Extracellular vesicles derived from head and neck squamous cells carcinoma inhibit NLRP3 inflammasomes

Abstract

The content of tumor-derived extracellular vesicles (EVs) can regulate the tumor microenvironment and functionally acts in favor of cancer aggressiveness. To better elucidate the role of EVs in the interplay between immune system and tumor microenvironment, the purpose of this study was to analyze the effect of head and neck squamous cells carcinoma (HNSCC)-derived EVs on the modulation of inflammasomes-mediators of pyroptosis and secretion of inflammatory factors by macrophages. Our results showed that macrophages treated with the Vesicular Secretome Fraction (VSF) isolated from patient-derived HNSCC presented a reduction in the secretion of mature IL-1β and caspase-1 without affecting cell viability. An analysis of the protein content of HNSCC-derived VSF by antibody array revealed that some of the most expressed proteins share a correlation with Transforming Growth Factor-beta (TGF-β) activity. Since TGF-β is related to the inhibition of the NF-kB-related pathways, including those required for the priming phase of the inflammasomes, we sought to evalute the interference of the VSF in the induction of inflammasome components. In fact, HNSCC-derived VSF inhibited the induction of pro-IL-1β and pro-caspase-1 proteins and NLRP3 gene expression during the priming phase of inflammasome activation. Thus, our findings contribute to a better understanding of how tumor-derived EVs modulate inflammatory response by demonstrating their role in inhibiting NLRP3 inflammasomes.

Full text

Current Research in Immunology 2 (2021) 175–183
2590-2555/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Extracellular vesicles derived from head and neck squamous cells
carcinoma inhibit NLRP3 inammasomes
Luiza Zainotti Miguel Fahur Bottino
a
, Dorival Mendes Rodrigues-Junior
b
,
d
,
Ingrid Sancho de Farias
a
, Laura Migliari Branco
a
, N. Gopalakrishna Iyer
c
,
Gabriela Estrela de Albuquerque
d
, Andr´
e Luiz Vettore
d
, Karina Ramalho Bortoluci
a
,
*
a
Departamento de Farmacologia, Universidade Federal de S˜
ao Paulo (UNIFESP), SP, Brazil
b
Institute of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden
c
Cancer Therapeutics Research Laboratory, National Cancer Centre of Singapore, 11 Hospital Drive, Singapore 169610, Singapore
d
Departamento de Ciˆ
encias Biol´
ogicas, UNIFESP, SP, Brazil
ARTICLE INFO
Keywords:
NLRP3
Extracellular vesicles
Exosomes
Inammasomes
Head and neck cancer
TGF-β
ABSTRACT
The content of tumor-derived extracellular vesicles (EVs) can regulate the tumor microenvironment and func-
tionally acts in favor of cancer aggressiveness. To better elucidate the role of EVs in the interplay between
immune system and tumor microenvironment, the purpose of this study was to analyze the effect of head and
neck squamous cells carcinoma (HNSCC)-derived EVs on the modulation of inammasomes - mediators of
pyroptosis and secretion of inammatory factors by macrophages. Our results showed that macrophages treated
with the Vesicular Secretome Fraction (VSF) isolated from patient-derived HNSCC presented a reduction in the
secretion of mature IL-1β and caspase-1 without affecting cell viability. An analysis of the protein content of
HNSCC-derived VSF by antibody array revealed that some of the most expressed proteins share a correlation with
Transforming Growth Factor-beta (TGF-β) activity. Since TGF-β is related to the inhibition of the NF-kB-related
pathways, including those required for the priming phase of the inammasomes, we sought to evalute the
interference of the VSF in the induction of inammasome components. In fact, HNSCC-derived VSF inhibited the
induction of pro-IL-1β and pro-caspase-1 proteins and NLRP3 gene expression during the priming phase of
inammasome activation. Thus, our ndings contribute to a better understanding of how tumor-derived EVs
modulate inammatory response by demonstrating their role in inhibiting NLRP3 inammasomes.
1. Introduction
The interactions between cancer cells and their microenvironment
are crucial for the tumor fate (Quatromoni and Eruslanov, 2012;
Schiavoni et al., 2013). Cell to cell communication can be mediated
through growth factors, hormones, cytokines, adhesion molecules, or by
extracellular vesicles (EVs)(Marar et al., 2021). EVs are lipid bilayer
membrane vesicles secreted by a great diversity of cells which content
includes nucleic acids, lipids, and proteins. The content of EVs shed by
cancer cells can play signicant roles in the recipient cells, by inducing
tumor progression (Bebelman et al., 2018; Yang et al., 2011; Zhang
et al., 2015), metastatic spread (Bebelman et al., 2018; Karp and
Zwicker, 2014; Wu et al., 2016; Mcgarty, 2013), and drug resistance
(Rodrigues-Junior et al., 2019a). Due to their presence in body uids
and their key role in cell communication (Bebelman et al., 2018; Karp
and Zwicker, 2014; Mcgarty, 2013; Raposo and Stoorvogel, 2013), EVs
have been considered an emerging hallmark of cancer, with the poten-
tial to reveal new tumor biomarkers (Rodrigues-Junior et al., 2019b).
Additionally, tumor-derived EVs also participate in several
pro-tumorigenic strategies of immune evasion - including manipulation
of immune cells phenotype and effector mechanisms (Bebelman et al.,
2018; Karp and Zwicker, 2014; Mcgarty, 2013).
Inammation has long been associated with tumor development and
can be triggered by a variety of immune cells, including macrophages
(Coussens and Werb, 2002). A central mechanism to drive inammation
on those cells is the assemble and activation of multimeric cytosolic
complexes termed inammasomes (Karki et al., 2017). One of the
best-characterized inammasomes is the NLRP3 (Nucleotide-binding
* Corresponding author. Departamento de Farmacologia, Escola Paulista de Medicina - Universidade Federal de S˜
ao Paulo, R. Pedro de Toledo, 669, Vila Clem-
entino, S˜
ao Paulo, SP, 04039-032, Brazil.
E-mail address: karina.bortolucci@unifesp.br (K.R. Bortoluci).
Contents lists available at ScienceDirect
Current Research in Immunology
journal homepage: www.sciencedirect.com/journal/current-research-in-immunology
https://doi.org/10.1016/j.crimmu.2021.10.005
Received 18 June 2021; Received in revised form 15 October 2021; Accepted 20 October 2021

Current Research in Immunology 2 (2021) 175–183
176
Oligomerization Domain (NOD)-, Leucine-rich repeat–containing Re-
ceptors (NLRs) family Pyrin domain containing 3), that acts through the
adapter molecule ASC (Apoptosis-associated Speck-like protein con-
taining CARD (Caspase Activating and Recruitment Domain)) for the
recruitment of caspase-1 (Malik and Kanneganti, 2017). Complete
activation of NLRP3 inammasome requires two steps: (i) a priming
phase induced by an activating stimulus of the nuclear factor-kB (NF-kB)
transcription factor for the expression of inammasome components
such as pro-interleukin (IL)-1β, pro-caspase-1 and NLRP3, and (ii) a
agonist-triggered stimuli inducing the platform oligomerization and
caspase-1 cleavage and activation (Swanson et al., 2019). Upon its as-
sembly, inammasomes activate caspase-1, leading to the maturation of
pro-inammatory cytokines IL-1β and IL-18 and the cleavage of Gas-
dermin D, the effector of pyroptosis (Swanson et al., 2019; Shi et al.,
2015; Kayagaki et al., 2015).
The NLRP3 inammasome is activated by a wide variety of unrelated
stimuli. Its activation has already been reported during bacterial, pro-
tozoan, viral, and fungal infections, as well as in sterile inammation
mediated by endogenous signals and exposure to environmental irri-
tants. A unifying factor of NLRP3 activators is that all of them induce
cellular stress, suggesting that the NLRP3 is activated indirectly,
reecting the homeostatic nature of the cell (Swanson et al., 2019).
Multiple signals upstream NLRP3 activation have already been pro-
posed, including potassium (Mu˜
noz-Planillo et al., 2013) and chloride
ions (Tang et al., 2017) efux, lysosomal disruption (Hornung et al.,
2008), mitochondrial dysfunction (Groß et al., 2016; Iyer et al., 2013),
metabolic changes (Sanman et al., 2016; Moon et al., 2015), release of
oxidized DNA (Shimada et al., 2012), and trans-Golgi disassembly (Chen
and Chen, 2018). Nevertheless, the precise molecular mechanism
responsible for NLRP3 activation remains to be elucidated.
The interaction between macrophages and inammasomes with
tumor cells represents an often-controversial scenario due to the enor-
mous macrophages plasticity and the various effects mediated by
inammasomes, which can be related to the better or worse prognosis of
cancer patients (Kolb et al., 2014; Liss et al., 2001). The NLRP3
inammasome has been described to contribute to the progression of
gastric cancer, lung cancer, prostate cancer, breast cancer among others
(Karki et al., 2017; He et al., 2018; Faria et al., 2021). However,
inammasome activation was also demonstrated to be protective in the
context of melanoma and the production of IL-18 was critically involved
in the protection against colorectal cancer (Karki et al., 2017). More-
over, the IL-1β signaling axis has been shown to lead an effective
adaptive immune response against dying tumor cells, driving an effec-
tive immune response against transplantable tumor cells (Ghiringhelli
et al., 2009).
As aforementioned, EVs and inammasomes activation can have
signicant roles in the tumor microenvironment (Bebelman et al., 2018;
Karp and Zwicker, 2014; Mcgarty, 2013; Kolb et al., 2014) and both
interact in multiple scenarios, with EVs acting as positive or negative
regulators of inammasomes activation (Wang et al., 2019, 2020; Chen
et al., 2019; Huang et al., 2020; Dai et al., 2020; Wu et al., 2020; Zhang
et al., 2019; Kohli et al., 2016). Nevertheless, it is still unknown how
head and neck squamous cell carcinoma (HNSCC)- derived EVs can
affect macrophages effector mechanisms, especially those mediated by
inammasomes. Therefore, we decided to evaluate the modulation of
NLRP3 inammasome by HNSCC-derived EVs. Here we show that
treatment of macrophages with HNSCC-derived VSF - which is enriched
in EVs – induced the inhibition of the NLRP3 inammasome.
HNSCC-derived VSF downregulates pro-caspase-1, pro-IL-1β, and
NLRP3 expression, thus affecting the priming phase of NLRP3 inam-
masome activation.
2. Material and methods
2.1. Cell culture and animals
The primary HNSCC (NCC–HN19) and the HEK293T cell lines were
grown in a suitable culture medium (RPMI-1640, ThermoFisher) sup-
plemented with 10% fetal bovine serum (FBS, ThermoFisher), 1%
Penicillin/Streptomycin (Gibco) and maintained at 37 ◦C in the presence
of 5% CO
2
.
C57BL/6 mice with 6–8 weeks of age were provided by the Center for
Development of Experimental Models for Medicine and Biology
(CEDEME) from Federal University of S˜
ao Paulo (UNIFESP). All animals
were maintained in specic pathogen-free conditions in microisolators
with free access to water and feed. The development of this project was
approved by the UNIFESP Ethics Committee on Animal Use (CEUA,
#9957100217). The mice were inoculated intraperitoneally with 2 mL
of 1.5% potato starch (Sigma) diluted in 1X PBS for the enrichment of
the peritoneum by macrophages. After 96h, the mice were euthanized in
a closed chamber by Halothane inhalation (Cristalia). After euthanasia,
5 mL of sterile and cold PBS (1X) were injected into the peritoneal cavity
to obtain cell suspension. These cells were centrifuged at 500×g for 5
min and resuspended with 5 mL of complete RPMI1640 medium
(ThermoFisher) supplemented with 3% FBS (LGC).
Further, according to the assays, cells were seeded and incubated for
4h at 37 ◦C in an atmosphere containing 5% CO
2
, to enrich the macro-
phage population by adhesion. PBS was used to remove non-adherent
cells. All the stimuli used (LPS, nigericin and VSF treatment) were pre-
pared using OptMem medium (Life Technologies) to avoid FBS inter-
ference in the results.
2.2. Vesicular Secretome Fraction
The NCC-HN19 and HEK293T cells-derived VSF was obtained as
previously described (Rodrigues-Junior et al., 2019a). The cells were
incubated for 72h in phenol red-free DMEM (Gibco), supplemented with
5% Insulin-Transferrin-Selenium-Ethanolamine (ThermoFisher), 10 mM
of Non-Essential Amino Acids (ThermoFisher), 500
μ
g of broblast
growth factor-basic (ThermoFisher), 100 mM of Sodium Pyruvate and
55 mM of 2-Mercaptoethanol (ThermoFisher). After incubation, the
culture medium was collected and centrifuged at 250×g for 5 min to
remove dead cells. Then, the supernatant was ltered on a 0.22
μ
m lter
and further concentrated 20X by tangential ow ltration on a 50 kDa
Ultra-15 Centrifugal Filter (Millipore) by centrifugation at 1200×g. This
concentrated conditioned medium, denominated as Vesicular Secretome
Fraction (VSF) is enriched in functional EVs (Rodrigues-Junior et al.,
2019a; Lai et al., 2010).
Hence, in order to characterize the EVs present in the VSF, rst the
size distribution was measured using the NanoSight system (NTA 3.1
analytic software; Malvern Panalytical) for nanoparticle tracking anal-
ysis (NTA). The concentration, mean and modal average diameter of the
nanoparticles were used for statistical analysis. The EVs morphology
present in the VSF was visualized by transmission electron microscopy
(TEM). Briey, the VSF from NCC-HN19 and HEK293T were incubated
with 50
μ
l of uranyl-oxalate solution for 5 min and washed four times
with H2O. Images were acquired on a JEM 1200 EX II (JEOL) at 80 kV at
the UNIFESP Electron Microscopy Center (CEME) (Th´
ery et al., 2006).
Furthermore, the expression of EVs markers (ALIX and CD9) were also
idened as detailed below in the immunoblotting section.
2.3. Cell culture and stimulation
To assess the role of HNSCC-derived VSF on peritoneal macrophages,
the cells (5 ×10
5
cells/well) isolated from wild-type C57BL/6 mice were
treated with different VSF concentrations overnight at 37 ◦C. The con-
centrations used in the experiments were 1:5 dilution (corresponding to
one part of VSF diluted in four parts of medium), 1:20 dilution
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Current Research in Immunology 2 (2021) 175–183
177
(corresponding to one part of VSF for nineteen parts of medium) and
1:50 dilution (corresponding to one part of VSF for forty-nine parts of
medium). The concentration of nanoparticles/ml obtained by NTA was
used to estimate the concentration of nanoparticles in each treatment.
The VSF dilutions were performed using the phenol red-free medium
used to isolate EVs (EVs-free culture medium). Then, the macrophages
were treated with 500 ng/mL of Lipopolysaccharide (LPS) (InvivoGen)
for 3h and stimulated with 10
μ
M nigericin (InvivoGen) for 2h.
2.4. Cell viability
For cell viability evaluation, 3 ×10
5
macrophages were cultured in
the presence of 5 nM Sytox Green (ThermoFisher) which can be incor-
porated by dead cells. The images were obtained using the Incucyte
Zoom microscope and the frequency of Sytox Green positive cells was
analyzed using IncuCyte® ZOOM Live-Cell Analysis system software
(Essen BioScience). Cell incorporation of ethidium bromide and acridine
orange (Sigma-Aldrich) was performed to evaluate cell death by uo-
rescence microscopy. Cell viability was also evaluated through lactate
dehydrogenase (LDH) release, using comercial kits (Sigma-Aldrich),
according to manufacturer’s instructions.
2.5. Cytokine measurment
IL-1β and IL-6 quantication in macrophages culture supernatants (5
×10
5
) was performed by capture sandwich ELISA (eBioscience), ac-
cording to the manufacturer’s instructions. Absorbance from ELISA
plates were read with a Spectra Max M2e microplate reader (Molecular
Devices) using Soft-Max Pro Version 5.4 software (Molecular Devices).
2.6. Immunoblotting
Western Blotting assays were performed as previously described
(Buzzo et al., 2017). Antibodies anti-ALIX and anti-CD9 were purchased
from Santa Cruz Biotechnology; anti-IL-1β from R&D Systems and
anti-β-actin from Sigma-Aldrich. Caspase-1 antibody was kindly pro-
vided by Dr. Vishva Dixit from Genentech. Image J software (Image J,
NIH) was used to determine densitometric quantication, according to
β-actin expression.
2.7. Antibody array
The VSF isolated from NCC-HN19 cells were lysed with cell lysis
buffer (#K269; Biovision) and 100
μ
L of the protein lysate was analyzed
using the RayBio L-Series Human Antibody Array 1000 Glass Slide Kit
(#AAH-BLG-1000-4, RayBiotech), according to manufacturer’s in-
structions. After the immune reaction, the array was scanned and
normalized using Gene-Pix Pro 7 software (Molecular Devices), the
analysis were performed as previously described (Rodrigues-Junior
et al., 2019b). The Fold Change >0.5 was used as a cut-off to identify the
altered proteins in the samples, according to their expression. The spe-
cic and differentially expressed proteins present in the VSF isolated
from NCC-HN19 were functionally clustered using the STRING algo-
rithm (Search Tool for the Retrieval of Interacting Genes/Proteins - htt
ps://string-db.org/). STRING was also used to identify the predicted
biological networks associated with the VSF related proteins.
2.8. Real time PCR
NLRP3 expression was evaluated by real-time PCR. Macrophages
were treated with the VSF and LPS-stimulated as previously described.
Cellular RNA was isolated using TRIzol (ThermoFisher Scientic, Inc).
The concentration and purication of mRNA were analyzed by spec-
trophotometry (NanoDrop 2000c – ThermoFisher Scientic, Inc). cDNA
was generated from 300 ng of total RNA, using M-MLV Reverse Tran-
scriptase (Invitrogen), according to manufacturer’s instructions. cDNA
(50 ng) was homogenized with TaqMan Universal PCR Master Mix
(Applied BioSystems) and standardized assays of NLRP3 expression
(Mm_00840904_m1) were carried out. NLRP3 expression levels were
normalized using the expression level of beta-actin, as an endogenous
control (Mm02619580_g1). Reactions were conducted in the Real-time
PCR System QuantumStudio 6 Flex (Applied BioSystems).
2.9. Statistical analyzes
The statistical analyzes were carried out with the aid of Graphpad
Prism software (GraphPad Software Incorporation, version 6.0). The
statistic test used was two-way ANOVA, followed by Sidak’s or Bon-
ferroni’s test. The value of p <0.05 was considered statistically
signicant.
3. Results
3.1. VSF characterization
To characterize the EVs secreted by NCC-HN19 and HEK293T cell
lines, the size of nanoparticles presented in their respective VSF was rst
determined by NTA. Our analysis showed that NCC-HN19-derived VSF is
composed mainly of nanoparticles up to 200 nm. The average mean and
modal sizes of EVs from NCC-HN19 present in the VSF were 171.63 ±
30.52 nm and 126.80 nm ±21.98, respectively (Fig. 1A). HEK293T is a
non-cancerigenous cell line that was used in some analysis as a control.
Similar to NCC-HN19, HEK293T-derived VSF is composed mainly of
nanoparticles up to 200 nm. In concern of this cell line, the average
mean and modal sizes of EVs from HEK293T present in the VSF were
152.56 ±17.65 nm and 134.66 nm ±6.7, respectively (Fig. 1A). The
TEM analysis of the VSF derived from NCC-HN19 and HEK293T cell
lines revealed spheroidal and rounded structures, as expected (Fig. 1B).
Moreover, CD9 and ALIX, classical EVs markers, were found highly
expressed in the VSF isolated from NCC-HN19, conrming the enrich-
ment of EVs (Fig. 1C). Finally, the concentration of nanoparticles/ml
present in NCC-HN19-derived VSF was determined through NTA. Since
24,2 ×10
8
±4,48 ×10
8
particles/ml were found in the VSF (Fig. 1D), it
was possible to estimate the concentration of nanoparticles present in
each treatment. Thus, the treatment of 5 ×10
5
cells with 200
μ
l of 1:5
VSF-diluted medium (40
μ
l of concentrated VSF and 160
μ
l of pure me-
dium) contains around 0,968 ×10
8
nanoparticles (or ≅193,6 nano-
particles per cell). Same logic applies to 1:20 and 1:50 concentrations,
which correspond to ≅48,4 and ≅19,3 nanoparticles/cell, respectively.
3.2. HNSCC-VSF treatment reduces the secretion of mature forms of IL-1β
and caspase-1 by macrophages
Next, we sought to evaluate the VSF impact in the regulation of
inammasomes. To this end, peritoneal macrophages were cultured in
the presence or absence of HNSCC-derived VSF prior to the LPS priming.
After 3h of LPS priming, cells were stimulated with 5
μ
M or 10
μ
M of
nigericin for 2h to induce inammasome activation. VSF treatment led
to a reduction of IL-1β levels secreted by nigericin-stimulated macro-
phages (Fig. 2A) in a concentration-dependent manner (Fig. 2B). It is
possible to note that the lowest dilution of the VSF (1:5, corresponding
to 193 nanoparticles/cell) was more efcient in inhibiting inamma-
tion, but other dilutions (1:20 and 1:50) were also capable of inducing
inammasome inhibition. Based on this, the 1:20 dilution was stan-
dardized for further assays. The inammasome activation was also
assessed through the presence of the mature forms of IL-1β and caspase-
1 by WB. Our data showed a 60% reduction of mature IL-1β (p17) and a
total inhibition of caspase-1 (p20) in the culture supernatant of VSF-
treated macrophages in comparison to those treated in the absence of
the vesicles (Fig. 2C), demonstrating that HNSCC-derived VSF inhibits
inammasome activation. Preliminary data obtained by our group
showed that treating the peritoneal macrophages with the non-
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Current Research in Immunology 2 (2021) 175–183
178
cancerigenous cell line HEK293T-derived VSF did not induce a decrease
in IL-1β secretion, raising the possibility that the observed inhibition
could be restricted to tumor-derived VSF (Supplementary Fig. 1).
3.3. HNSCC-VSF does not interfere in cell viability
To further evaluate the impact of VSF treatment on the inhibition of
inammasome signaling and to discard the effect of VSF-induced cell
death on reduced cytokine production, we evaluated the cell viability by
the uptake of ethidium bromide (Et/Br) and the incorporation of
Fig. 1. Characterization of EVs present in the VSF. (A) The particle size analysis performed by NTA showed that VSF is composed mainly of small particles up to
200 nm. The graph represents the mean and mode sizes obtained in the three independent assays. The values obtained indicated the modal particle size was 126 nm,
while the mean size was 171 nm. (B) Transmission electron microscopy of EVs present in the VSF of NCC-HN19 and HEK293T, scales bar =50 nm. (C) Western
Blotting assay of VSF isolated from the NCC-HN19 cells for two different classical EVs markers (ALIX; 95 kDa, and CD9; 24 kDa). (D) The nanoparticles concentration
values obtained through NTA showed that the VSF is enriched with approximately 24,2 ×10
8
nanoparticles/ml.
Fig. 2. Treatment of macrophages
with VSF obtained from HNSCC cells
reduces IL-1β and caspase-1 secretion.
Peritoneal macrophages from wild-type
C57BL/6 mice were initially treated
overnight with VSF (1:5) (A) or VSF (1:5,
1:20, and 1:50) (B). Then the macro-
phages were primed with LPS (500 ng/
mL) for 3h and stimulated with nigericin
(10
μ
M) for 2h. Secretion of IL-1β was
analyzed by ELISA. For the three ana-
lyzes, **p <0.005 and ***p <0.0005
when compared to control (EVs-free
culture medium). Data are representative
of four independent experiments with n
=3. (C) Protein expression of the active
forms of IL-1β and caspase-1 in culture
supernatant were evaluated by WB.
β-actin was used as an endogenous con-
trol. Data are representative of three in-
dependent experiments with n =3.
Below each band, the numbers represent
the respective densitometry quantica-
tion normalized in relation to β-actin
expression. To determine the magniti-
tude of reduction or enhancement of
protein expression, positive controls
were dened as 1.0 (100%) and were
compared to VSF treatments.
L.Z.M.F. Bottino et al.

Current Research in Immunology 2 (2021) 175–183
179
SytoxGreen. Our data indicated that VSF treatment did not affect the
uptake of Et/Br compared to cells cultured in the presence of EVs-free
culture medium, even in the presence of nigericin which promotes loss
of membrane integrity and consequent cell death (Fig. 3A).
To further conrm the data obtained with the Et/Br incorporation
assay and to perform a real-time analysis of cell viability, we also
observed the incorporation of SytoxGreen by permeable cells with the
aid of the Incucyte Zoom microscope. This assay conrmed that treat-
ment with the VSF does not induce cell death by itself (Fig. 3B) or alters
the frequency of nigericin-induced pyroptosis (Fig. 3A). Finally, no
signicant levels of LDH release were observed in macrophages treated
in the presence or absence of HNSCC-derived VSF, rulling out the
cytotoxic potential of VSF treatment, in contrast to that caused by
nigericin (Fig. 3C). The data obtained by multiple viability assays
corroborate the hypothesis that the inhibition of inammasome acti-
vation by the VSF treatment occurs independently of cell death.
3.4. The protein content of the HNSCC-derived VSF is enriched in TGF-β
modulatory factors
Tumor-derived EVs carry a variety of cargo that functions in immune
activation and suppression (Marar et al., 2021). To evaluate the
HNSCC-derived VSF protein content, the total protein was tested against
a commercial 1000-immobilized antibody array. Interestingly, some of
the most expressed proteins share a common correlation with Trans-
forming Growth Factor-beta (TGFβ) activity (Table 1). Among the
top-15 most expressed proteins we could identify Thrombospondin-1
(THBS1), Transferrin receptor (TRFC), and Plasminogen (PLG). Note-
worthy, TGF-β is a pleiotropic cytokine with established
anti-inammatory effects that can act as an inhibitor of the NF-kB
pathway, downregulating pro-inammatory cytokine production (Rus-
cetti et al., 1992; Lee et al., 2011; Shiou et al., 2013; Cho et al., 2006).
Our previous ndings suggest that HNSCC derived EVs can carry TGFβ
isoforms (Rodrigues-Junior et al., 2019a), and the results of this study
allow us to speculate a potential new role for the protein content of
HNSCC-derived VSF during the observed inhibition of the inamma-
some complex.
The top-15 most expressed proteins detected in this screening step
Fig. 3. HNSCC-VSF does not induce cell death. For the viability assays, peritoneal macrophages from wild-type C57BL/6 mice were treated overnight with VSF or
EVs-free medium followed by priming with LPS (500 ng/mL) for 3h and stimulation with nigericin (10
μ
M) for 120 min (A and C) or 70 min (B). (A) Frequency of
positive cells for ethidium bromide (Et/Br) was determined by uorescence microscopy. (B) Incorporation of Sytox Green by permeable cells was determined in a
Incucyte Zoom microscope. (C) The LDH release was measured using commercial kits, according to the manufacturer’s instructions ***p <0,0001 in comparison to
the control. Data are representative of three independent experiments with n =3. (For interpretation of the references to color in this gure legend, the reader is
referred to the Web version of this article.)
Table 1
List of the 15 mostly expressed proteins in the Vesicular Secretome Fraction
isolated from NCC-HN19 cell line.
Gene
Name
Full Protein Name Swissprot Expression
Level
ApoC3 Apolipoprotein C-III P02656 4,8003
THBS1 Trombospondin-1 P07996 3,0147
TFRC Transferrin receptor protein 1 P02786 2,2542
PLG Plasminogen P00747 2,2003
GZMA Granzyme A P12544 1,4665
MMP-10 Stromelysin-2 P09238 1,4326
FN1 Fibronectin P02751 1,4228
CLU Clusterin P10909 1,3740
MMP-12 Macrophage metalloelastase P39900 1,3367
TGFBI Transforming growth factor-beta-
induced protein ig-h3
Q15582 1,2812
CXCL1 Growth-regulated alpha protein P09341 1,2385
CXCL9 C-X-C motif chemokine 9 Q07325 1,1936
IL6 Interleukin-6 P05231 1,1257
IL13 Interleukin-13 P35225 1,1010
TXNIP Thioredoxin-interacting protein Q9H3M7 1,0851
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180
were functionally clustered using the STRING algorithm to determine
their contribution to biological processes. The most represented bio-
logical processes associated with the VSF protein cargo are response to
stimulus, regulation of cellular processes, regulation of response to
stimulus, regulation of molecular function, and regulation of cell pop-
ulation proliferation (Fig. 4). So, it is possible to suggest that those
proteins might also be involved in the NLRP3 inammasome inhibition.
3.5. HNSCC-derived VSF treatment interferes with the induction of
NLRP3 inammasome components, thus impacting its priming phase
Activation of NLRP3 inammasome requires two distinct steps. The
rst step – the priming – is associated with two main functions: (i) the
upregulation of NLRP3 and other inammasome components and (ii)
the induction of post-translational modications of NLRP3 (Swanson
et al., 2019; Kelley et al., 2019). The transcriptional upregulation can be
induced through the recognition of diverse pathogen-associated mo-
lecular patterns (PAMPs) or damage-associated molecular patterns
(DAMPs) that engage pattern recognition receptors (PRRs) and lead to
NF-kB activation and gene transcription (Swanson et al., 2019). The
agonist-triggered second step induces the full inammasome assembly
and activation. The stimuli that activate NLRP3 are diverse and unre-
lated but converge in the fact that they all induce cellular stress (Malik
and Kanneganti, 2017; Swanson et al., 2019).
The activation of NF-kB pathway that characterizes the priming step
of inammasomes activation is widely associated with the upregulation
of pro-inammatory genes involved in several physiological processes.
To explore the VSF mechanism of action during inammasome inhibi-
tion we rst evaluated the secretion of IL-6, a pro-inammatory cyto-
kine. HNSCC-derived VSF led to diminished secretion of IL-6 (Fig. 5A) in
comparison to the macrophages cultured in the absence of EVs, thus
suggesting a possible downregulation of NF-kB signaling pathway by
TGF-β-related molecules.
For a deeper comprehension of VSF inuence on the modulation of
inammasome activation and signaling, we evaluated the impact of VSF
treatment on the induction of precursor forms. Intersentingly, the
presence of HNSCC derived-VSF induced a 55% and 75% reduction in
the pro-IL-1β and pro-caspase-1, respectively (Fig. 5B). Moreover, a
signicant reduction in the NLRP3 gene expression was found after the
treatment with HNSCC derived-VSF (Fig. 5C), supporting the idea that
HNSCC derived-VSF treatment affects the priming phase of NLRP3
inammasome activation.
4. Discussion
Numerous studies have identied EVs as an essential means of
intercellular communication that plays a role in physiological or bio-
logical important processes (Rashed et al., 2017). EVs are key players in
cancer progression, being able to induce both pro-and anti-tumor re-
sponses (Bebelman et al., 2018; Mcgarty, 2013; D¨
orsam et al., 2018). For
instance, cancer-derived EVs are carriers of tumor-antigens that can
mediate antitumor immunity (Bebelman et al., 2018), as reported by
Wolfers et al. (2001). It was also demonstrated that DC-derived EVs can
directly induce apoptosis in various tumor cell lines and promote the
proliferation of natural killer cells (Viaud et al., 2009). On the other
hand, EVs are also capable of impairing the cytotoxic activity of TCD8
+
lymphocytes (Liu et al., 2013) and increase the proliferation of regula-
tory T cells (Treg) (Wieckowski et al., 2009), “educating” the innate
immune components towards a pro-tumorigenic phenotype (Boyiadzis
and Whiteside, 2015) and resulting in an immunoprivileged status for
tumor cells (Bebelman et al., 2018; Karp and Zwicker, 2014; Raposo and
Stoorvogel, 2013; D¨
orsam et al., 2018).
Inammation is a classic hallmark of cancer. Although there are
numerous studies on the involvement of TLRs or interferon pathways in
the chronic inammation that affects tumor development, the role of
inammasomes is controversial (Kantono and Guo, 2017). The inter-
action between inammasomes and EVs is cell and context-dependent.
In the literature, EVs have already been associated with both activa-
tion and inhibition of inammasomes. For example, EVs from cyclic
stretch-exposed periodontal ligament cells inhibited NLRP3 activation
in macrophages through inhibition of NK-kB signaling patway (Wang
et al., 2019). Similarly, in a model of myocardial ischemia/reperfusion
(MI/R), it was demonstrated that M2 macrophage-derived EVs inhibited
NLRP3 activation through the downregulation of
thioredoxin-interacting protein (TXNIP) signaling pathway (Dai et al.,
2020).
On the other hand, a recent work from Lee et al. demonstrated that
EVs released by Staphylococcus aureus induced NLRP3 activation in THP-
1 cells and human macrophages (Wang et al., 2020). Also, the ability of
plasma-derived EVs from acute pancreatitis (AP) mice to trigger
NLRP3-dependent pyroptosis of alveolar macrophages was described in
2020 (Wu et al., 2020). Furthermore, it was demonstrated that the in-
jection of pregnant mice with endothelial cells-derived EVs lead to the
development of characteristic hallmarks of preeclampsia (PE), with
NLRP3 activation in trophoblast cells (Kohli et al., 2016).
Based on the above discussion, the precise role of EVs in inamma-
some modulation is not fully elucidated. Despite the involvement of
inammasomes and EVs in both genesis and control of tumor progres-
sion (Kolb et al., 2014; D¨
orsam et al., 2018), there were no records in the
literature about a possible interaction between those two mechanisms in
HNSCC, which is one of the most common cancers, with more than 633,
000 new cases diagnosed per year worldwide (Bray et al., 2018). As far
as we are concerned, this is the rst time that inammasome modulation
induced by tumor-derived VSF was explored in the context of HNSCC.
Our data demonstrated that HNSCC-derived EVs-enriched VSF treat-
ment resulted in inammasome inhibition, as indicated by the reduction
in mature caspase-1 and IL-1β secretion in response to nigericin, a classic
agonist of NLRP3 inammasome. Of importance, the reduction in the
secretion of mature forms of caspase-1 and IL-1β could not be assigned to
a cytotoxic effect of VSF since the treatment by itself did not impact cell
viability and did not affect the nigericin-induced cell death. Conversely,
HNSCC-derived VSF treatment seems to prevent the priming step of
NLRP3 inammasome activation.
The bioactive components of tumor-derived EVs play key roles in
mediating tumor microenvironment reprogramming (Xiao et al., 2019),
drug resistance (Samuel et al., 2017), promoting cell migration and in-
vasion (Li et al., 2016), stimulating tumor innervation (Madeo et al.,
2018) and metastasis (Sento et al., 2016; Li et al., 2018), and inducing
M1-like polarization of tumor-associated macrophages (Chen et al.,
Fig. 4. The majority of VSF protein content is involved with regulation of
cellular processes. Gene-Ontology based mostly enriched Biological Process
related to the fteen-mostly expressed VSF proteins identied through
STRING algorithm.
L.Z.M.F. Bottino et al.

Current Research in Immunology 2 (2021) 175–183
181
2018). Of note, out of 772 proteins that were identied in the
HNSCC-derived VSF by antibody array, the ve most abundant were
Apolipoprotein C3 (ApoC3), THBS1, TRFC, PLG, and Granzyme A
(GZMA) and the majority of them can be identied as TGF-β activity
facilitators. For instance, THBS1 is known as the major activator of
TGF-β (Crawford et al., 1998; Daniel et al., 2004). THBS1 interacts with
the N-terminal region of the latency-associated peptide (LAP), leading to
conformational changes that will release TGF-β from the latent form,
allowing the interaction with its receptor and all the subsequent
signaling events (Schultz-Cherry et al., 1995; Khalil, 1999). Also, a study
using neural crest cells showed that the ablation of TRFC was associated
with the development of craniofacial defects with concomitant sup-
pression of TGF-β signaling in mandibular tissues, suggesting that TRFC
may act as a facilitator for the activation of this pathway (Lei et al.,
2016). Similar to THBS1, there are some reports about the activation of
TGF-β by PLG and the components of its activation system as plasmin
and urokinase plasminogen activator (Yee et al., 1993; Chu and
Kawinski, 1998; Lyons et al., 1990).
TGF-β is known to mediate the inhibition of NF-kB pathway (Lee
et al., 2011; Shiou et al., 2013; Cho et al., 2006). As aforementioned, the
NF-kB pathway is required to the induction of precursor forms of IL-1β
and IL-18 thus being indispensable for the inammasomes activation.
Thus, the observed downregulation in the induction of pro-IL-1β,
pro-caspase-1 precursors, and NLRP3 gene expression could be
accounted by the TGF- β-related protein cargo found in tumor-derived
VSF. Of importance, TGF-β along with programmed cell death-ligand
1 (PD-L1) and Fas ligand (FasL) are important players in the immuno-
suppressive interactions mediated by tumor-derived EVs (Marar et al.,
2021). TGF-β carried by hypoxia-induced EVs is involved with NK cell
supression (Berchem et al., 2016). Also, depletion of TGF-β in leukemia
or colon cancer-derived EVs resulted in enhanced antitumor immune
response (Huang et al., 2017; Rossowska et al., 2019), highlighting the
relevance of this molecule in the tumor immunobiology.
Although the data shown here is promising, further studies are
needed to elucidate the precise mechanism by which EV contents
interfere with the activity of inammasomes. Even showing that the
HNSCC-derived VSF plays a key role in modulating immune responses, it
is impossible to claim each VSF molecule’s specic contribution for the
observed modulation yet. Unrevealing the protein content of the VSF
brings important insights into the immunomodulatory role of EVs.
Nevertheless, it is essential to keep in mind that EVs carry a diverse
range of molecules including sugars, lipids, and RNAs besides proteins.
Therefore, to better understand the whole contribution of vesicles
secreted by HNSCC cells in the modulation of inammasomes, these
other molecules should also be examined in the future. Moreover, the
use of a unique patient-derived HNSCC cell line, considering the cell
plasticity, makes it hard to predict if the observed induction of an
inammasome inhibitory behavior would be phenocopied by EVs from
distinct HNSCC cells.
Throughout the literature on tumor immunomodulation, it is already
well established that vesicles derived from tumor cells usually take
advantage of the plasticity of immune cells to shape the immunosup-
pressive population that best suits them, supporting the tumor devel-
opment. Thus, it is possible to assume that inhibiting inammasome
activation could be one of the approaches taken during this manipula-
tion. Considering that the activation of inammasomes represents a
highly inammatory response leading to the recruitment of several
immune cells it’s is possible to hypothesize that, in a co-evolutionary
manner, the inhibition of inammasomes caused by the HNSCC-EVs
could be a suitable manipulation of the microenvironment to maintain
tumor progression, evading host defense.
Because tumor cells are the major EVs producers, therapeutical
strategies based on those nanoparticles are increasingly being proposed.
The main step to conclusively succeed in this search is to elucidate the
mechanisms triggered upon EVs incorporation, mainly on immune cells.
In this context, understanding the impact of EVs in the activation and
regulation of inammasomes is of extreme importance for the devel-
opment of appropriate therapies for HNSCC treatment. In addition,
exploring the behavior, composition, and biogenesis of EVs in different
tumor models promotes the search for successful therapies based on
Fig. 5. HNSCC-derived VSF inhibits
the induction of inammasomes
components. Peritoneal macrophages
(5 ×10 (Yang et al., 2011) cells/well for
ELISA and 1 ×10 (Zhang et al., 2015) for
WB assays) from wild-type C57BL/6
mice were initially treated with the
HNSCC-derived VSF (1:20) overnight.
Then the macrophages were primed with
500 ng/mL LPS for 3h and stimulated
with 10
μ
M nigericin for 2h. (A) The
secretion of IL-6 was evaluated by ELISA.
(B) Protein expression of the precursor
forms of IL-1β and caspase-1 in cell ly-
sates were analyzed by WB. β-actin was
used as endogenous control. Below each
band, the numbers represent the respec-
tive densitometry quantication
normalized in relation to β-actin expres-
sion. To determine the magnititude of
reduction or enhancement of protein
expression, positive controls were
dened as 1.0 (100%) and were
compared to VSF treatments. (C) NLRP3
gene expression in VSF-treated macro-
phages in comparison to non-treated
cells was determined by RT-PCR. All
values were normalized using the β-actin
as an endogenous control. Data are
representative of independent experi-
ments with n =3. **p <0.005 compared
to the control.
L.Z.M.F. Bottino et al.

Current Research in Immunology 2 (2021) 175–183
182
their activity and structure.
5. Conclusions
Our results demonstrated that HNSCC-derived VSF inhibits the
priming phase of inammasome activation, which could represent an
escape mechanism during the complex interplay between tumor envi-
ronment and immune system.
Ethics statement
All experimental procedures involving mice were carried out in
accordance to the Brazilian National Law 11.794 (2008), the Guide for
the Care and Use of Laboratory Animals of the Brazilian National
Council of Animal Experimentation (CONCEA) and the ARRIVE guide-
lines. This study was approved by the Institutional Animal Care and Use
Committees (IACUC) of the Federal University of S˜
ao Paulo (UNIFESP)
under the protocol #9957100217.
Funding
This study was supported by grants from Fundaç˜
ao de Amparo `
a
Pesquisa do Estado de S˜
ao Paulo [FAPESP, grant numbers 2017/25942-
0 and 2015/09182-0 to K.R.B. and A.L.V.], Conselho Nacional de
Desenvolvimento Cientíco e Tecnol´
ogico [CNPq, grant number
402100/2016-6 to K.R.B.], the Instituto Nacional de Ciˆ
encia e Tecno-
logia de Vacinas (INCTV/CNPq). N.G.I. acknowledges the National
Medical Research Council of Singapore Clinician Scientist Award
(NMRC/CSA-INV/011/2016). The sponsors had no role in the study
design, collection and analysis, interpretation of the data, decision to
publish, or writing the manuscript. L.Z.M.F.B. and L.M.B. received fel-
lowships from FAPESP/CNPq. D.M.R.Jr. received fellowships from
Coordenaç˜
ao de Aperfeiçoamento de Pessoal de Nível Superior
(CAPES)/FAPESP.
CRediT authorship contribution statement
Luiza Zainotti Miguel Fahur Bottino: Investigation, Methodology,
Formal analysis, Validation, Writing – original draft, Visualization.
Dorival Mendes Rodrigues-Junior: Investigation, Methodology, Vali-
dation, Writing – review & editing. Ingrid Sancho de Farias: Investi-
gation, Methodology, Validation. Laura Migliari Branco: Investigation,
Methodology, Validation, Writing – review & editing. N. Gopa-
lakrishna Iyer: Methodology, Resources. Gabriela Estrela de Albu-
querque: Methodology, Validation. Andr´
e Luiz Vettore:
Conceptualization, Methodology, Resources, Writing – review & editing,
Visualization, Supervision, Project administration. Karina Ramalho
Bortoluci: Conceptualization, Methodology, Resources, Writing – re-
view & editing, Visualization, Supervision, Project administration,
Funding acquisition.
Declaration of competing interest
The authors declare that they have no known competing nancial
interests or personal relationships that could have appeared to inuence
the work reported in this paper.
Acknowledgements
We thank FAPESP, CNPq and CAPES for funding. The Graphical
Abstract was created with Biorender.com.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.crimmu.2021.10.005.
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