title: Guidelines for the selection of functional assays to evaluate the hallmarks of cancer.
abstract: The hallmarks of cancer capture the most essential phenotypic characteristics of malignant transformation and progression. Although numerous factors involved in this multi-step process are still unknown to date, an ever-increasing number of mutated/altered candidate genes are being identified within large-scale cancer genomic projects. Therefore, investigators need to be aware of available and appropriate techniques capable of determining characteristic features of each hallmark. We review the methods tailored to experimental cancer researchers to evaluate cell proliferation, programmed cell death, replicative immortality, induction of angiogenesis, invasion and metastasis, genome instability, and reprogramming of energy metabolism. Selecting the ideal method is based on the investigator's goals, available equipment and also on financial constraints. Multiplexing strategies enable a more in-depth data collection from a single experiment - obtaining several results from a single procedure reduces variability and saves time and relative cost, leading to more robust conclusions compared to a single end point measurement. Each hallmark possesses characteristics that can be analyzed by immunoblot, RT-PCR, immunocytochemistry, immunoprecipitation, RNA microarray or RNA-seq. In general, flow cytometry, fluorescence microscopy, and multiwell readers are extremely versatile tools and, with proper sample preparation, allow the detection of a vast number of hallmark features. Finally, we also provide a list of hallmark-specific genes to be measured in transcriptome-level studies. Although our list is not exhaustive, we provide a snapshot of the most widely used methods, with an emphasis on methods enabling the simultaneous evaluation of multiple hallmark features.
authors.affiliation: MTA TTK Lendület Cancer Biomarker Research Group, Magyar tudósok körútja 2, H-1117 Budapest, Hungary.
authors.forename: Otília
authors.initials: O
authors.lastname: Menyhárt
authors.affiliation: 2nd Department of Pediatrics, Semmelweis University, H-1094 Budapest, Hungary.
authors.forename: Hajnalka
authors.initials: H
authors.lastname: Harami-Papp
authors.affiliation: Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
authors.forename: Saraswati
authors.initials: S
authors.lastname: Sukumar
authors.affiliation: German Cancer Consortium (DKTK), DKFZ, Im Neuenheimer Feld 280, D-69120 Heidelberg and Charité Comprehensive Cancer Center, Invalidenstr. 80, D-10115 Berlin, Germany.
authors.forename: Reinhold
authors.initials: R
authors.lastname: Schäfer
authors.affiliation: Department of Surgery and Cancer, Imperial College London, London W12 0NN, UK.
authors.forename: Luca
authors.initials: L
authors.lastname: Magnani
authors.affiliation: Group of Transcriptional Regulation of Gene Expression, Department of Oncology and Hematology, IDIBAPS, Barcelona, Spain.
authors.forename: Oriol
authors.initials: O
authors.lastname: de Barrios
authors.affiliation: MTA TTK Lendület Cancer Biomarker Research Group, Magyar tudósok körútja 2, H-1117 Budapest, Hungary; 2nd Department of Pediatrics, Semmelweis University, H-1094 Budapest, Hungary. Electronic address: gyorffy.balazs@ttk.mta.hu.
authors.forename: Balázs
authors.initials: B
authors.lastname: Győrffy
mesh_terms: D017209:Apoptosis; D020169:Caspases / Q000032:analysis; D049109:Cell Proliferation; D004734:Energy Metabolism; D044127:Epigenesis, Genetic; D042822:Genomic Instability; D017408:Guidelines as Topic; D006801:Humans; D009369:Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D009389:Neovascularization, Pathologic / Q000209:etiology; D016615:Telomere
chemical_list: D020169:Caspases
keywords: Cancer; Cell culture; Flow cytometry; Fluorescence microscopy; Functional assays; Gene chips; Hallmark; Immunohistochemistry; In vitro methods; Multiplexing; Next generation sequencing; PCR