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.