Introduction
Mucoepidermoid carcinoma (MEC) is the most prevalent
malignancy of the salivary glands, accounting for ~30% of all
salivary gland malignancies and 10–15% of all salivary gland tumors
(1,2). Tumor staging and grading serve as
notable prognostic indicators for patient survival. Notably, the
rate of cervical lymph node metastasis in MEC reaches as high as
36.9% globally, surpassing the regional lymph node metastasis rate
of 27.2% (3,4). Although surgery remains the primary
treatment modality for malignant MEC, neither surgery alone nor
chemotherapy alone yields favorable survival outcomes, leading to a
relatively low overall survival rate (5). For patients with advanced-stage
disease, distant metastasis or those who are ineligible for
surgery, effective therapeutic options are currently lacking.
Compared with conventional approaches, the research and application
of immunotherapy in various cancer types have markedly enhanced
treatment efficacy (6,7). Therapeutic strategies that induce
tumor cell death, elicit specific anti-tumor immune responses and
establish long-lasting immune memory are widely regarded as
superior choices for anti-tumor therapy (8–10).
As a central form of programmed cell death, the
initiation and execution of apoptosis are finely regulated by
various signals within the tumor microenvironment (TME). Extensive
research has demonstrated that cytokines and inflammatory mediators
secreted by immune effector cells can actively induce apoptosis in
target cells. This process is characterized by a series of
morphological and molecular alterations, such as the loss of
mitochondrial membrane potential, activation of the caspase cascade
and DNA fragmentation. These changes constitute the hallmarks of
induced apoptosis and provide the molecular foundation for
immune-mediated cell death (11).
Within the TME, cytokines such as tumor necrosis factor-α, released
by immune effectors, bind to death receptors such as CD95/Fas on
the target cell surface, directly activating extrinsic apoptotic
pathways and determining the fate of tumor cells (12). Such apoptosis triggered by immune
mediators is closely linked to immunogenic cell death (ICD). The
core feature of ICD is that dying tumor cells express or release a
series of damage-associated molecular patterns (DAMPs) (13–16),
including calreticulin (CRT), adenosine triphosphate (ATP), annexin
A1, type I interferons and high mobility group protein B1 (HMGB1).
These DAMPs activate the host immune system and promote the
establishment of anti-tumor immune responses (17,18).
Notably, CRT is a soluble protein localized in the endoplasmic
reticulum (ER) that translocates from the ER to the cell surface
via the Golgi apparatus under ER stress. This translocation is
typically mediated by protein kinase RNA-like ER kinase
(PERK)-induced phosphorylation of eukaryotic initiation factor 2α
(eIF2α). Subsequently, caspase-8-mediated BAP31 activation triggers
pro-apoptotic proteins such as BAX and BAK, leading to apoptosis.
Furthermore, C/EBP homologous protein (CHOP) serves as a notable
pro-apoptotic transcription factor during ER stress-induced
apoptosis, and the PERK/CHOP signaling pathway is recognized as a
classical axis in ER stress-related apoptotic research (19–22).
Various clinical drugs and treatment modalities have
been confirmed to induce ICD. Chemotherapeutic agents include
doxorubicin (DOX), oxaliplatin (OXA), cyclophosphamide and
paclitaxel (PTX) (23), while other
modalities encompass photodynamic therapy, photothermal therapy,
radiotherapy and sonodynamic therapy (24–27).
In this context, developing strategies that can effectively
eradicate MEC cells and induce ICD to elicit a systemic anti-tumor
immune response represents a research direction.
Thrombospondin-1 (TSP-1) is a multifunctional
protein that influences multiple aspects of tumor progression,
including angiogenesis, tumor growth, cell morphology, adhesion and
migration, serving as a key player in the TME (28). Miao et al (29) found that TSP-1 can inhibit the
growth of melanoma through its type 1 repeats, which exert
anti-angiogenic effects via both TGF-β-dependent and-independent
mechanisms. Additionally, Uscanga-Palomeque et al (30) developed the CD47 agonist peptide
PKHB1 based on the C-terminus of TSP-1 and discovered its ability
to induce ICD in T-cell acute lymphoblastic leukemia. However,
while most research on TSP-1 has focused on its anti-angiogenic and
growth-inhibitory properties, to the best of our knowledge, reports
regarding its role in inducing ICD are limited. Therefore, the
present study focuses on TSP-1 to explore its potential in
inhibiting growth and triggering ICD in MEC.
In our previous research, it was demonstrated that
TSP-1 inhibits the growth and migration of MEC cell lines and
promotes apoptosis, and subsequent investigations revealed that
TSP-1 increases the release of DAMPs and induces ICD in the MC-3
cell line (31). Given the
therapeutic advantages of TSP-1 in MC-3 cells, the present study
further investigated the molecular mechanism underlying
TSP-1-induced CRT translocation. Specifically, whether this process
is mediated by the PERK/CHOP signaling pathway. The present
research aimed to elucidate the mechanistic role of TSP-1 in MC-3
cells, thereby providing an experimental foundation for its future
therapeutic application in MEC.
Materials and methods
Cell culture
The MC-3 cell line was provided by the Department of
Biology, School of Stomatology, Air Force Medical University,
Xi'an, China (serial no. AFMU-BIO-003). The cells were maintained
in DMEM supplemented with 10% fetal bovine serum (both from Gibco;
Thermo Fisher Scientific, Inc.) and cultured in a humidified
incubator at 37°C with 5% CO2.
Flow cytometric detection of
surface-presented CRT
MC-3 cells were seeded into 6-well plates and
cultured overnight to allow for adherence. The cells were
subsequently assigned to five experimental groups: Isotype control,
blank control, TSP-1, TSP-1 + ISRIB (a PERK inhibitor) and TSP-1 +
CCT020312 (a PERK activator). TSP-1 (Novoprotein Scientific Inc.)
was used at a concentration of 0.1 µmol/l, ISRIB (cat. no.
548470-11-7; Medchemexpress) was used at 10 nmol/l and CCT020312
(cat. no. 324759-76-4; Medchemexpress) was used at 20 µmol/l.
Following treatment for either 4 or 72 h at 37°C, cells were
harvested and washed twice with ice-cold PBS. For surface staining,
the cells were incubated with a primary anti-CRT antibody (1:100;
cat. no. ET1068-60; HUABIO) or an isotype-matched control antibody
(1:100; cat. no. ab172730; Abcam) for 1 h at room temperature in
the dark. After washing, the cells were incubated with a
fluorescent secondary antibody (iFluor 647; 1:500; cat. no. HA1123;
HUABIO) under the same conditions for an additional 1 h. Finally,
the cells were resuspended in 0.3 ml of PBS and analyzed using the
APC channel of a flow cytometer (CytoFLEX S; Beckman Coulter).
Immunofluorescence
After treatment for the specified durations (4 and
72 h), MC-3 cells cultured on coverslips were pre-washed with PBS
and fixed with 4% paraformaldehyde at room temperature for 20 min.
Following the wash steps, all samples except those designated for
CRT detection were permeabilized with 0.5% Triton X-100 for 30 min
at room temperature. After blocking with goat serum (cat. no.
BL1092A; Biosharp Life Sciences) for 30 min, the cells were
incubated with primary antibodies (anti-PERK; cat. no. HA721510;
anti-CHOP; cat. no. HA722854; anti-CRT; cat. no. ET1068-60; HUABIO)
at a dilution of 1:200 at room temperature for 2 h in a humidified
incubator. Subsequently, the cells were incubated with
fluorescently labeled secondary antibodies (iFluor 594; 1:200; cat.
no. HA1122; HUABIO) for 1 h at room temperature in the dark,
followed by nuclear staining with DAPI at room temperature for 5
min. After mounting, images were captured using a fluorescence
microscope (EVOS M5000; Thermo Fisher Scientific, Inc.) at ×400
magnification.
Reverse transcription-quantitative
(RT-q)PCR
Total RNA was extracted from cells using the TRIzol
universal method (32) (DP424;
Tiangen Biotech Co., Ltd.). For cDNA synthesis, 1 µg of total RNA
was reverse-transcribed using a reverse transcription kit (Thermo
Fisher Scientific, Inc.). The reverse transcription reaction was
performed at 42°C for 15 min, followed by inactivation at 95°C for
5 min. qPCR was subsequently conducted using SYBR Green PCR Master
Mix (TaKaRa Biotechnology Co., Ltd.) on a CFX96 Real-Time PCR
Detection System (Bio-Rad, USA). The thermal cycling conditions
were as follows: Initial denaturation at 95°C for 3 min; 40 cycles
of denaturation at 95°C for 15 sec, annealing at 60°C for 30 sec,
and extension at 72°C for 30 sec; followed by melting curve
analysis from 65°C to 95°C with increments of 0.5°C per 5 sec to
confirm product specificity. The relative expression levels of
target genes were calculated using the 2−ΔΔCq method
(33), with GAPDH serving as the
endogenous reference gene. Currently, various molecular biology
techniques are widely employed to investigate genetic alterations
(34); in the present study, the
RT-qPCR method was selected to ensure high sensitivity and
accuracy. All primers were synthesized by Beijing Tsingke Biotech
Co., Ltd.. The primer sequences were as follows: PERK, forward
5′-AGCCAATTCAATGCCTGGGA-3′ and reverse 5′-ACTTCTCTGGTGGTGCTTCG-3′;
CHOP, forward 5′-TTAAAGATGAGCGGGTGGCA-3′ and reverse
5′-ACTTTCTTGAACACTCTCTCC-3′; CRT, forward
5′-AGCAGAACATCGACTGTGGG-3′ and reverse 5′-CCACAGATGTCGGGACCAAA-3′;
GAPDH forward 5′-GGAGTCCACTGGCGTCTTCA-3′ and reverse
5′-GTCATGAGTCCTTCCACGATACC-3′.
Western blotting (WB)
Total protein was extracted from MC-3 cells in each
group using RIPA lysis buffer (cat. no. AR0102S; (Wuhan Boster
Biological Technology, Ltd.). After determining protein
concentrations via the BCA assay, 25 µg of protein per lane were
separated by 12% SDS-PAGE (Beijing Solarbio Science &
Technology Co., Ltd.) and subsequently transferred onto PVDF
membranes (MilliporeSigma), following the protocol described by
Jurisic et al (35). The
membranes were blocked in TBS-Tween (TBST; 0.1% Tween 20)
containing 5% BSA (cat. no. ST023-50g; Beyotime Biotechnology) for
1 h at room temperature. Primary antibodies, including anti-PERK
(1:1,000; cat. no. MA8131; Abmart Pharmaceutical Technology Co.,
Ltd.), anti-CHOP (1:1,000; cat. no. WL00880; Wanleibio Co., Ltd.),
anti-CRT (1:800; cat. no. bs-5913R; BIOSS) and anti-β-actin
(1:5,000; cat. no. 66009-1-Ig; Proteintech Group Inc.), were
diluted in 5% BSA and incubated with the membranes overnight at
4°C. After washing with TBST, the membranes were incubated with
secondary antibodies (1:5,000; cat. nos. SA00001-1 and SA00001-2;
Proteintech Group Inc.) for 1 h at room temperature. Following
three additional washes with TBST, protein bands were visualized
using an ECL chemiluminescence detection kit (cat. no. BMU102;
Abbkine Scientific Co., Ltd.) in the dark and captured using an
imaging system.
Statistical methods
Statistical analysis was performed using GraphPad
Prism 10.0 software (Dotmatics). Data are presented as the mean ±
standard deviation. Comparisons between two groups were conducted
using the independent-samples t-test, whereas multiple group
comparisons were analyzed using one-way analysis of variance
(ANOVA) followed by Tukey's post hoc test. P<0.05 was considered
to indicate a statistically significant difference. All experiments
were repeated three times.
Results
TSP-1 upregulates cell surface CRT
expression in MC-3 cells
To investigate the mechanism by which TSP-1 induces
ICD in MC-3 cells, the surface expression of CRT was first
evaluated. Flow cytometry analysis revealed that following 72 h of
TSP-1 treatment, the mean fluorescence intensity of CRT on the MC-3
cell membrane was significantly increased (P<0.01) (Fig. 1). To further elucidate the
underlying mechanism, the regulatory role of ER stress and its
downstream PERK/CHOP signaling pathway in CRT translocation was
investigated. By employing the PERK inhibitor ISRIB and the
activator CCT020312, it was discovered that ISRIB partially
reversed the TSP-1-induced CRT expression (P<0.05), whereas
CCT020312 further synergistically enhanced this effect (P<0.05).
These results suggest that the PERK pathway is involved in the
process of TSP-1-induced surface translocation of CRT.
TSP-1 induces protein localization of
PERK, CHOP and CRT at 72 h
After treating MC-3 cells with TSP-1 in combination
with ISRIB or CCT020312 for 72 h, the expression levels of PERK,
CHOP and CRT were evaluated via immunofluorescence. The
fluorescence intensities reflected the cytoplasmic levels of PERK
and CHOP, as well as the surface expression of CRT, with higher
intensity indicating increased protein abundance. In the
TSP-1-treated group, PERK expression was significantly elevated
compared with the control group (P<0.01). The TSP-1 + ISRIB
group exhibited significantly reduced PERK expression compared with
the TSP-1 group (P<0.05). Notably, the combination of TSP-1 and
CCT020312 yielded the highest PERK fluorescence intensity
(P<0.01) (Fig. 2). The
expression patterns of CHOP and CRT across all groups were
consistent with those of PERK (Figs.
3 and 4). These findings
demonstrated that TSP-1 upregulated PERK/CHOP pathway components
and CRT expression, a process that is positively regulated by PERK
activity.
TSP-1 upregulates ER stress-related
genes
To clarify the impact of TSP-1 on the ER stress
pathway at the transcriptional level, RT-qPCR was performed to
evaluate the mRNA expression of PERK, CHOP and CRT in MC-3 cells
after 72 h of treatment. The results showed that the mRNA levels of
all three targets were significantly upregulated (P<0.05) with
TSP-1 treatment compared with control, consistent with the
previously observed protein fluorescence patterns (Fig. 5). Furthermore, analysis revealed
that inhibiting PERK resulted in a significant reduction in CHOP
expression (P<0.05; Fig. 5B),
whereas activating PERK led to a synchronized increase in both CHOP
and CRT expression (Fig. 5B and C;
P<0.05). These findings suggest that TSP-1 mediates CRT
expression in MC-3 cells via the PERK/CHOP signaling pathway.
Protein expression of CHOP and CRT
after 72 h treatment
WB analysis further confirmed these findings at the
protein level (Fig. 6). Compared
with the control group, TSP-1 treatment for 72 h significantly
increased the protein expression levels of CHOP and CRT
(P<0.01). Conversely, co-treatment with ISRIB reduced the
expression of these two proteins (P<0.05), whereas co-treatment
with CCT020312 further elevated their expression (P<0.05). These
results are consistent with the activation of ER stress and its
downstream signaling pathways, which are core features of ICD.
Studies have indicated that the PERK/CHOP pathway is a critical
signaling axis for ER stress-induced apoptosis and ICD, with
persistent high expression of CHOP serving as a major driver of
terminal apoptosis (36). In the
present study, TSP-1 effectively activated the PERK/CHOP pathway in
MC-3 cells, leading to the upregulation of the key ICD marker CRT.
This process was positively regulated by PERK kinase activity.
Collectively, these results establish the molecular mechanism by
which TSP-1 induces CRT expression via the PERK/CHOP pathway at the
protein level.
mRNA expression of PERK, CHOP and CRT
at 4 h
The mRNA expression levels of PERK, CHOP and CRT in
MC-3 cells were evaluated by RT-qPCR after 4 h of treatment
(Fig. 7). The results demonstrated
that no statistically significant differences in PERK, CHOP or CRT
mRNA levels were observed between the TSP-1 group and the control
group, suggesting that TSP-1 had a limited effect on MC-3 cells at
the 4 h time point. Furthermore, co-treatment with ISRIB did not
significantly downregulate the expression of these genes, whereas
co-treatment with CCT020312 resulted in significant upregulation
(P<0.05). These findings further support the conclusion that
PERK pathway activity is a factor regulating downstream gene
expression during the early stages of treatment.
Protein localization of PERK and CRT
at 4 h
At the early 4-h time point, immunofluorescence
results revealed that TSP-1 treatment alone failed to significantly
alter the protein expression levels of PERK and CRT (Fig. 8). Conversely, co-treatment with the
PERK inhibitor ISRIB significantly reduced the expression
(P<0.05) of both proteins, whereas co-treatment with CCT020312
significantly enhanced (P<0.05) them. These findings indicate
that, during the early stage, changes in CRT expression are
directly modulated by the activation status of PERK, and TSP-1
alone is insufficient to fully activate this signaling pathway.
Cell surface CRT expression following
TSP-1 treatment at 4 h
The results indicated that a 4-h treatment with
TSP-1 was insufficient to significantly enhance the cell surface
expression of CRT (Fig. 9),
suggesting that its effect may require a longer duration to
accumulate ER stress signals. Furthermore, co-treatment with the
PERK pathway activator CCT020312 significantly induced CRT
expression (P<0.01). This not only confirms that activation of
the PERK pathway is sufficient to drive CRT translocation but also
indicates that TSP-1 fails to effectively activate the PERK pathway
at this early stage. This stands in contrast to the significant
effects observed at 72 h, collectively revealing that the
regulation of CRT exposure by TSP-1 via the PERK pathway was a
time-dependent process.
Discussion
Based on preliminary findings (31) of the present group, the current
study utilized treatment with 0.1 µmol/l TSP-1 for 72 h as the
primary experimental condition to systematically explore the
potential molecular mechanisms by which TSP-1 induces ICD in human
MEC MC-3 cells. The present results demonstrated that TSP-1
significantly upregulated the expression of the ER stress-related
molecule PERK and its downstream transcription factor CHOP,
accompanied by elevated CRT expression. These findings suggest that
TSP-1 may regulate the immunogenic death of MC-3 cells by
activating the ER stress-PERK/CHOP signaling axis. Mechanistically,
this discovery aligns TSP-1 with classical ER stress-dependent ICD
inducers, such as DOX and OXA (37). However, TSP-1 may offer several
advantages, including lower systemic toxicity due to its endogenous
nature, the potential for targeted modulation of the tumor
microenvironment and a favorable safety profile for combination
therapy (28), highlighting its
potential therapeutic value in the treatment of MEC.
Cell death is not a solitary event but a dynamic
process characterized by distinct stages and morphological
features. Research indicates that apoptosis typically progresses
through early, middle and late stages, with morphological hallmarks
including cell shrinkage, chromatin condensation, nuclear
fragmentation, membrane blebbing and the formation of apoptotic
bodies, while membrane integrity is generally maintained during the
early and middle phases (38). By
contrast, necrosis is characterized by rapid cell swelling,
organelle rupture and loss of membrane integrity, leading to the
uncontrolled release of intracellular contents and a potent
inflammatory response (39).
Notably, ICD is not simply equivalent to apoptosis or necrosis;
rather, it is a specialized form of cell death that retains
apoptotic morphology while simultaneously releasing
immuno-stimulatory signals. The crux of ICD lies in specific
subcellular and molecular events, such as ER stress, CRT
translocation and the release of ATP and HMGB1, occurring in a
precise temporal sequence, thereby endowing the inherently
‘immuno-silent’ apoptotic process with immunogenicity (40). Therefore, understanding the stages
of cell death from both morphological and chronological
perspectives is essential for the correct interpretation of
ICD-related molecular events.
Previous studies have confirmed that the occurrence
of ICD is notably dependent on ER stress and the subsequent
unfolded protein response (UPR) (41,42).
As one of the three primary transmembrane sensors of the UPR, the
downstream PERK/eIF2α/CHOP signaling axis is considered the
classical pathway mediating the translocation of CRT from the ER
lumen to the cell membrane (43,44).
In the present study, it was observed that TSP-1 treatment
significantly upregulated PERK and CHOP expression in MC-3 cells.
Furthermore, pharmacological intervention in PERK signaling
correspondingly modulated the overall expression of CRT; the PERK
inhibitor ISRIB attenuated the TSP-1-induced upregulation of CHOP
and CRT, whereas the PERK activator CCT020312 further amplified
these effects. These results were consistent at both the
transcriptional and protein levels, supporting the pivotal role of
the PERK/CHOP pathway in the regulation of TSP-1-induced ER stress
markers.
Existing literature suggests that CRT dynamics
exhibit a distinct time-dependency. For instance, classical Type I
ICD inducers such as DOX and mitoxantrone can trigger CRT membrane
translocation within 2–4 h of treatment, whereas Type II ICD
inducers, such as photodynamic therapy, can induce CRT-related
changes as early as 1 h after treatment (45–47).
Based on these precedents, 4 h was selected as an early time point
to observe the initial molecular events of TSP-1-induced ICD.
However, at this interval, TSP-1 had not yet elicited significant
activation of the PERK/CHOP pathway or detectable changes in CRT
expression. Notably, even at this early stage, pharmacological
modulation of PERK signaling influenced CRT expression levels, as
evidenced by the finding that the PERK activator CCT020312
significantly upregulated CRT expression while the PERK inhibitor
ISRIB significantly downregulated it at 4 h, suggesting that CRT
regulation may be linked to PERK activity early on, though it had
not yet reached the threshold required to trigger significant
molecular shifts. These findings suggest that the molecular process
of TSP-1-induced ICD may possess ‘delayed-onset’ or ‘progressive
accumulation’ kinetic characteristics, distinguishing its mode of
action from that of classical small-molecule chemotherapeutics.
Furthermore, the possibility of PERK/CHOP-independent parallel or
alternative pathways, such as the IRE1α-XBP1 branch of the unfolded
protein response or the caspase-8/BAP31 pathway (19,22),
participating in initial CRT regulation cannot be excluded. Future
studies should incorporate finer time gradients and multi-pathway
intervention strategies to systematically elucidate the early
molecular networks and temporal features of TSP-1-induced ICD.
It is worth emphasizing that the role of the PERK
pathway in ICD may extend beyond CRT-related events. PERK-mediated
eIF2α phosphorylation globally regulates protein synthesis and
selectively induces transcription factors such as ATF4, thereby
coordinating the expression of various genes involved in stress,
apoptosis and immune modulation (48). Combined with our previous finding
that TSP-1 promotes the release of other DAMPs, such as ATP and
HMGB1 (31), the PERK/CHOP pathway
may serve as a ‘hub’ for coordinating the formation of the complete
immunogenic death phenotype.
From a clinical translation perspective, the present
results provide a theoretical foundation for the potential
application of TSP-1. First, the combination of TSP-1 with
chemotherapeutic agents such as PTX may enhance cytotoxicity while
simultaneously bolstering the ICD effect, potentially overcoming
chemoresistance in some cases of MEC. Furthermore, the DAMPs
released during TSP-1-induced ICD can promote dendritic cell
maturation and antigen presentation (31,49),
offering a strategy to remodel the tumor microenvironment from an
immunologically ‘cold’ state with low T cell infiltration to a
‘hot’ state with abundant activated T cells, and providing a
rationale for combination with immune checkpoint inhibitors.
Finally, the dual biological functions of TSP-1, encompassing both
anti-angiogenic potential via CD36 and immunomodulatory capacity
(50) may offer unique therapeutic
advantages in MEC, which is typically characterized by a rich blood
supply.
Despite these insights, the present study has
limitations. First, the present conclusions are primarily based on
in vitro cell models; whether TSP-1 can induce a functional
anti-tumor immune response in vivo requires validation in
animal models. Second, while PERK/CHOP axis was the primary focus,
the contributions of other UPR branches such as IRE1α-XBP1 and ATF6
(51) have not been systematically
evaluated. Furthermore, although immunofluorescence and flow
cytometry confirmed surface CRT expression, the temporal kinetics
of translocation and its impact on the strength and duration of the
in vivo immune response remain unclear. Finally, while
pharmacological agents demonstrated the involvement of the
PERK/CHOP pathway, a causal relationship has yet to be further
verified using genetic approaches.
Moving forward, future studies plan to construct a
humanized immune system MEC mouse model to systematically evaluate
the in vivo efficacy of TSP-1-induced ICD and the resulting
anti-tumor immune response. In vivo models will also be
combined with multi-pathway intervention strategies to resolve the
comprehensive molecular network of TSP-1-induced ICD and assess its
potential for combination therapy.
Acknowledgements
Not applicable.
Funding
The present study received funding from the Provincial Science
and Technology Plan Project of Sichuan Provincial Department of
Science and Technology (grant no. 2022SNZY001), Sichuan Provincial
Health Commission Science and Technology Project (Suitable
Technology Base) (grant no. 2022JDXM021), Clinical Research Fund
for Western Stomatology of the Chinese Stomatological Association
(grant no. CSA-W2023-0 3) and the General Program of Sichuan
Provincial Administration of Traditional Chinese Medicine (grant
no. 2024MS643).
Availability of data and materials
The data generated in the present study may be
requested from the corresponding author.
Authors' contributions
SY, SB and YZ designed the study and performed the
experiments. SY, SB, YZ and LG performed data analysis. SB and SY
drafted the manuscript and revised the manuscript. All authors read
and approved the final manuscript. SY and LG confirm the
authenticity of all the raw data.
Ethics approval and consent to
participate
Not applicable.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing
interests.
Glossary
Abbreviations
Abbreviations:
|
MEC
|
mucoepidermoid carcinoma
|
|
ICD
|
immuno-genic cell death
|
|
TSP-1
|
thrombospondin-1
|
|
CRT
|
calreticulin
|
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