Clonogenic Assay Data Research Articles

Modified peroxamide-based reactive oxygen species (ROS)-responsive doxorubicin prodrugs

Reactive oxygen species (ROS) plays a pivotal physiological role in intracellular signaling of any living organism. Due to the elevated levels of ROS in tumor microenvironment than normal tissues, an increasing number of ROS-responsive probes and prodrugs is being studied for the fight against cancer. This study describes the design and synthesis of a panel of novel modified peroxamide-based ROS-responsive prodrugs of doxorubicin, among which the OH-mOX-Dox prodrug showed very stable and highly specific ROS sensitivity. This novel Dox prodrug exerted potent anti-proliferation effects against the two breast cancer cell lines of MDA-MB-468 and MDA-MB-231 while it showed minimal toxicity toward the normal breast cell line, MCF-12A. The cytotoxicity of the OH-mOX-Dox prodrug was significantly enhanced at elevated ROS levels after co-incubation with l-buthionine sulfoximine (BSO). Our clonogenic assay data validated that enhanced intracellular ROS level upon X-ray irradiation resulted in an increase in the efficacy of the OH-mOX-Dox prodrug against the two breast cancer cell lines.

Comparison of Clonogenic Survival Data Obtained by Pre- and Post-Irradiation Methods.

Clonogenic assays are the gold standard to measure in vitro radiosensitivity, which use two cell plating methods, before or after irradiation (IR). However, the effect of the plating method on the experimental outcome remains unelucidated. By using common cancer cell lines, here we demonstrate that pre-IR and post-IR plating methods have a negligible effect on the clonogenic assay-derived photon sensitivity as assessed by SF2, SF4, SF6, SF8, D10, or D50 (N.B. SFx indicates the survival at X Gy; Dx indicates the dose providing X% survival). These data provide important biological insight that supports inter-study comparison and integrated analysis of published clonogenic assay data regardless of the plating method used.

Holistic View on Cell Survival and DNA Damage: How Model-Based Data Analysis Supports Exploration of Dynamics in Biological Systems.

In this work, a method is established to calibrate a model that describes the basic dynamics of DNA damage and repair. The model can be used to extend planning for radiotherapy and hyperthermia in order to include the biological effects. In contrast to “syntactic” models (e.g., describing molecular kinetics), the model used here describes radiobiological semantics, resulting in a more powerful model but also in a far more challenging calibration. Model calibration is attempted from clonogenic assay data (doses of 0–6 Gy) and from time-resolved comet assay data obtained within 6 h after irradiation with 6 Gy. It is demonstrated that either of those two sources of information alone is insufficient for successful model calibration, and that both sources of information combined in a holistic approach are necessary to find viable model parameters. Approximate Bayesian computation (ABC) with simulated annealing is used for parameter search, revealing two aspects that are beneficial to resolving the calibration problem: (1) assessing posterior parameter distributions instead of point-estimates and (2) combining calibration runs from different assays by joining posterior distributions instead of running a single calibration run with a combined, computationally very expensive objective function.

Dose response analysis program (DREAP): A user-friendly program for the analyses of radiation-induced biological responses utilizing established deterministic models at cell population and organ scales

PurposeTo develop a user-friendly program for biological modeling to analyze radiation-induced responses at the scales of the cell population and organ. MethodsThe program offers five established cell population surviving fraction (SF) models to estimate the SF and the relative biological effectiveness (RBE) from clonogenic assay data, and two established models to calculate the normal tissue complication probability (NTCP) and tumor control probability (TCP) from radiation treatment plans. Users can also verify the results with multiple types of quantitative analyses and graphical representation tools. ResultsUsers can verify the estimated SF, model parameters, RBE, and the respective uncertainties in the calculations of the SF and RBE modes. The qualities of the treatment plans can also be compared with at most three rival plans in terms of the NTCP, TCP, uncomplicated TCP (UCP), and user-dependent weight-based UCP (UUCP), in the calculation of the NTCP and TCP modes. Based on the validation study on accuracy and speed, the averaged mean relative errors (MREs) of the estimated parameters for all tested cell lines were not higher than 0.3% in each of the studied SF models, and the averaged MREs of the calculated NTCP and TCP for all tested treatment plans were not higher than 0.1%. The computation times for SF, RBE, NTCP, and TCP were less than 1.5 s. ConclusionsThe dose response analysis program can provide a trustworthy and convenient environment for researchers to analyze radiation-induced biological effects.

A microarray study of radiation-induced transcriptional responses and the role of Jagged 1 in two closely-related lung cancer cell lines

Background: Lung cancer is the most lethal and common cancer in the world. Non-small cell lung cancer accounts for 80% lung cancer cases and its five-year survival rate after radiotherapy is approximately 5%. Patients with lung cancer in the advanced stages are suitable for radiotherapy rather than surgical resection. Unfortunately, radiotherapy alone or in combination with chemotherapy does not significantly improve 5-year survival rates of lung cancer patients. The mechanism by which lung cancer responds to radiation is unclear and needed to be investigated. Methods: Two lung adenocarcinoma cell lines, CL1-0 and CL1-5, that have exhibited different metastasis capacities in previous research, were utilized herein to investigate cellular radio-sensitivity. Clonogenic assay was used to evaluate the radio-sensitivity of CL1-0 and CL1-5. Flow cytometry experiments were performed on both cell lines to understand the change in the cell cycle profile that followed radiation. Gene expression data with the untreated and radiation treated lung cancer cell samples was obtained using Illumina microarrays. Tight hierarchical clustering was conducted to group genes with similar expression profiles. Clonogenic assay and cell cycle distribution were performed with Jagged 1-overexpressed CL1-0 to investigate the role of Jagged 1 in the radio-sensitivity of the lung cancer cells. Results: Clonogenic assay results showed that CL1-5 was more radio-sensitive than CL1-0 after exposure to 10 Gy radiation. The proportion of the CL1-5 cell population in the G2/M phase was found to be markedly higher than that with the CL1-0 cell population following exposure to radiation. Based on the microarray experimental data, 1,595 genes were identified as differentially expressed and sub-grouped into six clusters by hierarchical clustering. The functions of the identified genes in the tight clusters were associated with cell death, cell cycle, cell growth and proliferation, cellular function and maintenance. Moreover, our microarray data suggested that the gene expression levels of Jagged 1 , a Notch ligand, differed significantly between CL1-0 and CL1-5. This finding was further verified by real-time PCR and western blot. Clonogenic assay results further revealed a lower survival rate of Jagged 1-overexpressed CL1-0 than that of CL1-0 following exposure to radiation. Conclusions: CL1-5 was shown to be more radio-sensitive than CL1-0 from our clonogenic assay data. The radiation-induced transcriptional responses in lung cancer cells were evaluated with gene expression microarrays in this work. Jagged 1, the Notch receptor ligand, may have an important role in increasing the radio-sensitivity of lung adenocarcinomas.

Inter-assay precision of clonogenic assays for radiosensitivity in cancer cell line A549.

Clonogenic assays are the gold standard for determining radiosensitivity, which governs tumor response to radiation therapy. Although multiple studies of clonogenic assays on cancer cell lines have been published, the robustness of this technique has not been examined by comparative analysis of data from different studies. To address this issue, we investigated the inter-assay precision of clonogenic assays by analyzing in-house and published data on A549, a cell line frequently studied in this context. The coefficients of variation for SF2, the surviving fraction after 2 Gy irradiation, and D10, the radiation dose that reduces survival to 10%, were below 30% for both in-house data obtained from 20 independent experiments performed under consistent experimental settings (i.e., radiation type, dose rate, and timing of cell seeding) and data collected from 192 publications using diverse experimental settings. Multivariate analyses of the published data revealed that timing of cell seeding significantly affected SF2. These data indicate that SF2 and D10 of clonogenic assay have acceptable inter-assay precision, and that timing of cell seeding influences the inter-assay precision of SF2. These results provide a rationale for combined analysis of published clonogenic assay data, which may help to discover robust biological properties associated with tumor radiosensitivity.

Raman micro-spectroscopy analysis of human lens epithelial cells exposed to a low-dose-range of ionizing radiation

Recent findings in populations exposed to ionizing radiation (IR) indicate dose-related lens opacification occurs at much lower doses (<2 Gy) than indicated in radiation protection guidelines. As a result, research efforts are now being directed towards identifying early predictors of lens degeneration resulting in cataractogenesis. In this study, Raman micro-spectroscopy was used to investigate the effects of varying doses of radiation, ranging from 0.01 Gy to 5 Gy, on human lens epithelial (HLE) cells which were chemically fixed 24 h post-irradiation. Raman spectra were acquired from the nucleus and cytoplasm of the HLE cells. Spectra were collected from points in a 3 × 3 grid pattern and then averaged. The raw spectra were preprocessed and principal component analysis followed by linear discriminant analysis was used to discriminate between dose and control for 0.25, 0.5, 2, and 5 Gy. Using leave-one-out cross-validation accuracies of greater than 74% were attained for each dose/control combination. The ultra-low doses 0.01 and 0.05 Gy were included in an analysis of band intensities for Raman bands found to be significant in the linear discrimination, and an induced repair model survival curve was fit to a band-difference-ratio plot of this data, suggesting HLE cells undergo a nonlinear response to low-doses of IR. A survival curve was also fit to clonogenic assay data done on the irradiated HLE cells, showing a similar nonlinear response.

Overexpression of HOXB7 protein reduces sensitivity of oral cancer cells to chemo-radiotherapy.

The upregulation of homeobox-B7 (HOXB7) in cancers has been reported. However, its role in oral cancer progression remains to be investigated. In current study, our data revealed that reconstitution of HOXB7 expression by transient transfection resulted in increased cell growth, migration and invasion in vitro. In addition, apoptosis and clonogenic assay data showed that overexpression of HOXB7 decreased the sensitivity of oral cancer cells to vincristine-induced apoptosis of HSC-4 and KB/VCR cells. Furthermore, overexpression of HOXB7 promoted oral cancer cells' migration and invasion through activation of TGFβ2/SMAD3 signaling pathway. Moreover, forced expression of HOXB7 increased Bcl-2 to Bax ratio, which would promote cell survival and induce drug and radiotherapy resistance. Altogether, our findings support the role of HOXB7 in the progression of oral cancer. Therefore, HOXB7 potentially can be a therapeutic target for oral cancer.

SU-E-T-264: Development of Dose Response Analysis Program for Basic Radiobiological Modeling

Purpose: To verify and quantify various dose responses including estimated survival fraction(SF) through clonogenic assay, estimated model parameters and its uncertainty, and relative biological effectiveness(RBE) and its uncertainty for each model. Methods: An user-friendly program, dose response analysis program(DREAP, ver.2.0) was developed by using Matlab. The program can provide two types of calculation modes at present. First, the “calculation of SF” mode is designed for giving estimated SF curves, model parameters, and its uncertainty at specific confidence level. it provides five types of established SF models such as linear quadratic(LQ), universal survival curve(USC), multi-target single-hit(MTSH) model, and repair kinetics models including repair mis-repair(RMR) and lethal potentially-lethal(LPL) model. Second, the “calculation of RBE” mode is made for giving estimated RBEs at a broad range of survival levels and its uncertainty at 95 percent confidence level. This mode gives three established SF models except for repair kinetics models. All the modes include basic analysis and comparison tools for each mode. And, all relevant and quantitative results can be easily exported to excel format by one mouse-click. Results: The “calculation of SF” mode can give SF curve through clonogenic assay data, and its uncertainty for each five SF models. Also, the “calculation of RBE” mode can provide RBE and its uncertainty for each three SF models. Since these modes have a feature of interactive and user-friendly structure, the user can easily quantify and access to a variety of dose responses results. Conclusion: The program can provide an easy way to access dose response results with interactive graphical user interfaces(GUIs). The features stated above will make the program practical to use for particle therapy center which needs to collect expected model parameters and its uncertainty, and manage meaningful RBE database.

Drug Repositioning Discovery for Early- and Late-Stage Non-Small-Cell Lung Cancer

Drug repositioning is a popular approach in the pharmaceutical industry for identifying potential new uses for existing drugs and accelerating the development time. Non-small-cell lung cancer (NSCLC) is one of the leading causes of death worldwide. To reduce the biological heterogeneity effects among different individuals, both normal and cancer tissues were taken from the same patient, hence allowing pairwise testing. By comparing early- and late-stage cancer patients, we can identify stage-specific NSCLC genes. Differentially expressed genes are clustered separately to form up- and downregulated communities that are used as queries to perform enrichment analysis. The results suggest that pathways for early- and late-stage cancers are different. Sets of up- and downregulated genes were submitted to the cMap web resource to identify potential drugs. To achieve high confidence drug prediction, multiple microarray experimental results were merged by performing meta-analysis. The results of a few drug findings are supported by MTT assay or clonogenic assay data. In conclusion, we have been able to assess the potential existing drugs to identify novel anticancer drugs, which may be helpful in drug repositioning discovery for NSCLC.

Abstract 3528: In vitro profiling of kinase inhibitors and targeted anticancer agents in panels of patient-derived xenografts using a clonogenic assay

Abstract Novel cancer therapeutics target proteins with key functions in cell survival and migration, protein turnover, mitosis regulation, or angiogenesis. These targets include receptor tyrosine (Y) kinases (e.g. BCR/ABL, EGF-R, FLT-3, Her-2, PDGF-R, KDR, c-KIT, Src, VEGF-R), serine-threonine (S/T) specific kinases (e.g. Aurora A/B (ARK), B-raf), and the chaperone Hsp90. At Oncotest, a unique profiling screen for anticancer agents is routinely done, using a clonogenic assay in a panel of 48 proprietary solid tumor xenografts. The xenograft models were in general established directly from patient tumors, representing all major tumor histotypes (non-small cell lung cancer/NSCLC, pancreatic, prostate, colon, gastric, breast, ovarian and renal cancer, melanoma and sarcoma) as well as niche tumors (pleuramesothelioma, bladder and head and neck cancer). After an initial profiling screen in the 48 tumor panel, follow-up testing in defined tumor histotypes are of high value for hypothesis generation and selection of a clinical development strategy. Here, activity profiles for inhibitors targeting multiple S/T and Y kinases (Dasatinib, Pazopanib, Sunitinib, Sorafenib), EGF-R (Erlotinib, Gefitinib), Her-2 (Lapatinib), Aurora A/B (VX-680), and Hsp90 (17-DMAG) are presented. Erlotinib and Gefitinib displayed a diverse pattern of activity with mean IC50 values of 36.5µM and 20.7µM, respectively. Selectivity was studied in an extended panel of NSCLC with tumors showing an activated EGF-R pathway being most sensitive. The Hsp90 inhibitor 17-DMAG was highly potent with a mean IC50 value of 19nM. As an example, tumor xenograft models selected based on clonogenic assay data responded very well in in vivo efficacy tests against 17-DMAG. The spectrum-kinase inhibitors Sunitinib and Sorafenib displayed a rather weak selectivity with mean IC50 values of 6.3µM and 10.5µM, respectively. In contrast the BCR/ABL and Src inhibitor Dasatinib was active with a mean IC50 of 5.4µM, and showed a highly differential activity against xenografts of solid tumors. Extended tests against 105 tumors of 16 different histotypes identified pancreatic, breast, small cell lung, and colon cancer to be the most sensitive ones. This implements that the use of this compound might not be limited to the treatment of chronic myelogenous leukaemia and gastrointestinal stromal tumors. In conclusion, the presented panel screen using a clonogenic assay, by retaining important characteristics of the original patient tumor, is of high value for profiling of traditional cytotoxic as well as new targeted cancer agents. By revealing diverse activity and resistance patterns, molecular characterization data including gene and protein expression are guiding the selection of tumor entities and patients likely to respond to therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3528. doi:10.1158/1538-7445.AM2011-3528

Abstract A173: Profiling of targeted anticancer agents in a panel of 48 patient-derived xenografts using a clonogenic assay

Abstract Novel experimental and approved cancer therapeutics target proteins with key functions in mitosis regulation, cell survival and migration, protein turnover or angiogenesis. These targets include receptor tyrosine (Y) kinases like EGF-R, PDGF-R, VEGF-R, c-KIT and FLT-3, serine-threonine (S/T) specific kinases like B-raf and Aurora A/B (ARK), mitotic kinesins like Eg5 (KSP), the chaperone Hsp90 and traditional targets, namely topoisomerase I / II and ß-tubulin. At Oncotest, a unique profiling screen for anticancer agents using a clonogenic assay using a panel of 48 proprietary solid tumor xenografts is routinely done. The xenograft models in general were established directly from patient tumor material, representing all major tumor histotypes (non-small cell lung cancer / NSCLC, pancreatic, prostate, colon, gastric, breast, ovarian and renal cancer, melanoma and sarcoma) as well as niche tumors (pleuramesothelioma, bladder and head and neck cancer). After an initial profiling screen in the 48 tumor panel, follow-up testing in defined tumor histotypes are of high value for hypothesis generation including selection of a clinical development strategy. Here, activity profiles for inhibitors targeting EGF-R (Erlotinib), Aurora A/B (VX-680), multiple S/T and Y kinases (Sunitinib, Sorafenib), Eg5 (Ispinesib) and tububiln (Vincristine), Hsp90 (17-DMAG) and topoisomerase I (SN-38, active metabolite of Irinotecan) are discussed. Erlotinib displayed a diverse pattern of activity with a mean IC50 = 20.7µM. Selectivity was studied in an extended panel of NSCLC with tumors showing activated EGF-R being mainly most sensitive. The spectrum-kinase inhibitors Sunitinib and Sorafenib displayed weak selectivity with mean IC50 values of 7.9µM and 11.8µM, respectively. The Hsp90 inhibitor 17-DMAG, the topoisomerase I inhibitor SN-38 and Vincristine were highly potent with mean IC50 values of 28nM, 45nM, and 100nM, respectively. For 17-DMAG and SN-38 as examples, tumor xenograft models selected based on clonogenic assay data responded very well in in vivo efficacy tests. Further evaluation of Vincristine in an extended melanoma panel revealed a strong correlation of sensitivity with a B-raf wild type genotype and resistance with the B-raf V600E mutation. This result was described recently for melanoma patients with B-raf mutations, showing a diminished duration of response to treatment with Dacarbazine, Vincristine, Bleomycin, Lomustine, and human leukocyte interferon. In conclusion, the Oncotest clonogenic assay by retaining important characteristics of the original patient tumor is of high value for profiling of traditional cytotoxic as well as new targeted cancer agents. By revealing diverse activity and resistance patterns, molecular characterization data including gene mutations and expression are guiding the selection of tumor entities and patients likely to respond to therapy. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A173.

Significance of hypoxia for tumor response to radiation: Mathematical modeling and analysis of local control and clonogenic assay data

Various hypotheses for radiation local tumorcontrol probability (ltcp) were modeled, and assessed against local tumorcontrol (LTC) and clonogenic assay (CA) data. For head-and-neck tumors receiving low-LET external-beam irradiation, the best model was a Poisson ltcp accounting for cell repopulation, hypoxia, and tumor volume dependence of radiosensitivity (α). This confirmed that hypoxia is limiting LTC of these tumors, with the magnitude depending upon tumor volume. However, LTC of cervical carcinoma receiving external-beam irradiation and brachytherapy was well described by a model not accounting for hypoxia. Furthermore, when the survival fraction at 2 Gy ( SF 2 ) and colony forming efficiency (CFE) measured for individual patients were incorporated into this model, very good correlation with LTC was seen (p=0.0004). After multivariate analysis, this model was the best independent prognostic factor for LTC and patient survival. Furthermore, no difference in prediction was seen when a model based on birth-and-death stochastic theory was used. Two forms of hypoxia are known to be present in tumors: diffusion-limited, chronic hypoxia (CH), and acute, transient hypoxia (TH). A modeling study on WiDr multicellular spheroids showed that the CH effect on LTC is significantly lower than expected from CA. This could arise from energy charge depletion accompanying CH, reducing the number of proliferating clonogenic cells that can repair radiation damage, and thus mitigating the radioresistance of CH cells. This suggests that TH, rather than CH, may be the limiting factor for in vivo LTC. Finally, by computing ltcp using Monte Carlo calculated dose distributions, it was shown that Monte Carlo statistical noise can cause an underestimation of ltcp, with the magnitude depending upon the model hypotheses.

N-myc amplification and its relationship to experimental therapy.

N-myc amplification correlates with poor prognosis in neuroblastoma patients. Although the reason for this is unclear, it is possible that amplified N-myc confers resistance to certain agents used in the therapy of the disease. The acquisition of resistance to cytotoxic drugs in human tumor cells is multifactorial. One mechanism involved in the development of drug resistance is an increased efficiency of DNA repair. This could reduce the effectiveness of both cisplatin and etoposide (VP-16). Previous studies on human neuroblastoma cells have shown a relationship between N-myc copy number and cisplatin sensitivity. We now report the response to VP-16 treatment of five human neuroblastoma cell lines with a range of N-myc gene copy numbers. After exposure of cells to drug for 24 hours, survival curves were constructed from clonogenic assay data and the iso-effective dose (the dose required to produce 1 log cell kill) was derived. The relationship between N-myc copy number or expression and response to VP-16 was assessed. A significant correlation was established between VP-16 resistance and copy number (r = 0.82; P < 0.05). However, no association was found between N-myc expression and isoeffective dose of VP-16. These results indicate that N-myc amplification may be responsible for treatment failure in those patients receiving cisplatin or VP-16.