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Background: Radiobiological experimental setups are challenged by precise sample positioning along depth dose profile, scattering conditions, and practical difficulties that must be addressed in individual designs. The aim of this study was to produce cell survival curves with several irradiation modalities, by using a setup designed at the Danish Centre for Particle Therapy (DCPT) for in vitro proton irradiations using a horizontal beam line and thereby evaluating the setups use for in vitro irradiations experiments. Materials and methods: The setup is a water phantom suitable for in vitro research with multiple irradiation modalities, in particular the pencil scanning proton beam available from a horizontal experimental beamline. The phantom included a water tank of 39.0 × 17.0 × 20.5 cm. Cell survival-curves were produced using the cell line V79 Chinese hamster lung fibroblast cells (V79s) in biological triplicates of clonogenic assays. Cell survival curves were produced with both a 18 MeV electron beam, 6 MV photon beam, and a Spread-Out Bragg Peak (SOBP) proton beam formed by pristine energies of 85–111 MeV where three positions were examined. Results: Survival curves with uncertainty areas were made for all modalities. Dosimetric uncertainty amounted to, respectively, 4%, 3% and 3% for proton, electron, and high energy photon irradiations. Cell survival fraction uncertainty was depicted as the standard deviation between replications of the experiment. Conclusion: Cell survival curves could be produced with acceptable uncertainties using this novel water phantom and cellular laboratory workflow. The setup is useful for future in vitro irradiation experiments.

Clonogenic assays are routinely used to evaluate the response of cancer cells to external radiation fields, assess their radioresistance and radiosensitivity, estimate the performance of radiotherapy. However, classic clonogenic tests focus on the number of colonies forming on a substrate upon exposure to ionizing radiation, and disregard other important characteristics of cells such their ability to generate structures with a certain shape. The radioresistance and radiosensitivity of cancer cells may depend less on the number of cells in a colony and more on the way cells interact to form complex networks. In this study, we have examined whether the topology of 2D cancer-cell graphs is influenced by ionizing radiation. We subjected different cancer cell lines, i.e. H4 epithelial neuroglioma cells, H460 lung cancer cells, PC3 bone metastasis of grade IV of prostate cancer and T24 urinary bladder cancer cells, cultured on planar surfaces, to increasing photon radiation levels up to 6 Gy. Fluorescence images of samples were then processed to determine the topological parameters of the cell-graphs developing over time. We found that the larger the dose, the less uniform the distribution of cells on the substrate—evidenced by high values of small-world coefficient (cc), high values of clustering coefficient (cc), and small values of characteristic path length (cpl). For all considered cell lines, s w > 1 for doses higher or equal to 4 Gy, while the sensitivity to the dose varied for different cell lines: T24 cells seem more distinctly affected by the radiation, followed by the H4, H460 and PC3 cells. Results of the work reinforce the view that the characteristics of cancer cells and their response to radiotherapy can be determined by examining their collective behavior—encoded in a few topological parameters—as an alternative to classical clonogenic assays. [ABSTRACT FROM AUTHOR]

• Integration of published radiosensitivity (RS) data is important but labor-intensive. • We developed deep learning-aided programs to extract RS data from the literature. • Programs #1–3 screen papers containing RS data obtained by clonogenic assays (CAs). • Program #4 extracts CA-derived SF 2 data from semi-logarithmic survival curves. • Programs #1–4 in combination help scientists mine CA-derived RS data from papers. Integrated analysis of existing radiosensitivity data obtained by the gold-standard clonogenic assay has the potential to improve our understanding of cancer cell radioresistance. However, extraction of radiosensitivity data from the literature is highly labor-intensive. To aid in this task, using deep convolutional neural networks (CNNs) and other computer technologies, we developed an analysis pipeline that extracts radiosensitivity data derived from clonogenic assays from the literature. Three classifiers (C1–3) were developed to identify publications containing radiosensitivity data derived from clonogenic assays. C1 uses Faster Regions CNN with Inception Resnet v2 (fRCNN-IRv2), VGG-16, and Optical Character Recognition (OCR) to identify publications that contain semi-logarithmic graphs showing radiosensitivity data derived from clonogenic assays. C2 uses fRCNN-IRv2 and OCR to identify publications that contain bar graphs showing radiosensitivity data derived from clonogenic assays. C3 is a program that identifies publications containing keywords related to radiosensitivity data derived from clonogenic assays. A program (iSF 2) was developed using Mask RCNN and OCR to extract surviving fraction after 2-Gy irradiation (SF 2) as assessed by clonogenic assays, presented in semi-logarithmic graphs. The efficacy of C1–3 and iSF 2 was tested using seven datasets (1805 and 222 publications in total, respectively). C1–3 yielded sensitivity of 91.2% ± 3.4% and specificity of 90.7% ± 3.6%. iSF 2 returned SF 2 values that were within 2.9% ± 2.6% of the SF 2 values determined by radiation oncologists. Our analysis pipeline is potentially useful to acquire radiosensitivity data derived from clonogenic assays from the literature. [ABSTRACT FROM AUTHOR]

We present a comprehensive software program, RAD-ADAPT, for the quantitative analysis of clonogenic assays in radiation biology. Two commonly used models for clonogenic assay analysis, the linear-quadratic model and single-hit multi-target model, are included in the software. RAD-ADAPT uses maximum likelihood estimation method to obtain parameter estimates with the assumption that cell colony count data follow a Poisson distribution. The program has an intuitive interface, generates model prediction plots, tabulates model parameter estimates, and allows automatic statistical comparison of parameters between different groups. The RAD-ADAPT interface is written using the statistical software R and the underlying computations are accomplished by the ADAPT software system for pharmacokinetic/pharmacodynamic systems analysis. The use of RAD-ADAPT is demonstrated using an example that examines the impact of pharmacologic ATM and ATR kinase inhibition on human lung cancer cell line A549 after ionizing radiation. [ABSTRACT FROM AUTHOR]

Few treatment options exist for metastatic prostate cancer (PC) that becomes hormone refractory (HRPC). In vitro, plant-derived MINA-05 caused dose-dependent decreases in cell numbers in HRPC cell lines LNCaPC4-2B and PC-3, and in androgen-sensitive LNCaP-FGC, DuCaP, and LAPC-4, by WST-1 assay. MINA-05 pretreatment significantly decreased clonogenic survival in agar and on plastic at 1 × and 2 × IC50 for PC-3 (P < .05 and P < .001, respectively), and at 1/2 ×, 1 ×, and 2 × IC50 for LNCaP-FGC cells (P < .001). MINA-05 also induced G2M arrest of LNCaP-FGC and PC-3 cells (by flow cytometry) and caused some apoptosis in LNCaPFGC (sub-G1, peak on flow, expression of activated caspase-3) but not in PC-3 cells. Western blotting indicated that these cell cycle changes were associated with decreased levels of regulatory proteins cyclin B1 and cdc25C. MINA-05 given daily by gavage for 39 days did not diminish primary orthotopic PC-3 growth in nude mice, but decreased the extent of lymph node invasion at higher doses. We conclude that MINA-05 induces G2M arrest, inhibits cell growth, reduces PC cell re-growth in vitro, and reduces lymph node invasion after orthotopic PC-3 cell implantation in vivo. It has potential as an adjuvant treatment for patients with PC.

Blood is a fluid connective tissue which plays a vital role for normal body function. It consist different type of blood cells which is continuously reproduce inside the bone marrow from hematopoietic system. Xenobiotics could be specifically toxic to the hematopoietic system and they can cause hematological disorders by disturbing the normal functions.In vitrohematopoietic colony-forming cell assays play a crucial role to evaluate potential toxic effects of new xenobiotics and also helpful in bridging the gap between preclinical toxicology studies in animal models and clinical investigations. Use of these assays in conjunction with, high-throughput screening reduces the cost and time associated with these assays. This article provides a critical view overin vitrohematopoietic colony-forming cell assays in assessment of hematotoxicity. [ABSTRACT FROM PUBLISHER]

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Hematotoxicology is concerned with the adverse effects of xenobiotics on hematopoietic processes in a living system. In vitro tests for hematotoxicity have been applied in toxicity assessment of chemicals, drugs, food supplements and environment related studies. Hematopoietic Progenitor Colony-Forming Cell (CFC) assays and Stromal Cell Assays (also called as Non-hematopoietic Progenitor Assays) are already being used to detect hematological toxicities induced by different contaminants. These in vitro tests have been very useful in reducing the number of animals required for hematotoxicity testing. In this review, applications, limitations and future prospective of in vitro tests for hematotoxicity with emphasis on the techniques involved in the Colony forming unit culture systems are described. [ABSTRACT FROM AUTHOR]

Background aims. The long-term stability of cryopreserved peripheral blood progenitor cells is an important issue for patients experiencing disease relapse. However, there is no consensus on how to evaluate the long-term effects of cryopreservation. We describe the effect of cryopreservation on viability and progenitor colony activity from 87 individual samples processed at the Scripps Green Hospital Stem Cell Processing Center (La Jolla, CA, USA). Methods. We randomly selected 87 peripheral blood hematopoietic stem cell (PBHSC) samples from 60 patients and evaluated the effect of cryopreservation on sample viability and red and white cell colony activity after < 24 h and 7, 10 and 15 years of cryopreservation. Viability was assayed via trypan blue dye exclusion and activity was measured following 14 days of culture. Results. An age at collection older than 50 years may result in suboptimal activity and viability following long-term cryopreservation, while gender and disease status had no effect. Cryopreservation did not significantly affect white or red cell activity following 10 years of cryopreservation. However, for samples stored longer than 10 years, viability and activity significantly decreased. We noted a positive association between higher pre-cryopreservation %CD34 count and colony activity. Conclusions. Cryopreservation of peripheral blood progenitor cells for up to 10 years results in no loss of clonogenic capacity, as determined by culture activity, although longer durations of storage may affect activity. Until validated methods are developed, cryopreserved grafts should be evaluated based on pre-freeze CD34+ cell counts as assayed by flow cytometry, and post-thaw sample evaluation should be reserved for patients identified as poor mobilizers. [ABSTRACT FROM AUTHOR]

Background: Many plant-derived products exhibit potent chemopreventive activity against animal tumor models as well as rodent and human cancer cell lines. They have low side effects and toxicity and presumably modulate the factors that are critical for cell proliferation, differentiation, senescence and apoptosis. The present study investigates the effects of some medicinal plant extracts from generally recognized as safe plants that may be useful in the prevention and treatment of cancer. Methods: Clonogenic assays using logarithmically-growing cells were performed to test the effect. The cytotoxic effects of Curcuma longa and Zingiber officinale were studied using sulforhodamine B assay, tetrazolium dye assay, colony morphology and microscopic analysis. Results: Out of the 13 lyophilized plant-derived extracts evaluated for growth-inhibitory effects on the PC-3M prostate cancer cell line, two extracts derived from C. longa and Z. officinale showed significant inhibitory effects on colony-forming ability. The individual and augmentative effects of these two extracts were tested for their narrow range effective lower concentration on PC-3M in clonogenic assays. At relatively lower concentrations, C. longa showed significant inhibition of colony formation in clonogenic assays; whereas at same concentrations Z. officinale showed only moderate inhibitory effects. However, when both the agents were tested together at the same concentrations, the combined effects were much more significant than their individual ones. On normal prostate epithelial cells both C. longa and Z. officinale had similar effects but at a lower magnitude. These observations were confirmed by several cytotoxicity assays involving the morphological appearance of the colonies, microscopic observations, per cent inhibition in comparison to control by sulforhodamine B and tetrazolium dye assay. Conclusions: From these observations, it was concluded that the combined effects of C. longa and Z. officinale are much greater than their individual effects, suggesting the role of multiple components and their synergistic mode of actions to elicit stronger beneficial effects. [ABSTRACT FROM AUTHOR]

We investigated the activity of ITF2357, a novel histone deacetylase inhibitor (HDACi) with antitumor activity, on cells carrying the JAK2V617F mutation obtained from polycythemia vera (PV) and essential thrombocythemia (ET) patients as well as the HEL cell line. The clonogenic activity of JAK2V617F mutated cells was inhibited by low concentrations of ITF2357 (IC50 0.001–0.01 μM), 100- to 250-fold lower than required to inhibit growth of normal or tumor cells lacking this mutation. Under these conditions, ITF2357 allowed a seven fold increase in the outgrowth of unmutated over mutated colonies. By western blotting we showed that in HEL cells, ITF2357 led to the disappearance of total and phosphorylated JAK2V617F as well as pSTAT5 and pSTAT3, but it did not affect the wild-type JAK2 or STAT proteins in the control K562 cell line. By real-time PCR, we showed that, upon exposure to ITF2357, JAK2V617F mRNA was not modified in granulocytes from PV patients while the expression of the PRV-1 gene, a known target of JAK2, was rapidly downmodulated. Altogether, the data presented suggest that ITF2357 inhibits proliferation of cells bearing the JAK2V617F mutation through a specific downmodulation of the JAK2V617F protein and inhibition of its downstream signaling.Leukemia (2008) 22, 740–747; doi:10.1038/sj.leu.2405049; published online 13 December 2007 [ABSTRACT FROM AUTHOR]

We investigated the activity of ITF2357, a novel histone deacetylase inhibitor (HDACi) with antitumor activity, on cells carrying the JAK2V617F mutation obtained from polycythemia vera (PV) and essential thrombocythemia (ET) patients as well as the HEL cell line. The clonogenic activity of JAK2V617F mutated cells was inhibited by low concentrations of ITF2357 (IC50 0.001–0.01 μM), 100- to 250-fold lower than required to inhibit growth of normal or tumor cells lacking this mutation. Under these conditions, ITF2357 allowed a seven fold increase in the outgrowth of unmutated over mutated colonies. By western blotting we showed that in HEL cells, ITF2357 led to the disappearance of total and phosphorylated JAK2V617F as well as pSTAT5 and pSTAT3, but it did not affect the wild-type JAK2 or STAT proteins in the control K562 cell line. By real-time PCR, we showed that, upon exposure to ITF2357, JAK2V617F mRNA was not modified in granulocytes from PV patients while the expression of the PRV-1 gene, a known target of JAK2, was rapidly downmodulated. Altogether, the data presented suggest that ITF2357 inhibits proliferation of cells bearing the JAK2V617F mutation through a specific downmodulation of the JAK2V617F protein and inhibition of its downstream signaling.Leukemia (2008) 22, 740–747; doi:10.1038/sj.leu.2405049; published online 13 December 2007 [ABSTRACT FROM AUTHOR]

Abstract: Damage to the stem cell progenitors caused by the chemotherapy received in patients diagnosed with non-Hodgkin’s lymphoma (NHL) may be an important factor limiting progenitor cell mobilization. The aim of the present analysis was to evaluate the effect of the chemotherapy on the different progenitor cell subpopulations obtained in the leukapheresis. For this purpose, a combination of immunophenotype and functional assays has been performed in 26 mobilized peripheral blood (PB) samples from NHL patients and 36 healthy donors. The different progenitor subpopulations analyzed by flow cytometry significantly correlated with the corresponding populations assessed by functional assays in both healthy donors and NHL patients (p <0.05, r >0.5). The number of committed CFU-GM was similar in both groups (p =0.246), but we found significant decrease in the number of BFU-E and more immature progenitors in PB from NHL patients as compared to donors (p <0.05). Moreover, the number of total CFU was significantly lower in NHL patients (p =0.007). Accordingly, CD34+ cells (p =0.018) and CD34+ subpopulations was decreased in NHL patients. Nevertheless, CD90 and CD34 intensity was significantly higher within CD34+ cells from NHL patients as compared to donors. However, although numerically reduced non-committed CD34+ cells are more immature in chemotherapy mobilized NHL patients. In summary, our results show that all NHL hematopoietic progenitors, analyzed by both immunophenotypical and functional approaches, are impaired in leukapheresis products. [Copyright &y& Elsevier]

Few treatment options exist for metastatic prostate cancer (PC) that becomes hormone refractory (HRPC). In vitro, plant-derived MINA-05 caused dose-dependent decreases in cell numbers in HRPC cell lines LNCaPC4- 2B and PC-3, and in androgen-sensitive LNCaP-FGC, DuCaP, and LAPC-4, by WST-1 assay. MINA-05 pretreatment significantly decreased clonogenic survival in agar and on plastic at 1 x and 2 x IC50 for PC-3 (P < .05 and P < .001, respectively), and at ½ x, 1 x, and 2 x IC50 for LNCaP-FGC cells (P < .001). MINA-05 also induced G2M arrest of LNCaP-FGC and PC-3 cells (by flow cytometry) and caused some apoptosis in LNCaPFGC (sub-G1 peak on flow, expression of activated caspase-3) but not in PC-3 cells. Western blotting indicated that these cell cycle changes were associated with decreased levels of regulatory proteins cyclin B1 and cdc25C. MINA-05 given daily by gavage for 39 days did not diminish primary orthotopic PC-3 growth in nude mice, but decreased the extent of lymph node invasion at higher doses. We conclude that MINA-05 induces G2M arrest, inhibits cell growth, reduces PC cell regrowth in vitro, and reduces lymph node invasion after orthotopic PC-3 cell implantation in vivo. It has potential as an adjuvant treatment for patients with PC. [ABSTRACT FROM AUTHOR]

As part of the early preclinical development of a new antipsychotic compound, Wistar rats of both sexes were dosed orally for upto 7 days. At high doses, expected changes in appearance and behavior, decreases in bodyweight gain and feed intake but also a fluid and pale bone marrow (BM) were observed. Blood cell counts were normal as were clinical chemical values. BM sections showed a red cell hypoplasia. Circulating reticulocytes and erythroblasts on BM smears were decreased suggesting that the compound might have a selective toxicity for the erythroid lineage. In a mechanistic experiment, rats were dosed for 9 days and phlebotomized after 7 days of exposure to stimulate erythroid regeneration. Red-blood cell mass, reticulocytes and erythropoietin (EPO) levels were monitored before and upto 48 h after bleeding. Results showed that an EPO-mediated pathogenesis could be excluded. The effect of the drug on the formation of Colony-forming units (CFU)-E and CFU-GM was then quantitatively measured in vitro after direct exposure to the compound. In two successive assays, rat or human BM cells were incubated with the drug dissolved in the collection medium at final concentrations of 0.3×10−7 –3×10−5 M. In the presence of adequate growth factors, CFU-E and CFU-GM were cultured and cell proliferation was compared between treated and control groups. Our results showed an expected inhibition by the drug of the growth of erythroid progenitors associated to a similar effect on myeloid progenitors. The CFU-E and CFU-GM of both human and rat sources were totally inhibited from the concentration of 3×10−5 M. The IC50 values were consistent with rat peak plasma levels reached in vivo by the drug. Therefore, the short-term cloning assays performed on rat BM cells were sensitive indicators of the hematotoxicity of the compound and were considered as predictive for human toxicity. [ABSTRACT FROM AUTHOR]

Herpesvirus-based gene therapy vectors offer an attractive alternative to retroviral vectors because of their episomal nature and ability to accommodate large transgenes. Saimiriine herpesvirus 2 (HVS) is a prototypical gamma-2 herpesvirus that can latently infect numerous different cell types. A cosmid-generated HVS vector in which transforming genes have been deleted and the marker gene encoding enhanced green fluorescent protein (HVS-GFP) has been incorporated was evaluated for its potential to transduce CD34+haemopoietic progenitors selected from cord blood. Expression of GFP could subsequently be readily detected in cells of the erythroid lineage in both CFU-GEMM assays and liquid differentiation cultures. These results confirm the potential of HVS as a candidate vector for gene therapy applications using primitive haemopoietic cells and suggest that it may be applicable to disorders affecting cells of the erythroid lineage.Gene Therapy (2005) 12, 373-379. doi:10.1038/sj.gt.3302422 Published online 23 December 2004 [ABSTRACT FROM AUTHOR]

The present work analyzes the hematopoietic progenitor cells (HPC) in myelodysplastic syndrome (MDS) patients using both an immunophenotypical and a functional approaches in order to know whether they are similar in patients with or without cytogenetic abnormalities. Among CD34+ HPC, the proportion of myeloid committed progenitors was higher in patients with an abnormal karyotype. Ninety MDS patients were studied. Patients with abnormal karyotype showed a similar platting efficiency than patients with normal cytogenetics. Trisomy 8 and 5q- showed a significant higher P.E. than patients with normal karyotype or monosomy 7. We observed that when the most immature HPC were studied, the total number of granulo-monocytic colonies produced by LTBMC was higher in the normal karyotype group. In summary, the present study shows that in MDS the HPC are impaired; this impairment is deeper in patients with abnormal karyotype. [Copyright &y& Elsevier]

Background: A new antipsychotic compound induced unexpected red cell hypoplasia (reticulocytopenia, red marrow hypoplasia) in rats dosed orally for 7 days. Materials and methods: Since an erythropoietin-mediated pathogenesis was excluded, In Vitro tests on rat and human bone marrow cells were performed with measurement of formation of late erythroid (CFU-E) and granulocyte-macrophage (CFU-GM) colony-forming units after incubation with the drug. CFU-E together with growth factors were cultured for 2 days (rat) or 7 days (human) and CFU-GM was cultured for 7 days (rat) or 10 days (human). Results: The drug induced inhibition of erythroid progenitors and myeloid progenitors for both species from 3 × 10-5 mol/L, with the concentration inhibiting the growth of 50% (IC50) consistent with drug plasma levels measured in rats. Conclusion: These cloning assays on rat bone-marrow cells were shown to be adequate models for determining the haematotoxicity of the agent and to be predictive of human toxicity. With only a small amount of compound required, they can be used as screening tools to detect haematotoxic potential of candidate drugs. Keywords: haematotoxicity, clonogenic assays, CFU-E, CFU-GM, screening, In Vitro model Résumé Contexte : Un nouvel antipsychotique induit une hypoplasie érythroïde (réticulocytopénie, hypoplasie de moelle rouge) chez le rat après administration orale pendant 7 jours. Matériels et méthodes : Un mécanisme érythropoïétine-dépendant étant écarté, des tests In Vitro sur cellules de moelle osseuse de rat et d’homme sont effectués, en évaluant la formation CFU-E (colony-forming units-erythrocyte) et CFU-GM (colony-forming units-granulocyte-macrophage) après incubation avec le produit. En présence de facteurs de croissance adéquats, les CFU-E sont cultivés pendant 2 (rat) ou 7 (homme) jours et les CFU-GM pendant 7 (rat) ou 10 (homme) jours. Résultats : Le produit inhibe la croissance des progéniteurs érythroïdes et myéloïdes dans les deux espèces à partir de 3 × 10-5 mol/L, avec des CI50 (concentration inhibant la croissance cellulaire de 50 %) cohérentes avec les taux plasmatiques mesurés chez le rat. Conclusion : Ces essais clonogéniques sur les cellules de moelle osseuse de rat sont donc des modèles adéquats pour l’hématotoxicité du produit et prédictifs pour l’homme. Consommant une faible quantité de produit, ils peuvent être utilisés pour sélectionner de futurs médicaments sur leur potentiel hématotoxique. Mots clés : hématotoxicité, essais clonogéniques, CFU-E, CFU-GM, screening, modèle In Vitro [ABSTRACT FROM AUTHOR]

The major side effect of anticancer drugs is damage to the hematopoietic system. These compounds may interfere with cell proliferation and differentiation in different blood lineages causing many diseases such as neutropenia, aplastic anaemia or trombocytopenia. The clonogenic assays are useful in vitro tools for evaluating and predicting acute xenobiotics myelotoxicity. A miniaturisation of these assays, in order to reduce costs and increase the number of compounds that could be tested, is under investigation. The in vitro sensitivity of human burst-forming unit erythroid (BFU-E) and colony-forming unit granulocyte-macrophage (CFU-GM) to three anticancer drugs: cyclophosphamide (CTX), 5-fluorouracil (5-FU) and taxol (TAX) was evaluated both in 35 mm plate and 96-well plate systems and the dose-response curves, IC50 values and IC90 values were compared. The correlation between in vitro data and clinical plasma levels confirms that severe hematotoxicity is the primary adverse effect of these drugs with an evident selectivity on erythroid progenitors for cyclophosphamide. IC50 and IC90 values, calculated on the basis of results obtained with the traditional assay, correlate with those obtained in microplate, as well as the dose-response curves, indicating that the 96 well plate assay could be a useful and reliable tool for high-throughput screening in early stages of drug development. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Radiotherapy treatment strategies should be personalized based on the radiosensitivity of individual tumors. Clonogenic assays are the gold standard method for in vitro assessment of radiosensitivity. Reproducibility is the critical factor for scientific rigor; however, this is reduced by insufficient reporting of methodologies. In reality, the reporting standards of methodologies pertaining to clonogenic assays remain unclear. To address this, we performed a literature search and qualitative analysis of the reporting of methodologies pertaining to clonogenic assays. A comprehensive literature review identified 1672 papers that report the radiosensitivity of human cancer cells based on clonogenic assays. From the identified papers, important experimental parameters (i.e. number of biological replicates, technical replicates, radiation source and dose rate) were recorded and analyzed. We found that, among the studies, (i) 30.5% did not report biological or technical replicates; (ii) 47.0% did not use biological or technical replicates; (iii) 3.8% did not report the radiation source; and (iv) 32.3% did not report the dose rate. These data suggest that reporting of methodologies pertaining to clonogenic assays in a considerable number of previously published studies is insufficient, thereby threatening reproducibility. This highlights the need to raise awareness of standardization of the methodologies used to conduct clonogenic assays., (© The Author(s) 2020. Published by Oxford University Press on behalf of The Japanese Radiation Research Society and Japanese Society for Radiation Oncology.)

The cytotoxicity of a dinuclear imine-copper (II) complex 2 , and its analogous mononuclear complex 1 , toward different melanoma cells, particularly human SKMEL-05 and SKMEL-147, was investigated. Complex 2 , a tyrosinase mimic, showed much higher activity in comparison to complex 1 , and its reactivity was verified to be remarkably activated by UVB-light, while the mononuclear compound showed a small or negligible effect. Further, a significant dependence on the melanin content in the tumor cells, both from intrinsic pigmentation or stimulated by irradiation, was observed in the case of complex 2. Similar tests with keratinocytes and melanocytes indicated a much lower sensitivity to both copper (II) complexes, even after exposition to UV light. Clonogenic assays attested that the fractions of melanoma cells survival were much lower under treatment with complex 2 compared to complex 1 , both with or without previous irradiation of the cells. The process also involves generation of reactive oxygen species (ROS), as verified by EPR spectroscopy, and by using fluorescence indicators. Autophagic assays indicated a remarkable formation of cytoplasmic vacuoles in melanomas treated with complex 2 , while this effect was not observed in similar treatment with complex 1. Monitoring of specific protein LC3 corroborated the simultaneous occurrence of autophagy. A balance interplay between different modes of cell death, apoptosis and autophagy, occurs when melanomas were treated with the dinuclear complex 2 , in contrast to the mononuclear complex 1. These results pointed out to different mechanisms of action of such complexes, depending on its nuclearity. Cytotoxicity of dinuclear imine-copper (II) complex is mediated by reactive oxygen species, but depends further on melanogenesis and in interaction with melanin. Image 1 • Reactivity dissimilarities between dinuclear and mononuclear imine-copper(II) complexes against melanomas were monitored. • Influence of UVB light and melanogenesis were particularly evaluated. • Cytotoxicity is mediated by reactive oxygen species but depends further on melanogenesis and interaction with melanin. • A remarkable sensitization of melanoma cells by the dinuclear complex pointed to diverse modes of action. • A balance interplay of apoptosis and autophagy seems to occur. [ABSTRACT FROM AUTHOR]

Photon radiation therapy is a major curative treatment for cancer. However, the lack of robust predictive biomarkers for radiosensitivity precludes personalized radiation therapy. Clonogenic assays are the gold standard method for measuring the radiosensitivity of cancer cells. Although a large number of publications describe the use of clonogenic assays to measure cancer cell radiosensitivity, the robustness of results from different studies is unclear. To address this, we conducted a comprehensive detailed literature search of 256 common cancer cell lines and identified the eight cell lines most-frequently examined for photon sensitivity using clonogenic assays. Survival endpoints and experimental parameters from all 620 relevant experiments were compiled and analyzed. We found that the coefficients of variation for SF2 (surviving fraction after 2 Gy irradiation) and for D10 (dose that yields a surviving fraction of 10%) were below 30% for all cell lines, indicating that SF2 and D10 have acceptable inter-assay precision. These data support further analysis of published data on clonogenic assays using SF2 and D10 as survival endpoints, which facilitates robust identification of biological profiles representative of cancer cell sensitivity to photons. [ABSTRACT FROM AUTHOR]

In the era of precision medicine, radiotherapy strategies should be determined based on genetic profiles that predict tumor radiosensitivity. Accordingly, pre-clinical research aimed at discovering clinically applicable genetic profiles is needed. However, how a given genetic profile affects cancer cell radiosensitivity is unclear. To address this issue, we performed a pilot in vitro study by utilizing EGFR mutational status as a model for genetic profile. Clonogenic assays of EGFR mutant (n = 6) and wild-type (n = 9) non-small cell lung carcinoma (NSCLC) cell lines were performed independently by two oncologists. Clonogenic survival parameters SF2, SF4, SF6, SF8, mean inactivation dose (MID), D10, D50, α, and β were obtained using the linear quadratic model. The differences in the clonogenic survival parameters between the EGFR mutant and wild-type cell lines were assessed using the Mann–Whitney U test. As a result, for both datasets, the p values for SF2, SF4, D50, α, and α/β were below 0.05, and those for SF2 were lowest. These data indicate that a genetic profile of NSCLC cell lines might be predictive for their radiation response; i.e., EGFR mutant cell lines might be more sensitive to low dose- and low fraction sized-irradiation. [ABSTRACT FROM AUTHOR]

Genomic editing methods, such as the CRISPR/Cas9 system, are routinely used to study gene function in somatic cells. Owing to the heterogeneity of mutations, it is necessary to purify cell clones grown from high dilution to the point of colony formation, which can be a time-consuming process. Here, we tested a modified approach in which we seeded cells at high dilution, together with non-edited carrier cells. As a comparison, cells were also grown at high dilution with conditioned medium from a high-density culture. When using carrier cells or conditioned medium, the formation of cell colonies is accelerated. Additionally, clones grown with carrier cells are more similar to the parental lines in terms of their tumorigenic properties. Surprisingly, key signaling cascades are highly divergent between clones isolated from low-density cultures, even with conditioned medium, in contrast to clones isolated with carrier cells. Thus, our study uncovers a significant limitation using the common approach of isolating cell clones following genetic modifications and suggests an alternative method that mitigates the problem of heterogeneity of gene expression between clones.This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Besides inactivating tumour suppressor activity in cells, mutations in p53 confer significant oncogenic functions and promote metastasis and resistance to anticancer therapy. A variety of therapies involving genetic and epigenetic signalling events regulate tumorogenesis and progression in such cases. Pharmacological interventions with HDAC inhibitors have shown promise in therapy. This work explores the changes in efficacy of the four HDAC inhibitors SAHA, MS-275, valproic acid and sodium butyrate on a panel of colon cancer cell lines - HCT116 (p53 wt), HCT116 p53-/-, HT29 and SW480 (with mutations in p53). Clonogenic assays, gene profiling and epigenetic expression done on these cells point to p53 dependent differential activity of the 4 HDAC inhibitors which also elevate methylation levels in p53 mutant cell lines. In silico modelling establishes the alterations in interactions that lead to such differential activity of valproic acid, one of the inhibitors considered for the work. Molecular Dynamic simulations carried out on the valproic acid complex ensure stability of the complex. This work establishes a p53 dependent epigenetic signalling mechanism triggered by HDAC inhibition expanding the scope of HDAC inhibitors in adjuvant therapy for p53 mutant tumours.

Objective: Epithelial-mesenchymal crosstalk (EMC) contributes to tumor progression, chemoresistance and acquisition of a mesenchymal phenotype (EMT) of cancer cells. This study aims to investigate the effects of EMC on radioresistance in head and neck squamous cell carcinoma (HNSCC) cells., Methods: In tumor cell lines, the response of HNSCC cells, stimulated with EMC conditioned medium (CM), to irradiation was evaluated with viability and clonogenic assays. Dose modifying factors (DMF) were calculated from the results of clonogenic assays. Potential pathways involved in radioresistance were analyzed with quantitative Real-Time PCR and western blot., Results: CM significantly reduced the doubling time of SCC-25 cells (from 32.8 hours to 16.8 hours, p=0.0001) and Detroit 562 cells (from 88.5 hours to 29.6 hours, p=0.014). Further it increased clonogenic survival after irradiation. The DMF of CM was 2.04 ± 0.43 (mean ± standard deviation) for SCC-25 cells (p=0.015) and 2.14 ± 0.34 for Detroit 562 cells (p=0.008). Treatment with CM more than tripled the ERCC1 and survivin gene expression in SCC-25 cells., Conclusion: EMC induced pathways involved in cell survival and DNA repair and led to increased radioresistance in HNSCC cells., Competing Interests: CONFLICTS OF INTEREST The authors declare that they have no competing interests.

Background: The current methodology for the Surviving Fraction (SF) measurement in clonogenic assay, which is a technique to study the anti-proliferative effect of treatments on cell cultures, involves manual counting of cell colony forming units. This procedure is operator-dependent and error-prone. Moreover, the identification of the exact colony number is often not feasible due to the high growth rate leading to the adjacent colony merging. As a matter of fact, conventional assessment does not deal with the colony size, which is generally correlated with the delivered radiation dose or the administered cytotoxic agent., Method: Considering that the Area Covered by Colony (ACC) is proportional to the colony number and size as well as to the growth rate, we propose a novel fully automatic approach exploiting Circle Hough Transform, to automatically detect the wells in the plate, and local adaptive thresholding, which calculates the percentage of ACC for the SF quantification. This measurement relies just on this covering percentage and does not consider the colony number, preventing inconsistencies due to intra- and inter-operator variability., Results: To evaluate the accuracy of the proposed approach, we compared the SFs obtained by our automatic ACC-based method against the conventional counting procedure. The achieved results (r = 0.9791 and r = 0.9682 on MCF7 and MCF10A cells, respectively) showed values highly correlated with the measurements using the traditional approach based on colony number alone., Conclusions: The proposed computer-assisted methodology could be integrated in laboratory practice as an expert system for the SF evaluation in clonogenic assays., (Copyright © 2017 Elsevier Ltd. All rights reserved.)