Pairs Of Cancer Cell Lines Research Articles

Abstract 4830: Predicting drug treatment response in African American lung cancer patients who smoke

Abstract Background: Lung cancer is the third most commonly diagnosed cancer in the US and the leading cause of cancer-related death. African Americans (AAs) have higher lung cancer incidence and mortality rates, and lower survival rates, when compared to their European American (EA) counterparts. It is widely known that cigarette smoking is the strongest risk factor for lung cancer development, but it also plays a significant role in clinical outcome. Cigarette smoking after lung cancer diagnosis (up to 30% patients) comes with several risks, including increased secondary tumors and mortality rates (by 20%), as well as poor treatment outcomes (higher radiation and chemotherapy failure and higher drug clearance). There are limited transcriptomic studies that describe molecular characterizations of AA smokers with lung cancer to predict treatment outcomes. Hypothesis: Cigarette smoking leads to greater drug resistance-related gene expression in AA lung cancer patients. Methods: mRNA expression changes in an AA (NCI-H2172) and EA (NCI-H1944) lung cancer cell line pair exposed to liquid cigarette smoke condensate (CSC) were profiled. The cell lines were matched based on age (62 years), sex (female), histology (lung adenocarcinoma, LUAD, the most common subtype), and culture properties (adherent). The CSC was derived from Murty Pharmaceuticals and prepared by smoking University of Kentucky's 3R4F Standard Research Cigarette on an FTC Smoke Machine. A total of 500,000 cells per well were treated for one hour under standard incubation conditions (37°C, pH 7.0, 5% CO2). Cells were pelleted, RNAs were isolated, treated with DNAse-I to remove contaminating genomic DNA, quantified, and sent for bulk mRNA-sequencing (Novogene Co). Three biological replicates and technical triplicates were performed. Partek Flow and Partek Genomics Suite workflows were used to identify population-specific differentially expressed gene (DEG) signatures and pathway enrichment after CSC exposure. DEG were profiled in LUAD tissues from current smokers in an AA and EA TCGA cohort (n = 18 AAs, 86 EAs). Results: Population-specific gene expression changes were discovered (n = 52 AA DEGs, n = 31 EA DEGs; P <0.001). All of the DEGs were mutually exclusive with no overlap. Some pathways were shared between both populations and others were unique. PI3K/Akt/mTOR signaling pathway enrichment was unique in AA LUAD cells and ECM receptor interactions were distinct in EA LUAD cells. AKT2 (which activates mTOR) had significantly higher expression in LUAD tumor tissues from current AA smokers compared to current EA smokers. Conclusion: Population-specific PI3K/Akt/mTOR pathway alterations may help explain drug resistance in LUAD tumors from AA patients. PI3K signaling is considered a significant cause of resistance to chemotherapy, targeted therapy, and immunotherapy. Future Directions: Expose AA and EA LUAD cell lines to CSC and PI3K/Akt/mTOR pathway inhibitors. Citation Format: Mia Ray, Isaiah Osei-Gyening, Kwabena Acheampong, Marisa Riebesell, Khadijah A. Mitchell. Predicting drug treatment response in African American lung cancer patients who smoke [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4830.

Abstract 213: Phylogenetic reconstruction across 303 metastatic tumor samples using Ultima whole-genome sequencing dramatically increases subclonal resolution

Abstract Comprehensive characterization of tumor evolution is essential for understanding the drivers of metastasis and treatment resistance, which are still largely unknown. Studying evolution and resistance requires large cohorts of patients; for each patient, a comprehensive phylogenetic tree is used to follow clones over time and space. Deep whole-genome sequencing (WGS) enables the creation of more robust phylogenies, since (i) each clone is characterized by far more clone-specific mutations (compared to whole-exome or panel sequencing); and (ii) WGS yields more accurate clonal and sub-clonal allelic copy-number estimates, enabling more precise estimation of the fraction of cancer cells that harbor each mutation. Due to recent development of more affordable WGS by Ultima Genomics, we set out to test our ability to construct phylogenetic trees based on Ultima WGS. Ultima sequencing has demonstrated high sensitivity and specificity for detecting germline polymorphisms, but its performance on somatic mutation calling has not yet been rigorously studied. First, we adjusted our somatic mutation detection pipeline to work with Ultima-generated WGS data and tested its performance using well-studied cell line tumor/normal pairs. We assembled a ground truth set of somatic mutations based on deep sequencing data from multiple platforms for the breast cancer cell line pairs (HCC1954/HCC1954-BL and HCC1143/HCC1143-BL) and the melanoma tumor/normal cell line pair (COLO829/COLO829-BL). We found that mutation detection performance was on-par with other platforms for single nucleotide variations. Next, we identified 32 patients from various cancer types (breast, lung, cholangiocarcinoma, and melanoma) treated with a variety of therapies. We collected between 4 to 14 samples per patient at autopsy (total of 303), and sequenced them with Ultima to an average coverage of 63x (range 48x-91x). We identified mutations using our Ultima-adjusted WGS pipeline and constructed phylogenetic trees using our PhylogicNDT suite of tools. We then compared the results to trees previously constructed from mostly whole-exomes (and a few whole-genomes) sequenced on Illumina. We demonstrate that the trees generated from Ultima WGS data are consistent with the previously generated trees, and proved to be much more detailed. At commercialization, we anticipate that WGS for projects like this will be performed at ~1/6th the cost of current NGS offerings (on par with current WES costs). Therefore, it will enable larger studies and more accurate phylogenetic reconstruction, which will advance the study of tumor evolution and resistance. Citation Format: Julian Hess, Elizabeth Martin, Ilya Soifer, Hila Benjamin, Mendy Miller, Carrie Cibulskis, Brian P. Danysh, Matthew Coole, Stacey Gabriel, Dejan Juric, Doron Lipson, Gad Getz. Phylogenetic reconstruction across 303 metastatic tumor samples using Ultima whole-genome sequencing dramatically increases subclonal resolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 213.

Fanconi anemia-isogenic head and neck cancer cell line pairs: A basic and translational science resource.

Fanconi anemia (FA) is a heritable malformation, bone marrow failure and cancer predisposition syndrome that confers an exceptionally high risk of squamous carcinomas. These carcinomas originate in epithelia lining the mouth, proximal esophagus, vulva and anus: their origins are not understood, and no effective ways have been identified to prevent or delay their appearance. Many FA-associated carcinomas are also therapeutically challenging: they may be multi-focal and stage-advanced at diagnosis, and most individuals with FA cannot tolerate standard-of-care systemic therapies such as DNA cross-linking drugs or ionizing radiation due to constitutional DNA damage hypersensitivity. We developed the Fanconi Anemia Cancer Cell Line Resource (FA-CCLR) to foster new work on the origins, treatment and prevention of FA-associated carcinomas. The FA-CCLR consists of Fanconi-isogenic head and neck squamous cell carcinoma (HNSCC) cell line pairs generated from five individuals with FA-associated HNSCC, and five individuals with sporadic HNSCC. Sporadic, isogenic HNSCC cell line pairs were generated in parallel with FA patient-derived isogenic cell line pairs to provide comparable experimental material to use to identify cell and molecular phenotypes driven by germline or somatic loss of Fanconi pathway function, and the subset of these FA-dependent phenotypes that can be modified, complemented or suppressed. All 10 FANC-isogenic cell line pairs are available to academic, non-profit and industry investigators via the "Fanconi Anemia Research Materials" Resource and Repository at Oregon Health & Sciences University, Portland OR.

Ruthenium(II) and Platinum(II) Complexes with Biologically Active Aminoflavone Ligands Exhibit In Vitro Anticancer Activity.

Continuing our studies on the mechanisms underlying the cytotoxicity of potential drugs, we have described several aspects of the in vitro anticancer activity of ruthenium(II) and platinum(II) complexes with bioactive, synthetic aminoflavone ligands. We examined the mechanism of proapoptotic activity of cis-dichlorobis(3-imino-2-methoxyflavanone)ruthenium(II), cis-dichlorobis(3-imino-2-ethoxyflavanone)ruthenium(II), and trans-dichlorobis(3-aminoflavone)platinum(II). Cisplatin was used as a reference compound. The cytotoxicity was investigated by MTT assay. The mechanism of proapoptotic activity of the tested compounds was investigated by evaluation of caspase-8 activity, cytometric analysis of annexin-V positive cells, and mitochondrial potential loss measurement. The results showed that ruthenium compounds break partially or completely the cisplatin resistance by activating the caspase 8-dependent apoptosis pathway and loss of mitochondrial membrane potential. Platinum compounds also have a cytostatic effect, but their action requires more exposure time. Potential mechanisms underlying drug resistance in the two pairs of cancer cell lines were investigated: total glutathione content, P-glycoprotein activity, and differences in the activity of DNA repair induced by nucleotide excision. Results showed that cisplatin-resistant cells have elevated glutathione levels relative to sensitive cells. Moreover, they indicated the mechanisms enabling cells to avoid apoptosis caused by DNA damage. Pg-P activity has no effect on the development of cisplatin resistance in the cell lines described.

Optimal Deep Belief Network with Opposition based Pity Beetle Algorithm for Lung Cancer Classification: A DBNOPBA Approach

Background and ObjectiveThis research proposes a successful method of extracting Gray-Level Co-occurrence Matrix (GLCM) picture handling models to classify low-and high-metastatic cancer organisms with five prevalent cancer cell line pairs, coupled with the scanning laser picture projection technique and the typical textural function, i.e. contrast, correlation, power, temperature and homogeneity. The most significant level of disease for highly metastatic cancer cells are the degree of disturbance, contrast as well as entropy refers to the energy and homogeneity. A texture classification scheme to quantify the emphysema in Computed Tomography (CT) pictures is performed. Local binary models (LBP) are used to characterize areas of concern as texture characteristics and intensity histograms. A wavelet filter is used to acquire the informative matrix of each picture and decrease the dimensionality of the function space in the suggested method. A four-layer profound creed network is also used to obtain characteristics of elevated stage. Local Tangent Space Alignment (LTSA) is then used to compress the multi-domain defect characteristics into low dimensional vectors as a dimension reduction method. An unmonitored deep-belief network (DBN) is intended for the second phase to learn the unmarked characteristics. The strategy suggested uses Opposition Based Teaching (OBL), Position Clamping (PC) and the Cauchy Mutation (CM) to improve the fundamental PBA efficiency. MethodsThis research presents a fresh meta-heuristic algorithm called Opposition-Based Pity Beetle Algorithm (OPBA), which assesses effectiveness against state-of-the-art algorithms. OBL speeds up the convergence of the technique as both PC and CM assist OPBA with escaping local optima. The suggested algorithm was motivated by the behaviour of the beetle, which had been named six-toothed spruce bark beetle to aggregate nests and meals. This beetle can be found and harvested from weakened trees ' bark in a forest, while its populace can also infest healthy and robust trees when it exceeds the specified threshold. Results & ConclusionThe methodology has been evaluated on CT imagery from the Lung Image Database Consortium and Image Resources Initiative (LIDC-IDRI), with a maximum sensitivity of 96.86%, precision of 97.24%, and an accuracy of 97.92%.

HDAC inhibition synergizes with ALK inhibitors to overcome resistance in a novel ALK mutated lung adenocarcinoma model

ObjectivesSomatic chromosomal rearrangements resulting in ALK fusion oncogenes are observed in 3–7 % of lung adenocarcinomas. ALK tyrosine kinase inhibitors (ALKi) induce initially response, however, various resistance mechanisms limit their efficacy. Novel therapeutic approaches are of utmost importance to tailor these targeted therapies. Materials and methodsA synchronous ALK-rearranged and mutated lung cancer cell line pair was established from malignant pleural effusion (PF240-PE) and carcinosis (PF240-PC) at time of ALKi resistance. Immunohistochemistry, FISH and sequencing were performed in pre- and post-treatment tumors and in both cell lines. Differentiation markers were measured by immunoblot. Viability was tested following treatment with ALKi and/or a pan-HDAC inhibitor. Additionally, a novel treatment-naïve ALK-rearranged cell line served as control. In vivo tumorigenicity was evaluated in subcutaneous xenografts. ResultsTwo distinct resistance mutations were identified in different carcinosis tissues at time of resistance, the previously described resistance mutation L1152R and the hitherto uncharacterized E1161K. Strikingly, PF240-PC cells carried E1161K and PF240-PE cells harbored L1152R. Immunohistochemistry and immunoblot identified epithelial-to-mesenchymal transition markers upregulated following ALKi resistance development both in carcinosis tissues and cell lines. While both lines grew as xenografts, they differed in morphology, migration, in vivo growth and sensitivity to ALKi in vitro. Strikingly, the combination of ALKi with SAHA yielded strong synergism. ConclusionUsing a patient-derived ALKi resistant lung cancer model we demonstrated the synergism of HDAC and ALK inhibition. Furthermore, our findings provide strong evidence for intratumoral heterogeneity under targeted therapy and highlight the importance of site-specific mutational analysis.

LKB1 loss is associated with glutathione deficiency under oxidative stress and sensitivity of cancer cells to cytotoxic drugs and γ-irradiation

The liver kinase B1 (LKB1) gene is a tumor suppressor associated with the hereditary Peutz-Jeghers syndrome and frequently mutated in non-small cell lung cancer and in cervical cancer. Previous studies showed that the LKB1/AMPK axis is involved in regulation of cell death and survival under metabolic stress. By using isogenic pairs of cancer cell lines, we report here that the genetic loss of LKB1 was associated with increased intracellular levels of total choline containing metabolites and, under oxidative stress, it impaired maintenance of glutathione (GSH) levels. This resulted in markedly increased intracellular reactive oxygen species (ROS) levels and sensitivity to ROS-induced cell death. These effects were rescued by re-expression of LKB1 or pre-treatment with the anti-oxidant and GSH replenisher N-acetyl cysteine. This role of LKB1 in response to ROS-inducing agents was largely AMPK-dependent. Finally, we observed that LKB1 defective cells are highly sensitive to cisplatin and γ-irradiation in vitro, suggesting that LKB1 mutated tumors could be targeted by oxidative stress-inducing therapies.

Intracellular Hyper-Acidification Potentiated by Hydrogen Sulfide Mediates Invasive and Therapy Resistant Cancer Cell Death.

Slow and continuous release of H2S by GYY4137 has previously been demonstrated to kill cancer cells by increasing glycolysis and impairing anion exchanger and sodium/proton exchanger activity. This action is specific for cancer cells. The resulting lactate overproduction and defective pH homeostasis bring about intracellular acidification-induced cancer cell death. The present study investigated the potency of H2S released by GYY4137 against invasive and radio- as well as chemo-resistant cancers, known to be glycolytically active. We characterized and utilized cancer cell line pairs of various organ origins, based on their aggressive behaviors, and assessed their response to GYY4137. We compared glycolytic activity, via lactate production, and intracellular pH of each cancer cell line pair after exposure to H2S. Invasive and therapy resistant cancers, collectively termed aggressive cancers, are receptive to H2S-mediated cytotoxicity, albeit at a higher concentration of GYY4137 donor. While lactate production was enhanced, intracellular pH of aggressive cancers was only modestly decreased. Inherently, the magnitude of intracellular pH decrease is a key determinant for cancer cell sensitivity to H2S. We demonstrated the utility of coupling GYY4137 with either simvastatin, known to inhibit monocarboxylate transporter 4 (MCT4), or metformin, to further boost glycolysis, in bringing about cell death for aggressive cancers. Simvastatin inhibiting lactate extrusion thence contained excess lactate induced by GYY4137 within intracellular compartment. In contrast, the combined exposure to both GYY4137 and metformin overwhelms cancer cells with lactate over-production exceeding its expulsion rate. Together, GYY4137 and simvastatin or metformin synergize to induce intracellular hyper-acidification-mediated cancer cell death.

Epigenomic and Functional Characterization of Junctophilin 3 (JPH3) as a Novel Tumor Suppressor Being Frequently Inactivated by Promoter CpG Methylation in Digestive Cancers.

Junctophilin (JPH) proteins stabilize junctional membrane complexes between plasma membrane and endoplasmic reticulum, also implicated in some human diseases. JPH3 mutations are linked to Huntington's disease-like 2 syndrome. Through epigenomic study of a colon cancer cell line pair (HCT116 and DKO), we identified JPH3 as a methylated novel tumor suppressor gene (TSG) candidate at 16q24. We further studied its epigenetic alterations and functions in digestive tumorigenesis. JPH3 expression at the RNA level was found to be frequently silenced or reduced in colorectal and gastric cancers due to its promoter CpG methylation, which is associated with tumor progression and poor survival of digestive cancer patients. Ectopic expression of JPH3 inhibited tumor cell growth in vitro and in vivo. JPH3 expression upregulated the cytosolic Ca2+ levels, and unfolded protein response gene expression upon endoplasmic reticulum stress. JPH3 also induced calpain activation and subsequent mitochondrial membrane depolarization and cell apoptosis. Thus, JPH3 was identified as a novel TSG methylated in colorectal and gastric tumors which promotes mitochondrial-mediated apoptosis, also as a potential metastasis and survival biomarker for digestive cancers.

Reversal of Chemoresistance in Ovarian Cancer by Co-Delivery of a P-Glycoprotein Inhibitor and Paclitaxel in a Liposomal Platform.

The overexpression of permeability-glycoprotein (P-gp), an ABC transporter involved in the cellular exclusion of chemotherapeutic drugs, is a major factor in paclitaxel-resistant ovarian cancer. However, in clinical trials, co-administration of P-gp inhibitors and anticancer drugs has not resulted in the efficient reversal of drug resistance. To improve administration, we encapsulated the third-generation P-gp inhibitor tariquidar (XR-9576, XR), alone or in combination with paclitaxel (PCT) in liposomes (LP). After optimization, the liposomes demonstrated favorable physicochemical properties and the ability to reverse chemoresistance in experiments using chemosensitive/chemoresistant ovarian cancer cell line pairs. Analyzing publicly available datasets, we found that overexpression of P-gp in ovarian cancer is associated with a shorter progression-free and overall survival. In vitro, LP(XR) significantly increased the cellular retention of rhodamine 123, a P-gp substrate. LP(XR,PCT) synergistically inhibited cell viability, blocked proliferation, and caused G2-M arrest in paclitaxel-resistant SKOV3-TR and HeyA8-MDR cell lines overexpressing P-gp. Holographic imaging cytometry revealed that LP(XR,PCT) treatment of SKOV3-TR cells induced almost complete mitotic arrest, whereas laser scanning cytometry showed that the treatment induced apoptosis. In proof-of-concept preclinical studies, LP(XR,PCT), when compared with LP(PCT), significantly reduced tumor weight (43.2% vs. 16.9%, P = 0.0007) and number of metastases (44.4% vs. 2.8%, P = 0.012) in mice bearing orthotopic HeyA8-MDR ovarian tumors. In the xenografts, LP(XR,PCT) efficiently induced apoptosis and impaired proliferation. Our findings suggest that co-delivery of a P-gp inhibitor and paclitaxel using a liposomal platform can sensitize paclitaxel-resistant ovarian cancer cells to paclitaxel. LP(XR,PCT) should be considered for clinical testing in patients with P-gp-overexpressing tumors. Mol Cancer Ther; 15(10); 2282-93. ©2016 AACR.

Dihydro-β-agarofuran sesquiterpene polyesters isolated from the stems of Tripterygium regelii

Twelve new dihydro-β-agarofuran polyesters, triptregelines A–J (1–3 and 5–11) and triptregelols A, B (4, 12), together with five known ones (13–17) were isolated from the stems of Tripterygium regelii. The structures were determined on the basis of extensive analysis of spectroscopic data (1D, 2D NMR, and UV etc.) and HRMS data. Cytotoxic effects of these compounds were evaluated against a pair of cancer cell lines, A549 and taxol-resistant A549T. Triptofordin B (14) showed cytotoxicity against both A549 and A549T cells with IC50 value of 21.2 and 10.8μM, respectively. In addition, triptregeline B (2), triptregeline C (3), triptregeline H (9) and 1α, 6β, 15-triacetoxy-8α-benzoyloxy-4β-hydroxy-9α-(3-nicotinoyloxy)-dihydro-β-agarofuran (17) exhibited weak cytotoxic effects on taxol-resistant A549T with IC50 values ranged from 29.4 to 54.4μM. The cytotoxic effect of triptofordin B (14) was much less than taxol with IC50 value of 0.08μM on A549 cancer cell.

Abstract C60: Pre-clinical efficacy of the ATR inhibitor AZD6738 in combination with the PARP inhibitor olaparib

Abstract The PARP inhibitor olaparib acts through both inhibition of DNA single-strand-break repair and trapping of PARP-DNA complexes creating DNA lesions which cause replication fork stalling and collapsed fork DNA breaks. Cells which have lost BRCA-dependent homologous recombination repair are highly sensitive to olaparib treatment and this has led to its approval in patients with tumors carrying BRCA mutations. In addition, ATM (Ataxia telangiectasia mutated) and ATR (Ataxia telangiectasia mutated and Rad3 related) dependent DNA repair processes are hypothesized to be important survival pathways to PARP inhibitor treatment. In pre-clinical studies cancer cells which have defects in either ATM or ATR have been shown to be sensitive to PARP inhibitors. Here we present data that the orally bioavailable ATR inhibitor AZD6738 (in Phase-I clinial trials) combines synergistically with olaparib leading to cell death and anti-tumour activity in pre-clinical models. The combinations are effective across a panel of gastric and lung cancer cell lines in vitro. In addition results from isogenic ATM−/-/− knockout versus ATM+/+/+FaDu head and neck cancer cell line pairs show enhanced combination activity in ATM knockout cells versus ATM wild-type cells. Studies in vivo show that through intermittent dosing the combination is tolerated while demonstrating significant anti-tumour efficacy and regressions across multiple human patient derived primary explant models. Together, these data support the notion of development of AZD6738 and olaparib combinations for the treatment of ATM-deficient cancers. Citation Format: Alan Lau, Elaine Brown, Andrew Thomason, Rajesh Odedra, Victoria Sheridan, Elaine Cadogan, Shirlian Xu, Andy Cui, Paul R. Gavine, Mark O'Connor. Pre-clinical efficacy of the ATR inhibitor AZD6738 in combination with the PARP inhibitor olaparib. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C60.

MiR-634 restores drug sensitivity in resistant ovarian cancer cells by targeting the Ras-MAPK pathway.

BackgroundDrug resistance hampers the efficient treatment of malignancies, including advanced stage ovarian cancer, which has a 5-year survival rate of only 30 %. The molecular processes underlying resistance have been extensively studied, however, not much is known about the involvement of microRNAs.MethodsDifferentially expressed microRNAs between cisplatin sensitive and resistant cancer cell line pairs were determined using microarrays. Mimics were used to study the role of microRNAs in drug sensitivity of ovarian cancer cell lines and patient derived tumor cells. Luciferase reporter constructs were used to establish regulation of target genes by microRNAs.ResultsMiR-634 downregulation was associated with cisplatin resistance. Overexpression of miR-634 affected cell cycle progression and enhanced apoptosis in ovarian cancer cells. miR-634 resensitized resistant ovarian cancer cell lines and patient derived drug resistant tumor cells to cisplatin. Similarly, miR-634 enhanced the response to carboplatin and doxorubicin, but not to paclitaxel. The cell cycle regulator CCND1, and Ras-MAPK pathway components GRB2, ERK2 and RSK2 were directly repressed by miR-634 overexpression. Repression of the Ras-MAPK pathway using a MEK inhibitor phenocopied the miR-634 effects on viability and chemosensitivity.ConclusionmiR-634 levels determine chemosensitivity in ovarian cancer cells. We identify miR-634 as a therapeutic candidate to resensitize chemotherapy resistant ovarian tumors.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0464-4) contains supplementary material, which is available to authorized users.

Optical detection of metastatic cancer cells using a scanned laser pico-projection system

Metastasis is responsible for 90% of all cancer-related deaths in humans. As a result, reliable techniques for detecting metastatic cells are urgently required. Although various techniques have been proposed for metastasis detection, they are generally capable of detecting metastatic cells only once migration has already occurred. Accordingly, the present study proposes an optical method for physical characterization of metastatic cancer cells using a scanned laser pico-projection system (SLPP). The validity of the proposed method is demonstrated using five pairs of cancer cell lines and two pairs of non-cancer cell lines treated by IPTG induction in order to mimic normal cells with an overexpression of oncogene. The results show that for all of the considered cell lines, the SLPP speckle contrast of the high-metastatic cells is significantly higher than that of the low-metastatic cells. As a result, the speckle contrast measurement provides a reliable means of distinguishing quantitatively between low- and high-metastatic cells of the same origin. Compared to existing metastasis detection methods, the proposed SLPP approach has many advantages, including a higher throughput, a lower cost, a larger sample size and a more reliable diagnostic performance. As a result, it provides a highly promising solution for physical characterization of metastatic cancer cells in vitro.

Label free quantification of time evolving morphologies using time-lapse video microscopy enables identity control of cell lines and discovery of chemically induced differential activity in iso-genic cell line pairs

Label free time-lapse video microscopy based monitoring of time evolving cell population morphology has potential to offer a simple and cost effective method for identity control of cell lines. Such morphology monitoring also has potential to offer discovery of chemically induced differential changes between pairs of cell lines of interest, for example where one in a pair of cell lines is normal/sensitive and the other malignant/resistant. A new simple algorithm, pixel histogram hierarchy comparison (PHHC), for comparison of time evolving morphologies (TEM) in phase contrast time-lapse microscopy movies was applied to a set of 10 different cell lines and three different iso-genic colon cancer cell line pairs, each pair being genetically identical except for a single mutation. PHHC quantifies differences in morphology by comparing pixel histogram intensities at six different resolutions. Unsupervised clustering and machine learning based classification methods were found to accurately identify cell lines, including their respective iso-genic variants, through time-evolving morphology. Using this experimental setting, drugs with differential activity in iso-genic cell line pairs were likewise identified. Thus, this is a cost effective and expedient alternative to conventional molecular profiling techniques and might be useful as part of the quality control in research incorporating cell line models, e.g. in any cell/tumor biology or toxicology project involving drug/agent differential activity in pairs of cell line models.

18FDG a PET tumor diagnostic tracer is not a substrate of the ABC transporter P-glycoprotein

2-[18F]fluoro-2-deoxy-d-glucose (18FDG) is a tumor diagnostic radiotracer of great importance in both diagnosing primary and metastatic tumors and in monitoring the efficacy of the treatment. P-glycoprotein (Pgp) is an active transporter that is often expressed in various malignancies either intrinsically or appears later upon disease progression or in response to chemotherapy. Several authors reported that the accumulation of 18FDG in P-glycoprotein (Pgp) expressing cancer cells (Pgp+) and tumors is different from the accumulation of the tracer in Pgp nonexpressing (Pgp−) ones, therefore we investigated whether 18FDG is a substrate or modulator of Pgp pump.Rhodamine 123 (R123) accumulation experiments and ATPase assay were used to detect whether 18FDG is substrate for Pgp. The accumulation and efflux kinetics of 18FDG were examined in two different human gynecologic (A2780/A2780AD and KB-3-1/KB-V1) and a mouse fibroblast (3T3 and 3T3MDR1) Pgp+ and Pgp− cancer cell line pairs both in cell suspension and monolayer cultures.We found that 18FDG and its derivatives did not affect either the R123 accumulation in Pgp+ cells or the basal and the substrate stimulated ATPase activity of Pgp supporting that they are not substrates or modulators of the pump. Measuring the accumulation and efflux kinetics of 18FDG in different Pgp+ and Pgp− cell line pairs, we have found that the Pgp+ cells exhibited significantly higher (p⩽0.01) 18FDG accumulation and slightly faster 18FDG efflux kinetics compared to their Pgp− counterparts. The above data support the idea that expression of Pgp may increase the energy demand of cells resulting in higher 18FDG accumulation and faster efflux.We concluded that 18FDG and its metabolites are not substrates of Pgp.

Lysosomotropic Properties of Weakly Basic Anticancer Agents Promote Cancer Cell Selectivity In Vitro

Drug distribution in cells is a fundamentally important, yet often overlooked, variable in drug efficacy. Many weakly basic anticancer agents accumulate extensively in the acidic lysosomes of normal cells through ion trapping. Lysosomal trapping reduces the activity of anticancer drugs, since anticancer drug targets are often localized in the cell cytosol or nucleus. Some cancer cells have defective acidification of lysosomes, which causes a redistribution of trapped drugs from the lysosomes to the cytosol. We have previously established that such differences in drug localization between normal and cancer cells can contribute to the apparent selectivity of weakly basic drugs to cancer cells in vitro. In this work, we tested whether this intracellular distribution-based drug selectivity could be optimized based on the acid dissociation constant (pKa) of the drug, which is one of the determinants of lysosomal sequestration capacity. We synthesized seven weakly basic structural analogs of the Hsp90 inhibitor geldanamycin (GDA) with pKa values ranging from 5 to 12. The selectivity of each analog was expressed by taking ratios of anti-proliferative IC50 values of the inhibitors in normal fibroblasts to the IC50 values in human leukemic HL-60 cells. Similar selectivity assessments were performed in a pair of cancer cell lines that differed in lysosomal pH as a result of siRNA-mediated alteration of vacuolar proton ATPase subunit expression. Optimal selectivity was observed for analogs with pKa values near 8. Similar trends were observed with commercial anticancer agents with varying weakly basic pKa values. These evaluations advance our understanding of how weakly basic properties can be optimized to achieve maximum anticancer drug selectivity towards cancer cells with defective lysosomal acidification in vitro. Additional in vivo studies are needed to examine the utility of this approach for enhancing selectivity.

521 Cell Cycle Regulation Changes in Drug-Resistant Cancers

Cell cycle perturbation is a common event in tumorigenesis and the development of anticancer drug resistance. Acquired drug resistance is a significant hindrance in achieving effective cancer treatment. Thus, understanding of mechanisms underlying acquired drug resistance is vital to develop strategies to overcome those processes and to improve the survival rates of cancer patients. Moreover, understanding drug resistance can lead to the discovery of potential druggable targets that can contribute towards increasing success of cancer intervention. In this study, molecular mechanisms leading to acquired drug resistance in various cancer cell lines were investigated. Informed by cDNA microarray screens, the cell cycle regulators p57Kip2, Weel and Stratifin (SFN) were shown to be frequently deregulated in drug resistance. Using Western blotting and qPCR, p57Kip2 was shown to be downregulated in platinum-resistant ovarian cancer cell lines PEO1CarbR and PEOlCisR in comparison with the platinum-sensitive counterpart. p57Kip2 downregulation was due to epigenetic silencing (hypermethylation of gene promoter region). Thus, p57Kip2 silencing with siRNA in otherwise drug sensitive cells recapitulated platinum resistance. Interestingly, p57Kip2 downregulation conferred collateral sensitivity towards the synthetic CDK inhibitor drug Seliciclib (CYC202). A similar approach was adopted in the study of Weel and SFN in taxane resistance. Weel and SFN were downregulated in Taxol-resistant SK-OV-3 and A2780 cell lines compared to drug-sensitive counterparts. Silencing of Weel in Taxol-sensitive SK-OV-3 decreased the apoptotic response to Taxol treatment, but this effect was not seen using SFN silencing. I conclude that Weel plays an important role in response to Taxol. However, p53 status is also associated with this response. Additionally, examination of several drug resistant and sensitive pairs of cancer cell lines indicated that DNA damage checkpoint and cell cycle regulation are significant factors in mediating anticancer drug resistance. Based on the results, the ATR-Chkl-Weel pathway was shown to be the most deregulated pathway in drug-resistant cancer cells. I conclude that deregulation of cell cycle is essential to the adaptive responses and enabling characteristics that result in the drug resistant phenotype.

MiR-141 regulates KEAP1 and modulates cisplatin sensitivity in ovarian cancer cells

Epithelial ovarian cancer is the most lethal gynecological malignancy in the Western world. A major impediment for the successful treatment is the development of drug resistance. The molecular processes that contribute to resistance have been extensively studied; however, there is not much known about regulation by microRNAs (miRNAs). We compared miRNA expression profiles of an isogenic cisplatin-sensitive and -resistant ovarian cancer cell line pair (A2780/A2780 DDP) and found 27 miRNAs to be differentially expressed (2-fold). Five of these, including the family members miR-141/200c, showed a correlation with cisplatin sensitivity in the NCI-60 panel. Overexpression of miR-141 resulted in enhanced resistance to cisplatin in ovarian cancer cell lines. We next correlated the expression level of miR-141 in 132 primary ovarian tumors (108 serous and 24 non-serous) with response to platinum-based chemotherapy. Although no differences were observed in the serous tumors, miR-141 levels were higher in non-serous ovarian tumors that did not respond well to therapy (platinum-free interval <6 months). We demonstrate that miR-141 directly targets KEAP1, and that downregulation of KEAP1 induces cisplatin resistance. Conversely, overexpression of KEAP1 significantly enhanced cisplatin sensitivity. Expression of KEAP1 with its 3'-UTR, and a 3'-UTR in which the miR-141 target site has been mutated, revealed that miR-141 regulates KEAP1 upon exposure to cisplatin. Finally, we show that the NF-κB pathway, which can be regulated by KEAP1, is activated upon miR-141 overexpression, and that inhibition of this pathway partially reverses miR-141-mediated cisplatin resistance. These findings demonstrate that the miR-141-mediated regulation of KEAP1 has a crucial role in the cellular response to cisplatin.

Cytoskeletal stiffness, friction, and fluidity of cancer cell lines with different metastatic potential

We quantified mechanical properties of cancer cells differing in metastatic potential. These cells included normal and H-ras-transformed NIH3T3 fibroblast cells, normal and oncoprotein-overexpressing MCF10A breast cancer cells, and weakly and strongly metastatic cancer cell line pairs originating from human cancers of the skin (A375P and A375SM cells), kidney (SN12C and SN12PM6 cells), prostate (PC3M and PC3MLN4 cells), and bladder (253J and 253JB5 cells). Using magnetic twisting cytometry, cytoskeletal stiffness (g') and internal friction (g″) were measured over a wide frequency range. The dependencies of g' and g″ upon frequency were used to determine the power law exponent x which is a direct measure of cytoskeletal fluidity and quantifies where the cytoskeleton resides along the spectrum of solid-like (x=1) to fluid-like (x=2) states. Cytoskeletal fluidity x increased following transformation by H-ras oncogene expression in NIH3T3 cells, overexpression of ErbB2 and 14-3-3-ζ in MCF10A cells, and implantation and growth of PC3M and 253J cells in the prostate and bladder, respectively. Each of these perturbations that had previously been shown to enhance cancer cell motility and invasion are shown here to shift the cytoskeleton towards a more fluid-like state. In contrast, strongly metastatic A375SM and SN12PM6 cells that disseminate by lodging in the microcirculation of peripheral organs had smaller x than did their weakly metastatic cell line pairs A375P and SN12C, respectively. Thus, enhanced hematological dissemination was associated with decreased x and a shift towards a more solid-like cytoskeleton. Taken together, these results are consistent with the notion that adaptations known to enhance metastatic ability in cancer cell lines define a spectrum of fluid-like versus solid-like states, and the position of the cancer cell within this spectrum may be a determinant of cancer progression.