Identification of a Highly Immunogenic Mouse Breast Cancer Sub Cell Line, 4T1-S
ABSTRACT: Recently, cancer immunotherapy has been paid much attention because of its improved efficacy and low frequency of adverse effects. A mouse breast cancer cell line, 4T1, has been known as poorly immunogeneic and highly metastatic cell line. In this study, we have identified a sub cell line of 4T1, designated as 4T1-Sapporo (4T1-S), which could induce a strong immune response against the same line. When 4T1-S was subcutaneously injected, striking enlargement of draining lymph nodes and increase of activated T cells were observed. The strong immune responses could not be observed when 4T1-S was injected to nude mice, indicating that this phenomenon is mediated by T cell response. Identification of 4T1-S characteristics may help to improve immunotherapy against breast cancer. 4T1-A1, 4T1-A2, 4T1-S1, 4T1-S2
Project description:Novel therapies targeting cancer stem cells (CSCs), which play critical roles in chemo- and radio-resistance, metastasis, and possibly resistance against cancer immunotherapy including granulocyte-macrophage colony-stimulating factor (GM-CSF) gene-transduced tumor cell vaccines, may provide beneficial clinical outcomes. Here, we used syngeneic immunocompetent mice that allowed precise evaluation of the immunogenicity of the side population (SP) isolated from 4T1 murine breast carcinoma (4T1-SP) cells as putative CSCs. 4T1-SP cells showed various stem cell properties including high capacities for colony formation and tumorigenicity as well as high expression of phosphorylated signal transducer and activator of transcription-3 and vascular endothelial growth factor that are inductive of immune tolerance. Despite these progressive malignant characteristics of 4T1-SP cells, subcutaneous injection of non-transmissible Sendai virus-mediated GM-CSF gene-transduced 4T1-SP (4T1-SP/GM) cells remarkably impaired their tumorigenicity compared with that of the controls. This impairment of tumorigenicity was partially dependent on CD8+ T cells in concert with CD4+ T cells and natural killer cells. Notably, therapeutic vaccinations using irradiated 4T1-SP/GM cells markedly suppressed tumor development of subcutaneously transplanted 4T1-SP cells compared with that of the controls including irradiated 4T1-non-SP/GM cells. Tumor suppression was accompanied by robust accumulation of mature dendritic cells at vaccination sites and systemic Th1-based cellular immunity. Moreover, vaccinations comprising primary 4T1-SP cells isolated from transplanted 4T1-SP tumors elicited antitumor effects. cDNA microarray analysis showed that 4T1-SP cells predominantly expressed genes of cancer-related antigens including cancer/testis antigens. Collectively, we demonstrate that SP cell-based vaccinations induce effective antitumor immunity that may improve the efficacy of SP cell-based immunotherapy. Gene expression profiles were compared between sorted 4T1-SP and 4T1-NSP cells.
Project description:Cesium-137 is a radionuclide of concern in fallout from reactor accidents or nuclear detonations. When ingested or inhaled, it can expose the entire body for an extended period of time, potentially contributing to serious health consequences ranging from acute radiation syndrome to increased cancer risks. In order to identify changes in gene expression that may be informative for detecting such exposure, and to begin examining the molecular responses involved, we have profiled global gene expression in mice injected with 137CsCl. We extracted RNA from the blood of control or 137CsCl-injected mice at 2, 3, 5, 20 or 30 days after exposure. Gene expression was measured using Agilent Whole Mouse Genome Microarrays, and the data was analyzed using BRB-ArrayTools. Three-month old male C57Bl/6 mice were injected intraperitoneally with 8.0 ± 0.3 MBq 137CsCl solution in a volume of 50 μL, or left as controls. Groups of treated and control mice were sacrificed at intervals during the first 2-30 days after exposure, and total blood was collected using cardiac puncture. RNA was extracted from the blood, globin-transcript reduced, and subjected to whole genome expression microarray analysis.
Project description:4T1 mouse mammary carcinoma cells have an autocrine FGFR active loop leading to constitutive activation of downstream signaling pathways. We found that FGFR inhibitors have a strong effect on 4T1 tumors in-vivo. We used microarray to understand the contribution of FGFR signaling to the tumor formation upon TKI258 treatment. Overall design: 4T1 cells were injected in the 4th mammary gland of Balb/C mice. After 7 days, daily treatment with TKI258 or water was performed for 14 days. At the end of the experiment, the RNA were extracted from three individual tumors per condition and hybridized on Affimetrix microarrays.
Project description:4T1 mouse mammary carcinoma cells have an autocrine FGFR active loop leading to constitutive activation of downstream signaling pathways. We found that FGFR inhibitors have a strong effect on 4T1 tumors in-vivo. We used microarray to understand the contribution of FGFR signaling to the tumor formation upon TKI258 treatment. 4T1 cells were injected in the 4th mammary gland of Balb/C mice. After 7 days, daily treatment with TKI258 or water was performed for 14 days. At the end of the experiment, the RNA were extracted from three individual tumors per condition and hybridized on Affimetrix microarrays.
Project description:To further analyze the change of microRNA(miRNA) between normal peritoneal macrophage(PEC) and TAM from early tumor(12 days after 4T1 cell injection) or TAM from late tumor(21 days after 4T1 cell injection) , we employed Agilent mouse microRNA microarray Rel 12.0 as a discovery platform to identify miRNAs Total RNA from peritoneal macrophage and TAM of early tumor (tumor formed for 12days after BALB/c mice injected wth 4T1 cell) or TAM of late tumor(tumor formed for 21days after BALB/c mice injected with 4T1 cell) were extracted and analyzed using Agilent mouse microRNA microarray platform, and changes of miRNA were screened out. Agilent mouse microRNA microarray is designed for the profiling of mouse miRNA .627 mouse miRNA and 39 mouse virus-related miRNA can be detected by our microRNA microarray.
Project description:Stabilizing mutations of NOTCH1 have been identified in about 10% of chronic lymphocytic leukemia (CLL) cases at diagnosis, with a higher frequency in unmutated IGHV (IGHV-UM) CLL, chemorefractory CLL and CLL in advanced disease phases. Clinically, the presence of NOTCH1 mutations is an independent predictor of overall survival in CLL and associates with resistance to anti-Cd20 immunotherapy. The Gene Expression Profile was generated to identify the peculiar molecular signatures of NOTCH1 mutated CLL in the context of IGHV-UM CLL. Constitutive gene expression in CLL cells bearing or not NOTCH1 mutation (c.7541_7542delCT). Five samples were selected for each category (WT vs MUT).
Project description:The transcriptional profile 23 cell lines derived from single clones of the 4T1 cell lines were assessed with RNAseq. The two clones with a strong propensity to intravasate were found to have 12 genes in common that were overexperessed relative to the other 21 clones. Overall design: Clone RNAseq 1) 23 clonal lines were established using single cell FACs sorting from the 4T1 mammary cancer cell line. 2) After establishing the lines the clones were assesed (in a pooled setting) for their capacity to intravasate the vascular system. 3) Transcriptional profiling was carried out using RNAseq. 4) Two clones were found to be strong intravasators and these were compared to the other clones to identify genes that were overexpressed (as compared to at least half of the other clones in both lines).
Project description:In order to understand the molecular mechanisms of changes seen in the adult mouse intestine in response to Sox2 overexpression, gene expression was analyzed in the ileum upon 2-day Doxycycline (DOX) treatment of mice containing Dox-inducible Sox2 transgene. Repression of Cdx2 and of its intestinal targets was observed and confirmed by Western blotting and immunostaining. This effect of Sox2 was distinct from the effect of overexpression of Oct4. 6-8 week old TetON-Sox2, rtTA controls (no TetOP-Sox2 transgene), TetON-Oct4, and TetON-Sox2 X TetON-Oct4 mice (2 replicates) were treated for 2 days with 2 mg/ml DOX or were left untreated. Intestines (ileum part) were collected and total mRNA was extracted.
Project description:In order to establish a profile of gene expression of tumours overexpressing PRH we performed agilient microarray analysis on four 4T1 tumour samples engineered to overexpress PRH, and four control tumours with normal PRH expression. cancer, colorectal metastasis to the liver and matched healthy tissues. The tumours had recieved no treatement prior to resection. The expression profiles were compared by one colour microarray analysis between PRH overexpression and control tumours. Genes consistently differentially expressed between the groups were taken forward for further analysis. Preparation of the tumours: 4T1 cells were infected with Adenoviral-PRH or empty Adenovirus at MOI 50 for 24 hrs. Adenoviral constructs described in Kershaw, R.M., et al., PRH/HHex inhibits the migration of breast and prostate epithelial cells through direct transcriptional regulation of Endoglin. Oncogene, 2014. 33(49): p. 5592-600. Cells were washed 4x with PBS, trypsinised, washed and resuspended in Opti-mem (Invitrogen) at 1.25x106 cells/ml and kept on ice for 20-30 min before injection. Aliquots 200 μl / 2.5x105 of the cells were injected into the no. 3 fat pad of 6-8 week old female BALB/C mice. Total RNA was extracted from each tumour using the Qiagen miRNAeasy kit, processed into cRNA using the Agilient low input, one colour labelling kit, hybridised onto the 8 x 66k whole human genome expression microarrays. Data was normalised in R64 and comparison matrices between matched Tumour and Healthy endothelum were constructed. Genes upregualated in all the comparisons were taken forward for further analysis.
Project description:To follow the earliest transcriptional changes in pre-metastatic tissues, we orthotopically injected 4T1 cells into the mammary fat pad of female nude mice. Control mice were orthotopically injected with PBS. 7 days post tumor engraftment, the mice were sacrificed and thoroughly perfused with ice cold PBS, and lung and liver samples were flash frozen in liquid nitrogen. Following RNA extraction, the lung and liver RNA samples were analyzed on MOE430A2 Affymetrix arrays using R/Bioconductor software. Expression array was performed on lung or liver tissue from control or tumor-bearing mice. Lung and liver tissues were isolated from 3 control mice (injected with PBS) and 3 tumor-bearing mice (injected with 4T1 cells) 7 days post injection.