Evaluation of cancer stemness in breast cancer and glioblastoma spheroids in vitro

Yılmazer, Açelya

doi:10.1007/s13205-018-1412-y

Cited by 9 publications

(6 citation statements)

References 30 publications

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“… 27 These spheroid cells exhibit CSC characteristics, such as enhanced migration, invasion and sphere formation, as confirmed by our group and other researchers. 28 , 29 Consistent with these findings, we showed that tumourspheres formed from ascites-derived cells express higher levels of stemness genes.…”

Section: Discussionsupporting

confidence: 86%

Ascites-derived ALDH+CD44+ tumour cell subsets endow stemness, metastasis and metabolic switch via PDK4-mediated STAT3/AKT/NF-κB/IL-8 signalling in ovarian cancer

et al. 2020

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Background Ovarian cancer is characterised by frequent recurrence due to persistent presence of residual cancer stem cells (CSCs). Here, we identify and characterise tumour subsets from ascites-derived tumour cells with stemness, metastasis and metabolic switch properties and to delineate the involvement of pyruvate dehydrogenase kinase 4 (PDK4) in such process. Methods Ovarian cancer cells/cell lines derived from ascites were used for tumourspheres/ALDH+CD44+ subset isolation. The functional roles and downstream signalling of PDK4 were explored. Its association with clinical outcome of ovarian cancer was analysed. Results We demonstrated enhanced CSC characteristics of tumour cells derived from ovarian cancer ascites, concomitant with ALDH and CD44 subset enrichment and high PDK4 expression, compared to primary tumours. We further showed tumourspheres/ALDH+CD44+ subsets from ascites-derived tumour cells/cell lines with CSC properties and enhanced glycolysis. Clinically, PDK4 expression was correlated with aggressive features. Notably, blockade of PDK4 in tumourspheres/ALDH+CD44+ subsets led to inhibition of CSC characteristics, glycolysis and activation of STAT3/AKT/NF-κB/IL-8 (signal transducer and activator of transcription 3/protein kinases B/nuclear factor-κB/interleukin-8) signalling. Conversely, overexpression of PDK4 in ALDH−CD44– subsets exerted the opposite effects. Conclusion Ascites-derived ALDH+CD44+ tumour cell subsets endow stemness, metastatic and metabolic switch properties via PDK4-mediated STAT3/AKT/NF-κB/IL-8 signalling, suggesting PDK4 as a viable therapeutic molecular target for ovarian cancer management.

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Section: Discussionsupporting

confidence: 86%

Ascites-derived ALDH+CD44+ tumour cell subsets endow stemness, metastasis and metabolic switch via PDK4-mediated STAT3/AKT/NF-κB/IL-8 signalling in ovarian cancer

et al. 2020

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“…LIF and Fas were also upregulated in both cell lines. To further explore the effect of topographical stimuli on stemness, the enriched factor bioprocess profiles of TNBC cells cultured on surfaces with Ra = 1.5 µm were compared to 3D spheroids as a standard control of enriched cancer stem cells (CSC) in TNBC ( Yilmazer, 2018 ). The factors and bioprocesses enriched in 3D spheroids were first identified relative to cell monolayers on TCP ( Supplementary Figures S2A, B ) prior to comparison with cells derived from rough surfaces.…”

Section: Resultsmentioning

confidence: 99%

Surface roughness modulates EGFR signaling and stemness of triple-negative breast cancer cells

Rosado-Galindo

Domenech²

2023

Front. Cell Dev. Biol.

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Introduction: Cancer stem cells (CSC), a major culprit of drug-resistant phenotypes and tumor relapse, represent less than 2 % of the bulk of TNBC cells, making them difficult to isolate, study, and thus, limiting our understanding of the pathogenesis of the disease. Current methods for CSC enrichment, such as 3D spheroid culture, genetic modification, and stem cell conditioning, are time consuming, expensive, and unsuitable for high-throughput assays. One way to address these limitations is to use topographical stimuli to enhance CSC populations in planar culture. Physical cues in the breast tumor microenvironment can influence cell behavior through changes in the mechanical properties of the extracellular matrix (ECM). In this study, we used topographical cues on polystyrene films to investigate their effect on the proteome and stemness of standard TNBC cell lines.Methods: The topographical polystyrene-based array was generated using razor printing and polishing methods. Proteome data were analyzed and enriched bioprocesses were identified using R software. Stemness was assessed measuring CD44, CD24 and ALDH markers using flow cytometry, immunofluorescence, detection assays, and further validated with mammosphere assay. EGF/EGFR expression and activity was evaluated using enzyme-linked immunosorbent assay (ELISA), immunofluorescence and antibody membrane array. A dose-response assay was performed to further investigate the effect of surface topography on the sensitivity of cells to the EGFR inhibitor.Results: Surface roughness enriched the CSC population and modulated epidermal growth factor receptor (EGFR) signaling activity in TNBC cells. Enhanced proliferation of MDA-MB-468 cells in roughness correlated with upregulation of the epidermal growth factor (EGF) ligand, which in turn corresponded with a 3-fold increase in the expression of EGFR and a 42% increase in its phosphorylation compared to standard smooth culture surfaces. The results also demonstrated that phenotypic changes associated with topographical (roughness) stimuli significantly decreased the drug sensitivity to the EGFR inhibitor gefitinib. In addition, the proportion of CD44+/CD24−/ALDH+ was enhanced on surface roughness in both MDA-MB-231 and MDA-MB-468 cell lines. We also demonstrated that YAP/TAZ activation decreased in a roughness-dependent manner, confirming the mechanosensing effect of the topographies on the oncogenic activity of the cells.Discussion: Overall, this study demonstrates the potential of surface roughness as a culture strategy to influence oncogenic activity in TNBC cells and enrich CSC populations in planar cultures. Such a culture strategy may benefit high-throughput screening studies seeking to identify compounds with broader tumor efficacy.

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“…When grown adherently, U87 cells typically have lower expression of stemness markers; compared to cells grown in serum‐free conditions, which exhibit higher expression of stemness markers 40 . In addition, other studies have found an increased expression of stemness markers (NESTIN, SOX2, CD133, NANOG, OCT 3/4, and CD44) in serum grown U87 cells when cultured as MCTS compared to when cultured as an adherent monolayer 41–43 . Here, we have utilized 1:4 GBM‐astrocytes co‐culture model, 1:9 GBM‐EC co‐culture model, and 1:4:9 GBM‐astrocytes‐EC triculture model as we had previously observed an enhanced expression of stemness markers‐ NESTIN, SOX2, CD133, NANOG, and OCT4 in these models 6 .…”

Section: Discussionmentioning

confidence: 99%

The impact of temozolomide and lonafarnib on the stemness marker expression of glioblastoma cells in multicellular spheroids

Nakod

Kondapaneni

Edney

et al. 2022

Biotechnology Progress

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Glioblastoma multiforme (GBM) is a highly malignant brain tumor with a poor prognosis. The GBM microenvironment is highly heterogeneous and is composed of many cell types including astrocytes and endothelial cells (ECs) along with tumor cells, which are responsible for heightened resistance to standard chemotherapeutic drugs such as Temozolomide (TMZ). Here, we investigated how drug treatments impact stemness marker expression of GBM cells in multicellular tumor spheroid (MCTS) models. Co‐ and tri‐culture MCTS constructed using U87‐MG GBM cells, astrocytes, and/or ECs were cultured for 7 days. At Day 7, 5 μM lonafarnib (LNF), 100 μM TMZ, or combination of 5 μM LNF + 100 μM TMZ was added and the MCTS were cultured for an additional 48 h. We assessed the spheroid sizes and expression of stemness markers‐ NESTIN, SOX2, CD133, NANOG, and OCT4‐ through qRT‐PCR and immunostaining. Following 48 h treatment with LNF, TMZ or their combination (LNF + TMZ), the spheroid sizes decreased compared to the untreated control. We also observed that the expression of most of the stemness markers significantly increased in the LNF + TMZ treated condition as compared to the untreated condition. These results indicate that drug treatment can influence the stemness marker expression of GBM cells in MCTS models and these aspects must be considered while evaluating therapies. In future, by incorporating other relevant cell types, we can further our understanding of their crosstalk, eventually leading to the development of new therapeutic strategies.

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Evaluation of cancer stemness in breast cancer and glioblastoma spheroids in vitro

Cited by 9 publications

References 30 publications

Ascites-derived ALDH+CD44+ tumour cell subsets endow stemness, metastasis and metabolic switch via PDK4-mediated STAT3/AKT/NF-κB/IL-8 signalling in ovarian cancer

Ascites-derived ALDH+CD44+ tumour cell subsets endow stemness, metastasis and metabolic switch via PDK4-mediated STAT3/AKT/NF-κB/IL-8 signalling in ovarian cancer

Surface roughness modulates EGFR signaling and stemness of triple-negative breast cancer cells

The impact of temozolomide and lonafarnib on the stemness marker expression of glioblastoma cells in multicellular spheroids

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