Characterization Of Cancer Cell Lines Research Articles

A Principal Componet Analysis to detect cancer cell line aggressiveness

In this paper, we propose the use of Principal Component Analysis (PCA) as a new post-processing method for the detection of breast and bone cancer cell lines cultured in vitro using a microwave biosensor. MDA-MB-231 and MCF7 breast cancer cell lines and SaOS-2 and 143B osteosarcoma cell lines were characterized using a circular patch resonator in the 1 MHz – 3 GHz frequency range. The return loss of each cancer cell line was analyzed, and the differences among each other were determined through Principal Component Analysis according to a protocol previously proposed mainly for electrocardiogram processing and X-ray photoelectron spectroscopy. Our results showed that the four cancer cell lines analyzed exhibited peculiar dielectric properties when compared to each other and the growth medium, confirming that PCA could be employed as an alternative methodology to analyze microwave characterization of cancer cell lines which, in turn, may be deeply exploited as a tool for the detection of cancer cells in healthy tissues.

Abstract 2025: Characterization of cancer cell lines made senescent by exposure to ribociclib, doxorubicin, or TGFβ1, and identification of genes required for entry into senescence and senescent cell survival

Abstract Cellular senescence is a stress-induced state of stable growth arrest characterized by high expression of cell cycle inhibitors; a dramatic change in cell morphology, including an increase in lysosomal content; and secretion of large numbers of proteins involved in immune signaling and extracellular matrix remodeling. The physiological importance of cellular senescence has been attributed to prevention of carcinogenesis, aging, development, and tissue repair, and tumor cells can undergo senescence in response to therapeutic agents. In this work, we sought to validate the senescence-inducing activity of two known inducers (doxorubicin and TGFβ1) and a CDK4/6 inhibitor (ribociclib) and to identify proteins that can kill senescent tumor cells (senolytic targets) if knocked out and to identify downstream components of the tumor cell senescence pathway. Huh7 hepatocellular carcinoma cells and SK-MEL-28 melanoma cells were induced to senescence by treatment with three different agents: ribociclib, low doses of doxorubicin, or TGFβ1. The induction of senescence was confirmed by observing growth arrest, an increase in SA-β-gal staining, dramatic cell morphology changes, loss of c-Myc protein, increased expression of p15, and increased expression of senescence-associated secretory phenotype (SASP) proteins. Induction of SASP components was measured by RNAseq and SOMAscan. All three agents induced a senescent state, with blockage at different stages of the cell cycle observed. Induction of known immune factors, including IL-8 and IL-11, were identified in senescent cells (Huh7). A whole-genome CRISPR screen identified proteins required to enter senescence and those that were incompatible with the senescent state if knocked out. Expected hits were observed (eg, TGFBR1/TGFBR2 for TGFβ1, RB for ribociclib, and TOP2A for doxorubicin) for guide DNAs (gDNAs) that blocked entry into the senescent state. No gDNA candidates for common downstream senescence pathway components were observed, suggesting that these components are essential genes or that they do not exist. gDNA-induced knockouts that were incompatible with the senescent state dropped out of the screen and represent potential senolytic targets. The screen identified BCL2L1 as the only common senolytic hit across multiple senescence-inducing reagents, confirming published reports suggesting it is a senolytic target. These data show that ribociclib, doxorubicin, and TGFβ1 induced senescence in cancer cell lines. Whole-genome CRISPR screens identified senescence pathway components for each of these agents, as well as a common senolytic target. Citation Format: Pasupuleti Rao, Jennifer Tullai, Peter Aspesi, Felipa Mapa, Nadire Cochran, Frederic Sigoillot, Guglielmo Roma, Scott Gleim, Jaison Jacob, Jason Marchese, Jonathan Solomon. Characterization of cancer cell lines made senescent by exposure to ribociclib, doxorubicin, or TGFβ1, and identification of genes required for entry into senescence and senescent cell survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2025.

Protein-gene Expression Nexus: Comprehensive characterization of human cancer cell lines with proteogenomic analysis.

Researchers have gained new therapeutic insights using multi-omics platform approaches to study DNA, RNA, and proteins of comprehensively characterized human cancer cell lines. To improve our understanding of the molecular features associated with oncogenic modulation in cancer, we proposed a proteogenomic database for human cancer cell lines, called Protein-gene Expression Nexus (PEN). We have expanded the characterization of cancer cell lines to include genetic, mRNA, and protein data of 145 cancer cell lines from various public studies. PEN contains proteomic and phosphoproteomic data on 4,129,728 peptides, 13,862 proteins, 7,138 phosphorylation site-associated genomic variations, 117 studies, and 12 cancer. We analyzed functional characterizations along with the integrated datasets, such as cis/trans association for copy number alteration (CNA), single amino acid variation for coding genes, post-translation modification site variation for Single Amino Acid Variation, and novel peptide expression for noncoding regions and fusion genes. PEN provides a user-friendly interface for searching, browsing, and downloading data and also supports the visualization of genome-wide association between CNA and expression, novel peptide landscape, mRNA-protein abundance, and functional annotation. Together, this dataset and PEN data portal provide a resource to accelerate cancer research using model cancer cell lines. PEN is freely accessible at http://combio.snu.ac.kr/pen.

High-Throughput Generation of Bispecific Binding Proteins by Sortase A-Mediated Coupling for Direct Functional Screening in Cell Culture.

High-throughput construction of multivalent binders and subsequent screening for biological activity represent a fundamental challenge: A linear increase of monovalent components translates to the square of possible bivalent combinations. Even high-efficiency cloning and expression methods become limiting when thousands of bispecific binders need to be screened for activity. In this study, we present an in vitro method for the efficient production of flexibly linked bispecific binding agents from individually expressed and purified monovalent binders. We established a sortase A-mediated coupling reaction to generate bispecific designed ankyrin repeat proteins (DARPins), with an optimized reaction maximizing the bivalent coupling product with low levels of monovalent side-products. These one-pot reaction mixtures could be used directly, without further purification, in cell-based assays. We generated a matrix of 441 different bispecific DARPins against the extracellular domains of the cancer-associated receptors EGFR, ErbB2, ErbB3, ErbB4, EpCAM, and c-MET and screened on two different ErbB2-positive cancer cells lines for growth-inhibitory effects. We identified not only known but also novel biologically active biparatopic DARPins. Furthermore, we found that the cancer cell lines respond in a highly reproducible and defined manner to the treatment with the 441 different bivalent binding agents. The generated response profiles can thus be used for functional characterization of cell lines because they are strongly related to the cell line-specific surface receptor landscape. Thus, our method not only represents a robust tool for screening and lead identification of bispecific binding agents, but additionally offers an orthogonal approach for the functional characterization of cancer cell lines.

Abstract LB-038: Development and characterization of cancer cell line exosomes as reference standards in cancer research

Abstract Exosomes are subcellular particles 50-200 nm in size released from cells through a fusion of multicellular bodies with the plasma membrane. Exosomes are currently being evaluated as potential diagnostic tools in a number of diseases, including cancer. Exosomes are stable carriers of cell-free cargo in the form of DNA, RNA, and protein, thereby making them an attractive candidate for early detection of cancer via liquid biopsy. Tumor-derived exosomes have been linked to stimulation of tumor cell growth, angiogenesis, metastasis, and suppression of the immune system. However, isolating a consistent population of exosomes can be challenging and there is an unmet need for highly characterized exosomes for use as reference standards for research and diagnostic applications. We have developed a novel method employing tangential flow filtration for the isolation of large quantities of highly purified, ready to use exosomes from cell culture supernatant. Exosomes from a variety of cancer cell lines representing the most prevalent cancer types, including prostate (LNCaP), colorectal (HCT116), breast (MDA-MB-231, MCF-7), lung (A549), cervical (HeLa), and glioblastoma (U87-MG) were isolated and characterized in this study. We employed a stringent quality control approach in order to define and characterize these exosomes. Our method generated a high yield (>1×1010 exosomes/mL) and average protein equivalent of 2 mg/mL. Our data demonstrated expression of a number of different characteristic exosomal proteins including tetraspanin, which was confirmed through western blot and PCR. Isolated exosomes had a median size of 102 nm through nanoparticle tracking analysis. Western blot data revealed cell line specific protein marker expression of CD63, CD81, flotillin 1, Alix and TSG101 protein. We assessed the functionality of these purified exosomes through cellular uptake, migration assay, and anchorage independent growth assay in this study. We also evaluated exosomal RNA from these purified exosomes for the presence of common mutations associated with cancer through PCR. Here we report that exosomes isolated by our method not only had high exosome yield and quality, but also retained relevant functionality and thereby make ideal reference standards/controls in the field of cancer research and diagnostics development. Citation Format: Siddhartha Paul, Nanda Mahashetty, Joy Wells, Dezhong Yin, Fang Tian. Development and characterization of cancer cell line exosomes as reference standards in cancer research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-038.

Multiplex Label Free Characterization of Cancer Cell Lines Using Surface Plasmon Resonance Imaging.

Rapid multiplex cell surface marker analysis can expedite investigations in which large number of antigens need to be analyzed. Simultaneous analysis of multiple surface antigens at the same level of sensitivity is however limited in the current golden standard analysis method, flow cytometry. In this paper we introduce a surface plasmon resonance imaging (SPRi)-based technique for 44-plex parameter analysis using a single sample, in less than 20 min. We analyzed the expression on cells from five different cancer cell lines by SPRi on a 44-plex antibody array including 4 negative controls and compared the output with flow cytometry. The combined correlation of the markers that showed expression by flow cytometry was 0.76. The results demonstrate as a proof of principle that SPRi can be applied for rapid semi-quantitative multiplex cell surface marker analysis.

Generation and partial characterization of cancer cell lines from Mexican breast cancer patients

Numerous cell models have been approached to study cancer biology. Some of those models are primary cell cultures, as well as, cancer cell lines derived from tumors. Several breast cancer cell lines are available for scientific research. Nevertheless, those cell lines do not represent all the cancer types and subtypes, neither their origin corresponds to most of the ethnic groups of the world. The main goal of this study is to generate and partially characterize cancer cell lines from tumor tissue obtained from Mexican patients with breast cancer, which can be used as models to study cancer biology and to characterize the phenotype and genotype of breast cancer affecting Mexican population. At present, four primary cell cultures (ID010, ID012, ID013 and ID014) have been established from primary tumor tissues representative of infiltrating ductal carcinomas, such as luminal B and HER‐enriched molecular subtypes. In addition, all the primary cell cultures showed the expression of cytokeratins, which suggests the presence of epithelial cells. The development and partial characterization of new breast cancer cell lines with different molecular subtypes, will allow us a better comprehension about the variations in morbidity, mortality and susceptibility to anti‐cancer drugs among different ethnicities.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract A42: Systematic modulation of the physical microenvironment for characterization of cancer cell lines and primary tissue

Abstract Introduction: The tumor microenvironment includes a range of physical, chemical, and cellular components such as pressure, hypoxia, acidosis, the extracellular matrix (ECM), stromal, and immune cells. These factors can influence the gene and protein expression of the various types of cells that make up the tumor as well as influence the selection of cells that can thrive in that environment; however, typical cancer cell culture rarely uses hypoxia and pressure nor utilizes substrates similar to the native ECM. We designed a system study the influence of hypoxia, pressure, and native ECM conditions on cancer cell lines and primary cells, with the goal of creating a more relevant culture environment for translational studies. Methods: Here, we comprehensively studied how hypoxia, pressure, and ECM composition and organization influences cell biology and gene expression, using transcriptome profiling across a range of physiologically-relevant culturing conditions to mimic various tumor microenvironments found within the body. To do this, we utilized Xcell's primary cell culture platform which includes a custom bioreactor that allows us to control both oxygen concentration (0.1%-20% O2) and hydrostatic pressure levels (26 to 260 mmHg / 0.5 psig to 5 psig). Additionally, we studied the influence of biomimetic substrates by controlling ECM composition and organization (aligned or unaligned collagen at concentrations from 1-2.5 mg/ml +/- fibronectin 0.1-10 microgram/ml) as both 2D ECM coatings and 3D hydrogels. Cell lines studied included models for brain cancer (U-87, A172), pancreatic cancer (PANC10.05), and prostate cancer (DU-145, PC-3, 22Rv1, LNCaP). We performed high-resolution immunofluorescence imaging and western blot protein expression analysis of key targets, including immunotherapy targets CTLA-4, PD-1, and PD-L1. Additionally, we applied our ability to tune culturing conditions to represent physiological conditions in the body for the successful culture of fresh primary tissues from normal volunteers as well as cancer patients for downstream analysis studies focused on biomarker discovery and as a clinical tool for disease monitoring and treatment decision-making. Conclusions: We identified both common and unique gene signatures across these different cells lines, with the hypoxic conditions activating HIF1 pathway activation having common across the cells studied whereas pressure results in more restricted signatures. Some cancer cell lines and PBMCs differentially express immunotherapeutic targets, at low oxygen and high pressure culturing conditions, resulting in reduced expression of key targets. Moreover, biomimetic ECM studies revealed modulation of ECM to represent a “tumor”-like ECM dramatically altered cell growth, morphology, and focal adhesion organization. In a 3D hydrogel context, a “tumor”-like ECM and a hypoxic environment dramatically enhanced tumor cell invasion. Consistently, analysis of mRNA-seq data revealed alterations in gene expression profiles of immunotherapeutic and drug-target pathways involving CTLA-4 and AR signaling as well as EMT targets. In contrast, we observed increased CD47 and CD44 expression at low oxygen and high pressure culturing conditions in cancer cell lines and PBMCs. Thus, supporting the notion that these are possible drug targets for tumors that are characterized by low oxygen levels and high interstitial fluid pressure. Citation Format: Luke Cassereau, Bruce Adams, Tianna Chow, Ajuni Sohota, Charles Ryan, James Lim. Systematic modulation of the physical microenvironment for characterization of cancer cell lines and primary tissue. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr A42.

ICAM1 Is a Potential Cancer Stem Cell Marker of Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) accounts for about 90% of esophageal cancer diagnosed in Asian countries, with its incidence on the rise. Cancer stem cell (CSC; also known as tumor-initiating cells, TIC) is inherently resistant to cytotoxic chemotherapy and radiation and associates with poor prognosis and therapy failure. Targeting therapy against cancer stem cell has emerged as a potential therapeutic approach to develop effective regimens. However, the suitable CSC marker of ESCC for identification and targeting is still limited. In this study, we screened the novel CSC membrane protein markers using two distinct stemness characteristics of cancer cell lines by a comparative approach. After the validation of RT-PCR, qPCR and western blot analyses, intercellular adhesion molecule 1 (ICAM1) was identified as a potential CSC marker of ESCC. ICAM1 promotes cancer cell migration, invasion as well as increasing mesenchymal marker expression and attenuating epithelial marker expression. In addition, ICAM1 contributes to CSC properties, including sphere formation, drug resistance, and tumorigenesis in mouse xenotransplantation model. Based on the analysis of ICAM1-regulated proteins, we speculated that ICAM1 regulates CSC properties partly through an ICAM1-PTTG1IP-p53-DNMT1 pathway. Moreover, we observed that ICAM1 and CD44 could have a compensation effect on maintaining the stemness characteristics of ESCC, suggesting that the combination of multi-targeting therapies should be under serious consideration to acquire a more potent therapeutic effect on CSC of ESCC.

Molecular Predictors of Sensitivity to the Insulin-like Growth Factor 1 Receptor Inhibitor Figitumumab (CP-751,871)

Figitumumab (CP-751,871), a potent and fully human monoclonal anti-insulin-like growth factor 1 receptor (IGF1R) antibody, has been investigated in clinical trials of several solid tumors. To identify biomarkers of sensitivity and resistance to figitumumab, its in vitro antiproliferative activity was analyzed in a panel of 93 cancer cell lines by combining in vitro screens with extensive molecular profiling of genomic aberrations. Overall response was bimodal and the majority of cell lines were resistant to figitumumab. Nine of 15 sensitive cell lines were derived from colon cancers. Correlations between genomic characteristics of cancer cell lines with figitumumab antiproliferative activity revealed that components of the IGF pathway, including IRS2 (insulin receptor substrate 2) and IGFBP5 (IGF-binding protein 5), played a pivotal role in determining the sensitivity of tumors to single-agent figitumumab. Tissue-specific differences among the top predictive genes highlight the need for tumor-specific patient selection strategies. For the first time, we report that alteration or expression of the MYB oncogene is associated with sensitivity to IGF1R inhibitors. MYB is dysregulated in hematologic and epithelial tumors, and IGF1R inhibition may represent a novel therapeutic opportunity. Although growth inhibitory activity with single-agent figitumumab was relatively rare, nine combinations comprising figitumumab plus chemotherapeutic agents or other targeted agents exhibited properties of synergy. Inhibitors of the ERBB family were frequently synergistic and potential biomarkers of drug synergy were identified. Several biomarkers of antiproliferative activity of figitumumab both alone and in combination with other therapies may inform the design of clinical trials evaluating IGF1R inhibitors.

High-Throughput RNA Interference Screening Identifies Synthetic Lethality Between Oncogenic KRAS Dependency and Suppression of STK33

Activating RAS mutations are among the most common pathogenetic events in a broad spectrum of hematologic malignancies and epithelial tumors. However, oncogenic RAS has thus far not proven to be a tractable target for therapeutic intervention. An alternative to direct targeting of known oncogenes is to perform “synthetic lethality” screens to identify genes that are selectively required for cell viability in the context of specific cancer-causing mutations. Using this approach, we have discovered a synthetic lethal interaction between mutant KRAS, the most frequently mutated oncogene in human cancer, and inactivation of the gene encoding the STK33 serine/threonine protein kinase. To identify genes that are essential for cell viability in the context of mutant KRAS, we performed high-throughput loss-of-function RNA interference (RNAi) screens in eight human cancer cell lines (mutant KRAS, n=4; wildtype KRAS, n=4), representing seven different tumor types (acute myeloid leukemia, multiple myeloma, colon cancer, breast cancer, ovarian cancer, prostate cancer, glioblastoma), as well as normal human fibroblasts and mammary epithelial cells. We screened each cell line with a subset of the short hairpin RNA (shRNA) library developed by the RNAi Consortium (http://www.broad.mit.edu/genome_bio/trc/rnai.html) that consists of 5,024 individual shRNA constructs targeting 1,011 human genes, including the majority of known and putative protein kinase and phosphatase genes and a selection of known cancer-related genes. In these cell lines, suppression of STK33 preferentially inhibited the viability and proliferation of cells that were dependent on mutant KRAS. The differential requirement for STK33 based on oncogenic KRAS dependency was confirmed in 16 additional cell lines using in vitro transformation assays and human tumor xenograft models. Biochemical analyses support the hypothesis that STK33 promotes cell growth and survival in a kinase activity-dependent manner by regulating the activity of S6K1 as well as BAD-induced apoptosis selectively in mutant KRAS-dependent cells. Notably, molecular genetic characterization of cancer cell lines and analysis of patient-derived genomic data sets indicate that STK33 is not frequently mutated or overexpressed in human tumors. These observations identify STK33 as a potential target for the treatment of mutant KRAS-driven cancers that may have a broad therapeutic index in normal versus malignant cells, and illustrate the potential of RNAi for discovering critical functional dependencies created by oncogenic mutations that cannot be identified using other genomic technologies.

Human papillomavirus type 16-immortalized endocervical cells selected for resistance to cisplatin are malignantly transformed and have a multidrug resistance phenotype.

Cis-diamminedichloroplatinum (II) (cisplatin, CDDP) is a highly effective chemotherapeutic agent against cervical cancer, but drug resistance is a major obstacle in its clinical application. The mechanism of drug resistance in human cervical cancer is not well understood. Here, we established an in vitro endocervical, cisplatin-resistant cell system that mimics the development of cisplatin resistance in the human cervix. Human papillomavirus (HPV) type 16-immortalized human endocervical cells (HEN-16-2) were treated with cisplatin, and the cisplatin-selected cells (HEN-16-2/CDDP) were resistant to cisplatin, paclitaxel, actinomycin D, doxorubicin, etoposide, and 5-fluorouracil, thus demonstrating a multidrug resistance (MDR) phenotype. Furthermore, compared with a similar passage of drug-sensitive HEN-16-2 cells, HEN-16-2/CDDP cells exhibited the general growth characteristics of cancer cell lines: faster growth in medium containing serum and high calcium levels, higher saturation density, anchorage-independent growth, and formation of tumors in nude mice. These results provided the first in vitro evidence that cisplatin selection can transform HPV-immortalized endocervical cells and cause a phenotype of MDR.