Proliferation by Many Other Names: Monitoring Cell Cycle Progression and Cell Division by Flow Cytometry

To the Editor

CYTO University (CYTO U;http://cytou.peachnewmedia.com) is an online educational resource created by ISAC for its members and the wider cytometry community. In addition to free webinars, CYTO U presents recorded courses and tutorials from the CYTO Conference, and interactive online courses on a variety of cytometry-related topics. These are available at no cost to ISAC members and for a nominal charge for non-members. In September 2015, CYTO U announced the launch of its second peer-reviewed e-learning course, entitled “Proliferation Monitoring by Flow Cytometry.” Lesson 1 (Basics of the Cell Cycle) is available online, with Lesson 2 (Monitoring Cell Cycle Progression) and Lesson 3 (Monitoring Cell Division by Dye Dilution) to follow.

Cytometric definitions of “proliferation” vary, but the study of normal growth processes and how they go awry in tumors has been of interest to cytometrists since the inception of the field [1]. Fluorescence based measurements of cellular DNA content have been used since the mid-1960's to study how rapidly both normal and neoplastic cells progress through the cell cycle and how those kinetics are affected by treatment with anti-proliferative agents in vitro and in vivo [2-4]. Clinicians have been particularly interested in determining whether a tumor's proliferative fraction (% of cells in S phase or S-phase fraction) has prognostic value for a given tumor type [5]. Modern multiparameter flow cytometry combines DNA content measurement with probes for phase specific antigens or regulatory proteins to give a detailed picture of progression through the entire cell cycle. These additional markers are particularly helpful in distinguishing between phases where there is no detectable change in DNA content such as G0 vs. G1 and G2 vs. M.

Measurement of S-phase fraction based on DNA content tells us what proportion of cells in a sample are preparing for cell division at a given point in time. It does not, however, allow us to say how many divisions a given cell may have undergone in response to a stimulus, how much a particular cell subset may have expanded during that response or what fraction of a starting cell population went on to divide during the response. Flow cytometry can be used to monitor the extent of cell division by: 1) staining cells with bright, stable, non-toxic fluorescent dyes that label bulk cell proteins or membranes; and 2) following the decrease in intensity (dye dilution) as the dyes are partitioned between daughter cells at each successive mitosis [6,7]. Over the last 30 years “proliferation dyes” with a broad range of spectral characteristics have become available for use in multiparameter flow cytometry, allowing this dye dilution technique to be productively applied to the study of normal immune system homeostasis, tumor stem cell behavior, improved vaccine strategies, and immunotherapies for cancer [8].

The CYTO U e-learning course on Cell Proliferation Monitoring covers both DNA-based and dye dilution-based methods for monitoring cell proliferation by flow cytometry and is divided into three different modules:

• Lesson 1 provides an introduction to (or review of) the basic cell cycle biology underlying specific techniques and applications presented in the other two lessons. Readers are assumed to be familiar with the basic principles of flow cytometry, including interpretation of single and dual-parameter data plots.

• Lessons 2 and 3 provide more in depth discussion of principles, data analysis strategies, practical problems and specific applications for each of the two major cytometric approaches to proliferation monitoring.

• Lesson 2 focuses on methods for assessing S phase fraction and monitoring the kinetics of cell progression through the different cell cycle phases, using DNA content alone or in combination with other phase specific probes.

• Lesson 3 focuses on methods for assessing extent of cell division based on dye dilution, with an emphasis on the use of multicolor methods to detect differential responses in complex cell populations without the necessity for manual isolation and enumeration.

Like Lesson 1, Lessons 2 and 3 assume a familiarity with the basic principles and techniques of flow cytometry. They will be of most benefit to individuals with some hands-on experience that wish to learn about (or review) the specific techniques discussed and examples of how they have been applied in basic, translational and/or clinical research.

Table 1 summarizes the major topics covered in each of the three modules. Four to six self-assessment questions are included at the end of each lesson to allow the reader to test their understanding of the material presented. Several of the questions include analysis of listmode data files using the reader's software of choice. The Proliferation Monitoring course is recognized as a continuing medical laboratory education activity and is accepted by both the ASCP Board of Registry Certification Maintenance Program and the ISAC/ICCS ICCE certification maintenance program. Successful completion of Lesson 1 is recognized by ASCP for 1 CMLE credit and by ICCE for 1 ICCE continuing education credit.

Table 1. Proliferation Monitoring by Flow Cytometry: Content Overview.

Module	Target Audience	Topics
Lesson 1 Basics of the Cell Cycle	Individuals with a basic understanding of flow cytometry who are unfamiliar with, or wish to review, the biology of the cell cycle.	Phases of the cell cycle and molecular events associated with each Cell cycle checkpoints and regulators (cyclins and cyclin dependent kinases) Relationship between the cell cycle phase distribution of a sample and DNA content histogram Use of DNA content histograms in combination with metabolic labeling or cellular synchronization to determine kinetics of progression through the different cell cycle phases
Lesson 2 Monitoring Cell Cycle Progression	Individuals with a basic understanding of flow cytometry who are unfamiliar with, or wish to review, how flow cytometry is used to monitor the distribution or progression of cells through the cell cycle.	Principles and methods for the use of DNA binding dyes alone, or in combination with thymidine analogs, proliferation related antigens, and/or phase specific expression vectors, to assess: How cells within a population of interest are distributed among the different cell cycle phases; and The kinetics with which cells of interest are progressing through the cell cycle. Strategies for analysis and interpretation of single and dual parameter DNA histograms to determine: The fraction of cells actively synthesizing DNA; and Whether a cell population is non-cycling, actively progressing through the cell cycle or blocked in a specific phase of the cell cycle. Practical considerations, common pitfalls, critical controls and troubleshooting tips Applications of DNA content analysis andcell cycle progression kinetics to:tumor diagnosis and prognosis; anti-tumormechanisms of therapeutic agents; and regulation of quiescence or senescence
Lesson 3 Monitoring Cell Division by Dye Dilution	Individuals with some hands-on experience with flow cytometry who are unfamiliar with, or wish to review, how flow cytometry is used to estimate the number of cell generations and daughter cells in each generation without manual counting	Principles and methods for the use of protein-reactive and/or membrane-intercalating dyes to assess extent of cell division in cell subsets of interest based on dye dilution Similarities, differences and considerations for selecting among commercially available “proliferation tracking dyes” Strategies for analysis and interpretation of dye dilution profiles to estimate: Number of generations present at the end of a test and number of daughter cells in each Total population expansion during the test period and frequency of responder cells in the starting population Practical considerations, common pitfalls, critical controls and troubleshooting tips Applications of dye dilution proliferation analysis to identification of: immunosuppressive effector cell potency; differential proliferative responses among immune cell subsets; whether/how early activation markers (antigen binding, CD69, cytokine expression) correlate with subsequent cell division; effective vaccination protocols for increasing precursor cell frequency; and quiescent or slow growing cell subpopulations with stem-like properties

By definition, an introductory course of this nature cannot be completely comprehensive and the authors therefore apologize to the many individuals who have contributed to our understanding of this topic but whose work is not specifically presented or referenced. We also wish to thank the many colleagues within ISAC and the wider flow cytometry community who have contributed to our own ongoing education on these topics, and to the extensive list of individuals involved with CYTO U who have contributed to making this course a reality. Proliferation monitoring by flow cytometry continues to be a vital and rapidly evolving field, and we look forward to adding new approaches and methods to this course in future updates.