clonogenic assays
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2021 ◽  
Vol 108 (Supplement_5) ◽  
Author(s):  
Rachael Elizabeth Clifford ◽  
Naren Govindarajah ◽  
David Bowden ◽  
Paul Sutton ◽  
Jason Parsons ◽  
...  

Abstract Introduction We have previously utilized proteomic and immuno-histochemical data to validate that high levels of acid ceramidase (AC) expression confers poorer neoadjuvant response in rectal cancer. Biological (siRNA, plasmid and CRISPR) AC manipulation altered radiosensitivity in-vitro. We aimed to assess the radiosensitising effect of pharmacological AC inhibitions and elucidate the potential underlying mechanism. Methods Optimal drug dosing was achieved using ELISA activity assays in multiple colorectal cancer cell lines (HCT116, HT29, LIM1215). Carmofur and a novel small molecular inhibitor (LCL521) were used as pharmacological inhibitors. Standard clonogenic assays assessed cell survival following increasing irradiation (2 D), volume change in 3 D spheroids and cell viability in patient derived organoids. Annexin V/PI staining was used to determine apoptosis. Results Carmofur clonogenic assays demonstrated reduced colony formation efficiency (CFE) and improved radiosensitivity across cell lines. HCT116 showed 0.438(CFE) control v 0.183(CFE) carmofur at 1 Gy, 0.140(CFE) control v 0.076(CFE) at 2 Gy (P = 0.000563). LCL521 dosing improved radiosensitivity in spheroid models. HCT116 volume day-15 2.36x10-5mm v control 4.15x10-5mm. siRNA-AC demonstrated increased apoptosis across time points compared to NT control (P = 0.035), and increased poly-ADP ribose polymerase-1 (PARP-1) cleavage in a p53-dependent process. Conclusion Initial work demonstrates that pharmacological inhibition of AC produces comparative radiosensitizing effects in these cell lines and cancer models. siRNA-AC increases apoptosis, suggestive of a potential underlying mechanism. This work further solidifies AC as a potential biomarker, however further recapitulation in more complex models and ultimately in-vivo is required to establish a translatable clinical role.


2021 ◽  
Vol 108 (Supplement_1) ◽  
Author(s):  
H Fowler ◽  
P Sutton ◽  
D Bowden ◽  
J Parsons ◽  
D Vimalachandran

Abstract Introduction Our proteomic data has validated that high levels of the protein myoferlin confers poorer response to neoadjuvant chemoradiotherapy in locally advanced rectal cancer. Myoferlin plays a role in membrane repair and VEGF signal transduction, and is associated with worse prognosis in numerous other epithelial cancers. We aim to assess the impact of myoferlin on the radiosensitivity of rectal cancer. Method Clonogenic assays were performed using immortalised colorectal cancer cells (HCT116,HT29,LIM,MDST8) to assess survival at escalating radiation doses following knockdown with myoferlin siRNA or a small molecular inhibitor(WJ460). 3D models (spheroids) were used to examine the effect of WJ460 on tumour growth. Result Quantification of myoferlin using immunoblotting demonstrated that MDST8 and LIM were higher expressors than HCT116 and HT29. Higher levels correlated with increasing radio-resistance as calculated by colony formation efficiency (CFE). Using clonogenic assays, cells treated with myoferlin siRNA or WJ460 demonstrated increased radiosensitivity compared to controls across all radiation doses, most significantly at 4Gy. Treatment of spheroids with WJ460 significantly reduced growth compared to controls at all radiation doses (p<0.05), with WJ460 limiting growth considerably more than treatment with the current gold standard 5-FU. HCT116 spheroid volume day 15; WJ460 4.96um3,5-FU 6.74um3,DMSO 24.9um3. Conclusion Inhibition of myoferlin is associated with increased radiosensitivity of colorectal cancer cells, and treatment with a small molecular inhibitor significantly reduces growth in spheroid models. Further work is required further validate its potential use as a biomarker in locally advanced rectal cancer. Take-home message We have found that myoferlin is a protein associated with poor response to neoadjuvant chemoradiotherapy in locally advanced rectal cancer. Manipulation of this protein sensitises the cancer cells to radiotherapy.


Author(s):  
José Manuel Bravo-San Pedro ◽  
Oliver Kepp ◽  
Allan Sauvat ◽  
Santiago Rello-Varona ◽  
Guido Kroemer ◽  
...  

2020 ◽  
Vol 61 (6) ◽  
pp. 828-831
Author(s):  
Takahiro Oike ◽  
Shuichiro Komatsu ◽  
Yuka Komatsu ◽  
Ankita Nachankar ◽  
Narisa Dewi Maulany Darwis ◽  
...  

Abstract 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.


2020 ◽  
Author(s):  
Ashley Robinson ◽  
Mackenzie Crow ◽  
Austin Kratz ◽  
Taylor Ritts ◽  
Yewseok K. Suh ◽  
...  

Clonogenic assays are a simple and robust method that allow researchers to characterize mammalian cell line features, including the ability of a single cell to grow into a colony. We have used this assay as a tool in the undergraduate biology laboratory, exposing students to a more specialized form of mammalian cell culture and helping them refine scientific research skills and knowledge. In this article, we share an easy and undergraduate-friendly method of using HeLa cells to carry out clonogenic assays. The methods described include the introduction of different treatments to assess their effect in HeLa cell colony formation. In this laboratory exercise, undergraduate students utilize different cell culture techniques such as growing, harvesting, counting, diluting, staining, and imaging cells. Clonogenic assay, Cytotoxic agents, HeLa cells, Mammalian cell colony formation, undergraduate laboratory, Open Inquiry-Based Curriculum


2020 ◽  
Author(s):  
Li Li ◽  
Ling Lin ◽  
Jamunarani Veeraraghavan ◽  
Yiheng Hu ◽  
Xian Wang ◽  
...  

AbstractBackgroundEndocrine therapy is the most common treatment for estrogen receptor (ER)-positive breast cancer, but its effectiveness is limited by high rates of primary and acquired resistance. There are likely many genetic causes and recent studies suggest the important role of ESR1 mutations and fusions in endocrine resistance. Previously we reported a recurrent ESR1 fusion called ESR1-CCDC170 in 6-8% of the luminal B breast cancers that has a worse clinical outcome after endocrine therapy. Despite being the most frequent ESR1 fusion, its functional role in endocrine resistance have not been studied in vivo, and the engaged mechanism and therapeutic relevance remain uncharacterized.MethodsThe endocrine sensitivities of HCC1428 or T47D breast cancer cells following genetic perturbations of ESR1-CCDC170 were assessed using clonogenic assays and/or xenograft mouse models. The underlying mechanisms were investigated by reverse phase protein array, western blotting, immunoprecipitation, and bimolecular fluorescence complementation assays. The sensitivity of ESR1-CCDC170 expressing breast cancer cells to concomitant treatments of tamoxifen and HER/SRC inhibitors was assessed by clonogenic assays.ResultsOur results suggested that different ESR1-CCDC170 fusions endow different levels of reduced endocrine sensitivity in vivo, resulting in significant survival disadvantages. Further investigation revealed a novel mechanism that ESR1-CCDC170 binds to HER2/HER3/SRC and activates SRC/PI3K/AKT signaling. Silencing of ESR1-CCDC170 in the fusion-positive cell line, HCC1428, downregulates HER2/HER3, represses pSRC/pAKT, and improves endocrine sensitivity. More important, breast cancer cells expressing ectopic or endogenous ESR1-CCDC170 are highly sensitive to treatment regimens combining endocrine agents with the HER2 inhibitor lapatinib and/or the SRC inhibitor dasatinib.ConclusionESR1-CCDC170 may endow breast cancer cell survival under endocrine therapy via maintaining/activating HER2/HER3/SRC/AKT signaling which implies a potential therapeutic strategy for managing these fusion positive tumors.


Author(s):  
Ouma Cisse ◽  
Muzthahid Quraishi ◽  
Federico Gulluni ◽  
Federica Guffanti ◽  
Ioanna Mavrommati ◽  
...  

Abstract Background Alteration of signalling pathways regulating cell cycle progression is a common feature of cancer cells. Several drugs targeting distinct phases of the cell cycle have been developed but the inability of many of them to discriminate between normal and cancer cells has strongly limited their clinical potential because of their reduced efficacy at the concentrations used to limit adverse side effects. Mechanisms of resistance have also been described, further affecting their efficacy. Identification of novel targets that can potentiate the effect of these drugs or overcome drug resistance can provide a useful strategy to exploit the anti-cancer properties of these agents to their fullest. Methods The class II PI3K isoform PI3K-C2β was downregulated in prostate cancer PC3 cells and cervical cancer HeLa cells using selective siRNAs and the effect on cell growth was determined in the absence or presence of the microtubule-stabilizing agent/anti-cancer drug docetaxel. Mitosis progression was monitored by time-lapse microscopy. Clonogenic assays were performed to determine the ability of PC3 and HeLa cells to form colonies upon PI3K-C2β downregulation in the absence or presence of docetaxel. Cell multi-nucleation was assessed by immunofluorescence. Tumour growth in vivo was assessed using a xenograft model of PC3 cells upon PI3K-C2β downregulation and in combination with docetaxel. Results Downregulation of PI3K-C2β delays mitosis progression in PC3 and HeLa cells, resulting in reduced ability to form colonies in clonogenic assays in vitro. Compared to control cells, PC3 cells lacking PI3K-C2β form smaller and more compact colonies in vitro and they form tumours more slowly in vivo in the first weeks after cells implant. Stable and transient PI3K-C2β downregulation potentiates the effect of low concentrations of docetaxel on cancer cell growth. Combination of PI3K-C2β downregulation and docetaxel almost completely prevents colonies formation in clonogenic assays in vitro and strongly inhibits tumour growth in vivo. Conclusions These data reveal a novel role for the class II PI3K PI3K-C2β during mitosis progression. Furthermore, data indicate that blockade of PI3K-C2β might represent a novel strategy to potentiate the effect of docetaxel on cancer cell growth.


BMC Cancer ◽  
2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Sathid Aimjongjun ◽  
Zimam Mahmud ◽  
Yannasittha Jiramongkol ◽  
Glowi Alasiri ◽  
Shang Yao ◽  
...  

Abstract Background Chemoresistance is an obstacle to the successful treatment of nasopharyngeal carcinoma (NPC). Lapatinib is a targeted tyrosine kinase inhibitor therapeutic drug also used to treat NPC, but high doses are often required to achieve a result. To investigate the mechanism for the development of Lapatinib resistance, we characterised a number of NPC cell lines to determine the role of FOXO3 and sirtuins in regulating NPC resistance. Methods Sulforhodamine B (SRB) assays, Clonogenic assays, Protein extraction, quantification and western blotting, RT qPCR, Co-immunoprecipitation assay. Results To explore novel treatment strategies, we first characterized the Lapatinib-sensitivity of a panel of NPC cell lines by SRB and clonogenic cytotoxic assays and found that the metastatic NPC (C666–1 and 5-8F) cells are highly resistant whereas the poorly metastatic lines (6-10B, TW01 and HK-1) are sensitive to Lapatinib. Western blot analysis of the Lapatinib-sensitive 6-10B and resistant 5-8F NPC cells showed that the expression of phosphorylated/inactive FOXO3 (P-FOXO3;T32), its target FOXM1 and its regulator SIRT2 correlate negatively with Lapatinib response and sensitivity, suggesting that SIRT2 mediates FOXO3 deacetylation to promote Lapatinib resistance. In agreement, clonogenic cytotoxic assays using wild-type and foxo1/3/4−/− mouse embryonic fibroblasts (MEFs) showed that FOXO1/3/4-deletion significantly attenuates Lapatinib-induced cytotoxicity, confirming that FOXO proteins are essential for mediating Lapatinib response. SRB cell viability assays using chemical SIRT inhibitors (i.e. sirtinol, Ex527, AGK2 and AK1) revealed that all SIRT inhibitors can reduce NPC cell viability, but only the SIRT2-specific inhibitors AK1 and AGK2 further enhance the Lapatinib cytotoxicity. Consistently, clonogenic assays demonstrated that the SIRT2 inhibitors AK1 and AGK2 as well as SIRT2-knockdown increase Lapatinib cytotoxicity further in both the sensitive and resistant NPC cells. Co-immunoprecipitation studies showed that besides Lapatinib treatment, SIRT2-pharmaceutical inhibition and silencing also led to an increase in FOXO3 acetylation. Importantly, SIRT2 inhibition and depletion further enhanced Lapatinib-mediated FOXO3-acetylation in NPC cells. Conclusion Collectively, our results suggest the involvement of SIRT2-mediated FOXO3 deacetylation in Lapatinib response and sensitivity, and that SIRT2 can specifically antagonise the cytotoxicity of Lapatinib through mediating FOXO3 deacetylation in both sensitive and resistant NPC cells. The present findings also propose that SIRT2 can be an important biomarker for metastatic and Lapatinib resistant NPC and that targeting the SIRT2-FOXO3 axis may provide novel strategies for treating NPC and for overcoming chemoresistance.


2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi209-vi210
Author(s):  
Ebin Sebastian ◽  
Tiantian Cui ◽  
Erica Hlavin Bell ◽  
Joseph McElroy ◽  
Benjamin Johnson ◽  
...  

Abstract BACKGROUND Glioblastoma is the most aggressive brain tumor with poor prognosis despite the best available treatment. MicroRNAs (miRNAs) are emerging as promising, novel prognostic biomarkers and therapeutic targets in glioblastoma. In a previous study, we demonstrated that miR-4516 predicts poor prognosis and functions as an oncogene in glioblastoma. Aim of the current study is to examine the role miR-4516 in radiation resistance and identify downstream targets contributing to this phenotype METHODS Radiosensitization was evaluated by cell viability and clonogenic assays. Cell apoptosis was evaluated using flow cytometry and immunoblotting. Potential targets of miR-4516 were identified using bioinformatic analysis (Targetscan and miRDB) and confirmed by luciferase reporter assays. Results were validated using immunoblotting. miR-4516 expression in glioblastoma cell lines after radiation treatment was quantified by qRT-PCR. RESULTS Expression of miR-4516 was increased up to 15 fold following radiation treatment, peaking at around 15min-60 min in primary and established glioblastoma cell lines including GBM 08-387, GBM 30 and U87-MG. Furthermore, inhibition of miR-4516 sensitized GBM 08-387, GBM30 and U87-MG cells to radiation in comparison to control groups as determined by cell viability and clonogenic assays. Further, miR-4516 inhibition induced apoptosis in these cell lines following radiation treatment. While conducting mechanistic studies, we found that the tumor-promoting function of miR-4516 was, in part, mediated by inhibition of p21 and PTPN14, two direct targets of miR-4516 CONCLUSION Our data suggest that radiation induces the expression of miR-4516 in glioblastoma cell lines. This miRNA plays a critical role in radio-resistance and promotes aggressive phenotypes in glioblastoma and therefore, functional analyses of its target pathways may uncover novel therapeutically vulnerable target(s) in glioblastoma. FUNDING: R01CA108633, R01CA169368, RC2CA148190, U10CA180850-01(NCI), Brain Tumor Funders Collaborative Grant, and OSU-CCC (all to AC). The Ton and Patricia Bohnenn Fund for Neuro_Oncology Research (to PR).


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