Microarray analysis of mouse gene expression in metronomic cyclophosphamide-treated rat glioma 9L xenografts implanted in scid mice
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ABSTRACT: Cyclophosphamide (CPA) treatment on a six-day repeating metronomic schedule induces a dramatic, innate immune cell-dependent regression of implanted gliomas. However, little is known about the underlying mechanisms of innate immune cell mobilization and recruitment, or about the role of DNA damage and cell stress response pathways in eliciting the anti-tumor immune responses linked to tumor regression. To address these questions, we compared untreated and 6-day metronomic cyclophosphamide-treated rat 9L gliosarcoma xenografts by mouse microarray analysis to identify responsive mouse (host) cell-specific factors. Rat glioma 9L tumors were implanted sc in scid immunodeficient mice then treated with cyclophosphamide at 140 mg/kg every 6 days. Tumors were collected 6 days after the fourth cyclophosphamide treatment. Tumor RNA was then analyzed on two color Agilent mouse expression microarrays comparing cyclophosphamide-treated RNA to untreated control tumor RNA.
Project description:Cyclophosphamide (CPA) treatment on a six-day repeating metronomic schedule induces a dramatic, innate immune cell-dependent regression of implanted gliomas. However, little is known about the underlying mechanisms of innate immune cell mobilization and recruitment, or about the role of DNA damage and cell stress response pathways in eliciting the anti-tumor immune responses linked to tumor regression. To address these questions, we compared untreated and 6-day metronomic cyclophosphamide-treated human U251 glioblastoma xenografts by human microarray analysis to identify responsive tumor cell-specific factors. Human glioma U251 tumors were implanted sc in scid immunodeficient mice then treated with cyclophosphamide at 140 mg/kg every 6 days. Tumors were collected 6 days after the second cyclophosphamide treatment and also 6 days after the third cyclophosphamide treatment. Tumor RNA was then analyzed on two color Agilent human expression microarrays comparing cyclophosphamide-treated RNA to untreated control tumor RNA.
Project description:Cyclophosphamide (CPA) treatment on a six-day repeating metronomic schedule induces a dramatic, innate immune cell-dependent regression of implanted gliomas. However, little is known about the underlying mechanisms of innate immune cell mobilization and recruitment, or about the role of DNA damage and cell stress response pathways in eliciting the anti-tumor immune responses linked to tumor regression. To address these questions, we compared untreated and 6-day metronomic cyclophosphamide-treated human U251 glioblastoma xenografts by mouse microarray analysis to identify responsive mouse (host) cell-specific factors. Human glioma U251 tumors were implanted sc in scid immunodeficient mice then treated with cyclophosphamide at 140 mg/kg every 6 days. Tumors were collected 6 days after the second cyclophosphamide treatment and also 6 days after the third cyclophosphamide treatment. Tumor RNA was then analyzed on two color Agilent mouse expression microarrays comparing cyclophosphamide-treated RNA to untreated control tumor RNA.
Project description:BACKGROUND: Cancer stem-like cells were isolated from human H460 non-small cell lung cancer cells by limiting dilution assays and by their holoclone morphology, followed by assessment of their self-renewal capacity, tumor growth, vascularity, and tumor blood perfusion. H460 holoclones were used to implant H460 holoclone-derived tumors, which grew slower than parental H460 tumors, but displayed significant increases in microvessel density and tumor blood perfusion. Microarray analysis identified differentially regulated genes in the holoclone-derived tumors, of which many were associated with angiogenesis. The differentially regulated genes include several small leucine-rich proteoglycans that may modulate angiogenesis and serve as novel therapeutic targets for inhibiting cancer stem cell-driven angiogenesis.(Cancer Letters Vol 346, Issue 1, 28 April 2014, Pages 63M-bM-^@M-^S73; PMID: 24334139; PMCID: PMC3947657). Implanted tumors derived from H460 parental cells, or from each of four independent H460 holoclones, were implanted subcutaneously in scid immunodeficient mice. Tumors derived from the 4 holoclones (designated H460/2E1, H460/2H3, H460/3F1, and H460/2E7) were used for global transcriptome/microarray analysis in direct comparison to H460 tumors grown from parental H460 cells. For each H460 holoclone-derived tumor line or H460 parental cell-derived tumor, total RNA was prepared from n=7 to n=10 individual tumors and used to prepare two independent pools of tumor RNA (each pool comprised of RNA isolated n=3-5 tumors) for use in a two color microarray analysis. Tumor RNA pools were prepared by combining equal amounts of RNA from each individual tumor to minimize the impasct of inter-tumoral variation on gene expression profiles. All RNAs had an RNA integrity number >8.0, determined using an Agilent Bioanalyzer 2100 instrument. cDNAs transcribed from pools of RNA for each holoclone-derived tumor line and for each parental H460 cell-derived tumor pool, were labeled with Alexa 647 or Alexa 555 dyes in a fluorescent reverse pair (dye swap) design for competitive hybridization to Agilent Whole Human Genome Microarrays.
Project description:Some neuroblastoma patients relapse after chemotherapy. Here, a Th-MYCNCPM32 mouse model usually have spontaneously tumours which are sensitive to chemotherapy. By treating mice with consecutive cycles of sublethal cyclophosphamide (CPM) using a personal dose escalation protocol (PDE), resistant tumours may develop. This experiment aims to compare the expression profiles (RNA-Seq) of sensitive and resistant MYCN driven tumours in order to understand the mechanisms behind resistance. Samples also presented cross-resistance to vincristine and doxorubicin.
Project description:The murine basal-like mammary carcinoma cell line H8N8 was transplanted into syngeneic WAP-T mice and growing tumors were subjected to a single dose of combination chemotherapy (100 mg/kg body weight cyclophosphamide, 5 mg/kg BW doxorubicin and 100 mg/kg BW 5-FU, short CAF). Mice of the control group were treated with one injection vehicle solution. Three cohorts of animals were generated: control, remission, and regrowth group. Tumor cells were isolated from lesions of each group, purified by flow cytometry to remove non-tumor cells and debris, and finally subjected to whole transcriptome analysis by mRNA-sequencing.
Project description:The EL4 mouse tumor model is widely utilized within the field of oncology due to its ease of use and excellent response to chemotherapy, with tumors typically experiencing a reduction in mass of 70% following chemotherapy treatment with cyclophosphamide and etoposide for three days. As not much is known regarding the biochemical changes that result in these drastic reductions in tumor mass, we decided to analyze treated and untreated tumors using shotgun proteomics to identify the changes that occur in relative protein abundance following chemotherapeutic treatment, which will help determine the biological processes resulting in tumor death.
Project description:This SuperSeries is composed of the following subset Series: GSE27421: Mouse bone marrow cyclophosphamide treated vs untreated GSE27422: Mouse peripheral blood leukocytes cyclophosphamide treated vs untreated GSE27423: Mouse spleen cyclophosphamide treated vs untreated Refer to individual Series
Project description:RNA-seq analysis was performed using RNA isolated from three tumor models (GL261 glioma, LLC Lewis lung carcinoma, B16F10 melanoma) implanted subcutaneousy in C57BL/6 mice, or in ICR scid mice. Mice were untreated or were treated with cyclophosphamide (CPA) given on a 6-day repeating metronomic schedule (CPA/6d), except as noted. Results from these global transcriptome analysis indicated substantial elevation of basal GL261 immune infiltration and strong activation by CPA/6d treatment of GL261 immune stimulatory pathways and their upstream regulators, but without preferential depletion of negative immune regulators compared to LLC and B16F10 tumors. In LLC tumors, where CPA/6d treatment was found to be anti-angiogenic, CPA/6d suppressed VEGFA target genes and down regulated cell adhesion and leukocyte transendothelial migration genes. In GL261 tumors implanted in adaptive immune-deficient scid mice, where CPA/6d-induced GL261 regression is incomplete and late tumor growth rebound can occur, T cell receptor signaling and certain cytokine-cytokine receptor responses seen in B6 mice were deficient. Extending the CPA treatment interval from 6 to 9 days (CPA/9d) â which results in a strong but transient natural killer cell response followed by early tumor growth rebound â induced fewer cytokines and increased expression of drug metabolism genes. Taken together, these findings elucidate molecular response pathways activated by intermittent metronomic CPA treatment and identify deficiencies that characterize immune-unresponsive tumor models and drug schedules. RNA isolated from various tumor cell lines implanted s.c in C57BL/6 mice or scid mice, untreated or treated with cyclophosphamide (CPA) given on a metronomic schedule, were prepared and used for stranded or unstranded RNA-seq.
Project description:Analysis of the effect of cyclophosphamide on peripheral blood leukocyte gene expression. Certain chemotherapeutic drugs such as cyclophosphamide can enhance the antitumor efficacy of immunotherapy because of their capacity to modulate innate and adaptive immunity. Indeed, it has been argued that this capacity may be more significant to chemotherapeutic efficacy in general than is presently appreciated. To gain insights into the core mechanisms of chemoimmunotherapy, we methodically profiled the effects of cyclophosphamide on gene expression in bone marrow, spleen and peripheral blood, and on cytokine expression in plasma and bone marrow of tumor-bearing mice. Gene and protein expression were modulated early and transiently by cyclophosphamide, leading to upregulation of a variety of immunomodulatory factors, including danger signals, pattern recognition receptors, inflammatory mediators, growth factors, cytokines, chemokines and chemokine receptors. These factors are involved in sensing cyclophosphamide myelotoxicity and activating repair mechanisms, which, in turn, stimulate immunoactivation events that promote efficacy. In particular, cyclophosphamide induced a T helper 17 (Th17)-related gene signature associated with an increase in Th17, Th1 and activated CD25+CD4+Foxp3- T lymphocytes and a slight recovery of regulatory T-cells. By analyzing gene and protein expression kinetics and their relationship to the antitumor efficacy of different therapeutic schedules of combination, we determined that optimal timing for performing adoptive immunotherapy is approximately 1 day after cyclophosphamide treatment. Together, our findings highlight factors that may propel the efficacy of chemoimmunotherapy, offering a mechanistic glimpse of the important immune modulatory effects of cyclophosphamide Four-condition experiment, Untreated mice - Cyclophosphamide-treated mice 1 day - Cyclophosphamide-treated mice 2 days - Cyclophosphamide-treated mice 5 days. Biological replicates: 4, controls: 4, independently harvested. Two replicates per array.