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SRP127564
Mus musculus
79 Downloadable Samples
Illumina HiSeq 2500
Description
Purpose: Eliciting effective anti-tumor immune responses in patients who fail checkpoint inhibitor therapy is a critical challenge in cancer immunotherapy, and in such patients, tumor-associated myeloid cells and macrophages (TAMs) are promising therapeutic targets. We demonstrate in an autochthonous, poorly immunogenic mouse model of melanoma that combination therapy with an agonistic anti-CD40 mAb and CSF1R inhibitor potently suppressed tumor growth. Microwell assays to measure multiplex protein secretion by single cells identified that untreated tumors have distinct TAM subpopulations secreting MMP9 or co-secreting CCL17/22, characteristic of an M2-like state. Combination therapy reduced the frequency of these subsets, while simultaneously inducing a separate polyfunctional inflammatory TAM subset co-secreting TNF?, IL-6, and IL-12. Tumor suppression by this combined therapy was partially dependent on T cells, TNF? and IFN?. Together, this study demonstrates the potential for targeting TAMs to convert a “cold” into an “inflamed” tumor microenvironment capable of eliciting protective T cell responses. Methods: Total RNA was purified with the use of QIAzol and RNeasy Mini kit (QIAGEN), in which an on-column DNase treatment was included. Purified RNA was submitted to the Yale Center for Genomic Analysis where it was subjected to mRNA isolation and library preparation. Non-strand specific libraries were generated from 50ng total RNA using the SMARTer Ultra Low Input RNA for Illumina Sequencing kit. Libraries were pooled, six samples per lane, and sequenced on an Illumina HiSeq 2500 (75-bp paired end reads), and aligned using STAR to the GRCm38 (mm10) reference genome. A count-based differential expression protocol was adapted for this analysis(Anders et al., 2013); mappable data were counted using HTSeq, and imported into R for differential expression analysis using the DESeq2.To find differentially regulated sets of genes for signature generation, a 1.5-Log2 fold-change difference between samples and p-adjusted (Holm-Sidak) = 0.01 was used. Results: To begin to understand how these treatments modulated T cells to control tumor growth, and to possibly illuminate additional biomarkers of response, we examined the transcriptomes of CD11b+ Ly6G- cells treated with CD40 or CSF1Ri, alone or in combination, relative to control, using high throughput RNA-sequencing. Principal components analysis (PCA) on the genome-wide dataset demonstrated that treating with CD40 and CSF1Ri individually caused largely non-overlapping changes in transcription, as indicated by their movement along orthogonal principal components (PC) relative to the control. Importantly, combination therapy was visualized as a systems-level combination of each individual treatment in PC space. We then examined the mRNAs most altered by either treatment alone or in combination relative to Controls (Log2FC>1.5, p<.01) by unsupervised hierarchical clustering. Five major gene patterns emerged from the clustering of genes. Cluster #1 comprises genes that are upregulated by CD40 and CSF1Ri+CD40 treatment but are mostly unaffected by CSF1Ri, suggesting that CD40 is the primary driver of this cluster in the combination treatment. Notable genes in this cluster include Tnfa, Ifng??Il12b and Cxcl9; interestingly, for Tnfa and Il12b, CSF1Ri+CD40 appears to have a synergistic effect on expression. In contrast to Cluster #1, Cluster #5 contains genes substantially downregulated by CSF1Ri and CSF1Ri+CD40 treatments, but are largely unaffected by CD40, suggesting that CSF1Ri is the driver of this cluster in the combination treatment. Cluster #5 genes include Cd36 and Fabp4, suggesting alterations in lipid homeostasis in the TAMs after treatment. Cluster #2 includes genes that are modestly upregulated by CD40 and CSF1Ri individually, leading to a stronger upregulation when combined. Finally, Clusters #3 and #4 include, for the most part, genes that are differentially affected by CD40 versus CSF1Ri and for which the combination treatment yields an intermediate response. In summary, these data show that CSF1Ri and CD40 agonism elicit predominantly distinct changes in gene expression in the CD11b+ cells, indicating they target different biological processes in myeloid cells. The net result of the changes in myeloid gene expression from the combination of CSF1Ri+CD40 treatment reveal additive effects by the individual treatments, but also synergy in the expression of several pro-inflammatory genes (e.g., Tnfa, Ifng, Il6 and Il12b). We further examined our dataset with Gene Set Enrichment Analysis (GSEA). Although CSF1Ri and CD40 treatments did not closely match any immunological signatures in the immunological database of MSigDb, combined CSF1Ri+CD40 had a strikingly similar signature to myeloid cells exposed to a variety of inflammatory stimulants, most closely reflected by BMDMs treated with lipopolysaccharide (LPS). This motivated us to look specifically at categories of NF-?B target genes that are significantly affected by LPS treatment, including transcription factors, cytokines and chemokines. Indeed, most of these NF-?B target genes associated with inflammation were strongly upregulated by CSF1Ri+CD40 treatment. Finally, Ingenuity Pathway Analysis identified TNFR1 and TNFR2 signaling and Acute phase response signaling among the top genetic signatures produced by the CSF1Ri+CD40 treatment combination, matching what we observed with GSEA. Thus, gene expression analysis not only revealed several biomarkers of response that may be relevant for assessing therapeutic activity in ongoing clinical trials using these drugs, but illuminated lead biological factors that may cause tumor regression. Conclusions: myeloid-targeted immunotherapies anti-CD40+CSF1R inhibition synergistically induce a pro-inflammatory microenviroment Overall design: mRNA profiles of tumor infiltrating lymphocytes (TILs) in mice were generated by deep sequencing, in triplicate, using Illumina.
Publication Title
Myeloid-targeted immunotherapies act in synergy to induce inflammation and antitumor immunity.
Sample Metadata Fields
Specimen part, Cell line, Subject
View Samples- Mus musculus
- 8 Downloadable Samples
- Illumina HiSeq 2000
Description
APs were isolated from naïve skin and day 5wounds from dorsal skin wound beds of 7-9 weeks old using FACS. This experiment describes changes in AP gene expression associated with injury and subsequent tissue repair. Overall design: APs were isolated by FACS.
Publication Title
Myofibroblast proliferation and heterogeneity are supported by macrophages during skin repair.
Sample Metadata Fields
Sex, Age, Cell line, Subject
View Samples- Mus musculus
- 4 Downloadable Samples
- Illumina HiSeq 2000
Description
Cells were isolated from day 5wounds from dorsal skin wound beds of 7-9 weeks old using FACS. This experiment describes the gene expression profile associated with different immune cell subsets during tissue repair. Overall design: Cells were isolated by FACS.
Publication Title
Myofibroblast proliferation and heterogeneity are supported by macrophages during skin repair.
Sample Metadata Fields
Sex, Age, Cell line, Subject
View Samples- Homo sapiens
- 12 Downloadable Samples
Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
This SuperSeries is composed of the SubSeries listed below.
Publication Title
ERα-dependent E2F transcription can mediate resistance to estrogen deprivation in human breast cancer.
Sample Metadata Fields
Specimen part, Cell line, Treatment
View Samples- Homo sapiens
- 12 Downloadable Samples
Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
A significant fraction of breast cancers exhibit de novo or acquired resistance to estrogen deprivation. To model resistance to aromatase inhibitor (AI) therapy, long-term estrogen-deprived (LTED) derivatives of MCF-7 and HCC-1428 cells were generated through culture for 3 and 7 months under hormone-depleted conditions, respectively. These LTED cells showed sensitivity to the ER downregulator fulvestrant under hormone-depleted conditions, suggesting continued dependence upon ER signaling for hormone-independent growth. To evaluate the role of ER in hormone-independent growth, LTED cells were treated +/- 1 uM fulvestrant x 48 h before RNA was harvested for gene expression analysis.
Publication Title
ERα-dependent E2F transcription can mediate resistance to estrogen deprivation in human breast cancer.
Sample Metadata Fields
Specimen part, Cell line, Treatment
View Samples- Homo sapiens
- 12 Downloadable Samples
- Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
RNA from circulating blood reticulocytes was utilized to provide a robust description of genes transcribed at the final stages of erythroblast maturation. After depletion of leukocytes and platelets, Affymetrix HG-U133Plus 2.0 arrays were hybridized with probe from total RNA isolated from blood sampled from 6 umbilical cords and 6 healthy adult humans.
Publication Title
Let-7 microRNAs are developmentally regulated in circulating human erythroid cells.
Sample Metadata Fields
Specimen part
View Samples- Mus musculus
- 22 Downloadable Samples
- Illumina HiSeq 2500
Description
Multiple sclerosis involves an aberrant autoimmune response and progressive failure of remyelination in the central nervous system. Prevention of neural degeneration and subsequent disability requires remyelination through the generation of new oligodendrocytes, but current treatments exclusively target the immune system. Oligodendrocyte progenitor cells are stem cells in the central nervous system and the principal source of myelinating oligodendrocytes. These cells are abundant in demyelinated regions of patients with multiple sclerosis, yet fail to differentiate, thereby representing a cellular target for pharmacological intervention. To discover therapeutic compounds for enhancing myelination from endogenous oligodendrocyte progenitor cells, we screened a library of bioactive small molecules on mouse pluripotent epiblast stem-cell-derived oligodendrocyte progenitor cells. Here we show seven drugs function at nanomolar doses selectively to enhance the generation of mature oligodendrocytes from progenitor cells in vitro. Two drugs, miconazole and clobetasol, are effective in promoting precocious myelination in organotypic cerebellar slice cultures, and in vivo in early postnatal mouse pups. Systemic delivery of each of the two drugs significantly increases the number of new oligodendrocytes and enhances remyelination in a lysolecithin-induced mouse model of focal demyelination. Administering each of the two drugs at the peak of disease in an experimental autoimmune encephalomyelitis mouse model of chronic progressive multiple sclerosis results in striking reversal of disease severity. Immune response assays show that miconazole functions directly as a remyelinating drug with no effect on the immune system, whereas clobetasol is a potent immunosuppressant as well as a remyelinating agent. Mechanistic studies show that miconazole and clobetasol function in oligodendrocyte progenitor cells through mitogen-activated protein kinase and glucocorticoid receptor signalling, respectively. Furthermore, both drugs enhance the generation of human oligodendrocytes from human oligodendrocyte progenitor cells in vitro. Collectively, our results provide a rationale for testing miconazole and clobetasol, or structurally modified derivatives, to enhance remyelination in patients. Overall design: RNA sequencing of oligodendrocyte progenitor cells treated with vehicle, miconazole or clobetasol for 0, 2, 6, or 12 hours. Cells were plated 1.5 hours prior to addition of drug.
Publication Title
Drug-based modulation of endogenous stem cells promotes functional remyelination in vivo.
Sample Metadata Fields
No sample metadata fields
View Samples- Homo sapiens
- 22 Downloadable Samples
- Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
Hyperactivation of phosphatidylinositol-3 kinase (PI3K) promotes escape from hormone dependence in estrogen receptor-positive breast cancer.
Publication Title
Hyperactivation of phosphatidylinositol-3 kinase promotes escape from hormone dependence in estrogen receptor-positive human breast cancer.
Sample Metadata Fields
Specimen part, Cell line
View Samples- Homo sapiens
- 9 Downloadable Samples
- Affymetrix HT HG-U133+ PM Array Plate (hthgu133pluspm)
Description
A significant fraction of breast cancers exhibit de novo or acquired resistance to estrogen deprivation.
Publication Title
A kinome-wide screen identifies the insulin/IGF-I receptor pathway as a mechanism of escape from hormone dependence in breast cancer.
Sample Metadata Fields
Cell line, Treatment
View Samples- Mus musculus
- 36 Downloadable Samples
- Illumina HiSeq 2500
Description
Translating ribosome affinity purification (TRAP) was performed on spinal cord dissections pooled from 3-4 mice 21 days post birth that were positive for the eGFP-L10A fusion ribosomal marker protein under the expression of either the Chat promoter (Tg(Chat-EGFP/Rpl10a)DW167Htz) or the Snap25 promoter (Tg(Snap25-EGFP/Rpl10a)JD362Jdd). RNA-sequencing was performed on both TRAP and pre-immunoprecipitation (PreIP) control RNA samples. Overall design: Three replicates of PreIP and TRAP for two transgenic lines.
Publication Title
MicroRNA Profiling Reveals Marker of Motor Neuron Disease in ALS Models.
Sample Metadata Fields
Specimen part, Cell line, Subject
View Samples