Tumor Volume In Xenograft Model Research Articles

CD4-/CD8- double-negative tumor-infiltrating lymphocytes expanded from solid tumor tissue suppress the proliferation of tumor cells in an MHC-independent way.

Tumor-infiltrating lymphocytes (TILs) have shown remarkable clinical responses in some patients with advanced solid tumors. As a rare subset of TILs, CD4-/CD8- double-negative T cells (DNTs) were poorly known. This study aims to investigate the characteristics and function of CD3+CD4-CD8- TILs (double-negative TIL, DN-TILs) derived from solid tumor. DN-TILs were derived and expanded ex vivo from resected gastric carcinoma tissue and phenotyped by flow cytometry. The cytotoxicity of DN-TILs was determined against established tumor cell lines in vitro or through in vivo adoptive transfer into xenograft models. K562 cells were transferred with the HLA gene to verify whether the cytotoxicity of DN-TILs was MHC-independent. Flow cytometric analysis revealed a high-purity population of DN-TILs (> 97%) within CD3+ TILs, which expanded more than 800-folds in 2weeks, consisting of a mixture of alpha-beta (αβ) and gamma-delta (γδ) T-cell receptor (TCR)-expressing cells (with the majority being αβ-TCR, > 95%). Using single-cell RNA sequencing, the expanded DN-TILs were categorized into four main subsets, Natural Killer T cells (approximately 80%, 5563 in 7028), Progenitor cells, Germ cells and T helper2 cells. DN-TILs exhibited a broad anticancer cytotoxicity in a donor-unrestricted manner against various cancer cell lines derived from pancreatic cancer (Panc-1), gastric cancer (HGC-27), ovarian cancer (SKOV-3), malignant melanoma (A375). The cytotoxicity was MHC-independent, which was not altered in K562 transferring with HLA gene or not. DN-TILs significantly reduced tumor volume in xenograft models with superior tumor-homing ability and low off-target toxicity. Gastric carcinoma derived DN-TIL can target tumor cells in vitro and in vivo. DN-TILs have the potential to be used as a adoptive cell therapy for solid cancers with both the advantages of DNT and TIL.

Catalpol induces apoptosis in breast cancer in vitro and in vivo: Involvement of mitochondria apoptosis pathway and post-translational modifications

Breast cancer is a fatal cancer with the highest mortality in female. New strategies for anti-breast cancer are still urgently needed. Catalpol, an iridoid glycoside extracted from the traditional Chinese medicinal plant Rehmannia glutinosa, has shown anticancer efficacy in various cancer cells. However, its effect on breast cancer remains unclear. In this study, we aim to investigate the anti-breast cancer activity of catalpol and elucidate its underlying mechanism. Cell counting kit-8 (CCK-8) and morphology change showed that catalpol could inhibit the proliferation and viability of MCF-7 cells. Catalpol administration reduced the tumor volume in xenograft model. Catalpol induced apoptosis in MCF-7 cells confirmed by Hoechst 33342 staining and Annexin V-FITC/PI double staining. In vivo, catalpol also induced apoptosis as seen from the increased level of terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) in tumor. According to JC-1 and Dichlorodi-hydrofluorescein Diacetate (DCFH-DA) staining, loss of mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) generation was found in MCF-7 cells treated with catalpol. Furthermore, catalpol also increased the level of cytoplasmic cytochrome c and activity of caspase-3 in MCF-7 cells. Likewise, histopathological and immunohistochemical (IHC) assay also found that catalpol enhanced the levels of cytochrome c and caspase-3 in breast cancer tissues. Ultimately, acetylation, 2-hydroxyisobutyrylation and lactylation were dramatically increased, whereas succinylation, malonylation and phosphorylation were markedly decreased in the breast cancer tumor treated with catalpol. Taken together, catalpol inhibited breast cancer in vitro and in vivo through induction of apoptosis via mitochondria apoptosis pathway and regulation of protein post-translational modifications (PTMs). Thus, it can be considered as an excellent candidate compound for treatment of breast cancer.

Therapeutic Effects of Hybrid Liposomes Against Xenograft Mouse Model of Colorectal Cancer In Vivo Due to Long-term Accumulation.

To examine therapeutic effects of hybrid liposomes (HL) composed of L-α-dimyristylphosphatidylcholine (DMPC) and polyoxyethylene (25) dodecyl ether (C12(EO)25) against human colorectal cancer (WiDr) cells in vitro and in vivo. HL composed of 90 mol% DMPC and 10 mol% C12(EO)25 were prepared by the sonication method. Therapeutic effects of HL in the subcutaneous xenograft model mice of colorectal cancer were examined in vivo. Inhibitory effects of HL on the growth of WiDr cells were obtained along with apoptosis measurement. Selective accumulation of HL, including a fluorescence probe (indocyanine green; (ICG)), was observed for WiDr cells using a fluorescence microscope. Remarkable reduction of tumor volume in xenograft model of mice topically HL-treated without drugs after the subcutaneous inoculation of WiDr cells was verified in vivo. Induction of apoptosis in tumor cells of xenograft mice topically administered with HL was observed in micrographs on the basis of terminal deoxynucleotidyl tranferase-mediated dUTP-biotin nick end labeling (TUNEL) method. Accumulation of HL, including ICG, for long-term into the tumor was obtained in the xenograft model. Therapeutic effects of HL without any drugs in the murine xenograft model after subcutaneous inoculation of human colorectal cancer were revealed for the first time in vivo due to long-term accumulation.

MiR-137 contributes to drug susceptibility and chromosomal instability of multiple myeloma through targeting AURKA

Background: MicroRNAs are non-coding small RNAs that modulate protein expression and implicated in the pathogenesis of much kind of cancers, including multiple myeloma (MM). Previous study revealed that mic-137 was significant down regulated in MM patients compared with health donors. The purpose of this study is to investigate how miR-137 involved in pathogenesis and drug resistance in MM and its potential as prognostic biomarker. Materials and Methods: Real-time RT-PCR was performed to identify the expression of miR-137 in 6 MM cell lines and CD138+ cell sorted from 21 MM patients and 10 healthy donors. The methylation status of miR-137 CpG island was determined by bisulfite pyrosequencing, methylation specific polymerase chain reaction （MSP）and bisulfite sequencing PCR (BSP). The functional roles of miR-137 in MM were characterized by CCK-8 assay, soft agar clonogenic- formation, standard apoptosis assay and CGH array (CytoScanHD Array, Affymetrix). Effect of miR-137 on MM progression in vivo was assessed in the NOD/SCID mice models. In addition, to further identify miR-137 targets, we used bioinformatics analysis and confirmed by luciferase reporter assay. In addition, a total of 19 paired sequential samples with GEP and clinical data, obtained from published studies, were analyzed. Results: The expression of miR-137 was down regulated in all 6 MM cell lines and MM patients (p=0.0017). Further study revealed that methylation of the miR-137 CpG islands was frequently observed in MM cell lines (p<0.0001) and patients (p=0.004) compared with healthy donor by MSP and BSP. Functional study of Cck-8 assay, soft agar clonogenic assay and standard apoptosis assay showed miR-137-OE NCI-H929 significantly inhibited cell proliferation and increased cell drug sensitivity compared with NCI-H929-EV in vitro. Meanwhile, the tumorigenicity of miR-137 overexpressed NCI-H929 reduces tumor volume in xenograft models at day 20 and prolonged the survival of NOD-SCID mice following tail vein injection of miR-137-OE NCI-H929 compared with control (p=0.0198). Overexpression of miR-137 inactivates both AKT and MAPK/ERK Signaling pathway by up-regulating p53 and down-regulating pAKT. Interestingly, CGH-array analysis indicated miR-137 overexpression could decrease chromosomal instability in NCI-H929, such as gain at 1q21, loss at 12p13.31 and 14q22.2 etc. To explore the mechanism of miR-137 involved in chromosome instability, our results demonstrated miR-137 mainly through up regulated AURKA expression and luciferase assay confirmed miR-137 targeted at position 374-380 of AURKA 3’UTR. Conclusion: Our data suggested that miR-137 was epigenetic silenced and targeted AURKA expression to contribute to drug susceptibility and chromosomal Instability in MM. Future studies will be performed on how miR-137 targets AURKA linked to the p53 pathway and evaluating miR-137 as prognostic biomarkers and targets for treatment in MM. Disclosures No relevant conflicts of interest to declare.

MiR-137 Contributes to Drug Susceptibility and Chromosomal Instability of Multiple Myeloma through Targeting AURKA

Background: MicroRNAs are non-coding small RNAs that modulate protein expression and implicated in the pathogenesis of much kind of cancers, including multiple myeloma (MM). Previous study revealed that mic-137 was significant down regulated in MM patients compared with health donors. The purpose of this study is to investigate how miR-137 involved in pathogenesis and drug resistance in MM and its potential as prognostic biomarker.Materials and Methods: Real-time RT-PCR was performed to identify the expression of miR-137 in 6 MM cell lines and CD138+ cell sorted from 21 MM patients and 10 healthy donors. The methylation status of miR-137 CpG island was determined by bisulfite pyrosequencing, methylation specific polymerase chain reaction （MSP）and bisulfite sequencing PCR (BSP). The functional roles of miR-137 in MM were characterized by CCK-8 assay, soft agar clonogenic- formation, standard apoptosis assay and CGH array (CytoScan"HD Array, Affymetrix). Effect of miR-137 on MM progression in vivo was assessed in the NOD/SCID mice models. In addition, to further identify miR-137 targets, we used bioinformatics analysis and confirmed by luciferase reporter assay. In addition, a total of 19 paired sequential samples with GEP and clinical data, obtained from published studies, were analyzed.Results: The expression of miR-137 was down regulated in all 6 MM cell lines and MM patients (p=0.0017). Further study revealed that methylation of the miR-137 CpG islands was frequently observed in MM cell lines (p<0.0001) and patients (p=0.004) compared with healthy donor by MSP and BSP. Functional study of Cck-8 assay, soft agar clonogenic assay and standard apoptosis assay showed miR-137-OE NCI-H929 significantly inhibited cell proliferation and increased cell drug sensitivity compared with NCI-H929-EV in vitro. Meanwhile, the tumorigenicity of miR-137 overexpressed NCI-H929 reduces tumor volume in xenograft models at day 20 and prolonged the survival of NOD-SCID mice following tail vein injection of miR-137-OE NCI-H929 compared with control (p=0.0198). Overexpression of miR-137 inactivates both AKT and MAPK/ERK Signaling pathway by up-regulating p53 and down-regulating pAKT. Interestingly, CGH-array analysis indicated miR-137 overexpression could decrease chromosomal instability in NCI-H929, such as gain at 1q21, loss at 12p13.31 and 14q22.2 etc. To explore the mechanism of miR-137 involved in chromosome instability, our results demonstrated miR-137 mainly through up regulated AURKA expression and luciferase assay confirmed miR-137 targeted at position 374-380 of AURKA 3’UTR.Conclusion: Our data suggested that miR-137 was epigenetic silenced and targeted AURKA expression to contribute to drug susceptibility and chromosomal Instability in MM. Future studies will be performed on how miR-137 targets AURKA linked to the p53 pathway and evaluating miR-137 as prognostic biomarkers and targets for treatment in MM. DisclosuresNo relevant conflicts of interest to declare.

Abstract B57: Distinct interactions of Ras superfamily members with common protein targets

Abstract Ras superfamily members act as molecular switches to many key signaling pathways, including proliferation cytoskeletal integrity, differentiation and cellular cargo transport. The Ras subfamily, consisting of KRas, NRas and HRas are the most widely studied Ras-family members due to the high instance of Ras activating mutation in human cancers. Rab subfamily members are important regulators of endocytosis, consisting of Rab5 (early endosome), Rab7 (recycling endosome) and Rab11 (late endosome). We have discovered that common binding partners exist between these Ras subfamilies, resulting in distinct interactions and regulatory effects. Two such commonalities are scaffold protein ITSN1 and lipid kinase PI3KC2β. There is evidence that the learning disabilities associated with Down syndrome may be somewhat attributed to deregulated vesicular transport in the brain. DS patients also have a 10-20 fold increased risk of developing acute myeloid leukemia (AML). ITSN1, a regulator of endocytosis and activator of the oncogenic AKT pathway (1), is overexpressed in Down syndrome patients. PI3KC2β is a member of the PI3K family of lipid kinases, which phosphorylate phosphatidylinositol leading to AKT activation. Treatment of neuroblastoma and AML cell lines with PI3KC2β-specific inhibitors greatly decreases proliferation (2) and silencing PI3KC2β in neuroblastoma cell lines results in a 50% reduction in tumor volume in xenograft models. Our previous work demonstrated that nucleotide-free HRas preferentially binds to PI3KC2β, inhibiting its activity (see poster by JP O'Bryan; 3). The addition of ITSN1 disrupts this interaction, presumably restoring the active state of both proteins. In contrast, constitutively activated Rab5 and KRas preferentially interact with PI3KC2β. Interestingly KRas sequesters PI3KC2β to the plasma membrane. The current dogma states that Ras proteins must be GTP-loaded and associated with the plasma membrane to activate their classic effectors suggesting that KRas would be active in this capacity and may therefore positively regulate PI3KC2β activity. Here we present evidence of three Ras superfamily members interacting in distinct ways with the same target protein and provide support for their related yet contrasting roles in disease states. Citation Format: Russell Spencer Smith, Katy A. Wong, Angela Russo, John P. O'Bryan. Distinct interactions of Ras superfamily members with common protein targets. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B57. doi: 10.1158/1557-3125.RASONC14-B57

Assessment of intravenous pbi-shRNA PDX1 nanoparticle (OFHIRNA-PDX1) in yucatan swine

PDX1 (pancreatic and duodenal homeobox 1) is overexpressed in pancreatic cancer, and its reduction results in tumor regression. Bi-functional pbi-shRNA PDX1 nanoparticle (OFHIRNA-PDX1) utilizes the endogenous micro-RNA biogenesis pathway to effect cleavage- and non-cleavage-dependent degradation of PDX1 mRNA. We have shown that OFHIRNA-PDX1 reduces pancreatic tumor volume in xenograft models. Thus, we are now exploring biorelevant large animal safety of OFHIRNA-PDX1. Mini pigs were chosen as the biorelevant species based on the similarity of human and pig PDX1 target sequence. In the initial study, animals developed fever, lethargy, hyporexia and cutaneous hyperemia following administration of OFHIRNA-PDX1. Twenty-one days later, the same animals demonstrated less toxicity with a second OFHIRNA-PDX1 infusion in conjunction with a prophylactic regimen involving dexamethasone, diphenhydramine, Indocin and ranitidine. In a new group of animals, PDX1 protein (31 kDa) expression in the pancreas was significantly repressed at 48 and 72 h (85%, P=0.018 and 88%, P=0.013; respectively) following a single infusion of OFHIRNA-PDX1 but recovered to normal state within 7 days. In conclusion, a single intravenous infusion of OFHIRNA-PDX1 in conjunction with premedication in pigs was well tolerated and demonstrated significant PDX1 knockdown.

Co-treatment with arsenic trioxide and ganciclovir reduces tumor volume in a murine xenograft model of nasopharyngeal carcinoma

We have previously shown that disruption of promyelocytic leukemia nuclear bodies (PML NBs) is sufficient to activate the EBV lytic cycle thus making infected cells susceptible to ganciclovir (GCV) mediated killing in vitro. Here we show that co-administration of GCV and arsenic trioxide (ATO), a PML NB disruptor, reduces tumor volume in a xenograft model of nasopharyngeal carcinoma utilizing CNE1 cells. When administered at pharmacologic levels, both GCV and ATO reduced tumor growth while co-treatment with GCV + ATO resulted in a diminution of tumor volume. Treatment with GCV or ATO individually resulted in an increased number of apoptotic cells while co-treatment with GCV + ATO synergistically induced apoptosis. Treatment with ATO or co-treatment with GCV + ATO resulted in expression of EBV lytic proteins. These data suggest that co-treatment with GCV + ATO may provide an effective treatment for nasopharyngeal carcinoma patients.