Emerging applications of flow cytometry in solid tumor biology Qing Chang, David Hedley ⇑ Research Division, Ontario Cancer Institute and Princess Margaret Hospital, 610 University Avenue, Toronto, Ontario, Canada M5G 2M9 article info Article history: Available online 6 April 2012 Communicated by David W. Galbraith Keywords: Flow cytometry Pancreatic cancer Cell cycle Cytokeratin Xenograft DNA content abstract Despite considerable interest during the early clinical development of flow cytometry, its application to solid tumours has been largely ignored in recent years. However, with rapid progress in cancer biology and molecular therapeutics, linked to technical developments in the areas of flow cytometry instrumen- tation, reagents, and data analysis, it is timely to re-evaluate this role. This article places emphasis on the unique potential of flow cytometry to analyze heterogeneous cell populations, and to provide information on the functional status of regulatory processes by the simultaneous measurement of multiple key ele- ments. Major obstacles to progress addressed include the acquisition of adequate clinical samples, tissue disaggregation to produce single cells suspensions suitable for flow cytometry, and protocols to label intracellular as well as cell surface antigens. Ó 2012 Elsevier Inc. All rights reserved. 1. Introduction Cancer is now the leading cause of death in developed coun- tries, with the overwhelming number of cases arising in the epithe- lium of the aerodigestive or genitourinary tracts. The mechanisms that initiate cancers and then drive their progression towards dis- seminated, treatment-resistant end-stage disease are fairly well understood. Consequently, a major effort is under way to develop novel treatments that target these mechanisms. Typically, genetic analysis of human cancers identifies multiple abnormalities in pathways regulating growth, survival and differentiation; consis- tent with the stepwise nature of cancer progression. This explains the clinical experience that treatments based on single agents that target specific abnormalities can slow tumour growth, but fail to cure. Furthermore, human cancers are extremely heterogeneous at the genetic level, and also show considerable intratumoral het- erogeneity with subpopulations likely to show wide differences in treatment sensitivity. The bulk of most human solid tumors consists of normal host cells that form the supportive stroma and vascular system, and inflammatory cells that can promote tumor growth as well as elicit an anticancer immune response. Dynamic interactions between these populations of host cells and the cancer are an area of intense study, and potentially fertile ground for the development of novel approaches to cancer treatment. Tumor heterogeneity largely explains why early attempts at cancer treatment based on simple cell line models failed to work in the clinic. In the era of genomics-based medicine it remains a major obstacle to progress. Thus individualized treatment of patients, which is the long term goal of molecular oncology, will require new analytical tools applied directly to patient samples or to clinically relevant models, in order to direct patient treatment selection and monitoring. Since flow cytometry is well suited to the study of complex biological processes occurring in heteroge- neous cell populations, it would appear well suited to this task. Cancer diagnosis and treatment were major goals during the early development of flow cytometry in the 1970’s, and pathology departments made extensive use of DNA content analysis of solid tumours during the 1980’s. However, clinical flow cytometry is now almost exclusively concerned with disorders of the immune and hematopoietic systems, with very little attention being paid to the analysis of solid tumours. This article reviews the potential for the development of high level flow cytometry applications to study the fundamental processes of cancer, based on recent ad- vances in basic science and technology. Obstacles to progress in the development of solid tumor flow cytometry and its clinical implementation, and possible approaches to overcoming these, are discussed. 2. Biological processes in cancer potentially amenable to flow cytometric analysis In this article we will be mainly concerned with biological pro- cesses. Deranged functionality, which is a hallmark feature of can- cer, is better appreciated using applications that measure multiple components of a cellular process rather than specific markers, and flow cytometry is uniquely capable of doing this. Table 1 lists cel- lular processes that can be altered in cancers, for which flow cyto- metric methods might be developed in order provide new insights 1046-2023/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ymeth.2012.03.027 ⇑ Corresponding author. E-mail address: David.Hedley@uhn.on.ca (D. Hedley). Methods 57 (2012) 359–367 Contents lists available at SciVerse ScienceDirect Methods journal homepage: www.elsevier.com/locate/ymeth