Project description:CD4+ helper T (Th) cells are critical regulators of immune responses but their role in breast cancer is currently unknown. This work aims to characterize Th cells infiltrating invasive primary human breast tumors, analyze the influence by the tumor microenvironment and identify Th cell specific prognostic gene signatures. CD4+ T cells isolated from the tumor (TIL), axillary lymph node (LN) and blood (PB) of 10 patients were analyzed on Affymetrix U133 Plus 2.0 arrays. A confirmation set of 60 patients were studied by flow cytometry, qRT-PCR or immunohistochemistry and analyzed according to the extent of the tumor immune infiltrate. Gene expression profiles of freshly isolated TIL were also compared with TIL that had been rested overnight or with CD4+ T cells [non-stimulated (NS) or stimulated (S)] from healthy donor PB treated with tumor supernatant (SN). Comparing gene expression profiles of donor blood derived memory CD4+ T cells [non-stimulated (NS) or stimulated (S)] with and without tumor supernatant (SN) treatment Memory CD4+ T cells isolated from a healthy donor blood (NS or S) were treated (and as control: untreated samples in biological triplicate) with SN obtained from fresh breast tumor homogenates of 4 patients and analyzed on Affymetrix U133 Plus 2.0 arrays
Project description:CD4+ helper T (Th) cells are critical regulators of immune responses but their role in breast cancer is currently unknown. This work aims to characterize Th cells infiltrating invasive primary human breast tumors, analyze the influence by the tumor microenvironment and identify Th cell specific prognostic gene signatures. CD4+ T cells isolated from the tumor (TIL), axillary lymph node (LN) and blood (PB) of 10 patients were analyzed on Affymetrix U133 Plus 2.0 arrays. A confirmation set of 60 patients were studied by flow cytometry, qRT-PCR or immunohistochemistry and analyzed according to the extent of the tumor immune infiltrate. Gene expression profiles of freshly isolated TIL were also compared with TIL that had been rested overnight or with CD4+ T cells [non-stimulated (NS) or stimulated (S)] from healthy donor PB treated with tumor supernatant (SN). Comparing gene expression profiles of donor blood derived total CD4+ T cells [non-stimulated (NS)] with and without tumor supernatant (SN) treatment Total CD4+ T cells from a healthy donor blood (NS) were treated (and as control: untreated samples in biological triplicate) with SN from fresh breast tumor homogenates of 3 patients and analyzed on Affymetrix U133 Plus 2.0 arrays
Project description:CD4+ helper T (Th) cells are critical regulators of immune responses but their role in breast cancer is currently unknown. This work aims to characterize Th cells infiltrating invasive primary human breast tumors, analyze the influence by the tumor microenvironment and identify Th cell specific prognostic gene signatures. CD4+ T cells isolated from the tumor (TIL), axillary lymph node (LN) and blood (PB) of 10 patients were analyzed on Affymetrix U133 Plus 2.0 arrays. A confirmation set of 60 patients were studied by flow cytometry, qRT-PCR or immunohistochemistry and analyzed according to the extent of the tumor immune infiltrate. Gene expression profiles of freshly isolated TIL were also compared with TIL that had been rested overnight or with CD4+ T cells [non-stimulated (NS) or stimulated (S)] from healthy donor PB treated with tumor supernatant (SN). Analysis of CD4+ TIL with or without 24h ex-vivo rest, including donor blood memory CD4+ T cells treated in the same conditions as control CD4+ T cells isolated from primary tumors of 2 patients and memory CD4+ T cells from a healthy donor blood were immediately analyzed or incubated for 24h without stimulation before being analyzed on Affymetrix U133 Plus 2.0 arrays
Project description:The DNA exonuclease TREX1 degrades endogenous cytosolic DNA. Cytosolic DNA triggers the cGAS/STING pathway which increases type I interferon. To investigate the physiological significance of TREX1 loss on in vivo tumor growth, we implanted control and TREX1-deficient CT26 tumor cells into immunocompetent BALB/c hosts.Tumor cells were collected 7 days after tumors reached around 200mm3.
Project description:Meg3 is a long non-coding RNA. It's target genes are unknown. The mouse pancreatic beta cell line MIN6-4N was used to assess the expression of genes upon stable Meg3 overexpression Stable cell lines were isolated that have integated pcDNA3.1 or pcDNA3.1-mMeg3.The cells were processed for RNA isolation. The level of Meg3 expression was assessed by RT-PCR. RNA preps were used for microarray analysis.
Project description:Peripheral nerve injuries due to physical insults or chronic diseases are quite common, yet no pharmacological therapies are available for the effective repair of injured nerves. The slow growth rate of adult nerves and insufficient access to growth factors pose major hurdles in timely reinnervating target tissues and restoring functions after nerve injuries. A better understanding of the molecular changes that occur during the immediate regenerative reprogramming of neurons, stated here as in vivo priming, following nerve injury may reveal ideal candidates for future therapies. Hence, molecular profiling of neuronal soma within the first week of nerve injury has been the gold standard for revealing molecular candidates critical for nerve regeneration. A complementary in vitro regenerative priming approach was recently shown to induce enhanced outgrowth in adult sensory neurons. In this work, we exploited the in vitro priming model to reveal novel candidates for adult nerve regeneration. We performed the whole tissue proteomics analysis of in vitro primed DRGs and compared their molecular profile with that of the in vivo primed, and control DRGs. Through this approach, we identified several commonly and uniquely altered molecules in the in vitro and in vivo primed DRGs that have the potential to modulate adult nerve regrowth. We further validated the growth inducing potential of mesencephalic astrocyte-derived neurotrophic factor (MANF), one of the hits identified in our proteomics analysis, in primary adult sensory neurons. Overall, this study showed that in vitro priming partially reproduces the molecular features in in vivo primed adult sensory neurons. The shortlisted candidates presented here from the two priming approaches may serve as potential therapeutic targets for adult nerve regeneration.
Project description:We investigated changes to the head and neck tumour microenvironment immune cell transcriptome following treatment with an adenovirus encoding TNFa and IL-2 in the context of a antiPD-L1 refractory model. Murine head and neck cancer cell line was engrafted subcutaneously onto the right flank of immunocompetent mice. Tumours were treated with antiPD-L1 until 'refractory' status as determined by tumour volume. The tumours were than treated with or without an adenovirus encoding TNFa and IL2. Tumours were digested and CD45+/- were isolated using magnetic bead isolation.
Project description:Given that TREX1-deficient tumor cells showed a growth delay in immunocompetent but not immunodeficient hosts, we characterize the consequences of CT26 tumor-intrinsic TREX1 loss on the host immune system by performing single-cell RNA sequencing on intra-tumoral immune cells sorted from control and TREX1 KO CT26 tumors.
Project description:One of the most detrimental hallmarks of glioblastoma multiforme (GBM) is cellular invasiveness, considered a potential cause of tumor recurrence. Infiltrated GBM cells are difficult to completely eradicate surgically and with local therapeutic modalities. Although much effort has focused on understanding the various mechanisms controlling GBM invasiveness, the nature of the invasiveness remains poorly characterized. Here, we established a highly invasive glioma cell line (U87R4 cells) and a non-invasive cell line (U87L4 cells) from U87MG glioma cells following four rounds of serial in vivo intracranial transplantation. Compared to U87L4 cells, U87R4 cells were highly invasive and had glioma stem cell-like properties. Microarray analysis showed that apoptosis-inducing genes (caspase3 and PDCD4) were downregulated, whereas several cancer stem cell-relevant genes (Wnt10A, Frizzled 4, and CD44) were upregulated in U87R4 cells compared to U87L4 cells. U87R4 cells were resistant to anticancer drug-induced cell death, which was partially due to downregulation of caspase3 and PDCD4. U87R4 cells retained activated Wnt/β-catenin signaling through Frizzled 4, which was sufficient to control neurosphere formation. In addition, Frizzled 4 promoted expression of the epithelial to mesenchymal transition regulator, SNAI1, and acquisition of a mesenchymal phenotype. Taken together, our results indicate that Frizzled 4 may be a member of the Wnt signaling family that governs both stemness and invasiveness of glioma stem cells, and may be a major cause of GBM recurrence and poor prognosis. We established a highly invasive glioma cell line (U87R4 cells) and a non-invasive cell line (U87L4 cells) from U87MG glioma cells following four rounds of serial in vivo intracranial transplantation to characterize the mRNA expression profile of highly invasive glioma cells compared to non-invasive/parental glioma cell lines.