2D ligands, including MHC class I-related chain A/B (MICA/B) and UL16-binding proteins (ULBPs), to kill tumor cells. However, despite their potent antitumor effects, the treatment protocols specifically targeting ovarian tumors require further improvements. Ovarian cancer is one of the most lethal and challenging female malignancies worldwide because of delayed diagnoses and resistance to traditional chemotherapy. In this study, we successfully enriched and expanded γδ-T-cells up to ~78% from peripheral blood mononuclear cells (PBMCs) with mostly the Vγ9Vδ2-T-cell subtype in the circulation. We showed that expanded γδ-T-cells alone exerted significant cytotoxic activities towards specific epithelial-type OVCAR3 and HTB75 cells, whereas the combination of γδ-T cells and pamidronate (PAM), a kind of aminobisphosphonates (NBPs), showed significantly enhanced cytotoxic activities towards all types of ovarian cancer cells in vitro. Furthermore, in tumor xenografts of immunodeficient NSG mice, γδ-T-cells not only suppressed tumor growth but also completely eradicated preexisting tumors with an initial size of ~5 mm. Thus, we concluded that γδ-T-cells alone possess dramatic cytotoxic activities towards epithelial ovarian cancers both in vitro and in vivo. These results strongly support the potential of clinical immunotherapeutic application of γδ-T-cells to treat this serious female malignancy.

The overexpression of MYST4 in human solid tumors is associated with increased aggressiveness and decreased overall survival.

MYST4 (also called MORF and KAT6B) is one of the histone acetyltransferases with transcriptional regulatory activity. It was found to be overexpressed in ovarian cancer by a serial analysis of gene expression assays that focused on plant homeodomain-linked domain-containing genes. Compared to ovarian clear cell carcinomas and endometrioid carcinomas, MYST4 is significantly overexpressed in ovarian high-grade serous carcinomas (HGSCs) and was correlated with diminished patient survival in advanced stage HGSCs. Due to limited data on MYST4 in tumorigenesis and tumor progression, we explored the functional roles of MYST4 in human tumors. Besides the ovarian cancer cell line A2780, we chose two other types of human cancer cell lines expressing high mRNA levels of MYST4, SKBR3 and Huh7, for further in vitro investigation. Athymic nu/nu mice were utilized to facilitate the in vivo xenograft study. To search for potentially regulated genes, a microarray study comparing the expression profile before and after MYST4 knockdown was performed. Overexpression of MYST4 in HCCs was significantly associated with decreased survival. The knockdown of MYST4 significantly reduced cellular proliferation, migration, and cell cycle progression in all three cancer cell lines. Moreover, the knockdown of MYST4 in Huh7 cells suppressed tumor growth in a mouse xenograft model. Furthermore, based on our microarray study, we identified several downstream genes important in regulating tumor behaviors. Collectively, our results suggest that MYST4 is involved in cancer progression and contributes to a more aggressive behavior in human solid tumors. Targeting MYST4 represents an appealing strategy for the effective treatment of advanced solid tumors overexpressing MYST4.

The dataset from administration of single or combined immunomodulation agents to modulate anti-FVIII antibody responses in FVIII plasmid or protein primed hemophilia A mice.

Hemophilia A mice with pre-existing inhibitory antibodies against factor VIII (FVIII) were treated with single agents, AMD3100 and GCS-F, respectively. Inhibitor titers in treated mice and control HemA inhibitors mice were followed over time. Total B cells and plasma cells (PCs) were characterized by flow cytometry. HemA inhibitor mice were then treated with a combination regimen of IL-2/IL-2mAb complexes plus rapamycin and AMD3100. Finally, HemA inhibitor mice were treated with a new combination therapy using include IL-2/IL-2mAb complexes + Anti-CD20+AMD3100+G-CSF. The timeline of combination therapy was illustrated. Inhibitor titers following treatment in FVIII plasmid or protein induced inhibitor mice were evaluated overtime. A representative figure and gating strategies to characterize the subsets of Treg cells and B cells are presented. Please see http://dx.doi.org/10.1016/j.cellimm.2016.01.005 [1] for interpretation and discussion of these data and results.

Strategies to target long-lived plasma cells for treating hemophilia A inhibitors.

Long-lived plasma cells (LLPCs) can persistently produce anti-factor VIII (FVIII) antibodies which disrupt therapeutic effect of FVIII in hemophilia A patients with inhibitors. The migration of plasma cells to BM where they become LLPCs is largely controlled by an interaction between the chemokine ligand CXCL12 and its receptor CXCR4. AMD3100 combined with G-CSF inhibit their interactions, thus facilitating the mobilization of CD34(+) cells and blocking the homing of LLPCs. These reagents were combined with anti-CD20 to reduce B-cells and the specific IL-2/IL-2mAb (JES6-1) complexes to induce Treg expansion for targeting anti-FVIII immune responses. Groups of mice primed with FVIII plasmid and protein respectively were treated with the combined regimen for six weeks, and a significant reduction of anti-FVIII inhibitor titers was observed, associated with the dramatic decrease of circulating and bone marrow CXCR4(+) plasma cells. The combination regimens are highly promising in modulating pre-existing anti-FVIII antibodies in FVIII primed subjects.

Anti-CD20 as the B-Cell Targeting Agent in a Combined Therapy to Modulate Anti-Factor VIII Immune Responses in Hemophilia a Inhibitor Mice.

Neutralizing antibody formation against transgene products can represent a major complication following gene therapy with treatment of genetic diseases, such as hemophilia A. Although successful approaches have been developed to prevent the formation of anti-factor VIII (FVIII) antibodies, innovative strategies to overcome pre-existing anti-FVIII immune responses in FVIII-primed subjects are still lacking. Anti-FVIII neutralizing antibodies circulate for long periods in part due to persistence of memory B-cells. Anti-CD20 targets a variety of B-cells (pre-B-cells to mature/memory cells); therefore, we investigated the impact of B-cell depletion on anti-FVIII immune responses in hemophilia A mice using anti-CD20 combined with regulatory T (Treg) cell expansion using IL-2/IL-2mAb complexes plus rapamycin. We found that anti-CD20 alone can partially modulate anti-FVIII immune responses in both unprimed and FVIII-primed hemophilia A mice. Moreover, in mice treated with anti-CD20+IL-2/IL-2mAb complexes+rapamycin+FVIII, anti-FVIII antibody titers were significantly reduced in comparison to mice treated with regimens targeting only B or T cells. In addition, titers remained low after a second challenge with FVIII plasmid. Treg cells and activation markers were transiently and significantly increased in the groups treated with IL-2/IL-2mAb complexes; however, significant B-cell depletion was obtained in anti-CD20-treated groups. Importantly, both FVIII-specific antibody-secreting cells and memory B-cells were significantly reduced in mice treated with combination therapy. This study demonstrates that a combination regimen is highly promising as a treatment option for modulating anti-FVIII antibodies and facilitating induction of long-term tolerance to FVIII in hemophilia A mice.

In vivo expansion of regulatory T cells with IL-2/IL-2 mAb complexes prevents anti-factor VIII immune responses in hemophilia A mice treated with factor VIII plasmid-mediated gene therapy.

Generation of transgene-specific immune responses can constitute a major complication following gene therapy treatment. An in vivo approach to inducing selective expansion of Regulatory T (Treg) cells by injecting interleukin-2 (IL-2) mixed with a specific IL-2 monoclonal antibody (JES6-1) was adopted to modulate anti-factor VIII (anti-FVIII) immune responses. Three consecutive IL-2 complexes treatments combined with FVIII plasmid injection prevented anti-FVIII formation and achieved persistent, therapeutic-level of FVIII expression in hemophilia A (HemA) mice. The IL-2 complexes treatment expanded CD4(+)CD25(+)Foxp3(+) Treg cells five- to sevenfold on peak day, and they gradually returned to normal levels within 7-14 days without changing other lymphocyte populations. The transiently expanded Treg cells are highly activated and display suppressive function in vitro. Adoptive transfer of the expanded Treg cells protected recipient mice from generation of high-titer antibodies following FVIII plasmid challenge. Repeated plasmid transfer is applicable in tolerized mice without eliciting immune responses. Mice treated with IL-2 complexes mounted immune responses against both T-dependent and T-independent neoantigens, indicating that IL-2 complexes did not hamper the immune system for long. These results demonstrate the important role of Treg cells in suppressing anti-FVIII immune responses and the potential of developing Treg cell expansion therapies that induce long-term tolerance to FVIII.

L2dtl is essential for cell survival and nuclear division in early mouse embryonic development.

l(2)dtl (lethal (2) denticleless), is an embryonic lethal homozygous mutation initially identified in Drosophila melanogaster that produces embryos that lack ventral denticle belts. In addition to nucleotide sequence, bioinformatic analysis has revealed a conservation of critical functional motifs among the human L2DTL, mouse L2dtl, and Drosophila l(2)dtl proteins. The function of the L2DTL protein in the development of mammalian embryos was studied using targeted disruption of the L2dtl gene in mice. The knock-out resulted in early embryonic lethality. L2dtl-/- embryos were deformed and terminated development at the 4-8-cell stage. Microinjection of a small interfering RNA (siRNA) vector (siRNA-L2dtl) into the two-cell stage nuclei of wild-type mouse embryos led to cell cycle progression failure, termination of cell division, and, eventually, embryonic death during the preimplantation stage. Morphological studies of the embryos 54 h after injection showed fragmentation of mitotic chromosomes and chromosomal lagging, hallmarks of mitotic catastrophe. The siRNA-L2dtl-treated embryos eventually lysed and failed to develop into blastocysts after 72 h of in vitro culturing. However, the embryos developed normally after they were microinjected into one nucleus of the two-celled embryos. The siRNA studies in HeLa cells showed that L2dtl protein depletion results in multinucleation and down-regulation of phosphatidylinositol 3-kinase, proliferating cell nuclear antigen, and PTTG1/securin, which might partially explain the mitotic catastrophe observed in L2dtl-depleted mouse embryos. Based on these findings, we conclude that L2dtl gene expression is essential for very early mouse embryonic development.

Role of L2DTL, cell cycle-regulated nuclear and centrosome protein, in aggressive hepatocellular carcinoma.

L2DTL is a human ortholog of Drosophila lethal (2) denticleless, l(2)dtl. This study is to elucidate its function and clinicopathological significance in hepatocellular carcinoma (HCC) progression. We used RT-PCR, immunostaining, Western blotting, and centrosome isolation to determine the L2DTL expression and protein localization, and RNAi to analyze its role in tumor cell growth. L2DTL protein located to the nucleus in interphase and centered to centrosomes, with colocalization of gamma-tubulin and Aurora-A, throughout the cell cycle, and cofractionated with gamma-tubulin. L2DTL gene expression increased during G1/S phase, and the DNA synthesis in liver regeneration. L2DTL protein decreased in mitosis via degradation by the APC/C-Cdh1 complex. L2DTL was downregulated in the induced differentiation of HepG2 and NT2 cells. L2DTL downregulation by RNAi oligos led to reduced cancer cell growth and invasion capability in vitro, in which microarray analysis disclosed dysregulation of genes involved in cell cycle regulation, chromosome segregation, and cell division. L2DTL overexpressed in 59% of 270 resected, unifocal, primary HCCs. L2DTL overexpression correlated with bigger tumor (p=0.000003), high-grade (p=0.00003), and high-stage tumors with portal vein invasion (p=1x10(-8)). L2DTL overexpression was associated with a lower 10-year survival, particularly in the p53-mutated HCCs (p=0.00006). In conclusion, L2DTL encodes a nuclear protein with centrosome targeting in mitosis, and plays important roles in DNA synthesis, cell cycle progression, cytokinesis, proliferation, and differentiation. L2DTL overexpression is associated with enhanced metastatic potential of HCC, and contributes synergistically with p53 mutation, which leads to the loss of p53-governed checkpoints, toward advanced HCC with poor prognosis.