Increasing tumor cell apoptosis and tumor-infiltrating cells in
nasopharyngeal cancer cell line-derived spheroids upon IDX treatment
To mimic in vivo conditions, we examined the cytotoxicity of IDX
and cis on NPC cells using three-dimensional (3D) tumor cultures
(Supplementary Figure 3A). We monitored the effect of increasing
concentrations of IDX (Figure 5A, left panel) and cis (Figure 5A, right
panel) on apoptosis using fluorescent probes (non-toxic CellEvent
Caspase-3/7 Green and LysoTracker Deep Red) in combination with
bright-field microscopy to perform persistent real-time spheroid
imaging. The drug effect in the 3D “CellEvent,” was used to build a
dose-response curve to determine the IC50 values for cis
at 1.4 µM and IDX at 2.1 µM (Figure 5B). Overall, the
IC50 values were comparable between the spheroid-derived
cells in 3D and 2D conditions.
We next explored the interactions between tumor spheroids and PBMCs
through heterotypic cocultures. Spheroids generated from an NPC cell
line cocultured with PBMCs obtained from healthy donors that were
HLA-matched whenever possible. After coculture, we studied the cellular
composition within infiltrated tumor spheroid by mechanically separating
infiltrating cells (IN) and cells remaining in the medium (OUT) (Figure
5C). An increase in PBMCs infiltration in the tumor spheroids upon
treatment was detected as early as 2 hours, while the number of
infiltrated cells remained constant in the controls (DMSO) over 72 hours
of coculture. We found that IDX alone and combination treatment
significantly increased the influx of PBMC into the spheroids when
compared to cis alone. Combination treatment resulted in elevated
infiltrated PBMCs (mean 164.5±4.5) within the spheroids after 72 hours,
compared to cis alone (mean 53.0±6.0), representing a significant
(p=0.0149) 3.1-fold increase. Strikingly, PBMCs infiltrated in
cis-treated tumor spheroids started to decline after 24 hours of
incubation, where infiltrated PBMCs (mean 75.5±.5) within the control
spheroids (DMSO) after 72 hours was higher when compared to cis alone.
(1.4-fold increase; Figure 5D-E). Moreover, we also performed CellEvent
staining on the coculture to confirm the induction of apoptosis and
observed that spheroid infiltration was proportional to an active
apoptosis process in tumor cells (Figure 5D, F). In agreement with our
previous in vitro results (Figure 2D), the infiltrated PBMCs in
IDX2+cis1.4-treated tumor spheroids showed increased levels, albeit
modest, of CD107a compared to control (Supplementary Figure 3B).
We next hypothesized that IDX-conditioned tumor cells might activate
PBMCs that in turn, infiltrate the tumor under the influence of
chemokine gradients. Exposure of NPC cell lines to IDX resulting in the
expression of the T-cell chemokines CXCL8, 9 and 10 supports this
hypothesis (Figure 4G). To explore further, neutralizing antibodies
against one of the chemoattractants, CXCL10, were added to the tumor
spheroid cultures, which significantly reduced IDX-induced PBMC
migration, and also, to a lesser extent, in the combination treatment,
towards tumor spheroids. Collectively, these experiments refine our
previous conclusions that IDX not only arrest tumor growth, but also
induces the expression of T-cell chemoattractants, thereby enhancing
T-cell infiltration, possibly augmenting further tumor killing.