Optimization of Extracellular Flux Assay to Measure Respiration of Anchorage-independent Tumor Cell Spheroids

Javed, Zaineb; Worley, Beth L.; Stump, Coryn L.; Shimko, Sara; Crawford, LaTaijah C.; Mythreye, Karthikeyan; Hempel, Nadine

doi:10.21769/bioprotoc.4321

Cited by 8 publications

(4 citation statements)

References 18 publications

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“…In terms of OvCa, this method has been utilised in toxicity screening assays for monitoring chemoresistance in drugs, such as cisplatin and Niraparib [ 17 , 31 ]. Grown in ultra-low-attachment plates, OvCa cells show altered mitochondrial function through augmented extracellular acidification rates [ 32 ]. Re-sensitisation to treatments in cell lines previously thought resistant is also evident using this method, with a number of BRCA wildtype epithelial OvCa cell lines responding to platinum-based therapeutics and showing an increased rate in apoptosis [ 33 ].…”

Section: Discussionmentioning

confidence: 99%

Transcriptional Landscape of 3D vs. 2D Ovarian Cancer Cell Models

Kerslake

Belay

Panfilov

et al. 2023

Cancers

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Three-dimensional (3D) cancer models are revolutionising research, allowing for the recapitulation of an in vivo-like response through the use of an in vitro system, which is more complex and physiologically relevant than traditional monolayer cultures. Cancers such as ovarian (OvCa) are prone to developing resistance, are often lethal, and stand to benefit greatly from the enhanced modelling emulated by 3D cultures. However, the current models often fall short of the predicted response, where reproducibility is limited owing to the lack of standardised methodology and established protocols. This meta-analysis aims to assess the current scope of 3D OvCa models and the differences in the genetic profiles presented by a vast array of 3D cultures. An analysis of the literature (Pubmed.gov) spanning 2012–2022 was used to identify studies with paired data of 3D and 2D monolayer counterparts in addition to RNA sequencing and microarray data. From the data, 19 cell lines were found to show differential regulation in their gene expression profiles depending on the bio-scaffold (i.e., agarose, collagen, or Matrigel) compared to 2D cell cultures. The top genes differentially expressed in 2D vs. 3D included C3, CXCL1, 2, and 8, IL1B, SLP1, FN1, IL6, DDIT4, PI3, LAMC2, CCL20, MMP1, IFI27, CFB, and ANGPTL4. The top enriched gene sets for 2D vs. 3D included IFN-α and IFN-γ response, TNF-α signalling, IL-6-JAK-STAT3 signalling, angiogenesis, hedgehog signalling, apoptosis, epithelial–mesenchymal transition, hypoxia, and inflammatory response. Our transversal comparison of numerous scaffolds allowed us to highlight the variability that can be induced by these scaffolds in the transcriptional landscape and identify key genes and biological processes that are hallmarks of cancer cells grown in 3D cultures. Future studies are needed to identify which is the most appropriate in vitro/preclinical model to study tumour microenvironments.

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Section: Discussionmentioning

confidence: 99%

Transcriptional Landscape of 3D vs. 2D Ovarian Cancer Cell Models

Kerslake

Belay

Panfilov

et al. 2023

Cancers

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show abstract

“…Hanging drop is particularly useful for assessing diffusion gradients in an accessible format [31]. In terms of OvCa this method has been utilised in toxicity screening assays for monitoring chemoresistance in drugs such as cisplatin and Niraparib [18], [32]. Grown in ultra-low attachment plates, OvCa cells show altered mitochondrial function through augmented extracellular acidification rates [33].…”

Section: Discussionmentioning

confidence: 99%

A Meta-analysis of 2D vs. 3D Ovarian Cancer Cellular Models

Kerslake

Belay

Panfilov

et al. 2022

Preprint

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Three dimensional (3D) cancer models are revolutionizing research, allowing for the recapitulation of in vivo like response through the use of an in vitro system, more complex and physiologically relevant than traditional mono-layer culture. Cancers such as ovarian (OvCa), are prone to developing resistance and are often lethal, and stand to benefit greatly from the enhanced modelling emulated by 3D culture. However current models often fall short of predicted response where reproducibility is limited owing to the lack of standardized methodology and established protocols. This meta-analysis aims to assess the current scope of 3D OvCa models and the differences in genetic profile presented by a vast array of 3D cultures. A meta-analysis of the literature (Pubmed.gov) spanning 2012 to 2022, was used to identify studies with comparable monolayer (2D) counterparts in addition to RNA sequencing and microarray data. From the data 19 cell lines were found to show differential regulation in their gene expression profiles depending on the bioscaffold (i.e. agarose, collagen or Matrigel) compared to 2D cell cultures. Top genes differentially expressed 2D vs. 3D include C3, CXCL1, 2 and 8, IL1B, SLP1, FN1, IL6, DDIT4, PI3, LAMC2, CCL20, MMP1, IFI27, CFB, and ANGPTL4. Top Enriched Gene sets for 2D vs. 3D include IFN-alpha and IFN-gamma response, TNF-alpha signalling, IL-6-JAK-STAT3 signalling, angiogenesis, hedgehog signalling, apoptosis, epithelial mesenchymal transition, hypoxia, and inflammatory response. Our transversal comparison of numerous scaffolds allowed us to highlight the vari-ability that can be induced by these scaffolds in the transcriptional landscape as well as identifying key genes and biological processes that are hallmarks of cancer cells grown in 3D cultures. Future studies are needed to identify which is the most appropriate in vitro/preclinical model to study tumour microenvironment.

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“…The Agilent Seahorse XFp Metabolic Analyzer (Agilent, model S7802A) was used to assess mitochondrial respiration of OVCA433 and SKOV3 as described previously for attached cells 75 . Briefly, prior to the start of the experiment, cells were evenly seeded and cultured overnight in a Seahorse XFp cell culture plate (Agilent, 103022-100) at a density of 10,000 and 8,000 cells/well for OVCA433 and SKOV3, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…The Agilent Seahorse XFp Metabolic Analyzer (Agilent, model S7802A) was used to assess mitochondrial respiration of OVCA433 and SKOV3 as described previously for attached cells 77 .…”

Section: Bioenergetic Analysis Of Oxygen Consumption Rate (Ocr) and E...mentioning

confidence: 99%

Alternative splice variants of the mitochondrial fission proteinDNM1L/Drp1 regulate mitochondrial dynamics and tumor progression in ovarian cancer

Javed,

Shin,

Pan

et al. 2023

Preprint

Self Cite

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Aberrant mitochondrial fission/fusion dynamics have previously been reported in cancer cells. While post translational modifications are known regulators of GTPases of the mitochondrial fission/fusion machinery, we show for the first time that alternate splice variants of the fission protein Drp1 (DNM1L) have specific and unique roles in ovarian cancer, adding to the complexity of mitochondrial fission/fusion regulation in tumor cells. We find that ovarian cancer specimens express a Drp1 alternate splice transcript variant lacking exon 16 of the variable domain. High expression of Drp1 lacking exon 16 relative to other transcripts is associated with poor patient outcome. Unlike the unspliced variant, expression of Drp1 lacking exon 16 leads to decreased association of Drp1 to mitochondrial fission sites, more fused mitochondrial networks, enhanced respiration and TCA cycle metabolites, and is associated with a more tumorigenic phenotype. These effects can also be reversed by specific siRNA-mediated inhibition of the endogenously expressed transcript lacking exon 16. Moreover, lack of exon 16 abrogates mitochondrial fission in response to pro-apoptotic stimuli and leads to decreased sensitivity to chemotherapeutics. These data emphasize the significance of the pathophysiological consequences of Drp1 alternate splicing and divergent functions of Drp1 splice variants, and strongly warrant consideration of Drp1 splicing in future studies.

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Optimization of Extracellular Flux Assay to Measure Respiration of Anchorage-independent Tumor Cell Spheroids

Cited by 8 publications

References 18 publications

Transcriptional Landscape of 3D vs. 2D Ovarian Cancer Cell Models

Transcriptional Landscape of 3D vs. 2D Ovarian Cancer Cell Models

A Meta-analysis of 2D vs. 3D Ovarian Cancer Cellular Models

Alternative splice variants of the mitochondrial fission proteinDNM1L/Drp1 regulate mitochondrial dynamics and tumor progression in ovarian cancer

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