Gene Expression In Xenografts Research Articles

The Zebrafish Xenograft Platform-A Novel Tool for Modeling KSHV-Associated Diseases.

Kaposi’s sarcoma associated-herpesvirus (KSHV, also known as human herpesvirus-8) is a gammaherpesvirus that establishes life-long infection in human B lymphocytes. KSHV infection is typically asymptomatic, but immunosuppression can predispose KSHV-infected individuals to primary effusion lymphoma (PEL); a malignancy driven by aberrant proliferation of latently infected B lymphocytes, and supported by pro-inflammatory cytokines and angiogenic factors produced by cells that succumb to lytic viral replication. Here, we report the development of the first in vivo model for a virally induced lymphoma in zebrafish, whereby KSHV-infected PEL tumor cells engraft and proliferate in the yolk sac of zebrafish larvae. Using a PEL cell line engineered to produce the viral lytic switch protein RTA in the presence of doxycycline, we demonstrate drug-inducible reactivation from KSHV latency in vivo, which enabled real-time observation and evaluation of latent and lytic phases of KSHV infection. In addition, we developed a sensitive droplet digital PCR method to monitor latent and lytic viral gene expression and host cell gene expression in xenografts. The zebrafish yolk sac is not well vascularized, and by using fluorogenic assays, we confirmed that this site provides a hypoxic environment that may mimic the microenvironment of some human tumors. We found that PEL cell proliferation in xenografts was dependent on the host hypoxia-dependent translation initiation factor, eukaryotic initiation factor 4E2 (eIF4E2). This demonstrates that the zebrafish yolk sac is a functionally hypoxic environment, and xenografted cells must switch to dedicated hypoxic gene expression machinery to survive and proliferate. The establishment of the PEL xenograft model enables future studies that exploit the innate advantages of the zebrafish as a model for genetic and pharmacologic screens.

Effects of high-intensity focused ultrasound on apoptosis-associated gene expression in xenografts with human pancreatic cancer

Objective: To identify the expression of apoptosis-associated genes of high- intensity focused ultrasound(HIFU) in xenograft with human pancreatic cancer. Methods: Mice implated with human pancreatic cancer cells (YY-1) were divided into HIFU group or control group. Tumor cell apoptosis was verified by TUNEL. The expression of the apoptosis-associated genes was analyzed by Agilent Human Gene Expression. Selected genes was validated by quantitative real-time PCR(RT-PCR)and Western blot. Results: The rate of tumor cell apoptosis in HIFU group was higher than that of control group at 7, 14 days after HIFU treatment ((63.6%±15.2%)vs (19.0%±2.4%), P<0.01)and ((41.4%±7.3%)vs(18.0%±2.4%), P<0.01). Gene expression profiling revealed a total of 69 differentially expressed genes in related to apoptosis pathway, among which 44 genes were up-regulated, and 25 genes down-regulated. The RT-PCR results of selected 4 genes were consistent with those of gene expression profiling. The results of Western blot analysis at 7, 14 days after HIFU treatment showed that the expressions level of Bax protein in HIFU group was greater that of in control group ((0.39±0.11)vs (0.20±0.09), P<0.05)and ((0.46±0.12)vs(0.24±0.10), P<0.05), while the expressions level of Bcl-2 protein in HIFU group was lower than that of in control group ((0.68±0.14)vs(1.56±0.21), P<0.05)and((0.51±0.16)vs(1.57±0.22), P<0.05). Conclusions: HIFU could induce apoptosis and results in dramatic changes in gene expression, indicating that multiple pathways are involved. Although intrinsic pathway might be predominantly involved in HIFU-elicited apoptosis, further research is needed to clarify the detailed mechanisms.

Abstract 2439: Inhibition of STAT3 with the generation 2.5 antisense oligonucleotide, AZD9150 increases the chemosensitivity and decreases tumor-initiating potential of neuroblastoma cells

Abstract Neuroblastoma (NB) is the most common extra-cranial solid tumor in childhood. The long-term, event-free survival of high-risk NB remains ∼ 50% despite intensive multi-modality treatments. Activated JAK/STAT3 pathway plays an important role in many human cancers. In NB patients, cytokines activating STAT3 have been associated with poor patient outcome and have been implicated in the survival of a rare population of NB tumor initiating cells. Thus targeting STAT3 may be a promising therapeutic strategy for high-risk NB. To evaluate the biologic consequences of genetic targeting of STAT3, we assessed the effects of inhibition of STAT3 in NB cell lines containing a tetracycline (Tet)-inducible STAT3 expression plasmid. Additionally we evaluated pharmacogenomic inhibition of STAT3 using AZD9150, a generation 2.5, 16-nucleotide, antisense oligonucleotide (ASO) that is now in Phase I/II clinical trials. Studies were conducted in 3 representative NB cell line models (AS (MYCN-WT) and NGP and IMR32 (MYCN-Amplified)). Both the Tet-inducible STAT3 shRNA and AZD9150 reduced endogenous STAT3 mRNA and protein levels causing decreased transcription and expression of STAT3 target genes, such as CyclinD1, D3, and MYC/MYCN. In functional in vitro studies Tet-inducible STAT3 shRNA and AZD9150 decreased NB cell migration and clonogenicity in soft agar. In vivo, STAT3 inhibition by Tet-inducible STAT3 shRNA or AZD9150 alone had little effect on the growth of established tumors nor did it alter the survival of tumor-bearing mice, despite decreases in STAT3, P-STAT3 and target gene expression in xenografts from AZD9150-treated mice compared to those from ASO-treated mice. To assess whether inhibition of STAT3 altered the tumor initiating potential of NB cells, NB tumor xenograft cells from ASO or AZD9150 treated mice were re-implanted and secondary tumor growth assessed. At 200,000 and 20,000 ASO-treated NB cells, 100% of mice had tumors while only 40 and 20%, respectively of AZD9150-treated mice had tumors. These results indicate that the inhibition of STAT3 decreased the frequency of tumor initiating cells in the NB xenograft from AZD9150 treated mice. Since tumor initiating or stem-like cells are frequently more resistant to cytotoxic agents, we next evaluated a combination therapy with cisplatin. We found that in vitro either genetic shSTAT3 or AZD9150 mediated pharmacogenomic inhibition of STAT3 significantly increased the sensitivity of NB cells to cisplatin. Furthermore, in established NB tumors xenografts, the combination of STAT3 inhibition with cisplatin caused a 30% decrease in tumor size (P = 0.0092) and increased the survival of AZD9150 treated tumor-bearing mice compared to ASO-treated mice (P = 0.026). Our study supports the development of strategies targeting STAT3 in combination with conventional chemotherapy for patients with high-risk NB. Citation Format: Seiichi Odate, Shuang Yan, Veronica Veschi, Norris Lam, Zhihui Liu, Carol J. Thiele. Inhibition of STAT3 with the generation 2.5 antisense oligonucleotide, AZD9150 increases the chemosensitivity and decreases tumor-initiating potential of neuroblastoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2439.

Developmental patterns of mucin gene expression in human fetal small intestinal xenografts maintained in severe-combined immunodeficient mice.

The lack of a suitable animal model that expresses human intestinal mucin genes limits the study of mucin function. The aim of this study was to examine whether human fetal intestinal xenografts, known to model host-restricted interactions with human-specific pathogens, express mucin genes in an appropriate developmental pattern when transplanted into severe-combined immunodeficient (scid) mice. Expression profiles for eight mucin genes were examined in human fetal ileal xenografts transplanted ectopically into scid mice for 10 wk. In situ hybridization was performed on fetal, xenograft, and adult intestinal tissue sections with 35S-labeled oligonucleotides specific to human tandem repeat sequences for MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC6, and MUC7. Hybridization patterns observed with the MUC2, MUC3, MUC4, and MUC5AC probes demonstrated that mucin gene expression in xenografted fetal intestine was comparable to third trimester fetal and/or adult tissues. MUC2 and MUC5AC were expressed in a developmental-specific fashion. MUC5AC, expressed in first and early second trimester fetal bowel, was never detected in intestinal xenografts. MUC2 expression displayed a late fetal and/or adult-type hybridization pattern. MUC3 and MUC4 were not developmentally expressed. Appropriate developmental regulation of known intestinal mucin genes was recorded in ectopically grafted human fetal intestinal xenografts. Adult-like patterns of mucin gene expression in this model system will permit future studies aimed at characterizing cis/trans-acting factors that regulate mucin gene expression and function during development, disease, and wound healing and also in mucin-pathogen interactions during host defense.

Expression of the MDR1 Gene in Human Gastric and Colorectal Carcinomas

We measured expression of the MDR1 gene (also known as the PGY1 gene) in the human gastrointestinal tract. MDR1 messenger RNA (mRNA) levels were elevated in 13 of 15 colorectal carcinoma specimens and in six of 13 gastric carcinoma specimens. Well-differentiated colorectal carcinomas contained significantly higher concentrations of MDR1 mRNA than moderately differentiated colorectal carcinomas. Similarly, moderately differentiated gastric carcinomas contained higher concentrations of MDR1 mRNA than poorly differentiated gastric carcinomas. MDR1 gene expression in normal colorectal and gastric tissues adjacent to carcinomas was similar to that in the carcinomas. MDR1 gene expression in xenografts of colorectal and gastric carcinomas in nude mice was also investigated. Elevated expression of the MDR1 gene was seen in only four of 18 xenografts of colorectal carcinoma and was not seen in any xenografts of gastric carcinoma. P-glycoprotein was distributed over the luminal surface of the colorectal carcinoma. These results imply that the higher levels of MDR1 mRNA found in well-differentiated carcinomas derived from colorectal tissues are the results of increased expression of the MDR1 gene in the luminal surface cells. The level of expression of the MDR1 gene in colorectal and gastric carcinomas appears to correlate with the degree of differentiation and also appears to be affected by transplantation into nude mice.