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Page 722 Bijnsdorp et al. Cancer Drug Resist 2021;4:719-27 https://dx.doi.org/10.20517/cdr.2021.21

Immunofluorescent staining of autophagic vesicles
Immunofluorescent staining was performed as described previously [24,32] . In brief, cells were seeded in six-
well plates on a cover slip and exposed to rapamycin, TPI, and/or TdR for 72 h. After exposure, cells were
fixed for 15 min in 4% formaldehyde and permeabilized with methanol for 10 min at -20 °C. Subsequently,
cells were blocked and the LC3B antibody was added (1:200, Cell Signaling; overnight at 4 °C), after which
the secondary antibody goat-anti-rabbit conjugated with Alexa Fluor was added together with Hoechst
33342 (1:1000) for 1 h at RT. The coverslips were mounted onto microscope slides using Vectashield
(Vector, Burlingame, CA, USA). Fluorescence microscopy was carried out using an inverted Leica
DMIRB/E fluorescence microscope (Leica Cambridge, Cambridge, UK). Images were collected using
Q500MC Quantimet software V01.01 (Leica Cambridge).

RESULTS
Rapamycin cytotoxicity is decreased by TdR
Colo320 and Colo320TP1 had comparable levels of sensitivity to rapamycin, as determined by the SRB-
assay [Table 1]. TPI addition to rapamycin hardly affected the sensitivity to rapamycin in both Colo320 and
Colo320TP1 cells. However, TdR addition protected both the Colo320 and Colo320TP1 cells against the
cytotoxicity induced by rapamycin, which resulted in 13.3-fold resistance in Colo320 but even 50-fold
resistance in Colo320TP1 cells. To determine whether this increase was related to the breakdown of TdR by
TP, TPI was added to this combination which has been previously been reported to effectively inhibit this
[7]
conversion . As expected, in Colo320 cells (which do not express TP), TPI addition to rapamycin and TdR
did not affect the resistance, indicating that in Colo320 cells protection is related to anabolism of TdR to
TdR nucleotides. In Colo320TP1 cells, TPI addition to rapamycin and TdR completely reversed the
protective effect, indicating a role for TP in the protection.

Effects on cell cycle and cell death
To determine whether the protective effect of TdR was related to specific cell cycle effects, FACS analysis of
PI-stained cells was performed. To study this, 20 and 200 nM rapamycin were used, 20 nM being a
concentration slightly above that inducing 50% cell growth inhibition, but lower than the IC concentration
50
of rapamycin + TdR in Colo320TP1 cells, while 200 nM is a concentration causing complete growth
inhibition [Table 1]. In both cell lines, 20 nM rapamycin did not significantly affect the cell cycle
[Figure 1A], although the G1-phase was slightly increased after rapamycin exposure. TdR addition to
rapamycin slightly increased the S- and G2/M-phases in Colo320 cells, which might be related to activation
of TdR by thymidine kinase. In Colo320TP1 cells, addition of both TdR and TPI to rapamycin increased
accumulation of cells in the S- and G2/M-phase. At 200 nM rapamycin alone (data not shown), the effects
on cell cycle distribution in both Colo320 and Colo320TP1 cells were comparable to that at 20 nM. In
addition, the combinations showed a similar effect in Colo320 cells, but, in Colo320TP1 cells, the
combinations of 200 nM rapamycin with TPI and TdR increased the fraction of cells in S-phase to 20% and
25%, respectively, with a similar effect on the G2/M-phase (25%). In the triple combination of rapamycin,
TPI, and TdR, the effect on the S-phase was around 25%, but that on the G2/M-phase increased to 40%.

To determine whether the protective effect against rapamycin was mediated by a decreased cell death
induction, we analyzed the sub-G1-fraction of PI-stained cells at 20 nM rapamycin. As expected, the effect
of 20 nM rapamycin was limited and only present in Colo320 cells and absent in Colo320TP1 cells
[Figure 1B]. In Colo320 cells, the addition of TdR increased cell kill, but not significantly, while addition of
TPI reduced the cell death induction.