Fluorescent Protein-Based Biosensors and Their Clinical Applications - Progress in Molecular Biology and Translational Science - page 339

low noise and high potential of our system, indicating that when we are able
to increase the number of analyzed cells, we will able to identify less resistant
cells. Therefore, we are currently intensely engaged in increasing the ana-
lyzed cell number by (1) automating image acquisition with computer-
controlled architecture and (2) measuring lifetime using Flicyme (see
Section 6.4 ).
The single-cell analysis made possible by Pickles also increases our un-
derstanding of the mechanisms underlying the disease. The common mu-
tations in BCR-ABL and their relationship to drug susceptibility have
been extensively examined; thus, if the mutation observed in a given pa-
tient is a common one, it is relatively straightforward to determine the
most effective drug. However, the effectiveness of the second-generation
drug NL on cells containing the Gly250Glu BCR-ABL mutant (hereafter
referred to as G250E) remains controversial. 119,120 We thus attempted to
investigate this issue using Pickles. Each individual cell expressing the
G250E mutant of BCR-ABL showed diverse NL sensitivities, resulting
in a large standard deviation. When we examined the dose-dependent
responses of BCR-ABL, we found that drug responsiveness decreased in
a manner that was dependent on the expression level of BCR-ABL
only for the G250E mutant and NL combination. Therefore, the
expression level of BCR-ABL with the G250E mutation in the patients'
tumor cells might have been low in the study that described this
mutation as being sensitive to NL but high in the report that concluded
that G250E is resistant to NL. Although it has previously been reported
that drug susceptibility decreases with an increase in BCR-ABL
expression, 121 this tendency is especially important for the G250E
mutant. In either case, it is necessary to take the expression level of
BCR-ABL into consideration, specifically when this mutation is
detected, and to note that drug optionsmayvarydependingonBCR-
ABL expression levels.
7.3. Evaluation of drug efficacy in patient-derived cells
Approval from the Internal Review Board in our research institute has al-
ready been obtained for this system, and drug efficacy is being evaluated
using patient-derived tumor cells. More than 50 cases have been examined
over the past 4 years. This project is still ongoing and includes many cases
that will require careful follow-up. However, from the patients who were
incorporated into the study at earlier stages, we have learned that our system