Analysis of clonogenic growth in vitro

Brix, Nikko; Samaga, Daniel; Belka, Claus; Zitzelsberger, Horst; Lauber, Kirsten

doi:10.1038/s41596-021-00615-0

Cited by 59 publications

(49 citation statements)

References 71 publications

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“…Colonies containing >50 cells were counted. The plating efficiency (PE) was defined as the number of colonies observed/the number of cells plated, and the surviving fraction was calculated as (colonies counted/cells seeded) × (PE/100) [ 56 , 57 , 58 ].…”

Section: Methodsmentioning

confidence: 99%

ATX-101, a Peptide Targeting PCNA, Has Antitumor Efficacy Alone or in Combination with Radiotherapy in Murine Models of Human Glioblastoma

Gravina

Colapietro

Mancini

et al. 2022

Cancers

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Cell proliferation requires the orchestrated actions of a myriad of proteins regulating DNA replication, DNA repair and damage tolerance, and cell cycle. Proliferating cell nuclear antigen (PCNA) is a master regulator which interacts with multiple proteins functioning in these processes, and this makes PCNA an attractive target in anticancer therapies. Here, we show that a cell-penetrating peptide containing the AlkB homolog 2 PCNA-interacting motif (APIM), ATX-101, has antitumor activity in a panel of human glioblastoma multiforme (GBM) cell lines and patient-derived glioma-initiating cells (GICs). Their sensitivity to ATX-101 was not related to cellular levels of PCNA, or p53, PTEN, or MGMT status. However, ATX-101 reduced Akt/mTOR and DNA-PKcs signaling, and a correlation between high Akt activation and sensitivity for ATX-101 was found. ATX-101 increased the levels of γH2AX, DNA fragmentation, and apoptosis when combined with radiotherapy (RT). In line with the in vitro results, ATX-101 strongly reduced tumor growth in two subcutaneous xenografts and two orthotopic GBM models, both as a single agent and in combination with RT. The ability of ATX-101 to sensitize cells to RT is promising for further development of this compound for use in GBM.

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Section: Methodsmentioning

confidence: 99%

ATX-101, a Peptide Targeting PCNA, Has Antitumor Efficacy Alone or in Combination with Radiotherapy in Murine Models of Human Glioblastoma

Gravina

Colapietro

Mancini

et al. 2022

Cancers

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“…To evaluate the antitumor potential of 12 more comprehensively, its effects at non-toxic concentrations (25–500 nM; cell survival of ≥90%) on a range of metastatic-related processes in glioblastoma cells were studied. Firstly, we investigated the inhibitory activity of 12 on the clonogenic capacity of tumor cells in vitro, which reflects their ability to form colonies from single cells which characterizes the stemness of tumor cells and plays a crucial role in metastasis [ 74 ]. It was found that treatment of U87 and U118 cells seeded at low density with 12 resulted in marked suppression of the clonogenic activity of glioblastoma cells ( Figure 7 A).…”

Section: Resultsmentioning

confidence: 99%

Novel Soloxolone Amides as Potent Anti-Glioblastoma Candidates: Design, Synthesis, In Silico Analysis and Biological Activities In Vitro and In Vivo

et al. 2022

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The modification of natural or semisynthetic triterpenoids with amines can be explored as a promising strategy for improving their pharmacological properties. Here, we report the design and synthesis of 11 novel amide derivatives of soloxolone methyl (SM), a cyano enone-bearing derivative of 18βH-glycyrrhetinic acid. Analysis of their bioactivities in vitro and in silico revealed their high toxicity against a panel of tumor cells (average IC50(24 h) = 3.7 µM) and showed that the formation of amide moieties at the C-30 position of soloxolone did not enhance the cytotoxicity of derivatives toward tumor cells compared to SM, though it can impart an ability to pass across the blood–brain barrier. Further HPLC–MS/MS and mechanistic studies verified significant brain accumulation of hit compound 12 (soloxolone tryptamide) in a murine model and showed its high anti-glioblastoma potential. It was found that 12 induced ROS-dependent and autophagy-independent death of U87 and U118 glioblastoma cells via mitochondrial apoptosis and effectively blocked their clonogenicity, motility and capacity to form vessel-like structures. Further in vivo study demonstrated that intraperitoneal injection of 12 at a dosage of 20 mg/kg effectively inhibited the growth of U87 glioblastoma in a mouse xenograft model, reducing the proliferative potential of the tumor and leading to a depletion of collagen content and normalization of blood vessels in tumor tissue. The obtained results clearly demonstrate that 12 can be considered as a promising leading compound for drug development in glioblastoma treatment.

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“…Clonogenic survival was determined by colony formation assays as described [ 21 , 22 ]. Briefly, cells were detached with Trypsin/EDTA (ThermoScientific), counted with a Neubauer counting chamber, and up to 55,000 cells per well were seeded into 6-well plates yielding a range of 10–200 colonies after treatment.…”

Section: Methodsmentioning

confidence: 99%

Integrative analysis of therapy resistance and transcriptomic profiling data in glioblastoma cells identifies sensitization vulnerabilities for combined modality radiochemotherapy

et al. 2022

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Background Inherent resistance to radio/chemotherapy is one of the major reasons for early recurrence, treatment failure, and dismal prognosis of glioblastoma. Thus, the identification of resistance driving regulators as prognostic and/or predictive markers as well as potential vulnerabilities for combined modality treatment approaches is of pivotal importance. Methods We performed an integrative analysis of treatment resistance and DNA damage response regulator expression in a panel of human glioblastoma cell lines. mRNA expression levels of 38 DNA damage response regulators were analyzed by qRT-PCR. Inherent resistance to radiotherapy (single-shot and fractionated mode) and/or temozolomide treatment was assessed by clonogenic survival assays. Resistance scores were extracted by dimensionality reduction and subjected to correlation analyses with the mRNA expression data. Top-hit candidates with positive correlation coefficients were validated by pharmacological inhibition in clonogenic survival assays and DNA repair analyses via residual γH2AX/53BP1-foci staining. Results Inherent resistance to single-shot and similarly also to fractionated radiotherapy showed strong positive correlations with mRNA expression levels of known vulnerabilities of GBM, including PARP1, NBN, and BLM, as well as ATR and LIG4—two so far underestimated targets. Inhibition of ATR by AZD-6738 resulted in robust and dose-dependent radiosensitization of glioblastoma cells, whereas LIG4 inhibition by L189 had no noticeable impact. Resistance against temozolomide showed strong positive correlation with mRNA expression levels of MGMT as to be expected. Interestingly, it also correlated with mRNA expression levels of ATM, suggesting a potential role of ATM in the context of temozolomide resistance in glioblastoma cells. ATM inhibition exhibited slight sensitization effects towards temozolomide treatment in MGMT low expressing glioblastoma cells, thus encouraging further characterization. Conclusions Here, we describe a systematic approach integrating clonogenic survival data with mRNA expression data of DNA damage response regulators in human glioblastoma cell lines to identify markers of inherent therapy resistance and potential vulnerabilities for targeted sensitization. Our results provide proof-of-concept for the feasibility of this approach, including its limitations. We consider this strategy to be adaptable to other cancer entities as well as other molecular data qualities, and its upscaling potential in terms of model systems and observational data levels deserves further investigation.

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Analysis of clonogenic growth in vitro

Cited by 59 publications

References 71 publications

ATX-101, a Peptide Targeting PCNA, Has Antitumor Efficacy Alone or in Combination with Radiotherapy in Murine Models of Human Glioblastoma

ATX-101, a Peptide Targeting PCNA, Has Antitumor Efficacy Alone or in Combination with Radiotherapy in Murine Models of Human Glioblastoma

Novel Soloxolone Amides as Potent Anti-Glioblastoma Candidates: Design, Synthesis, In Silico Analysis and Biological Activities In Vitro and In Vivo

Integrative analysis of therapy resistance and transcriptomic profiling data in glioblastoma cells identifies sensitization vulnerabilities for combined modality radiochemotherapy

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