Abstract

Results of in vitro cell culture drug testing are correlated
with clinical response to chemotherapy: The benefit of using in vitro models
lies in the ability to probe cellular response in a controlled closed system,
where the effects of drug concentrations, treatment duration, drug efflux
kinetics and multidrug combinations can be assessed using a variety of cell
biology techniques. However, obtaining highly multiplexed data for surface,
intracellular and functional markers for each cell within a test well has been
challenging using standard immunofluorescence techniques.

Imaging mass cytometry (IMC) (1,2) allows us to investigate
complex effects of chemotherapy drugs as well as the intracellular localization
of metal-containing drugs (e.g., cisplatin and oxaliplatin),
ruthenium-containing drugs (e.g., NAMI) and cytostatic agents (e.g., nocodazole
and etoposide) (Table 2). A large panel of metal-tagged antibodies (Table 1)
was used to analyze responses to drug treatments at the single-cell level.
Proteins involved in DNA damage repair, apoptosis, cell proliferation,
substrate adhesion, organelle morphology and signaling pathways, as well as
surface and cytoskeletal markers, were studied. S-phase cells were visualized
by detection of iodine (127I) from 5-iodo-2′-deoxyuridine (IdU) incorporated by
growing cells.

Imaging mass cytometry workflow. The immunostained and dried
samples of tissue sections (FFPE or cryosections) and cells attached to glass
slides are inserted into the ablation chamber of the Hyperion™ Imaging System
(Fluidigm), where a 1 µm spot-size pulsed UV laser ablates the tissue. Isotopes
associated with each spot are transferred into the mass cytometer, detected and
indexed against the source location, yielding an intensity map of the target
proteins throughout the tissue.