OBJECTIVE: To establish a MM patient-derived tumor xenograft model (MM-PDX) in zebrafish, and to evaluate the anti-myeloma activity of indirubin-3'-monoxime(I3MO) using this model. METHODS: Zebrafish embryos 2 days after fertilization were transplanted with fluorescence labeled myeloma primary tumor cells, the survival of primary tumor cells in zebrafish was observed at 0,16 and 24 hours after cell injection. The zebrafish embryos after tumor cell transplantation were randomly divided into control group, BTZ treatment and I3MO treatment group. Before and 24 hours after treatment with BTZ and I3MO, the positive area with calcein or Dil in zebrafish were observed under fluorescence microscope to reflect the survival of tumor cells, and it was verified. RESULTS: MM patient derived tumor cells survived in zebrafish. The construction of MM-PDX was successful. Compared with control group, the fluo- rescence area of the BTZ and I3MO treatment groups in zebrafish were significantly decreased(P<0.05), and BTZ and I3MO significantly inhibited the survival of MM cells in zebrafish. CONCLUSION MM-PDX model was successfully established. Zebrafish model derived from tumor cells of MM patients can be used as a tool for drug screening of MM.
Animals ; Humans ; Bortezomib/therapeutic use* ; Cell Line, Tumor ; Disease Models, Animal ; Drug Evaluation, Preclinical ; Heterografts ; Multiple Myeloma/pathology* ; Xenograft Model Antitumor Assays ; Zebrafish

Cabozantinib, mainly targeting cMet and vascular endothelial growth factor receptor 2, is the second-line treatment for patients with advanced hepatocellular carcinoma (HCC). However, the lower response rate and resistance limit its enduring clinical benefit. In this study, we found that cMet-low HCC cells showed primary resistance to cMet inhibitors, and the combination of cabozantinib and mammalian target of rapamycin (mTOR) inhibitor, rapamycin, exhibited a synergistic inhibitory effect on the in vitro cell proliferation and in vivo tumor growth of these cells. Mechanically, the combination of rapamycin with cabozantinib resulted in the remarkable inhibition of AKT, extracellular signal-regulated protein kinases, mTOR, and common downstream signal molecules of receptor tyrosine kinases; decreased cyclin D1 expression; and induced cell cycle arrest. Meanwhile, rapamycin enhanced the inhibitory effects of cabozantinib on the migration and tubule formation of human umbilical vascular endothelial cells and human growth factor-induced invasion of cMet inhibitor-resistant HCC cells under hypoxia condition. These effects were further validated in xenograft models. In conclusion, our findings uncover a potential combination therapy of cabozantinib and rapamycin to combat cabozantinib-resistant HCC.
Anilides/pharmacology* ; Animals ; Carcinoma, Hepatocellular/drug therapy* ; Cell Line, Tumor ; Cell Proliferation ; Endothelial Cells/metabolism* ; Humans ; Liver Neoplasms/drug therapy* ; Pyridines/pharmacology* ; Sirolimus/pharmacology* ; Xenograft Model Antitumor Assays

Peroxisome proliferator-activated receptors γ (PPARγ) is a master regulator that controls energy metabolism and cell fate. PPARγ2, a PPARγ isoform, is highly expressed in the normal prostate but expressed at lower levels in prostate cancer tissues. In the present study, PC3 and LNCaP cells were used to examine the benefits of restoring PPARγ2 activity. PPARγ2 was overexpressed in PC3 and LNCaP cells, and cell proliferation and migration were detected. Hematoxylin and eosin (H&E) staining was used to detect pathological changes. The genes regulated by PPARγ2 overexpression were detected by microarray analysis. The restoration of PPARγ2 in PC3 and LNCaP cells inhibited cell proliferation and migration. PC3-PPARγ2 tissue recombinants showed necrosis in cancerous regions and leukocyte infiltration in the surrounding stroma by H&E staining. We found higher mixed lineage kinase domain-like (MLKL) and lower microtubule-associated protein 1 light chain 3 (LC3) expression in cancer tissues compared to controls by immunohistochemistry (IHC) staining. Microarray analysis showed that PPARγ2 gain of function in PC3 cells resulted in the reprogramming of lipid- and energy metabolism-associated signaling pathways. These data indicate that PPARγ2 exerts a crucial tumor-suppressive effect by triggering necrosis and an inflammatory reaction in human prostate cancer.
Animals ; Cell Proliferation ; Humans ; Male ; Mice ; PC-3 Cells ; PPAR gamma/genetics* ; Prostatic Neoplasms/genetics* ; Signal Transduction ; Xenograft Model Antitumor Assays

BACKGROUND: Gene promoter methylation is a major epigenetic change in cancers, which plays critical roles in carcinogenesis. As a crucial regulator in the early stages of B-cell differentiation and embryonic neurodevelopment, the paired box 5 (PAX5) gene is downregulated by methylation in several kinds of tumors and the role of this downregulation in esophageal squamous cell carcinoma (ESCC) pathogenesis remains unclear. METHODS: To elucidate the role of PAX5 in ESCC, eight ESCC cell lines, 51 primary ESCC tissue samples, and eight normal esophageal mucosa samples were studied and The Cancer Genome Atlas (TCGA) was queried. PAX5 expression was examined by reverse transcription-polymerase chain reaction and western blotting. Cell apoptosis, proliferation, and chemosensitivity were detected by flow cytometry, colony formation assays, and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assays in ESCC cell lines with PAX5 overexpression or silencing. Tumor xenograft models were established for in vivo verification. RESULTS: PAX5 methylation was found in 37.3% (19/51) of primary ESCC samples, which was significantly associated with age (P = 0.007) and tumor-node-metastasis stage (P = 0.014). TCGA data analysis indicated that PAX5 expression was inversely correlated with promoter region methylation (r = -0.189, P = 0.011 for cg00464519 and r = -0.228, P = 0.002 for cg02538199). Restoration of PAX5 expression suppressed cell proliferation, promoted apoptosis, and inhibited tumor growth of ESCC cell lines, which was verified in xenografted mice. Ectopic PAX5 expression significantly increased p53 reporter luciferase activity and increased p53 messenger RNA and protein levels. A direct interaction of PAX5 with the p53 promoter region was confirmed by chromatin immunoprecipitation assays. Re-expression of PAX5 sensitized ESCC cell lines KYSE150 and KYSE30 to fluorouracil and docetaxel. Silencing of PAX5 induced resistance of KYSE450 cells to these drugs. CONCLUSIONS As a tumor suppressor gene regulated by promoter region methylation in human ESCC, PAX5 inhibits proliferation, promotes apoptosis, and induces activation of p53 signaling. PAX5 may serve as a chemosensitive marker of ESCC.
Animals ; Carcinoma, Squamous Cell/genetics* ; Cell Line, Tumor ; Cell Proliferation/genetics* ; Epithelial Cells/metabolism* ; Esophageal Neoplasms/genetics* ; Esophageal Squamous Cell Carcinoma/genetics* ; Gene Expression Regulation, Neoplastic ; Humans ; Mice ; PAX5 Transcription Factor/genetics* ; Tumor Suppressor Protein p53/genetics* ; Xenograft Model Antitumor Assays

OBJECTIVE: To investigate the effects and mechanism of bromodomain and extra-terminal (BET) inhibitor JQ1 on the double-expressor lymphoma (DEL) cell lines. METHODS: Protein expressions of cMyc and BCL-2 in 3 lymphoma cell lines were checked by Western blot so as to identify DEL cell lines. CCK-8 assay was used to detect the effects of JQ1 on anti-proliferation in the DEL cell lines. Western blot and RT-PCR were used to measure the protein and mRNA expressions of cMyc, BCL-2 and BCL-6 in DEL cell lines which treated by JQ1. Flow cytometry was used to detect the effect of JQ1 on cell apoptosis. RESULTS: Based on the expressions of cMyc and BCL-2, the SU-DHL6 and OCILY3 cell lines were confirmed as DEL cell lines. CCK-8 assay showed that the proliferation of DEL cell lines was inhibited by JQ1, which was similar to non-DEL cell lines and mainly regulated the expression of cMyc and BCL-6 but not BCL-2. JQ1 had no effects on apoptosis in the DEL cell lines. CONCLUSION BET inhibitor JQ1 has anti-tumor effect in the DEL cell lines, thus providing evidence for the therapeutic potential of BET inhibitor JQ1.
Apoptosis ; Azepines/pharmacology* ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Proto-Oncogene Proteins c-myc/metabolism* ; Sincalide/pharmacology* ; Triazoles/pharmacology* ; Xenograft Model Antitumor Assays

Head and neck cancer is the seventh common cancer in the world, and various existing treatment strategies provide modest benefit for most patients with head and neck cancer. Meanwhile, therapeutic strategies lacking molecular typing significantly hinder the development of individualized treatment for head and neck cancer. In recent years, connected by preclinical models, the novel ideal has gradually reached a consensus in terms of facilitating inter-transformation of clinical problems and basic achievements. As a bridge between basic research and clinical transformation, patient-derived xenografts (PDX) models precisely replicate genetic characteristics and tumor evolution, which are displaying great vitality in elucidating the mechanism of tumorigenesis and progression. Moreover, cohorts composed of several PDX models highlight the unique advantages of mice for drug screening and biomarker analysis for patients. This ideal preclinical model explores potential treatment strategies suited the ethical standards as much as possible for patients.
Animals ; Disease Models, Animal ; Head and Neck Neoplasms ; Heterografts ; Humans ; Mice ; Xenograft Model Antitumor Assays

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and is the fourth-leading cause of cancer-related deaths worldwide. HCC is refractory to many standard cancer treatments and the prognosis is often poor, highlighting a pressing need to identify biomarkers of aggressiveness and potential targets for future treatments. Kinesin family member 2C (KIF2C) is reported to be highly expressed in several human tumors. Nevertheless, the molecular mechanisms underlying the role of KIF2C in tumor development and progression have not been investigated. In this study, we found that KIF2C expression was significantly upregulated in HCC, and that KIF2C up-regulation was associated with a poor prognosis. Utilizing both gain and loss of function assays, we showed that KIF2C promoted HCC cell proliferation, migration, invasion, and metastasis both in vitro and in vivo. Mechanistically, we identified TBC1D7 as a binding partner of KIF2C, and this interaction disrupts the formation of the TSC complex, resulting in the enhancement of mammalian target of rapamycin complex1 (mTORC1) signal transduction. Additionally, we found that KIF2C is a direct target of the Wnt/β-catenin pathway, and acts as a key factor in mediating the crosstalk between Wnt/β-catenin and mTORC1 signaling. Thus, the results of our study establish a link between Wnt/β-catenin and mTORC1 signaling, which highlights the potential of KIF2C as a therapeutic target for the treatment of HCC.
Adult ; Aged ; Animals ; Carcinoma, Hepatocellular/pathology* ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Epithelial-Mesenchymal Transition/genetics* ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Intracellular Signaling Peptides and Proteins/metabolism* ; Kinesins/metabolism* ; Liver Neoplasms/pathology* ; Male ; Mice ; Mice, Inbred BALB C ; Middle Aged ; Neoplasm Staging ; Prognosis ; Protein Binding ; RNA, Small Interfering/metabolism* ; Survival Analysis ; Tumor Burden ; Wnt Signaling Pathway ; Xenograft Model Antitumor Assays ; beta Catenin/metabolism*

Adoptive immunotherapy based on chimeric antigen receptor-modified T cells (CAR-T) is one of the most promising strategies to treat malignant tumors, but its application in solid tumors is still limited. Glypican-3 (GPC3) is a meaningful diagnostic, therapeutic, and prognostic biomarker for hepatocellular carcinoma (HCC). The second/third generation GPC3-targeted CAR-T cells are generated to treat HCC. In order to improve the therapeutic effect, we constructed a fourth-generation lentiviral vector to express GPC3 CAR, human interleukin-7 (IL-7) and CCL19. Then the lentiviral vector and packaging plasmids were co-transfected into HEK293T cells to generate CAR lentiviral particles. Human T lymphocyte cells were transduced with CAR lentiviral to develop the fourth-generation GPC3-targeted CAR-T cells (GPC3-BBZ-7×19). In vitro, we used cell counting, transwell assay, luciferase bioluminescence assay and flow cytometry to compare the proliferation, chemotaxis, cytotoxicity and subtype distribution between GPC3-BBZ-7×19 CAR-T cells and the second generation GPC3-targeted CAR-T cells (GPC3-BBZ). In vivo, we established GPC3-positive HCC xenograft model in immunodeficient mice, then untransduced T cells (non-CAR-T) or GPC3-BBZ-7×19 CAR-T cells were injected. Tumor growth in mice was observed by bioluminescence imaging. Results showed that compared with GPC3-BBZ CAR-T, GPC3-BBZ-7×19 CAR-T cells had stronger proliferation, chemotactic ability, and higher composition of memory stem T cells (Tscm) (P values<0.05). However, there were no significant difference in cytotoxicity and cytokine secretion between them. In addition, GPC3-BBZ-7×19 CAR-T cells could significantly eliminate GPC3-positive HCC xenografts established in immunodeficient mice. Therefore, the fourth-generation GPC3-targeted CAR-T cells (secreting IL-7 and CCL19) are expected to be more durable and effective against HCC and produce tumor-specific memory, to provide a preclinical research basis for future clinical trials.
Animals ; Carcinoma, Hepatocellular ; Cell Line, Tumor ; Chemokine CCL19 ; metabolism ; Glypicans ; metabolism ; HEK293 Cells ; Humans ; Interleukin-7 ; metabolism ; Lentivirus ; genetics ; Liver Neoplasms ; Mice ; Receptors, Chimeric Antigen ; metabolism ; T-Lymphocytes ; metabolism ; Xenograft Model Antitumor Assays

OBJECTIVE: To investigate the difference of tumor formation in different mouse strains bearing patient-derived xenograft of esophageal squamous cell carcinoma(ESCC) and establish a better animal model for preclinical study of individualized treatment of ESCC. METHODS: The tumor tissues collected from 22 ESCC patients were used to establish tumor-bearing mouse models in B-NDG (NSG) mice and BALB/c nude mice. The tumor formation rate and tumor formation time were compared between the two mouse models, and HE staining, immunohistochemistry and genome sequencing were carried out to assess the consistency between transplanted tumor tissues in the models and patient-derived tumor tissues. RESULTS: The tumor-bearing models were established successfully in both NSG mice (50%, 11/22) and BALB/c nude mice (18.18%, 4/22). The average tumor formation time was significantly shorter in NSG mice than in BALB/c nude mice (75.95 91.67 days, < 0.001). In both of the mouse models, the transplanted tumors maintained morphological characteristics identical to those of patient-derived ESCC tumors. Genetic analysis showed that the xenografts in NSG mice had a greater genetic similarity to the patients' tumors than those in BALB/c nude mice ( < 0.0001). CONCLUSIONS Mouse models bearing xenografts of patient-derived ESCC can be successfully established in both NSG mice and BALB/c nude mice, but the models in the former mouse strain can be more reliable.
Animals ; Cell Line, Tumor ; Cell Proliferation ; Esophageal Neoplasms ; Esophageal Squamous Cell Carcinoma ; Heterografts ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Xenograft Model Antitumor Assays

We constructed the CS1-targeted second- and third-generation CAR-T cells with genetic engineered 4-1BB or/and ICOS as a costimulatory signaling molecule by use of lentiviral platform. The CS1-targeted second-generation CAR-T cells with ICOS or 4-1BB had similar anti-neoplastic activity. When effector/target ratio was 1:1, the CAR-T cells with ICOS showed better killing effect on IM9-lucgfp cells than those with 4-1BB. However, The CS1-targeted third-generation CAR-T cells exihibited lower cytolytic capacity against IM9-lucgfp cells than the CS1-targeted second-generation CAR-T cells when the ratio of effector/target was 1:1, 2:1 or 5:1. When the ratio of effector/target was 10:1, the killing efficacy of both the second- and third-generation CAR-T cells against IM9-lucgfp cells was more than 85%, significantly higher than that of the control T cells. Taken together, both the CS1-targeted second- and third-generation CAR-T cells with ICOS or/and 4-1BB could efficiently kill CS1-positive multiple myeloma cells, but the CS1-targeted second-generation CAR-T cells had more potent killing effect on CS1-positive multiple myeloma cells than the CS1-targeted third-generation CAR-T cells.
4-1BB Ligand/metabolism* ; Cell Line, Tumor ; Genetic Engineering ; Humans ; Inducible T-Cell Co-Stimulator Protein/metabolism* ; Multiple Myeloma/therapy* ; Signal Transduction ; T-Lymphocytes/chemistry* ; Xenograft Model Antitumor Assays