RNA-seq of FACS-enriched mammary subpopulations (basal, luminal progenitor, mature luminal) from WT and ID4 knockout mice
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PROVIDER: PRJNA630710 | ENA |
REPOSITORIES: ENA
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Project description:The goal of this study was to determine the genes regulated by ID4 in mammary epithelial subpopulations. Basal, luminal progenitor, and mature luminal mammary epithelial subpopulations were FACS-sorted from adult mice that were either WT or ID4 KO in quadruplicate. RNA-seq and differential gene expression analysis was performed.
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Project description:The cellular heterogeneity of one patient derived orthotopic breast cancer xenograft model (PDBCX) was investigated using flow cytometry , combined with assessment of in vivo tumorigenicity and whole genome expression profiling. Epithelial cell adhesion molecule (EpCAM) was revealed as a highly specific cell surface marker of the human tumor cell population in both xenografts. Based on expression patterns observed in primary tumor tissue, SSEA-4 and CD24 were chosen as markers to further subdivide the luminal tumor cells into four subpopulations. FACS sorting was used to isolate four cell subpopulations. Results: In vivo tumorigenicity assay showed that SSEA-4+/CD24+ cells were non-tumorigenic, while the three other subpopulations were tumorigenic. Tumors resulting from the SSEA-4+/CD24- subpopulation of luminal cancer cells, did not express CD24, while tumors arising from the SSEA-4-/CD24-, and SSEA-4-/CD24+ populations both recapitulated the original tumor containing all four subpopulations. Whole genome expression analysis revealed distinct transcriptional profiles, and 44 genes were significantly differentially expressed when comparing the tumorigenic vs non-tumorigenic populations. Several interesting genes putatively suppressing the cancer cells ability to initiate tumors in vivo were upregulated in the non-tumorigenic population. We here show that tumor initiating cells within one primary tumor evidently included more than one phenotype. Furthermore, with respect to cell surface marker expression, one of the subpopulations produced tumors unlike both the originating cells, and the original tumor. Discussion: Our results imply that subpopulations from one primary tumor can give rise to dissimilar daughter tumors. These tumors may not necessarily respond to the same targeted treatment, and thereby represent a therapy escape mechanism. This study highlights that to remove the risk of breast cancer recurrence, inhibition of the molecules critical for driving the tumor progression in several tumor cell subpopulations might be essential Gene expression was measured in four cell subpopulations isolated from Patient derived human luminal-like breast cancer xenograft. Four replicates from three subpopulations and three replicates from one subpopulation.
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Project description:To delineate epithelial subpopulations in mouse mammary tissue, hematopoietic and endothelial cells were depleted from freshly isolated cell suspensions derived from mammary glands using fluorescence-activated cell sorting. The resultant Lin- population was fractionated into four distinct subpopulations using antibodies against CD29, CD24 and CD61. Based on the immunohistochemical phenotype, and in vivo and in vitro functional assays, these subpopulations were identified as fibroblast-containing stromal (CD29loCD24-), mammary stem cell (MaSC)-enriched (CD29hiCD24+CD61+), luminal progenitor (CD29loCD24+CD61+), and mature luminal (CD29loCD24+CD61-) cell subpopulations. Microarray profiling was used to derive gene expression signatures of these 4 subpopulations. The four mammary cell subpopulations were found to have distinct gene expression profiles. Four mammary cell subpopulations from 3-5 pooled mouse tissues were analysed. MS is for the MaSC-enriched cell subpopulation. LP is for the Luminal Progenitor subpopulation. ML is for the Mature Luminal subpopulation.
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Project description:The cellular heterogeneity of one patient derived orthotopic breast cancer xenograft model PDX was investigated using flow cytometry, combined with assessment of in vivo tumorigenicity and SNP genotyping array for copy number analysis. Epithelial cell adhesion molecule (EpCAM) was revealed as a highly specific cell surface marker of the human tumor cell population in both xenografts. Based on expression patterns observed in primary tumor tissue, SSEA-4 and CD24 were chosen as markers to further subdivide the luminal tumor cells into four subpopulations. FACS sorting was used to isolate four cell subpopulations. Results: In vivo tumorigenicity assay showed that SSEA-4+/CD24+ cells were non-tumorigenic, while the three other subpopulations were tumorigenic. Tumors resulting from the SSEA-4+/CD24- subpopulation of luminal cancer cells, did not express CD24, while tumors arising from the SSEA-4-/CD24-, and SSEA-4-/CD24+ populations both recapitulated the original tumor containing all four subpopulations. Copy number analysis comparing the four subpopulations between eachother, did reveal high similarity. Although there were some minor differencies between the subpopulations, no differences in genome aberrations could explain their difference in tumorigenicity. Discussion: Our results imply that subpopulations from one primary tumor can give rise to dissimilar daughter tumors. These tumors may not necessarily respond to the same targeted treatment, and thereby represent a therapy escape mechanism. This study highlights that to remove the risk of breast cancer recurrence, inhibition of the molecules critical for driving the tumor progression in several tumor cell subpopulations might be essential Four subpopulations were analysed using Infinium HumanOmniExpressExome BeadChip array. Replicates are not included, the HumanOmniExpressExome-8v1-2_A.egt file was used as a reference.
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Project description:Differentiation of stem cells embedded within the mammary epithelium is orchestrated by lineage-specifying transcription factors. Unlike the well-defined luminal hierarchy, dissection of the basal lineage has been hindered by a lack of specific markers. Inhibitor of Differentiation 4 (ID4) is a basally-restricted helix-loop-helix (HLH) transcription factor essential for mammary development. Here we show that ID4 is highly expressed in basal stem cells and decreases during myoepithelial differentiation. By integrating transcriptomic, proteomic and chromatin-association data we reveal that ID4 is required to suppress myoepithelial gene expression and cell fate.
2021-01-25 | PXD017517 | Pride
Project description:Highly purified subpopulations of primitive bipotent and committed luminal progenitor cells as well as mature luminal and myoepithelial cells from normal human mammary tissue were isolated and compared their transcriptomes obtained using the Affymetrix GeneChip Human X3P Array. Experiment Overall Design: 12 Afffymetrix array hybridizations were performed on RNA samples extracted from highly purified subpopulations of primitive bipotent and committed luminal progenitor cells as well as mature luminal and myoepithelial cells isolate from 3 different normal human mammary tissue.
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Project description:To reveal distinct transcriptomes associated with spermatogonial stem cell renewal vs. initiation of differentiation, single-cell transcriptomes from Adult ID4-EGFP+ spermatogonia were subdivided into subpopulations that displayed distinct fates when assayed by transplantation, with ID4-EGFPbright cells highly enriched for SSCs, and ID4-EGFPdim cells enriched for progenitors. We used the Fluidigm C1 instrument to capture individual spermatogonia for SMART-Seq2 single-cell RNA-seq.
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Project description:To reveal distinct transcriptomes associated with spermatogonial stem cell renewal vs. initiation of differentiation, single-cell transcriptomes from P6 ID4-EGFP+ spermatogonia were subdivided into subpopulations that displayed distinct fates when assayed by transplantation, with ID4-EGFPbright cells highly enriched for SSCs, and ID4-EGFPdim cells enriched for progenitors. We used the Fluidigm C1 instrument to capture individual spermatogonia for SMART-Seq2 single-cell RNA-seq.
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