| 1. |
- Akrap, Nina, et al.
(författare)
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Identification of Distinct Breast Cancer Stem Cell Populations Based on Single-Cell Analyses of Functionally Enriched Stem and Progenitor Pools
- 2016
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Ingår i: Stem Cell Reports. - : Elsevier BV. - 2213-6711. ; 6:1, s. 121-136
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Tidskriftsartikel (refereegranskat)abstract
- The identification of breast cancer cell subpopulations featuring truly malignant stem cell qualities is a challenge due to the complexity of the disease and lack of general markers. By combining extensive single-cell gene expression profiling with three functional strategies for cancer stem cell enrichment including anchorage-independent culture, hypoxia, and analyses of low-proliferative, label-retaining cells derived from mammospheres, we identified distinct stem cell clusters in breast cancer. Estrogen receptor (ER)alpha+ tumors featured a clear hierarchical organization with switch-like and gradual transitions between different clusters, illustrating how breast cancer cells transfer between discrete differentiation states in a sequential manner. ER alpha- breast cancer showed less prominent clustering but shared a quiescent cancer stem cell pool with ER alpha+ cancer. The cellular organization model was supported by single-cell data from primary tumors. The findings allow us to understand the organization of breast cancers at the single-cell level, thereby permitting better identification and targeting of cancer stem cells.
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| 2. |
- Andersson, Daniel, 1979, et al.
(författare)
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Properties of targeted preamplification in DNA and cDNA quantification
- 2015
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Ingår i: Expert Review of Molecular Diagnostics. - : Informa UK Limited. - 1473-7159 .- 1744-8352. ; 15:8, s. 1085-1100
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Forskningsöversikt (refereegranskat)abstract
- Objective: Quantification of small molecule numbers often requires preamplification to generate enough copies for accurate downstream enumerations. Here, we studied experimental parameters in targeted preamplification and their effects on downstream quantitative real-time PCR (qPCR). Methods: To evaluate different strategies, we monitored the preamplification reaction in real-time using SYBR Green detection chemistry followed by melting curve analysis. Furthermore, individual targets were evaluated by qPCR. Result: The preamplification reaction performed best when a large number of primer pairs was included in the primer pool. In addition, preamplification efficiency, reproducibility and specificity were found to depend on the number of template molecules present, primer concentration, annealing time and annealing temperature. The amount of nonspecific PCR products could also be reduced about 1000-fold using bovine serum albumin, glycerol and formamide in the preamplification. Conclusion: On the basis of our findings, we provide recommendations how to perform robust and highly accurate targeted preamplification in combination with qPCR or next-generation sequencing.
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| 3. |
- Berger, Karoline, 1991, et al.
(författare)
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Interleukin-6 Induces Stem Cell Propagation through Liaison with the Sortilin-Progranulin Axis in Breast Cancer.
- 2023
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Ingår i: Cancers. - 2072-6694. ; 15:24
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Tidskriftsartikel (refereegranskat)abstract
- Unraveling the complex network between cancer cells and their tumor microenvironment is of clinical importance, as it might allow for the identification of new targets for cancer treatment. Cytokines and growth factors secreted by various cell types present in the tumor microenvironment have the potential to affect the challenging subpopulation of cancer stem cells showing treatment-resistant properties as well as aggressive features. By using various model systems, we investigated how the breast cancer stem cell-initiating growth factor progranulin influenced the secretion of cancer-associated proteins. In monolayer cultures, progranulin induced secretion of several inflammatory-related cytokines, such as interleukin (IL)-6 and -8, in a sortilin-dependent manner. Further, IL-6 increased the cancer stem fraction similarly to progranulin in the breast cancer cell lines MCF7 and MDA-MB-231 monitored by the surrogate mammosphere-forming assay. In a cohort of 63 patient-derived scaffold cultures cultured with breast cancer cells, we observed significant correlations between IL-6 and progranulin secretion, clearly validating the association between IL-6 and progranulin also in human-based microenvironments. In conclusion, the interplay between progranulin and IL-6 highlights a dual breast cancer stem cell-promoting function via sortilin, further supporting sortilin as a highly relevant therapeutic target for aggressive breast cancer.
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| 4. |
- Busch, Susann, et al.
(författare)
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Cellular organization and molecular differentiation model of breast cancer-associated fibroblasts
- 2017
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Ingår i: Molecular Cancer. - : Springer Science and Business Media LLC. - 1476-4598. ; 16:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: The role of cancer-associated fibroblasts (CAFs) during tumour progression is obscured by the inherently complex, heterotypic nature of fibroblast cells and behaviours in various subtypes of malignancies. Therefore, we sought to identify distinct fibroblast subpopulations at the single-cell level. Methods: Using single-cell quantitative PCR as a powerful tool to study heterogeneity and rare cell events, in a high-throughput manner a panel of gene targets are run simultaneously on transcripts isolated from single cells obtained by fluorescence-activated cell sort. Assessment of cells with stem-like characteristics was attained by anchorage-independent, anoikis-resistant culture. Results: Single-cell analysis of fibroblasts and their tumour-activated counterparts demonstrated molecularly distinct cell types defined by differential expression of characteristic mesenchymal and fibroblast activation markers. Identified subpopulations presented overlapping gene expression patterns indicating transitional molecular states during fibroblast differentiation. Using single-cell resolution data we generated a molecular differentiation model which enabled the classification of patient-derived fibroblasts, validating our modelling approach. Remarkably, a subset of fibroblasts displayed expression of pluripotency markers, which was enriched for in non-adherent conditions. Yet the ability to form single-cell derived spheres was generally reduced in CAFs and upon fibroblast activation through TGF beta 1 ligand and cancer cell-secreted factors. Hence, our data imply the existence of putative stem/progenitor cells as a physiological feature of undifferentiated fibroblasts. Conclusions: Within this comprehensive study we have identified distinct and intersecting molecular profiles defining fibroblast activation states and propose that underlying cellular heterogeneity, fibroblasts are hierarchically organized. Understanding the molecular make-up of cellular organization and differentiation routes will facilitate the discovery of more specific markers for stromal subtypes and targets for anti-stromal therapies.
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| 5. |
- Dolatabadi, Soheila, et al.
(författare)
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Cell Cycle and Cell Size Dependent Gene Expression Reveals Distinct Subpopulations at Single-Cell Level
- 2017
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Ingår i: Frontiers in Genetics. - : Frontiers Media SA. - 1664-8021. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Cell proliferation includes a series of events that is tightly regulated by several checkpoints and layers of control mechanisms. Most studies have been performed on large cell populations, but detailed understanding of cell dynamics and heterogeneity requires single-cell analysis. Here, we used quantitative real-time PCR, profiling the expression of 93 genes in single-cells from three different cell lines. Individual unsynchronized cells from three different cell lines were collected in different cell cycle phases (GO/G1 - S - G2/M) with variable cell sizes. We found that the total transcript level per cell and the expression of most individual genes correlated with progression through the cell cycle, but not with cell size. By applying the random forests algorithm, a supervised machine learning approach, we show how a multi-gene signature that classifies individual cells into their correct cell cycle phase and cell size can be generated. To identify the most predictive genes we used a variable selection strategy. Detailed analysis of cell cycle predictive genes allowed us to define subpopulations with distinct gene expression profiles and to calculate a cell cycle index that illustrates the transition of cells between cell cycle phases. In conclusion, we provide useful experimental approaches and bioinformatics to identify informative and predictive genes at the single-cell level, which opens up new means to describe and understand cell proliferation and subpopulation dynamics.
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| 6. |
- Dolatabadi, Soheila, et al.
(författare)
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JAK–STAT signalling controls cancer stem cell properties including chemotherapy resistance in myxoid liposarcoma
- 2019
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Ingår i: International Journal of Cancer. - : Wiley. - 0020-7136 .- 1097-0215. ; 145:2, s. 435-449
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Tidskriftsartikel (refereegranskat)abstract
- Myxoid liposarcoma (MLS) shows extensive intratumoural heterogeneity with distinct subpopulations of tumour cells. Despite improved survival of MLS patients, existing therapies have shortcomings as they fail to target all tumour cells. The nature of chemotherapy-resistant cells in MLS remains unknown. Here, we show that MLS cell lines contained subpopulations of cells that can form spheres, efflux Hoechst dye and resist doxorubicin, all properties attributed to cancer stem cells (CSCs). By single-cell gene expression, western blot, phospho-kinase array, immunoprecipitation, immunohistochemistry, flow cytometry and microarray analysis we showed that a subset of MLS cells expressed JAK–STAT genes with active signalling. JAK1/2 inhibition via ruxolitinib decreased, while stimulation with LIF increased, phosphorylation of STAT3 and the number of cells with CSC properties indicating that JAK–STAT signalling controlled the number of cells with CSC features. We also show that phosphorylated STAT3 interacted with the SWI/SNF complex. We conclude that MLS contains JAK–STAT-regulated subpopulations of cells with CSC features. Combined doxorubicin and ruxolitinib treatment targeted both proliferating cells as well as cells with CSC features, providing new means to circumvent chemotherapy resistance in treatment of MLS patients. © 2019 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC
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| 7. |
- Garre, Elena, 1978, et al.
(författare)
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Breast Cancer Patient-Derived Scaffolds Can Expose Unique Individual Cancer Progressing Properties of the Cancer Microenvironment Associated with Clinical Characteristics
- 2022
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Ingår i: Cancers. - : MDPI AG. - 2072-6694. ; 14:9
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Tidskriftsartikel (refereegranskat)abstract
- Simple Summary Despite huge progress in cancer diagnostics and medicine we still lack optimal cancer treatments for patients with aggressive diseases. This problem can be influenced by the biological heterogeneity of cancer cells as well as poorly understood cancer promoting effects of the cancer microenvironment being an important part of the cancer niche. In this study we have specifically monitored the activity of the cancer microenvironment in breast cancer patients using cell-free scaffolds repopulated with reporter cancer cells sensing the activity of the patient environment. The data show that scaffold induced changes in epithelial-mesenchymal transition and pluripotency markers were linked to clinical and prognostic properties of the original cancer and the information was even more precise when matching estrogen receptor status in our system. The findings highlight that patient-derived scaffolds uncover important information about varying malignant promoting properties in the cancer niche and can be used as a complementary diagnostic tool. Breast cancer is a heterogeneous disease in terms of cellular and structural composition, and besides acquired aggressive properties in the cancer cell population, the surrounding tumor microenvironment can affect disease progression and clinical behaviours. To specifically decode the clinical relevance of the cancer promoting effects of individual tumor microenvironments, we performed a comprehensive test of 110 breast cancer samples using a recently established in vivo-like 3D cell culture platform based on patient-derived scaffolds (PDSs). Cell-free PDSs were recellularized with three breast cancer cell lines and adaptation to the different patient-based microenvironments was monitored by quantitative PCR. Substantial variability in gene expression between individual PDS cultures from different patients was observed, as well as between different cell lines. Interestingly, specific gene expression changes in the PDS cultures were significantly linked to prognostic features and clinical information from the original cancer. This link was even more pronounced when ER alpha-status of cell lines and PDSs matched. The results support that PDSs cultures, including a cancer cell line of relevant origin, can monitor the activity of the tumor microenvironment and reveal unique information about the malignancy-inducing properties of the individual cancer niche and serve as a future complementary diagnostic tool for breast cancer.
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| 8. |
- Gustafsson, Anna, et al.
(författare)
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Patient-derived scaffolds as a drug-testing platform for endocrine therapies in breast cancer
- 2021
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- Three-dimensional cell culture platforms based on decellularised patient-based microenvironments provide in vivo-like growth conditions allowing cancer cells to interact with intact structures and components of the surrounding tissue. A patient-derived scaffold (PDS) model was therefore evaluated as a testing platform for the endocrine therapies (Z)-4-Hydroxytamoxifen (4OHT) and fulvestrant as well as the CDK4/6-inhibitor palbociclib, monitoring the treatment responses in breast cancer cell lines MCF7 and T47D adapted to the patient-based microenvironments. MCF7 cells growing in PDSs showed increased resistance to 4OHT and fulvestrant treatment (100- and 20-fold) compared to 2D cultures. Quantitative PCR analyses of endocrine treated cancer cells in PDSs revealed upregulation of pluripotency markers further supported by increased self-renewal capacity in sphere formation assays. When comparing different 3D growth platforms including PDS, matrigel, gelatin sponges and 3D-printed hydrogels, 3D based cultures showed slightly varying responses to fulvestrant and palbociclib whereas PDS and matrigel cultures showed more similar gene expression profiles for 4OHT treatment compared to the other platforms. The results support that the PDS technique maximized to provide a multitude of smaller functional PDS replicates from each primary breast cancer, is an up-scalable patient-derived drug-testing platform available for gene expression profiling and downstream functional assays.
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| 9. |
- Jacobsson, Hanna, et al.
(författare)
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Hypoxia-induced secretion stimulates breast cancer stem cell regulatory signalling pathways
- 2019
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Ingår i: Molecular Oncology. - : Wiley. - 1574-7891 .- 1878-0261. ; 13:8, s. 1693-1705
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Tidskriftsartikel (refereegranskat)abstract
- It is well known that tumour cells are dependent on communication with the tumour microenvironment. Previously, it has been shown that hypoxia (HX) induces pronounced, diverse and direct effects on cancer stem cell (CSC) qualities in different breast cancer subtypes. Here, we describe the mechanism by which HX-induced secretion influences the spreading of CSCs. Conditioned media (CM) from estrogen receptor (ER)-α-positive hypoxic breast cancer cell cultures increased the fraction of CSCs compared to normal growth conditions, as determined using sets of CSC assays and model systems. In contrast, media from ERα-negative hypoxic cell cultures instead decreased this key subpopulation of cancer cells. Further, there was a striking overrepresentation of JAK-STAT-associated cytokines in both the ERα-positive and ERα-negative linked hypoxic responses as determined by a protein screen of the CM. JAK-STAT inhibitors and knockdown experiments further supported the hypothesis that this pathway is critical for the CSC-activating and CSC-inactivating effects induced by hypoxic secretion. We also observed that the interleukin-6-JAK2-STAT3 axis was specifically central for the ERα-negative hypoxic behaviour. Our results underline the importance of considering breast cancer subtypes in treatments targeting JAK-STAT or HX-associated processes and indicate that HX is not only a confined tumour biological event, but also influences key tumour properties in widespread normoxic microenvironments. © 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
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| 10. |
- Jonasson, Emma, 1987, et al.
(författare)
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Identification of breast cancer stem cell related genes using functional cellular assays combined with single-cell RNA sequencing in MDA-MB-231 cells
- 2019
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Ingår i: Frontiers in Genetics. - : Frontiers Media SA. - 1664-8021. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Breast cancer tumors display different cellular phenotypes. A growing body of evidence points toward a population of cancer stem cells (CSCs) that is important for metastasis and treatment resistance, although the characteristics of these cells are incomplete. We used mammosphere formation assay and label-retention assay as functional cellular approaches to enrich for cells with different degree of CSC properties in the breast cancer cell line MDA-MB-231 and performed single-cell RNA sequencing. We clustered the cells based on their gene expression profiles and identified three subpopulations, including a CSC-like population. The cell clustering into these subpopulations overlapped with the cellular enrichment approach applied. To molecularly define these groups, we identified genes differentially expressed between the three subpopulations which could be matched to enriched gene sets. We also investigated the transition process from CSC-like cells into more differentiated cell states. In the CSC population we found 14 significantly upregulated genes. Some of these potential breast CSC markers are associated to reported stem cell properties and clinical survival data, but further experimental validation is needed to confirm their cellular functions. Detailed characterization of CSCs improve our understanding of mechanisms for tumor progression and contribute to the identification of new treatment targets. © 2019 Jonasson, Ghannoum, Persson, Karlsson, Kroneis, Larsson, Landberg and Ståhlberg. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
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