Explore the vast range of titles available.

The clonogenic assay measures the capacity of single cells to form colonies in vitro. It is widely used to identify and quantify self-renewing mammalian cells derived from in vitro cultures as well as from ex vivo tissue preparations of different origins. Varying research questions and the heterogeneous growth requirements of individual cell model systems led to the development of several assay principles and formats that differ with regard to their conceptual setup, 2D or 3D culture conditions, optional cytotoxic treatments and subsequent mathematical analysis. The protocol presented here is based on the initial clonogenic assay protocol as developed by Puck and Marcus more than 60 years ago. It updates and extends the 2006 Nature Protocols article by Franken et al. It discusses different strategies and principles to analyze clonogenic growth in vitro and presents the clonogenic assay in a modular protocol framework enabling a diversity of formats and measures to optimize determination of clonogenic growth parameters. We put particular focus on the phenomenon of cellular cooperation and consideration of how this can affect the mathematical analysis of survival data. This protocol is applicable to any mammalian cell model system from which single-cell suspensions can be prepared and which contains at least a small fraction of cells with self-renewing capacity in vitro. Depending on the cell system used, the entire procedure takes ~2–10 weeks, with a total hands-on time of <20 h per biological replicate. This Protocol Extension discusses several approaches to analyzing clonogenic growth of mammalian cells in vitro, using a modular framework to facilitate the use of various formats to fully optimize clonogenic growth.

Background The clonogenic assay is a versatile and frequently used tool to quantify reproductive cell survival in vitro. Current state-of-the-art analysis relies on plating efficiency-based calculations which assume a linear correlation between the number of cells seeded and the number of colonies counted. The present study was designed to test the validity of this assumption and to evaluate the robustness of clonogenic survival results obtained. Methods A panel of 50 established cancer cell lines was used for comprehensive evaluation of the clonogenic assay procedure and data analysis. We assessed the performance of plating efficiency-based calculations and examined the influence of critical experimental parameters, such as cell density seeded, assay volume, incubation time, as well as the cell line-intrinsic factor of cellular cooperation by auto-/paracrine stimulation. Our findings were integrated into a novel mathematical approach for the analysis of clonogenic survival data. Results For various cell lines, clonogenic growth behavior failed to be adequately described by a constant plating efficiency, since the density of cells seeded severely influenced the extent and the dynamics of clonogenic growth. This strongly impaired the robustness of survival calculations obtained by the current state-of-the-art method using plating efficiency-based normalization. A novel mathematical approach utilizing power regression and interpolation of matched colony numbers at different irradiation doses applied to the same dataset substantially reduced the impact of cell density on survival results. Cellular cooperation was observed to be responsible for the non-linear clonogenic growth behavior of a relevant number of cell lines and the impairment of survival calculations. With 28/50 cell lines of different tumor entities showing moderate to high degrees of cellular cooperation, this phenomenon was found to be unexpectedly common. Conclusions Our study reveals that plating efficiency-based analysis of clonogenic survival data is profoundly compromised by cellular cooperation resulting in strongly underestimated assay-intrinsic errors in a relevant proportion of established cancer cell lines. This severely questions the use of plating efficiency-based calculations in studies aiming to achieve more than semiquantitative results. The novel approach presented here accounts for the phenomenon of cellular cooperation and allows the extraction of clonogenic survival results with clearly improved robustness.

The limiting dilution assay (LDA) is a key method to quantify clonogenic cells with self‐renewing capacity in vitro, crucial for preclinical cancer research and therapy response assessment. It estimates the frequency of individual clonogenic, stem‐like cells within a population based on their ability to form colonies with ≥50 cells at limiting cell numbers. Standard LDA analysis relies on linear, single‐hit Poisson models, yet clonogenic growth under single‐cell conditions often involves cooperative or competitive dynamics, violating this linearity assumption. Here, we present a modeling framework incorporating non‐linear population dynamics into LDA analysis and introduce LDAcoop, an R‐based tool for universal quantification of clonogenic cells in LDA formats. Across multiple cancer cell types, we benchmarked LDA against the colony formation assay (CFA) and show that LDA outperforms CFA, especially for patient‐derived organoids, suspension cultures, and higher throughput applications. This renders the LDA format particularly suitable for larger‐scale pharmacogenomic screening and drug sensitivity testing in complex models. Our results establish LDA and LDAcoop as versatile, scalable tools for robust quantification of clonogenic growth, supporting preclinical drug development and molecular precision oncology research. Limiting dilution assays (LDAs) quantify clonogenic growth by seeding serial dilutions of cells and scoring wells for colony formation. The fraction of negative wells is plotted against cells seeded and analyzed using the non‐linear modeling of LDAcoop. LDAs perform comparably to colony formation assays (CFAs) but require less scoring time and are better suited for suspension cells, 3D cultures, and higher throughput formats.

Despite intensive basic scientific, translational, and clinical efforts in the last decades, glioblastoma remains a devastating disease with a highly dismal prognosis. Apart from the implementation of temozolomide into the clinical routine, novel treatment approaches have largely failed, emphasizing the need for systematic examination of glioblastoma therapy resistance in order to identify major drivers and thus, potential vulnerabilities for therapeutic intervention. Recently, we provided proof-of-concept for the systematic identification of combined modality radiochemotherapy treatment vulnerabilities via integration of clonogenic survival data upon radio(chemo)therapy with low-density transcriptomic profiling data in a panel of established human glioblastoma cell lines. Here, we expand this approach to multiple molecular levels, including genomic copy number, spectral karyotyping, DNA methylation, and transcriptome data. Correlation of transcriptome data with inherent therapy resistance on the single gene level yielded several candidates that were so far underappreciated in this context and for which clinically approved drugs are readily available, such as the androgen receptor (AR). Gene set enrichment analyses confirmed these results, and identified additional gene sets, including reactive oxygen species detoxification, mammalian target of rapamycin complex 1 (MTORC1) signaling, and ferroptosis/autophagy-related regulatory circuits to be associated with inherent therapy resistance in glioblastoma cells. To identify pharmacologically accessible genes within those gene sets, leading edge analyses were performed yielding candidates with functions in thioredoxin/peroxiredoxin metabolism, glutathione synthesis, chaperoning of proteins, prolyl hydroxylation, proteasome function, and DNA synthesis/repair. Our study thus confirms previously nominated targets for mechanism-based multi-modal glioblastoma therapy, provides proof-of-concept for this workflow of multi-level data integration, and identifies novel candidates for which pharmacological inhibitors are readily available and whose targeting in combination with radio(chemo)therapy deserves further examination. In addition, our study also reveals that the presented workflow requires mRNA expression data, rather than genomic copy number or DNA methylation data, since no stringent correlation between these data levels could be observed. Finally, the data sets generated in the present study, including functional and multi-level molecular data of commonly used glioblastoma cell lines, represent a valuable toolbox for other researchers in the field of glioblastoma therapy resistance.

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.

Purpose A reliable method for regional in vivo imaging of radiation-induced cellular damage would be of great importance for the detection of therapy-induced injury to healthy tissue and the choice of adequate treatment of radiation emergency patients in both civilian and military events. This study aimed to investigate in a mouse model if positron emission tomography (PET) imaging with proliferation and apoptosis markers is potentially suitable for this purpose. Methods Four groups, including twenty mice (wild-type C57BL/6) each, were whole-body irradiated with 0 Gy, 0.5 Gy, 1 Gy, and 3 Gy and examined by PET over a six-month period at defined time points. 3'-[ 18 F]fluoro-3'-deoxythymidine ([ 18 F]FLT) and 2-(5-[ 18 F]fluoropentyl)-2-methyl malonic acid ([ 18 F]ML-10) were used to visualise proliferation and apoptosis. Regional standard uptake values were compared with respect to irradiation dose over time. Histologic data and peripheral blood cell values were correlated with the PET results. Results The hematopoietic bone marrow showed a significantly increased [ 18 F]FLT signal at early time points after radiation exposure (day 3 and day 7). This correlated with blood parameters, especially leukocytes, and histological data. A significantly increased [ 18 F]FLT signal also occurred in the gastrointestinal tract and thymus at early time points. An increased [ 18 F]ML-10 signal related to irradiation doses was observed in the bone marrow on day 8, but there was a high variability of standard uptake values and no correlation with histological data. Conclusion [ 18 F]FLT showed potential to visualise the extent, regional distribution and recovery from radiation-induced cellular damage in the bone marrow, gastrointestinal tract and thymus. The potential of [ 18 F]FLT imaging to assess the extent of bone marrow affected by irradiation might be especially useful to predict the subsequent severity of hematopoietic impairment and to adapt the therapy of the bone marrow reserve. [ 18 F]ML-10 PET proved to be not sensitive enough for the reliable detection of radiation induced apoptosis.

Background Radiotherapy, administered in fractionated as well as ablative settings, is an essential treatment component for breast cancer. Besides the direct tumor cell death inducing effects, there is growing evidence that immune mechanisms contribute - at least in part - to its therapeutic success. The present study was designed to characterize the type and the extent of cell death induced by fractionated and ablative radiotherapy as well as its impact on the release of monocyte migration stimulating factors by dying breast cancer cells. Methods Cell death and senescence assays were employed to characterize the response of a panel of breast cancer cell lines with different receptor and p53 status towards γ-irradiation applied in a fractionated (daily doses of 2 Gy) or ablative setting (single dose of 20 Gy). Cell-free culture supernatants were examined for their monocyte migration stimulating potential in transwell migration and 2D chemotaxis/chemokinesis assays. Irradiation-induced transcriptional responses were analyzed by qRT-PCR, and CD39 surface expression was measured by flow cytometry. Results Fast proliferating, hormone receptor negative breast cancer cell lines with defective p53 predominantly underwent primary necrosis in response to γ-irradiation when applied at a single, ablative dose of 20 Gy, whereas hormone receptor positive, p53 wildtype cells revealed a combination of apoptosis, primary, and secondary (post-apoptotic) necrosis. During necrosis the dying tumor cells released apyrase-sensitive nucleotides, which effectively stimulated monocyte migration and chemokinesis. In hormone receptor positive cells with functional p53 this was hampered by irradiation-induced surface expression of the ectonucleotidase CD39. Conclusions Our study shows that ablative radiotherapy potently induces necrosis in fast proliferating, hormone receptor negative breast cancer cell lines with mutant p53, which in turn release monocyte migration and chemokinesis stimulating nucleotides. Future studies have to elucidate, whether these mechanisms might be utilized in order to stimulate intra-tumoral monocyte recruitment and subsequent priming of adaptive anti-tumor immune responses, and which breast cancer subtypes might be best suited for such approaches.

Radiotherapy, administered in fractionated as well as ablative settings, is an essential treatment component for breast cancer. Besides the direct tumor cell death inducing effects, there is growing evidence that immune mechanisms contribute - at least in part - to its therapeutic success. The present study was designed to characterize the type and the extent of cell death induced by fractionated and ablative radiotherapy as well as its impact on the release of monocyte migration stimulating factors by dying breast cancer cells. Cell death and senescence assays were employed to characterize the response of a panel of breast cancer cell lines with different receptor and p53 status towards [gamma]-irradiation applied in a fractionated (daily doses of 2 Gy) or ablative setting (single dose of 20 Gy). Cell-free culture supernatants were examined for their monocyte migration stimulating potential in transwell migration and 2D chemotaxis/chemokinesis assays. Irradiation-induced transcriptional responses were analyzed by qRT-PCR, and CD39 surface expression was measured by flow cytometry. Fast proliferating, hormone receptor negative breast cancer cell lines with defective p53 predominantly underwent primary necrosis in response to [gamma]-irradiation when applied at a single, ablative dose of 20 Gy, whereas hormone receptor positive, p53 wildtype cells revealed a combination of apoptosis, primary, and secondary (post-apoptotic) necrosis. During necrosis the dying tumor cells released apyrase-sensitive nucleotides, which effectively stimulated monocyte migration and chemokinesis. In hormone receptor positive cells with functional p53 this was hampered by irradiation-induced surface expression of the ectonucleotidase CD39. Our study shows that ablative radiotherapy potently induces necrosis in fast proliferating, hormone receptor negative breast cancer cell lines with mutant p53, which in turn release monocyte migration and chemokinesis stimulating nucleotides. Future studies have to elucidate, whether these mechanisms might be utilized in order to stimulate intra-tumoral monocyte recruitment and subsequent priming of adaptive anti-tumor immune responses, and which breast cancer subtypes might be best suited for such approaches.