In vivo anti-tumor effect is associated with increased T
cells infiltrations
To evaluate possible antitumor effects of IDX on the growth of
nasopharyngeal NPC43+ve tumors in nude mice, test animals were treated
every-other-day with IDX and cis at a dosage of 10 and 20mg/kg and
5mg/kg weight/day, respectively. The growth curves demonstrated that
high combination-treatment (IDX20+cis5) significantly decreased tumor
growth, compared to the control group (Figure 7A, B). Although athymic
nude mice are grossly deficient in peripheral T cells, the number of
lymphocytes bearing T-cell markers (CD4, CD8) increases steadily with
age (Kennedy, Pierce & Lake, 1992).
Analogously, in our model, while CD14+ (monocytes) and
CD19+ (B cells) lymphocytes represented as the main
two subsets, CD4+ and CD8+ T cells
were also detectable in the circulation across
all experimental groups.
(Supplementary Figure 4A-C; before and after drug treatment for
CD4+ T cells was 19.47±5.877 and 18.01±5.411,
respectively; CD8+ T cells was 4.971±1.390 and
9.675±5.213, respectively). In relation to tumor-infiltrating
lymphocytes (TILs), we observed significantly higher number of
infiltrated CD8+ T cells within
CD19- population in the tumor of IDX20+Cis-treated
mice than that detected in the cis group (Figure 7C; 2.860±1.548 and
8.482±1.758, respectively). Importantly, the percentages of
CD8+ T cells, albeit at low levels among all live
TILs, was significantly higher in IDX20+cis-treated mice than that
observed in the DMSO group (Figure 7D; 1.668±0.5130 and 5.400±1.426,
respectively), which was confirmed by immunostaining (Supplementary
Figure 4D). However, there were no significant differences in the
percentage of infiltrating CD4+ T cells among all
groups.
Finally, we evaluated the clinical relevance of our signature findings
by investigating the expression of ENOX2 as a potential link to IDX’s
pronounced anti-cancer effect in NPC. From a publicly available
sequencing cohort GSE150430, we observed that malignant cells were the
main cell type expressed ENOX2 at single-cell resolution (Figure 7E-F),
this feature was further confirmed at protein level by histological
staining (Figure 7G). Another three publicly microarray GEO datasets
(GSE12452, GSE53819 and GSE34573) illustrated that ENOX2 gene was
present at all stages of cancer progression (I–IV) and expressed at a
significantly higher levels in tumors as compared to normal tissues,
which was associated with decreased DNA methylation as evident in the
GSE52068 dataset (Figure 7G). As such, the antitumor-responsive ENOX2
activity might be closely associated with NPC cancer, and thus IDX
treatment could potentially elicit a tumor-killing and immune-mediated
destruction in NPC tumor cells, while adding cis in combination could
further enhance this effect.
Discussion and
Conclusions
Previously, most studies with IDX focused on its anticancer properties
in association with cell cycle arrest and via a range of apoptotic
pathways in tumor cells, with little attention given to the drugs
immuno-oncology potential. Therefore, for the first time, we have
discovered that low-dose IDX acts as an immune stimulus that can induce
immunogenic tumor cell death by facilitating T-cell priming and
trafficking into tumors. Three experimental challenges were overcome to
allow this finding: determining
the appropriate concentration of IDX enabling an effective immune
response in addition to its anti-tumor effect, elucidating the functions
of IDX on tumor-lymphocyte interactions and finally, seeking the
activation pathways involved in fighting NPC disease. The
immune-stimulating properties of IDX might
therefore lay a new foundation
toward giving this unique molecule a second chance at contributing to
the future of cancer treatment.
We first determined the cytotoxicity of IDX against a range of NPC
cancer cell lines. Although IDX was found to be less cytotoxic than cis,
the relative restricted range of IC50 values reflected
its homogenous response across the various cancer cell lines.
Additionally, we confirmed previous findings showing that IDX further
sensitizes these cells to apoptosis in combination with a
chemotherapeutic drug. Our interest in determining how IDX impacts
immune function stems in part from results obtained during a past human
clinical study. In 2004,
phenoxodiol gained fast track
approval from the FDA and was tested in phase II/III trials in humans
(Mor, Fu & Alvero, 2006) against various
types of primary and metastatic cancers. However, lymphocytopenia was
associated with phenoxodiol use (Herst,
Davis, Neeson, Berridge & Ritchie, 2009), suggesting that dosing was
not fully optimised in these studies. Herein, we confirmed previous
findings (Georgaki et al., 2009) and
demonstrated that whilst IDX at high concentrations (4µM) could severely
inhibit T cell proliferation in vitro , if given at appropriate
dosages (1-2µM), IDX can act as an
immune-enhancer in addition to its
anticancer effects by allowing proliferating T cells to unleash their
cytotoxic ability. This finding further supports the application of this
agent in future therapeutic interventions aiming to activate
tumor-reactive lymphocytes.
As T cells are one of the main effectors of the antitumor immune
response and are often suppressed during the disease, our finding that
IDX positively modulates tumor-lymphocyte interactions is of
significance. In our 2D coculture assay, we observed that conditioning
of tumors with IDX, and to a larger extent upon combination treatment of
IDX and cis, drives the expansion, activation and migration of T-cells.
The effect is thought to be partially type I IFN/CXCL10-dependent in
this study. To date, the STING field has been focused on the concept
that it activates a predominant IRF3 response which has the potential to
help recruit dendritic cells and CD8+ T cells to the
TME, thereby promoting anti-tumor activities. While activation of STING
in the myeloid compartment clearly promotes antitumor effects, the role
of STING in tumor cells, and the role it may play in the antitumor
response is starting to gain attention. Herein, we observed that low
concentrations of IDX had no impact on endogenous STING expression,
whilst higher IDX concentrations in combination with cisplatin
down-regulated STING protein in NPC cell lines. Unexpectedly, a strong
reduction in IFNα which is responsible for the upregulation of T-helper
1 cell chemokines, such as CXCL10, was only observed following cis
treatment in NPC cells. Accordingly, the expressions of CXCL10, an
important chemokine for the homing of antigen-presenting cells and
trafficking of CD8+ T-cells, were attenuated. This
raised an important question as to why IDX is capable of sustaining IFNα
and drive up-regulation of CXCL10 expressions in NPC cells given that
the STING pathway was partially impaired. As such, we drew our attention
toward a recent discovery pointing at caspase cascade functions during
apoptosis to prevent dying cells from producing IFN
(White et al., 2014). Likewise, we
observed that IDX modestly induced the release of mitochondrial DNA
(mtDNA, data not shown), while cis was more likely to trigger cleaved
apoptotic caspase-7, and the latter fed back to inhibit IFNα production
by the dying cell. This finding highlights that, at least when it comes
to the mode of action, IDX and cis exert their functions through
different apoptotic downstream signaling pathways. Whether the elevation
in IFNα caused by loss of the caspase cascade leading to the induction
of chemoattractant is the result of upstream mitochondrial damage or an
alternative signaling mechanism, and whether perturbation in STING
activity might contribute to the immunological impact an apoptotic cell
has on the NPC cells by inducing mtDNA remain to be established.
We further developed heterotypic cocultures of NPC tumor spheroids with
T cells to study the infiltration, phenotype and function of
infiltrates. Critically, this study showed that activated/memory DP
CD4+CD8+ T cells, acquiring lower
expression of CD62L receptor and PD-1 marker, were able to infiltrate
spheroids and kill tumor cells, and that CXCL10 was involved in these
processes. Expression of CD45RO was the classical marker of primed
memory CD8 T cells, which could be further divided into
CCR7+CD62L+ central memory and
CCR7-CD62L- effector memory cells
based on the assumption that the presence of CD62L and CCR7 would
facilitate entry of central memory cells to secondary lymphoid tissue
from blood, whereas effector memory cells home to peripheral tissues. As
such, our results pointed at another potential mechanism through which
IDX could render the accumulation of exhausted PD1+TILs into tumor cells. Given the memory phenotype, cytotoxic profile,
and migratory capacity that we observed for DP T lymphocytes, we could
speculate that multiple immune activation circumstances exerted by IDX
could favor the presence of these cells in the periphery of, and
potentially inside, the TME. Further studies are deemed vital to better
understand the mechanisms controlling the co-expression of CD4 and CD8
lymphocytes and whether such co-expression confers any functional
advantage. We further confirmed our results in an in vivotherapeutic animal tumor model, confirming that this combination
strategy was capable of inducing
the accumulation of T cells within tumor sites and abrogate tumor
growth.
Collectively, the multifaceted
behavior of IDX provides the potential to synergize with, or complement,
a wide range of cancer therapies, and overcome resistance mechanisms. It
is therefore possible that IDX could condition or sensitize NPC patients
to chemotherapy by converting “cold” or “altered” tumors into
“hot” tumors, thereby increasing the chance of a durable response to
targeted therapy.