RECENT ADVANCES IN FLOW CYTOMETRY: PLATFORMS, TOOLS, AND CHALLENGES FOR DATA ANALYSIS - Flow Cytometry in Drug Discovery and Development - page 37

FIGURE2.2 Analysis of cell cycle arrest in humanA549 lung carcinoma cells induced by the
candidate anticancer prodrug AQ4N (100 nM exposure) under normoxic or hypoxic conditions
(1%O 2 , 4 days). The prodrug AQ4N is converted to a cytotoxic form under hypoxic conditions
generating cell cycle arrest and a concomitant increase in cellular light scatter andCYO staining
(20 m M CyTRAK Orange, 10min). CYO staining permits the differentiation, for example, by
quadrant analysis, of arrested cells (UR) versus nonarrested (LR) cells compared to unstained
cells (LL and UL). Quadrants of the flow cytometric contour plots show percent gated cells
(P.J. Smith et al., unpublished data).
factor for tumor aggressiveness andmetastasis by the activation of signal transduction
pathways and gene regulatory mechanisms [122]. Under such conditions, the meta-
bolic environment of the tumor cell is altered and an adaptation response is mounted
through HIF-1 (hypoxia-inducible factor-1) induction [122]. Recent attention has
been directed toward hypoxia-targeted therapeutics, for example, through a prodrug
concept exemplified by the aliphatic amine N-oxide AQ4N (Oncotherics Ltd,
UK) [123]. The operating principle is that AQ4N, a di-N-oxide anticancer prodrug
with little intrinsic cytotoxicity but having tissue penetrant properties [124], will
undergo bioreduction in hypoxic regions of solid tumors andmicrometastatic deposits
generating the active parent amine [125]. In early clinical trials, intratumoral
concentrations of AQ4 exceeded those required for activity in animal models and
support the evaluation of AQ4N as a novel tumor targeting agent [126]. Further, there
is evidence that AQ4N possesses antiangiogenic effects in normoxic conditions,
which could potentiate overall antitumor activity [127]. Flow cytometry has had an
important role in the tracking of cell targeting of such prodrugs [128] and the intended
pharmacodynamic effects in terms of long-term cell cycle arrest generated under
hypoxia-driven prodrug activation. Figure 2.2 outlines the analysis of long-termarrest
that cannot be tracked using cellular DNA content alone. The concept is to take into