Paired image‐ and FACS‐based toxicity assays for high content screening of spheroid‐type tumor cell cultures

Trumpi, Kari; Egan, David A.; Vellinga, Thomas T.; Rinkes, Inne H.M. Borel; Kranenburg, Onno

doi:10.1016/j.fob.2015.01.003

Cited by 12 publications

(6 citation statements)

References 27 publications

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“…We have demonstrated the versatility and robustness of DeathPro in drug screens of heterogeneous OC cells in monolayer and organoid culture, co‐cultures with fibroblasts, PDX‐derived cells as well as in lung cancer cells as a second cancer entity. Unlike most image‐based viability assays, which detect viable cells by cytoplasmic staining with Calcein AM (Celli et al , ; Trumpi et al , ), DeathPro directly compares the area of nuclei of dead and live cells and generates drug efficacy measures over time independent of cellular morphology and cytoplasmic stains. Counting dead and live cells as an alternative to area measurements would require detailed, time‐consuming imaging of organoids unfeasible in high‐throughput drug screens.…”

Section: Discussionmentioning

confidence: 99%

Screening drug effects in patient‐derived cancer cells links organoid responses to genome alterations

Jabs

Zickgraf

Park

et al. 2017

Molecular Systems Biology

172

186

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Cancer drug screening in patient‐derived cells holds great promise for personalized oncology and drug discovery but lacks standardization. Whether cells are cultured as conventional monolayer or advanced, matrix‐dependent organoid cultures influences drug effects and thereby drug selection and clinical success. To precisely compare drug profiles in differently cultured primary cells, we developed DeathPro, an automated microscopy‐based assay to resolve drug‐induced cell death and proliferation inhibition. Using DeathPro, we screened cells from ovarian cancer patients in monolayer or organoid culture with clinically relevant drugs. Drug‐induced growth arrest and efficacy of cytostatic drugs differed between the two culture systems. Interestingly, drug effects in organoids were more diverse and had lower therapeutic potential. Genomic analysis revealed novel links between drug sensitivity and DNA repair deficiency in organoids that were undetectable in monolayers. Thus, our results highlight the dependency of cytostatic drugs and pharmacogenomic associations on culture systems, and guide culture selection for drug tests.

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Section: Discussionmentioning

confidence: 99%

Screening drug effects in patient‐derived cancer cells links organoid responses to genome alterations

Jabs

Zickgraf

Park

et al. 2017

Molecular Systems Biology

172

186

View full text Add to dashboard Cite

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“…Spheroids were made into single cells 3 d before the start of each experiment as previously described 40 . However, 1 mL trypsin was used instead of Accumax and inactivated using medium containing 11% of fetal calf serum (Bodinco BV).…”

Section: Methodsmentioning

confidence: 99%

“…CRC spheroids were cultured in SCM and 10 ng/ml FGF was added. p7T, p25T and p26T organoids were made into single cells 2–4 d before the start of each experiment in a similar manner as described for the spheroids elsewhere 40 . However, TrypLE was used for dissociation of the organoids and organoid medium was used to dilute TrypLE after establishment of a single cell suspension.…”

Section: Methodsmentioning

confidence: 99%

Differential anti-tumour effects of MTH1 inhibitors in patient-derived 3D colorectal cancer cultures

Waals

Laoukili

Raats

et al. 2019

Sci Rep

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Reactive oxygen species (ROS) function as second messengers in signal transduction, but high ROS levels can also cause cell death. MTH1 dephosphorylates oxidized nucleotides, thereby preventing their incorporation into DNA and protecting tumour cells from oxidative DNA damage. Inhibitors of MTH1 (TH588 and (S)-crizotinib) were shown to reduce cancer cell viability. However, the MTH1-dependency of the anti-cancer effects of these drugs has recently been questioned. Here, we have assessed anti-tumour effects of TH588 and (S)-crizotinib in patient-derived 3D colorectal cancer cultures. Hypoxia and reoxygenation – conditions that increase intracellular ROS levels – increased sensitivity to (S)-crizotinib, but not to TH588. (S)-crizotinib reduced tyrosine phosphorylation of c-MET and ErbB3 whereas TH588 induced a mitotic cell cycle arrest, which was not affected by adding ROS-modulating compounds. Furthermore, we show that both compounds induced DNA damage that could not be prevented by adding the ROS inhibitor N-acetyl-L-cysteine. Moreover, adding ROS-modulating compounds did not alter the reduction in viability in response to TH588 and (S)-crizotinib. We conclude that TH588 and (S)-crizotinib have very clear and distinct anti-tumour effects in 3D colorectal cancer cultures, but that these effects most likely occur through distinct and ROS-independent mechanisms.

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“…The cytotoxicity assays on colosphere cultures have been described extensively elsewhere. [ 36 ] Briefly, 80–100 spheroids were plated per well in a 96-well plate with RK-33 or DMSO. After 72 hours of drug exposure the total cell population was labelled with DRAQ5™ (Abcam, Cambridge, UK) and live cells were labelled with Calcein Green AM (LifeTechnologies, Carlsbad, CA, USA).…”

Section: Methodsmentioning

confidence: 99%

Identification of the DEAD box RNA helicase DDX3 as a therapeutic target in colorectal cancer

et al. 2015

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Identifying druggable targets in the Wnt-signaling pathway can optimize colorectal cancer treatment. Recent studies have identified a member of the RNA helicase family DDX3 (DDX3X) as a multilevel activator of Wnt signaling in cells without activating mutations in the Wnt-signaling pathway. In this study, we evaluated whether DDX3 plays a role in the constitutively active Wnt pathway that drives colorectal cancer.We determined DDX3 expression levels in 303 colorectal cancers by immunohistochemistry. 39% of tumors overexpressed DDX3. High cytoplasmic DDX3 expression correlated with nuclear β-catenin expression, a marker of activated Wnt signaling. Functionally, we validated this finding in vitro and found that inhibition of DDX3 with siRNA resulted in reduced TCF4-reporter activity and lowered the mRNA expression levels of downstream TCF4-regulated genes. In addition, DDX3 knockdown in colorectal cancer cell lines reduced proliferation and caused a G1 arrest, supporting a potential oncogenic role of DDX3 in colorectal cancer.RK-33 is a small molecule inhibitor designed to bind to the ATP-binding site of DDX3. Treatment of colorectal cancer cell lines and patient-derived 3D cultures with RK-33 inhibited growth and promoted cell death with IC50 values ranging from 2.5 to 8 μM. The highest RK-33 sensitivity was observed in tumors with wild-type APC-status and a mutation in CTNNB1.Based on these results, we conclude that DDX3 has an oncogenic role in colorectal cancer. Inhibition of DDX3 with the small molecule inhibitor RK-33 causes inhibition of Wnt signaling and may therefore be a promising future treatment strategy for a subset of colorectal cancers.

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Paired image‐ and FACS‐based toxicity assays for high content screening of spheroid‐type tumor cell cultures

Cited by 12 publications

References 27 publications

Screening drug effects in patient‐derived cancer cells links organoid responses to genome alterations

Screening drug effects in patient‐derived cancer cells links organoid responses to genome alterations

Differential anti-tumour effects of MTH1 inhibitors in patient-derived 3D colorectal cancer cultures

Identification of the DEAD box RNA helicase DDX3 as a therapeutic target in colorectal cancer

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