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- Landberg, Göran, 1963, et al.
(författare)
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Characterization of cell-free breast cancer patient-derived scaffolds using liquid chromatography-mass spectrometry/mass spectrometry data and RNA sequencing data
- 2020
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Ingår i: Data in Brief. - : Elsevier BV. - 2352-3409. ; 31
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Tidskriftsartikel (refereegranskat)abstract
- Patient-derived scaffolds (PDSs) generated from primary breast cancer tumors can be used to model the tumor microenvironment in vitro . Patient-derived scaffolds are generated by repeated detergent washing, removing all cells. Here, we analyzed the protein composition of 15 decellularized PDSs using liquid chromatography-mass spectrometry/mass spectrometry. One hundred forty-three proteins were detected and their relative abundance was calculated using a reference sample generated from all PDSs. We performed heatmap analysis of all the detected proteins to display their expression patterns across different PDSs together with pathway enrichment analysis to reveal which processes that were connected to PDS protein composition. This protein dataset together with clinical information is useful to investigators studying the microenvironment of breast cancers. Further, after repopulating PDSs with either MCF7 or MDA-MB-231 cells, we quantified their gene expression profiles using RNA sequencing. These data were also compared to cells cultured in conventional 2D conditions, as well as to cells cultured as xenografts in immune-deficient mice. We investigated the overlap of genes regulated between these different culture conditions and performed pathway enrichment analysis of genes regulated by both PDS and xenograft cultures compared to 2D in both cell lines to describe common processes associated with both culture conditions. Apart from our described analyses of these systems, these data are useful when comparing different experimental model systems. Downstream data analyses and interpretations can be found in the research article "Patient-derived scaffolds uncover breast cancer promoting properties of the microenvironment" [1] . (C) 2020 The Authors. Published by Elsevier Inc.
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| 2. |
- Jonasson, Emma, 1987, et al.
(författare)
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Total mRNA Quantification in Single Cells: Sarcoma Cell Heterogeneity.
- 2020
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Ingår i: Cells. - : MDPI AG. - 2073-4409. ; 9:3
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Tidskriftsartikel (refereegranskat)abstract
- Single-cell analysis enables detailed molecular characterization of cells in relation to cell type, genotype, cell state, temporal variations, and microenvironment. These studies often include the analysis of individual genes and networks of genes. The total amount of RNA also varies between cells due to important factors, such as cell type, cell size, and cell cycle state. However, there is a lack of simple and sensitive methods to quantify the total amount of RNA, especially mRNA. Here, we developed a method to quantify total mRNA levels in single cells based on global reverse transcription followed by quantitative PCR. Standard curve analyses of diluted RNA and sorted cells showed a wide dynamic range, high reproducibility, and excellent sensitivity. Single-cell analysis of three sarcoma cell lines and human fibroblasts revealed cell type variations, a lognormal distribution of total mRNA levels, and up to an eight-fold difference in total mRNA levels among the cells. The approach can easily be combined with targeted or global gene expression profiling, providing new means to study cell heterogeneity at an individual gene level and at a global level. This method can be used to investigate the biological importance of variations in the total amount of mRNA in healthy as well as pathological conditions.
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| 3. |
- Jonasson, Emma, 1987
(författare)
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Tumor cell heterogeneity profiling using single-cell analysis
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cancer is a diverse disease with large variations between tumor types and patients regarding tumor progression and prognosis. Additionally, most individual tumors are heterogeneous, containing subpopulations of cells with various characteristics. Numerous factors affect the differences observed between tumor cells, such as variations in genetics, epigenetics, cellular states and the microenvironment surrounding the individual cells. One clinically relevant subpopulation, commonly referred to as cancer stem cells, consists of cells with stem cell characteristics. These are present in many tumor types and are known to be important for tumor development and treatment resistance. The tumor microenvironment is a key factor affecting the cellular phenotype, including the cancer stem cell subpopulation. Analysis at cell population level will not capture the true variations between individual cells. Instead, single-cell analysis offers new means to study and understand cellular and molecular differences between tumor subpopulations. The main objective of this thesis was to study tumor cell heterogeneity in myxoid liposarcoma and breast cancer with the help of single-cell gene expression analysis methods. We could generate a flexible workflow to measure gene expression, including the assessment of total mRNA amounts in each cell, using several diverse approaches developed from already existing protocols. Subsequently, we combined a number of functional cell culture methods to enrich for tumor cells with characteristic cellular properties together with single-cell gene expression profiling methods, to match phenotype with the corresponding transcription pattern. Single-cell analysis of myxoid liposarcoma cells, sorted based on the cell-cycle, identified a number of genes previously not reported as cell-cycle regulated and defined two subgroups of cells within the G1 phase. In the same tumor type, we identified a subpopulation of cells with cancer stem cell- and chemotherapy resistance properties associated with an active JAK-STAT signaling pathway. Here, a combination treatment of chemotherapy and JAK-STAT inhibition was in vitro shown to be more effective against tumor cells than chemotherapy alone. In breast cancer cells, we identified a number of potential biomarkers overexpressed in a subpopulation of cells with cancer stem cell characteristics. We also developed a new in vivo-like culture system based on decellularized human tumors to study the effect of the microenvironment on breast cancer cells. We demonstrated that the gene expression profiles of cells cultured in these patient-derived scaffolds closely mimic the profiles of in vivo cells. Furthermore, gene expression patterns changed differently depending on the patient-derived scaffold, which could be linked to patient recurrence. In conclusion, we developed single-cell analysis methods as well as a new in vivo-like model system. Furthermore, we identified genes and pathways connected to different subpopulations of myxoid liposarcoma or breast cancer cells that potentially can be used as biomarkers and future drug targets.
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| 4. |
- Walsh, Claire, 1983, et al.
(författare)
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The mevalonate precursor enzyme HMGCS1 is a novel marker and key mediator of cancer stem cell enrichment in luminal and basal models of breast cancer
- 2020
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 15:7
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Tidskriftsartikel (refereegranskat)abstract
- The definitive characterization of common cancer stem cell (CSCs) subpopulations in breast cancer subtypes with distinct genotypic and phenotypic features remains an ongoing challenge. In this study, we have used a non-biased genome wide screening approach to identify transcriptional networks that may be specific to the CSC subpopulations in both luminal and basal breast cancer subtypes. In depth studies of three CSC-enriched breast cancer cell lines representing various subtypes of breast cancer revealed a striking hyperactivation of the mevalonate metabolic pathway in comparison to control cells. The upregulation of metabolic networks is a key feature of tumour cells securing growth and proliferative capabilities and dysregulated mevalonate metabolism has been associated with tumour malignancy and cellular transformation in breast cancer. Furthermore, accumulating evidence suggests that Simvastatin therapy, a mevalonate pathway inhibitor, could affect breast cancer progression and reduce breast cancer recurrence. When detailing the mevalonate pathway in breast cancer using a single-cell qPCR, we identified the mevalonate precursor enzyme, HMGCS1, as a specific marker of CSC-enriched subpopulations within both luminal and basal tumour subtypes. Down-regulation of HMGCS1 also decreased the CSC fraction and function in various model systems, suggesting that HMGCS1 is essential for CSC-activities in breast cancer in general. These data was supported by strong associations between HMGCS1 expression and aggressive features, such as high tumour grade, p53 mutations as well as ER-negativity in lymph node positive breast cancer. Importantly, loss of HMGCS1 also had a much more pronounced effect on CSC-activities compared to treatment with standard doses of Simvastatin. Taken together, this study highlights HMGCS1 as a potential gatekeeper for dysregulated mevalonate metabolism important for CSC-features in both luminal and basal breast cancer subtypes. Pharmacological inhibition of HMGCS1 could therefore be a superior novel treatment approach for breast cancer patients via additional CSC blocking functions.
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