Research Article

Hepatic and Biliary Cancer

Patient pIgR-enriched extracellular vesicles drive cancer

stemness, tumorigenesis and metastasis in
hepatocellular carcinoma

Graphical abstract Authors

Sze Keong Tey, Samuel Wan Ki Wong,

Janice Yuen Tung Chan, ., Yi Gao,
Hepatocellular Anti-pIgR antibody Jing Ping Yun, Judy Wai Ping Yam
carcinoma (HCC)

Correspondence
Cyto D judyyam@pathology.hku.hk
Self-renewal
EV-pIgR
(J.W.P. Yam).
P PDK1
Lay summary
P
Akt1
Liver Tumorigenesis The World Health Organization es-
β-catenin timates that more than 1 million
P GSK3β
patients will die from liver cancer,
mostly hepatocellular carcinoma
(HCC), in 2030. Understanding the
Metastasis
underlying mechanism by which
HCC acquires aggressive attributes
is crucial to improving the diag-
nosis and treatment of patients.
Highlights Herein, we demonstrated that
 Elevated pIgR levels are found in circulating extracellular vesicles nanometer-sized extracellular vesi-
from patients with liver cancer. cles released by tumors promote
cancer stemness and tumorigen-
 EV-pIgR promotes cancer stemness and cancerous phenotypes in
esis. Within these oncogenic vesi-
recipient liver cancer cells.
cles, we identified a key component
 EV-pIgR promotes liver cancer cell aggressiveness by activating the that functions as a potent modu-
PDK1/Akt/GSK3b/b-catenin signaling axis. lator of cancer aggressiveness. By
inhibiting this functional compo-
 Combined treatment using sorafenib and anti-pIgR antibody atten-
nent of EVs using a neutralizing
uates growth of patient-derived xenografts in mice.
antibody, tumor growth was pro-
foundly attenuated in mice. This
hints at a potentially effective
therapeutic alternative for patients
with cancer.

https://doi.org/10.1016/j.jhep.2021.12.005
© 2021 The Author(s). Published by Elsevier B.V. on behalf of European Association for the Study of the Liver. This is an open access article
under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). J. Hepatol. 2022, 76, 883–895
Research Article
Hepatic and Biliary Cancer

Patient pIgR-enriched extracellular vesicles drive cancer stemness,

tumorigenesis and metastasis in hepatocellular carcinoma
Sze Keong Tey1,†, Samuel Wan Ki Wong1,†, Janice Yuen Tung Chan1, Xiaowen Mao1,
Tung Him Ng1, Cherlie Lot Sum Yeung1, Zoe Leung1, Hiu Ling Fung1, Alexander Hin Ning Tang1,
Danny Ka Ho Wong2,3, Lung-Yi Mak2,3, Man-Fung Yuen2,3, Chun-Fung Sin1, Irene Oi-Lin Ng1,3,
Stephanie Kwai Yee Ma3,4, Terence Kin Wah Lee5, Peihua Cao6,7, Kebo Zhong7, Yi Gao7,
Jing Ping Yun8, Judy Wai Ping Yam1,3,*
1
Department of Pathology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong; 2Department of Medicine, Li Ka Shing
Faculty of Medicine, The University of Hong Kong, Hong Kong; 3State Key Laboratory of Liver Research (The University of Hong Kong), Hong
Kong; 4School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong; 5Department of Applied
Biology and Chemical Technology, The Hong Kong Polytechnic University, Hong Kong; 6Clinical Research Center, Zhujiang Hospital, Southern
Medical University, Guangzhou, Guangdong, PR China; 7Department of Hepatobiliary Surgery II, Zhujiang Hospital, Southern Medical
University, Guangzhou, Guangdong, PR China; 8Department of Pathology, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong,
PR China

See Editorial, pages 768–770

Background & Aims: Extracellular vesicles (EVs) play a pivotal
role in connecting tumor cells with their local and distant mi-
croenvironments. Herein, we aimed to understand the role (on a
molecular basis) patient-derived EVs play in modulating cancer
stemness and tumorigenesis in the context of hepatocellular
carcinoma (HCC).
Methods: EVs from patient sera were isolated, quantified and
characterized. The EVs were vigorously tested, both in vitro and
in vivo, through various functional assays. Proteomic analysis was
performed to identify the functional components of EVs. The
presence and level of polymeric immunoglobulin receptor (pIgR)
in circulating EVs and tumor and non-tumorous tissues of pa-
tients with HCC were determined by ELISA, immunoblotting,
immunohistochemistry and quantitative PCR. The functional role
and underlying mechanism of EVs with enhanced pIgR expres-
sion were elucidated. Blockade of EV-pIgR with neutralizing
antibody was performed in nude mice implanted with patient-
derived tumor xenografts (PDTXs).
Results: Circulating EVs from patients with late-stage HCC (L-HCC)
had significantly elevated pIgR expression compared to the EVs
released by control individuals. The augmenting effect of L–HCC–
EVs on cancer stemness and tumorigenesis was hindered by an
anti-pIgR antibody. EVs enriched with pIgR consistently promoted
cancer stemness and cancerous phenotypes in recipient cells.
Mechanistically, EV-pIgR-induced cancer aggressiveness was
abrogated by Akt and b-catenin inhibitors, confirming that the role
of EV-pIgR depends on the activation of the PDK1/Akt/GSK3b/b-
Keywords: Liver cancer; Tumor microenvironment; Intercellular communication;
Proteomics; Neutralizing antibody.
Received 24 April 2021; received in revised form 24 November 2021; accepted 7
December 2021; available online 16 December 2021
* Corresponding author. Address: Department of Pathology, 7/F Block T, Queen Mary
Hospital, Pokfulam, Hong Kong, China; Tel.: 852-22552681, fax: 852-22185212.
E-mail address: judyyam@pathology.hku.hk (J.W.P. Yam).
†
Contributed equally
https://doi.org/10.1016/j.jhep.2021.12.005
catenin signaling axis. Furthermore, an anti-pIgR neutralizing
antibody attenuated tumor growth in mice implanted with PDTXs.
Conclusions: This study illustrates a previously unknown role of
EV-pIgR in regulating cancer stemness and aggressiveness: EV-
pIgR activates PDK1/Akt/GSK3b/b-catenin signaling cascades.
The blockade of the intercellular communication mediated by
EV-pIgR in the tumor microenvironment may provide a new
therapeutic strategy for patients with cancer.
Lay summary: The World Health Organization estimates that
more than 1 million patients will die from liver cancer, mostly
hepatocellular carcinoma (HCC), in 2030. Understanding the
underlying mechanism by which HCC acquires aggressive attri-
butes is crucial to improving the diagnosis and treatment of
patients. Herein, we demonstrated that nanometer-sized extra-
cellular vesicles released by tumors promote cancer stemness
and tumorigenesis. Within these oncogenic vesicles, we identi-
fied a key component that functions as a potent modulator of
cancer aggressiveness. By inhibiting this functional component
of EVs using a neutralizing antibody, tumor growth was pro-
foundly attenuated in mice. This hints at a potentially effective
therapeutic alternative for patients with cancer.
© 2021 The Author(s). Published by Elsevier B.V. on behalf of Euro-
pean Association for the Study of the Liver. This is an open access
article under the CC BY-NC-ND license (http://creativecommons.org/
licenses/by-nc-nd/4.0/).
Introduction
Hepatocellular carcinoma (HCC) accounts for the majority of
primary liver cancers and is currently the fourth leading cause of
cancer-related death worldwide.1 HCC is often diagnosed at an
advanced stage, thus precluding curative surgical resection.
Therapeutic options for advanced HCC are limited in availability
and efficacy. Therefore, understanding how HCC acquires
aggressive traits is important to revamp the current state of early
diagnosis and treatment.

Journal of Hepatology 2022 vol. 76 j 883–895

Research Article Hepatic and Biliary Cancer

A Migration assay Invasion assay

Huh7 PLC/PRF/5 Huh7 PLC/PRF/5

No. of migrated cells ( 102)

No. of migrated cells ( 102)

PBS PBS PBS PBS

No. of invaded cells ( 102)

No. of invaded cells ( 10)

10 6 3 20
**
8 ** 5 ** **
Normal Normal ** Normal Normal 15 ***
-EV ** ** -EV 4 -EV 2 ** -EV * **
6 *
3 10
Cirrhosis Cirrhosis Cirrhosis Cirrhosis
4
-EV -EV 2 -EV 1 -EV
5
2 1
E-HCC E-HCC E-HCC E-HCC
-EV 0 -EV 0 -EV 0 -EV 0
S V V V V S V V V V S V V V V S V V V V
L-HCC PB al-Esis-E C-E C-E L-HCC PB al-Esis-E C-E C-E L-HCC PB al-Esis-E C-E C-E L-HCC PB al-Esis-E C-E C-E
r m ho HC HC r m ho HC HC r m ho HC HC r m ho HC HC
-EV NoCirr E- L- -EV NoCirr E- L- -EV NoCirr E- L- -EV NoCirr E- L-

B 8
Huh7
8
PLC/PRF/5 C
PBS PBS 3 weeks 6 weeks
No. of colonies ( 10)

No. of colonies ( 10)

7 7 **
6 6
5 5
EVs
Normal 4 *** Normal 4 n.s.
(one injection/week;
-EV 3 n.s. -EV 3 15 μg/dose)
2 2
1 1
L-HCC 0 L-HCC 0
S V V S V V Orthotopic Lung
-EV PB al-E C-E -EV PB al-E C-E liver implantation metastasis
r m HC r m HC (MHCC97L tumor seed)
No L- No L-
D E
Bioluminescence signal ( 108)
**

Bioluminescence signal ( 104)

Bioluminescence signal ( 108)

PBS Normal-EV L-HCC-EV Liver Lung **

20 *** 8 ** 10 **
***
6 8
15
6
10 4
4
* n.s.
5 2 **
2

0 0 0
S V V S V V S V V S V V S V V
PB al-E CC-E PB al-E C-E PB al-E CC-E PB al-E C-E PB al-E CC-E
r m H r m HC r m - H r m HC r m - H
No L- No L- No L No L- No L
F G I PBS Normal-EV

Relative vascular area (Dextran+)

Bioluminescence signal (x109)

6
**
PBS

EVs (10 g) ** 5 ***

+ p53-/-;Myc mouse
hepatoblasts (1x105) 4
4
L-HCC-EV Normal-EV

3
14 days n.s.
n.s.
2 L-HCC-EV LectinDextranDAPI 2

1
0
Lung S V V S V V 0
PB al-E CC-E PB al-E CC-E S V V
colonization
rm -H rm L-H PB al-E C-E
of hepatoblasts No L No rm C
No L-H
H PBS Normal-EV L-HCC-EV

Fig. 1. Circulating EVs from patients with HCC promote HCC cell aggressiveness. (A) Migration and invasion assays of HCC cells pretreated with the indicated
EVs. (B) Colony formation assay of EV-treated cells. (C) Schematic diagram of the EV education model. (D) Bioluminescence imaging of animals at the end of the
experiment (n = 5). (E) Ex vivo bioluminescence imaging of liver and lung tissues. (F) Schematic diagram of the experimental metastasis assay. (G) Biolumi-
nescence imaging of excised lungs (left). The intensity of signals in the lung were plotted (middle). Image of lungs after fixation (right). Tumor nodules are
indicated by arrows. (H) H&E staining of lung tissues. Scale bar, 200 lm. (I) Analysis of lung vessel leakiness. Arrowhead indicates areas with diffused dextran.
Scale bar: 20 lm. Data are presented as the mean ± SEM. Student’s t test was used for 2 groups. *p <0.05; **p <0.01; ***p <0.001; n.s., not significant. Cirrhosis-EV,
EV from patients with HBV-related cirrhosis; E–HCC–EV, EV from patients with early HCC; EVs, extracellular vesicles; HCC, hepatocellular carcinoma; L–HCC–EVs,
EVs from patients with late HCC; Normal-EV, EVs from healthy individuals.

884 Journal of Hepatology 2022 vol. 76 j 883–895

A Huh7 30
** ***
B Huh7
100 ** 5

Number of spheroids
PBS Normal-EV L-HCC-EV PBS Normal-EV L-HCC-EV
* ***
79.5% 84.1% 91.5% 80 4

CD133
20

CD133 (%)

CD90 (%)
P1 n.s. 60 3
n.s.
10 n.s. 40 2

1.54% 1.28% 3.80%

20 1

CD90
0
P2 S V V S V V 0 0
PB al-E C-E PB al-E C-E S V V
PB al-E C-E
S V V
PB al-E C-E
orm -HC orm -HC rm -HC rm -HC
N L N L o
N L o
N L
P1 P2
PLC/PF/5 60 PLC/PRF/5
25 5
Number of spheroids

PBS Normal-EV L-HCC-EV *** PBS Normal-EV

V L-HCC-EV ***
50 ** ***
20 4
13.6% 16.1% 23.9%

CD133
40

CD133 (%)

CD90 (%)
n.s. n.s.
P1 30 15 3
n.s.
20 n.s. 10 2
10 5 1
2.75% 2.57% 4.06%

CD90
0
P2 S V V S V V 0 0
PB al-E C-E PB al-E C-E S V V S V V
r m -HC r m HC PB al-E C-E PB al-E C-E
No L No L- r m HC r m HC
No L- No L -
P1 P2

C Huh7 40 **
n.s. PLC/PRF/5 60 n.s.
**
Apoptotic cells (%)

Apoptotic cells (%)

PBS Normal-EV L-HCC-EV PBS Normal-EV L-HCC-EV
30
40
20
20
10
PI

PI

34.5% 34.7% 25.9% 41.2% 38.2% 29.3%

Annexin V 0 Annexin V 0
S V V S V V
PB al-E C-E PB al-E C-E
orm - HC orm -HC
N L N L
D PBS
E Primary implantation Secondary implantation
1,000 cells

120 120
Tumor-free survival (%)

Tumor-free survival (%)

Normal-EV
100 100
L-HCC-EV
80 80
60 n.s. 60 n.s.
PBS 40 * 40
5,000 cells

n.s.
PBS PBS * **
Normal-EV 20 Normal-EV 20 Normal-EV
L- HCC-EV L-HCC-EV
L-HCC-EV 0 0
0 10 20 30 40 50 60 0 5 10 15 20
Day Day
Primary implantation Secondary implantation

Fig. 2. Circulating EVs from patients with late-stage HCC enhance the cancer stemness properties of HCC cells. (A) Hepatosphere formation and serial
passage of HCC cells pretreated with the indicated EVs before plating. Representative photographs of spheroids formed after incubation. The histogram indicates
the number of spheres formed per 1,000 cells. P1 and P2 represent passage 1 and 2, respectively. (B) Flow cytometry analysis of CD90 and CD133 expression in
HCC cells treated with PBS or EVs. (C) Apoptosis assay in which EVs and sorafenib-treated cells were stained with PI and annexin V-FITC. Flow cytometry was
conducted to determine the percentage of apoptotic cells. (D) Image of tumors formed by 1,000 and 5,000 PLC/PRF/5 cells treated with or without EVs before
subcutaneous injection into NOD/SCID mice (n = 8). (E) Tumor-free survival curves of mice injected with 5,000 cells in the primary (left) and secondary (right)
implantation are shown. Data are presented as the mean ± SEM. Student’s t test was used for 2 groups, and a log-rank (Mantel-Cox) test was used for survival
curves. *p <0.05; **p <0.01; ***p <0.001; n.s., not significant. EVs, extracellular vesicles; HCC, hepatocellular carcinoma; L–HCC–EVs, EVs from patients with late
HCC; Normal-EV, EVs from healthy individuals; PI, propidium iodide.

Growing evidence has revealed that intercellular communi- cancer cell-derived EVs are critical messengers that connect tu-
cation is mediated not only by direct cellular contact and soluble mor cells with their local and distant microenvironments,
factors, but also by extracellular vesicles (EVs). EVs are orchestrating multiple systemic pathophysiological processes to
membrane-derived nanometer-sized vesicles that function by facilitate cancer progression.
transferring donor cell-derived bioactive molecules, including Despite the multifarious effect of EVs in cancer development,
lipids, nucleic acids and proteins, into recipient cells. The tumor the role of EVs associated with cancer stem cells (CSCs) remains
microenvironment is a heterogeneous and composite milieu that uncertain, and the function of EVs in hepatic CSCs is scarcely
supports cancer cell growth, survival and metastasis. Evidently, reported. In a chemically induced HCC rat model, EVs derived

Journal of Hepatology 2022 vol. 76 j 883–895 885

Research Article Hepatic and Biliary Cancer

A Expression of proteins of L-HCC-EV B V V

C 1 10
-4 D 2 10
-5

EV -E V -5
compared to Normal-EV 1 10
al- -EV osis CC-ECC-E ***

EV-pIgR level ( g/ g)
EV-pIgR level ( g/ g)
-6
m 5 10
r V h
Upregulated Downregulated No HB Cirr E-H L-H -5 -6
1 10 3 10
-6
IGHG1 ADIPOQ C5 n.s. n.s. * 1 10
-7
IGHG3 F2 APOB -6
8 10
-7
pIgR C4BPA ITIH4 75 pIgR 1 10 7 10
-7
6 10
IGLC6 HRG C3 5 10
-7

LGALS3BP FCN3 MASP1 -7 4 10

-7

1 10 -7
CP THBS1 3 10
-7
VWF FN1 2 10
-7
GAPDH 1 10
IGHA1 C4B -8
37 1 10 0
IGHV5 -51 TTR is Before After
al V os C CC
SERPINA1 C1S rm HB Cirrh HC
Fold difference >2-fold No E- L-H

E Normal-EV HBV-EV Cirrhosis-EV E-HCC-EV L-HCC-EV

50 nm 50 nm 50 nm 50 nm 50 nm

CD63 pIgR

F G H
Copy number of pIgR/HPRT

6 10-5 3
F/5 7L M3
EV-pIgR level ( g/ g)

*** PR CC9 CCL

HA E h7 C/
4 10-5 2 ** MI HL Hu PL MH MH
***
pIgR
75
2 10-5 1
*
**
50 α-tubulin
0 0
HA HLE uh7 RF/5 97L LM3 HA HLE uh7 RF/5 97L LM3
MI H /P CC C MI H /P CC C
C H HC C H HC
PL M M PL M M
I J K
Copy number of pIgR/100 HPRT

Expression of pIgR, log2 (T/NT)

4 1 105
** ** 15
EV-pIgR level (w/w)

1 104
3 10
1 103

2 1 102
5

1 101 0
1
1 100
-5
0 1 10-1 Upregulation, 56% (28/50 cases)
0 1 2 3 4 5 NT T -10 Downregulation, 20% (10/50 cases)

L
Week
Non-tumourous tissues Tumor
Score 0 Score 1 Score 2 Score 3 Score 0 Score 1 Score 2 Score 3

Fig. 3. pIgR-enriched circulating EVs are found in patients with HCC. (A) Proteins modulated by >2-fold in L–HCC–EVs when compared to Normal-EVs
analyzed from proteomic profiling are listed. (B) Immunoblot showing pIgR expression in the indicated EVs, with GAPDH as the loading standard. (C) ELISA
of pIgR levels in circulating EVs from control individuals (Normal, n = 36) and patients with HBV (HBV, n = 20), HBV-related cirrhosis (Cirrhosis, n = 9), early HCC
(E-HCC, n = 49) or late HCC (L-HCC, n = 22). (D) Determination of EV-pIgR in patients with HCC before and after surgery (n = 25). Late-stage cases are indicated
in red and early-stage cases are marked in black. (E) Representative electron micrograph of EVs subjected to immunogold labeling using anti-CD63 and anti-
pIgR antibodies followed by secondary antibodies conjugated to 5 and 10 nm gold particles, respectively. Scale bar: 50 nm. (F) The expression of pIgR in EVs
obtained from conditioned medium of cells was analyzed via ELISA. The expression of pIgR in normal liver cell line (blue) and non-metastatic (orange) and
metastatic (red) HCC cell lines was determined using qPCR (G) and western blotting (H). (I) pIgR levels in circulating EVs collected from mice implanted with
MHCC97L tumor seeds in the liver (n = 4). (J) pIgR mRNA expression in 50 paired T and NT liver tissues was determined by qPCR. (K) Relative fold change in pIgR
mRNA expression between T and NT liver tissues (n = 50). A fold difference >2 was considered to indicate upregulation or downregulation. (L) Representative

886 Journal of Hepatology 2022 vol. 76 j 883–895

from hepatic CSCs augmented tumor growth and metastasis.2 In
addition, EVs derived from CD90+ liver cancer cells modulated
endothelial cells to facilitate angiogenesis in HCC.3 The signifi-
cant impact of CSCs and EVs in HCC underscores the need for a
better understanding of the link between EVs and liver CSCs.
Polymeric immunoglobulin receptor (pIgR) is widely
expressed in mucosal epithelial cells and regulates transcytosis
of dimeric IgA and pentameric IgM, which are the first-line an-
tibodies against initial infection. Expression of pIgR is upregu-
lated by proinflammatory cytokines upon viral or bacterial
infection, thus bridging innate and adaptive immunity.4
Emerging findings have revealed the aberrant expression of
pIgR in cancerous tissues. However, the clinical relevance and
functions of pIgR in tumor cells remain uncertain. Intriguingly,
studies have yet to report on the functional role of pIgR when
expressed by tumor-derived EVs. For the first time, we identified
pIgR as a key molecule that confers oncogenic effects on circu-
lating EVs from patients with HCC. Herein, we uncover the role of
pIgR-enriched EVs in enhancing cancer stemness and aggres-
siveness, and provide insight into the clinical relevance of pIgR as
a promising biomarker and therapeutic target in HCC.
Materials and methods
Human samples
Serum samples were randomly collected from healthy donors
with non-liver disease backgrounds (as controls), individuals
with chronic HBV infection and individuals with liver disease
(cirrhosis, early HCC and late HCC) who had not received any
treatment. Information on serum donors is listed in Table S1. The
collection of serum samples was executed at Queen Mary Hos-
pital, Hong Kong and Zhujiang Hospital, Guangzhou, China with
informed consent from all donors. Procedure approval was ob-
tained from the Institutional Review Board of The University of
Hong Kong/Hospital Authority Hong Kong West Cluster (HKU/HA
HKW IRB) and Zhujiang Hospital of Southern Medical University.
All experiments involving human samples were handled in
accordance with relevant ethical regulations.
Statistical analysis
The readings of all assays were calculated as the mean ± SEM.
Student’s t test, ANOVA and log-rank (Mantel-Cox) tests per-
formed using Prism software (version 8.0.1, GraphPad) were used
for statistical analysis. A p value of less than 0.05 was considered
statistically significant.
For further details regarding the materials and methods used,
please refer to the CTAT table and supplementary information.
Results
HCC patient-derived EVs promote aggressive phenotypes in
HCC cells
EVs derived from HCC cell lines have been shown to play pivotal
roles in enhancing HCC aggressiveness5,6; however, the physio-
logical relevance of their functional effect in patients remains
ambiguous. Circulating EVs were collected from healthy
=
images of pIgR expression with intensity scores (0-3). Scale bar, 100 lm. (M) The number of cases with each intensity score (left) and the H-scores of pIgR
expression (right) were plotted (n = 143). (N) Representative cases with pIgR overexpression. Scale bar, 100 lm. Alteration of pIgR expression in paired samples are
depicted in the pie chart. Data are presented as the mean ± SEM. Student’s t test for 2 groups was used. *p <0.05; **p <0.01; ***p <0.001; n.s., not significant.
Cirrhosis-EV, EV from patients with HBV-related cirrhosis; E–HCC–EV, EV from patients with early HCC; EVs, extracellular vesicles; HCC, hepatocellular carci-
noma; L–HCC–EVs, EVs from patients with late HCC; Normal-EV, EVs from healthy individuals; NT, non-tumorous; pIgR, polymeric immunoglobulin receptor;
T, tumor.
Journal of Hepatology 2022 vol. 76 j 883–895
individuals (Normal-EVs), and from patients with chronic HBV
infection, HBV-related cirrhosis, early HCC (E–HCC–EVs) or late
HCC (L–HCC–EVs) for functional analyses. Based on the Minimal
Information for Studies of Extracellular Vesicles (MISEV) guide-
lines,7 the isolated EVs were validated by their expression of
small EV markers, size and morphology (Fig. S1A-S1C). Notably,
the greatest amount of EVs was obtained from late-stage patients
compared to other serum donors (Fig. S1D).
EVs from patients with HCC but not Normal-EVs increased the
growth and motility of Huh7 and PLC/PRF/5 cells (Fig. S2A-S2D).
Compared to Normal-, Cirrhosis- and E–HCC–EVs, L–HCC–EVs
exhibited the highest potency in promoting cell migration,
invasiveness and colony formation (Fig. 1A-B) and in enhancing
tumor development (Fig. S2E). In an orthotopic liver implanta-
tion model, repeated injection of L–HCC–EVs into mice resulted
in enhanced liver tumor formation and distant lung metastasis
(Fig. 1C-E). A consistent effect of L–HCC–EVs in promoting
metastasis was observed in an experimental metastasis assay
involving coinjection of EVs and murine p53-/-;Myc-transduced
hepatoblasts (Fig. 1F-H). L–HCC–EVs were shown to reduce
expression of the tight junction protein VE-cadherin in human
umbilical vein endothelial cells (Fig. S3) and enhance endothelial
permeability in mice (Fig. 1I). These findings suggest that the
promotion of metastasis could be ascribed to leakiness in the
pulmonary vasculature induced by L–HCC–EVs, thus facilitating
extravasation and colonization of tumor cells in the lungs.
HCC patient-derived EVs promote cancer stemness in
HCC cells
In addition to modulating the cancer phenotypes of tumor cells,
L–HCC–EVs increased the cancer stemness properties of HCC
cells. HCC cells treated with L–HCC–EVs but not Normal-EVs
showed a marked increase in the ability to form tumorspheres
in a spheroid formation assay (Fig. 2A) and enhanced expression
of the CSC markers CD90 and CD133 (Fig. 2B). L–HCC–EVs also
increased the expression of CD24, CD47 and EpCAM in Huh7 cells
but not in PLC/PRF/5 cells (Fig. S4). In addition, L–HCC–EVs
reduced the sensitivity of cells to sorafenib (Fig. 2C). Limiting
dilution analysis performed in NOD/SCID mice showed that HCC
cells treated with L–HCC–EVs displayed a significant enhance-
ment in tumorigenicity, with an increased tumor incidence,
shorter tumor latency and increased estimated tumor-initiating
cell frequency compared to cells treated with PBS or Normal-
EVs (Table S2). The self-renewal ability of L–HCC–EV-treated
cells was further expedited as revealed by serial transplantation
of primary xenografts into secondary mouse recipients (Fig. 2D).
L–HCC–EV-treated cells showed significantly worse tumor-free
survival than cells treated with PBS or Normal-EVs after both
primary and secondary implantation (Fig. 2E).
pIgR is upregulated in circulating EVs of patients with HCC
To ascertain differences in biological activities between EVs of
healthy controls and those of patients, their proteomic profiles
were compared. Among candidates that were modulated by at
887
Research Article
M 150 400
***
300
Number of cases
IHC H-score
100
200
100
50
0
0 -100
NT T NT T
Score 0
Score 1
Score 2
Score 3
Fig. 3.
least 2-fold in L–HCC–EVs compared to Normal-EVs, pIgR –
which has been implicated in HCC8,9 – was chosen for further
investigation (Fig. 3A). Upregulation of pIgR in E–HCC– and L–
HCC–EVs was corroborated by immunoblotting (Fig. 3B). A sig-
nificant increase in pIgR was consistently detected in a cohort of
patients’ circulating EVs (Fig. 3C). The level of EV-pIgR was
reduced in 72% (18/25) of patients after surgery (Fig. 3D).
Immunogold labeling revealed the presence of pIgR on the sur-
face of CD63+ circulating EVs (Fig. 3E). A higher level of pIgR was
detected in EVs from metastatic HCC cells than in EVs from
normal liver and non-metastatic cells (Fig. 3F). Similar pIgR
expression profiles were also observed at both the transcript
(Fig. 3G) and protein (Fig. 3H) level in HCC cell lines. In mice
implanted with tumor seeds derived from metastatic
MHCC97L cells, the level of circulating EV-pIgR progressively
increased during tumor development (Fig. 3I). These data indi-
cate that tumors were likely the major source of EV-pIgR and
that its level could be an indicator of tumor development. Similar
to EV-pIgR, overall pIgR mRNA expression was significantly
upregulated in tumorous tissues compared to non-tumorous
tissues (Fig. 3J). Notably, the pIgR transcript was upregulated in
56% (28/50) of patients with HCC (Fig. 3K). In a tissue microarray
comprising 143 paired HCC samples, strong (score 3) and mod-
erate (score 2) positive staining of pIgR was detected in 91.6%
(131/143) of tumorous tissues, whereas 62.9% (90/143) of non-
tumorous tissues showed weak positive (score 1) or negative
staining (score 0) (Fig. 3L-M). The H-score revealed a signifi-
cantly higher immunoreactivity of pIgR in tumorous tissue
(Fig. 3M). Frequent overexpression of pIgR was found in 77.6%
(111/143) of cases (Fig. 3N).
pIgR is functionally responsible for the tumor-promoting
effect of HCC patient-derived EVs
Due to the presence of pIgR on the surface of EVs (Fig. 3E), we
examined whether neutralizing antibody against pIgR was able
to block the tumor-promoting effect of L–HCC–EVs. Indeed,
cotreatment with anti-pIgR antibody blocked the effect of L–
HCC–EVs on colony formation, migration and invasion of PLC/
PRF/5 cells (Fig. 4A,B). Furthermore, the metastasis of murine
p53-/-;Myc-transduced hepatoblasts induced by L–HCC–EVs was
significantly hindered by coinjection with anti-pIgR antibody in
the experimental metastasis assay (Fig. 4C-F). Anti-pIgR antibody
also abrogated L–HCC–EVs’ ability to promote the in vitro self-
renewal and the percentage of CD90-and CD133-expressing
888
Hepatic and Biliary Cancer
N NT
Case # 595
T NT
Case # 611
T
4
28
111
Total = 143
T < NT
T = NT
T > NT
PLC/PRF/5 cells (Fig. 4G,H), as well as to reduce the sensitivity
of HCC cells to sorafenib (Fig. 4I). The augmenting activity of L–
HCC–EVs was also observed in Huh7 cells and was neutralized by
anti-pIgR antibody (Fig. S5). These findings suggest that circu-
lating EVs from patients with HCC augment the cancer stemness
properties of cells through the functions of pIgR.
To identity the functional role of pIgR in EVs, pIgR was stably
expressed in Huh7 cells using CRISPR synergistic activation medi-
ators (SAM-pIgR) and an EV-targeting expression vector (XP-pIgR)
(Fig. S6A-S6B). EVs collected from the conditioned medium of SAM-
pIgR and XP-pIgR cells showed upregulated pIgR compared to EVs
collected from the respective control SAM-CTL and XP-CTL cells.
The purity and integrity of the isolated EVs were validated (Fig. S6A,
S6C,D). As revealed by various in vitro functional assays, SAM-pIgR-
EVs and XP-pIgR-EVs enhanced the cancer properties of PLC/PRF/5
and Huh7 cells (Fig. 5A-C; Fig. S7A-S7C). This augmenting effect of
SAM-pIgR-EVs and XP-pIgR-EVs in promoting Huh7 cell migration
and invasion was abolished by the addition of anti-pIgR antibody
(Fig. S7D-S7E). XP-pIgR-EVs were capable of promoting the
metastasis of murine p53-/-; Myc-transduced hepatoblasts (Fig. 5D-
F). Furthermore, XP-pIgR-EVs promoted in vitro self-renewal,
increased the CD90-and CD133-expressing PLC/PRF/5 population
(Fig. 5G-H) and suppressed the sensitivity of HCC cells to sorafenib
(Fig. 5I). Serial transplantation assays showed that PLC/PRF/5 cells
treated with XP-pIgR-EVs displayed a significant enhancement in
tumorigenicity, with an increased tumor incidence, shorter tumor
latency and increased estimated tumor-initiating cell frequency,
compared to cells treated with PBS or XP-CTL-EVs (Table S3). The
self-renewal ability of XP-pIgR-EV-treated cells was further
increased in secondary implants compared to primary xenografts
(Fig. 5J). XP-pIgR-EV-treated cells showed worse tumor-free sur-
vival than cells treated with PBS or XP-CTL-EVs (Fig. 5K). Taken
together, these findings demonstrate that the functional capacity of
EV-pIgR was consistent with the clinical results showing an upre-
gulation of EV-pIgR in patients with HCC. Importantly, these data
suggest a previously undiscovered role of EV-enriched pIgR in
promoting HCC aggressiveness, which prompted us to investigate
the underlying molecular basis of this observation.
EV-pIgR activates b-catenin signaling through PDK1/Akt/
GSK3b signaling
The expression of genes that are involved in cancer stemness and
drug resistance regulation was analyzed in PLC/PRF/5 and Huh7
cells treated with Normal-EVs and L–HCC–EVs (Fig. 6A). L–HCC–
Journal of Hepatology 2022 vol. 76 j 883–895
A B

No. of migrated cells ( 10)

No. of invaded cells ( 10)

PBS + IgG
g 8 PBS + IgG 50 PBS + IgG 10

No. of colonies ( 10)

7 *** *** * ** *** **
40 8
6
5 30 6
4
L-HCC-EV L-HCC-EV 3 L-HCC-EV L-HCC-EV
20 L-HCC-EV L-HCC-EV
4
+ IgG + pIgR Ab 2 + IgG + pIgR Ab + IgG + pIgR Ab
10 2
1
0 0 0
b b b
IgG IgG A IgG IgG A IgG IgG A
S+ V+ IgR S+ V+ IgR S+ V+ IgR
PB C-E V+p PB C-E V+p PB C-E V+p
H C -E H C -E H C -E
L- CC L- C C L- CC
L-H L-H L- H
C D E

Bioluminescence signal (x1010)

Bioluminescence signal (x109)
PBS 2.0 * ** 2.0
L-HCC-EVs (10 g) +IgG
+ p53-/-;Myc mouse ** ***
hepatoblasts (1x105) 1.5 n.s. 1.5 n.s.
+ pIgR Ab (10 g) PBS
14 days +pIgR Ab 1.0 1.0

0.5 0.5
L-HCC-EV
+IgG
0 0
Lung IgG R A +Ig
b G Ab b b
IgG R Ab +IgG R A IgG R Ab +IgG R A
colonization S+ Ig V IgR S+ Ig V Ig S+ Ig V Ig
of hepatoblasts L-HCC-EV PB S+p C-E V+p PB S+p C-E V+p PB S+p C-E V+p
C -E C C
+pIgR Ab PB L - H PB L-H C-
E PB L-H C-
E
CC
L -H HC HC
L- L-
F PBS+IgG L-HCC-EV+IgG
G PBS L-HCC-EV L-HCC-EV 50
*** ***

Number of spheroids
+ IgG + IgG + pIgR Ab ** **
40

30
P1
20

PBS+pIgR Ab L-HCC-EV+pIgR Ab 10

0
b b
IgG IgG A IgG IgG A
P2 S+ V+ IgR S+ V+ IgR
PB C-E V+p PB C-E V+p
C -E C -E
L- H CC L-H CC
L- H L- H
P1 P2

H PBS
+ IgG
L-HCC-EV
+ IgG
L-HCC-EV
+ pIgR Ab
20 8 ** * I 40
Apoptotic cells (%)

11.0% 15.9% 8.0%

*** *** *** ** PBS L-HCC-EV L-HCC-EV
15 6 30 + IgG + IgG + pIgR Ab
CD133 (%)
CD133

CD90 (%)

10 4 20
30.38% 24.41% 29.73%
5 2 10
2.26% 5.86% 3.54%
PI
CD90

0 0 0
b b b
IgG IgG A IgG IgG A IgG IgG A Annexin V
S+ V+ IgR S+ V+ IgR S+ V+ IgR
PB C-E V+p PB C-E V+p PB C-E V+p
C -E C -E C -E
L-H CC L-H CC L-H CC
H H H
L- L- L-

Fig. 4. Anti-pIgR antibody hinders the ability of EVs from patients with HCC to enhance the cancer and cancer stemness properties of HCC cells. PLC/PRF/5
cells treated with L–HCC–EVs in the presence of control IgG or anti-pIgR antibody were subjected to colony formation (A), migration and invasion (B) assays. (C)
Schematic diagram of the experimental metastasis assay. (D) Bioluminescence imaging of animals at the end of experiment and quantification of the intensity of
signals (n = 5). (E) Ex vivo bioluminescence imaging of lungs. (F) Representative image of H&E staining of lung tissues. Scale bar, 200 lm. Cells treated with L–
HCC–EVs and with control IgG or anti-pIgR antibody were seeded for spheroid formation assays (G) and flow cytometry analysis to detect CD90 and CD133
expression (H). Histogram indicating the number of spheres formed per 1,000 cells. P1 and P2 represent passage 1 and 2, respectively. The percentage of
positively stained CD90 and CD133 cells was plotted. (I) Cells pretreated as described and treated with sorafenib were stained with PI and annexin V-FITC. Flow
cytometry analysis was conducted to determine the percentage of apoptotic cells. Data are presented as the mean ± SEM. Student’s t test for 2 groups was used. *p
<0.05; **p <0.01; ***p <0.001. EVs, extracellular vesicles; HCC, hepatocellular carcinoma; L–HCC–EVs, EVs from patients with late HCC; PI, propidium iodide; pIgR,
polymeric immunoglobulin receptor.

EVs exerted the highest potency in activating b-catenin expres- were further confirmed by immunofluorescence microscopy
sion, highlighting its potential in prompting malignant behav- which revealed a notable increase in nuclear translocation of b-
iors. The TOP/FOP reporter system demonstrated the activation catenin in HCC cells following treatment with EVs with enhanced
of b-catenin activity by L–HCC–EVs (Fig. 6B). These observations pIgR expression (Fig. 6C,D; Fig. S8A,B). Notably, the nuclear

Journal of Hepatology 2022 vol. 76 j 883–895 889

Research Article Hepatic and Biliary Cancer

A B

No. of migrated cells (x102)

No. of migrated cells (x102)
4 8 ** 2.5 ** 4
** *

colonies (x102)

No. of colonies (x10)

7 * **
PBS ** PBS n.s. PBS 2.0 n.s.
PBS
3 *
3 n.s. 6

No. of colonies
5 n.s.
1.5
2 4 2
SAM-CTL 1.0 XP-CTL
SAM-CTL
S XP -CTL 3 -EV -EV
-EV 1 -EV 2 1
0.5
1
N

0 0 0 0
SAM-pIgR XP-pIgR

XP TL S
-p -EV

V
SA -CT BS

XP TL S

SA -CT BS
V

V
-p EV

-p -EV

-p EV

-C PB

-E
-C PB
-E

-E

-E
SAM-pIgR
S X pIgR
XP- -EV -EV
M P

M P

R
M L-

M L-
R

Ig
-EV
EV Ig -EV
EV

Ig

Ig

XP
XP
SA

SA
C D

Bioluminescence signal ( 108)

*** XP-CTL XP-pIgR *
*
No. of invaded cells (x102)

No. of invaded cells (x10)

1.0 6 PBS -EV -EV 20 *
** *
PBS 0.8 PBS 5
15
4
0.6 ** n.s.
3 10
SAM-CTL 0.4 XP-CTL
-EV -EV 2
5 n.s.
0.2 1
0 0 0
SAM-pIgR XP-pIgR

S
SA -CT BS

XP TL S
-p EV

V
V

-p -EV

V
-C PB

XP PB

-E

-E
-E

-E
-EV -EV
M P
M L-

R
R

TL

Ig
Ig

Ig

-C

-p
XP

XP
SA

E F G
Bioluminescence signal ( 109)

XP-CTL XP-pIgR XP-CTL XP-pIgR **

8
*** PBS -EV -EV PBS -EV -EV 60 60 **
*** ** **

Number of spheroid

Number of spheroid
50 50

6 n.s.
40 40
P1 n.s.
30 30
4
n.s. 20 20

2 10 10

P2 0 0
0

XP TL S

XP TL S
V

V
-p -EV

-p -EV
-C PB

-C PB
-E

-E
S

S
V
V

V
PB

XP PB
-E
-E

R
XP L-E

-E

Ig

Ig
TL

R
T
Ig

Ig
C

-C

XP

XP
-p

-p
-
XP

XP

H
P1 P2
PBS XP-CTL-EV XP-pIgR-EV 4 *** 5 25
** ** **
1.96% 2.29% 3.22% *
I ***

Apoptotic cells (%)

4 20
CD90

3 n.s. PBS XP-CTL-EV XP-pIgR-EV

CD133 (%)
CD90 (%)

3 15
2
2 n.s. 10
1.11% 1.47% 3.72% 1 22.64% 19.90% 13.98%
1 5
PI
CD133

0 0 0
S V V S V V Annexin V
PB TL-E R-E PB TL-E R-E S V V
PB TL-E R-E
- C -pIg - C -pIg C - pIg
XP XP XP XP -
XP XP
J PBS K Primary implantation Secondary implantation
1,000 cells

120 120
Tumor-free survival (%)

Tumor-free survival (%)

XP-CTL-EV
100 100
XP-pIgR-EV
80 80
60 n.s. 60 n.s.
PBS 40 40
5,000 cells

PBS ** ** PBS * **
XP-CTL-EV 20 XP-CTL-EV 20 XP-CTL-EV
XP-pIgR-EV XP-pIgR-EV
XP-pIgR-EV 0 0
0 5 10 15 20 25 30 0 2 4 6 8 10 12
Day Day
Primary implantation Secondary implantation

Fig. 5. pIgR-enriched EVs exert enhanced promoting effects on HCC cancer and cancer stemness properties. Colony formation (A), migration (B) and invasion
(C) assays were performed on PLC/PRF/5 cells treated with the indicated EVs from Huh7 cells and corresponding control EVs. (D) Luciferase-labeled murine
p53-/-;Myc hepatoblasts were coinjected with the indicated EVs via the tail vein. Bioluminescence imaging of animals was performed 14 days post injection, and
the luciferase signals were quantified. (E) Bioluminescence imaging of excised lungs. (F) Representative image of H&E staining of lung tissues. Scale bar, 200 lm.
Cells treated with the indicated EVs were subjected to hepatosphere formation (G), flow cytometry analysis of the markers CD90 and CD133 (H) and apoptosis
assays (I). Histogram indicating the number of spheres formed per 1,000 cells. P1 and P2 represent passage 1 and 2, respectively. The percentages of positively
stained CD90+ and CD133+ cells and apoptotic cells were analyzed. (J) Image of tumors formed by PLC/PRF/5 cells pretreated with the indicated EVs in 2 rounds of
implantation (n = 8). (K) Tumor-free survival curves of mice injected with 5,000 cells in the primary and secondary implantation are shown. Data are presented as
the mean ± SEM. Student’s t test was used for 2 groups, and a log-rank (Mantel-Cox) test was used for the survival curve. *p <0.05; **p <0.01; ***p <0.001; n.s., not
significant. EVs, extracellular vesicles; HCC, hepatocellular carcinoma; pIgR, polymeric immunoglobulin receptor.

890 Journal of Hepatology 2022 vol. 76 j 883–895

A 2.5
PBS ***
2.5
PBS
***
Normal-EV Normal-EV
2.0 2.0
Late HCC-EV Late HCC-EV

Fold change
Fold change

1.5 * * * 1.5
* ** * * * ** * ***
1.0 1.0

0.5 0.5

0 0
B1 BCC1 BCG2 DHA1 tenin MI1 ANOG ESTIN TCH1 CT4 B1 C1 BCG2 DHA1 atenin MI1 ANOG ESTIN TCH1 CT4
ABC A A AL β-c
a B N N NO O ABC ABC A AL β-c B N N NO O

PLC/PRF/5 Huh7

B C 200 200
D PLC/PRF/5
150 150 PBS XP-CTL-EV
*

Mean fluorescent intensity

Mean fluorescent intensity
*** *** *** ***
***
Luciferase activity

Luciferase activity

n.s. 150 150

100 n.s.
100
100 100
n.s.
n.s.
50 50
50 50

0 0 0 0
S V V D S V V D
S V
PB al-E C-E
V S V
PB al-E C-E
V PB L-E R-E yto PB L-E R-E yto XP-pIgR-EV XP-pIgR-EV+CytoD
T C T C
rm C rm C -C P-pIg EV+ -C P-pIg EV+
No L-H No L-H P
X X R- P
X X R-
g g
PLC/PRF/5 Huh7 -p I -p I
XP XP
PLC/PRF/5 Huh7

E V V V +C
yto
D
V V V +C
yto
D

-E -E -E -E -E -E
TL pIgR pIgR TL pIgR pIgR
S -C - - S -C - -
PB XP XP XP PB XP XP XP

p-PDK1
50 Huh7
PBS XP-CTL-EV
PDK1
50

p-Akt
50

Akt
50
50
XP-pIgR-EV XP-pIgR-EV+CytoD
p-GSK3β

50
GSK3β

50
β-actin
37
PLC/PRF/5 Huh7

Fig. 6. pIgR-enriched EVs activate b-catenin signaling through the PDK1/Akt/GSK3b axis in HCC cells. (A) Expression of genes regulating cancer stemness and/
or drug resistance in HCC cells treated with the indicated EVs was quantified by qPCR. Expression levels are expressed as the fold change compared to the PBS
group. (B) The activity of b-catenin in HCC cells treated with the indicated EVs was analyzed using a TOP/FOP reporter assay. (C) HCC cells treated with EVs
derived from stable XP-CTL and XP-pIgR cells in the absence or presence of CytoD were stained with anti-b-catenin antibody and DAPI. Scale bar, 20 lm. His-
togram indicating the mean fluorescence intensity of the average nuclear b-catenin fluorescence captured by confocal microscopy (n = 10). (D) Representative
fluorescence images. (E) Immunoblotting of PDK1, p-PDK1 (S241), Akt, p-Akt (S473), GSK3b, and p-GSK3b (S9) expression in PLC/PRF/5 cells treated with the
indicated EVs; b-actin was used as the loading standard. Data are presented as the mean ± SEM. Student’s t test was used for 2 groups was used. *p <0.05; **p
<0.01; ***p <0.001; n.s., not significant. CytoD, cytochalasin D; EVs, extracellular vesicles; HCC, hepatocellular carcinoma; L–HCC–EVs, EVs from patients with late
HCC; Normal-EV, EVs from healthy individuals; XP, expression vector.

translocation of b-catenin activated by L–HCC–EVs was sup- was required. In PLC/PRF/5 cells, the phosphorylation of Akt and
pressed by treatment with an endocytosis inhibitor, cytochalasin GSK3b induced by XP-pIgR-EV was diminished by the Akt in-
D, indicating that EVs were taken up by cells via endocytosis. hibitor IV and the b-catenin inhibitor PRI-724 (Fig. 7A). Func-
Phosphorylation of PDK1 leads to activation of Akt, which tionally, treatment with these inhibitors abrogated the
subsequently phosphorylates GSK3b, leading to stabilization and promoting effect of XP-pIgR-EVs on HCC cell growth and motility
nuclear translocation of b-catenin. b-catenin signaling has been (Fig. 7B-D). Moreover, the effect of XP-pIgR-EVs on enhancing
reported to regulate tumorigenesis, cancer stemness and che- the in vitro self-renewal ability of PLC/PRF/5 cells, the percentage
moresistance.10 XP-pIgR-EVs increased PDK1, Akt and GSK3b of CD90+ and CD133+ cells (Fig. 7E-F) and the sensitivity of cells
phosphorylation (Fig. 6E; Fig. S8C), and this activation was to sorafenib (Fig. 7G) was diminished by cotreatment with the
blocked by cytochalasin D, suggesting that cellular uptake of EVs inhibitors. These findings demonstrate that pIgR-enriched EVs

Journal of Hepatology 2022 vol. 76 j 883–895 891

Research Article Hepatic and Biliary Cancer

V
h I 24
A +
SO MSO t In I-7
DM +D +Ak +PR
V -EV -EV -EV
B C
-E
TL gR gR gR
-C -pI -pI -pI
XP XP XP XP XP-CTL-EV XP-pIgR-EV XP-CTL-EV XP-pIgR-EV

Number of migrated cells ( 102)

+DMSO +DMSO 10 +DMSO +DMSO 5 **
DMSO
pAkt (S473)
73) * DMSO

Number of colonies ( 10)

Akt Inh IV * **
50
8 ** ** 4 Akt Inh IV
PRI -724
Akt PRI -724
50 6 3
50
pGSK3β (S9) XP-pIgR-EV XP-pIgR-EV 4 XP-pIgR-EV XP-pIgR-EV 2
+Akt Inh IV +PRI-724 +Akt Inh IV +PRI-724
50
2 1
GSK3β
50 0 0
β-actin XP-CTL XP-pIgR XP-CTL XP-pIgR
37 -EV -EV -EV -EV

D XP-CTL-EV XP-pIgR-EV E XP-CT

XP-CTL-EV XP-pIgR-EV XP-pIgR-EV XP-pIgR-EV 6 *** 8
+DMSO +DMSO * +DMS
+DMSO +DMSO +Akt Inh IV +PRI-724

Number of spheroid ( 10)

Number of spheroid ( 10)

Number of invaded cells ( 10)

8 ** ***
* * 5 **
6 *** ***
6 4
P1
3 4
4
XP-pIgR-EV XP-pIgR-EV 2
+Akt Inh IV +PRI-724 DMSO 2
2 1
Akt Inh IV
PRI-724 0 0
0 XP-CTL XP-pIgR XP-CTL XP-pIgR
XP-CTL XP-pIgR P2 -EV -EV -EV -EV
-EV -EV P1 P2
DMSO Akt Inh IV PRI -724

F XP-CTL-EV
+DMSO
XP-pIgR-EV
+DMSO
XP-pIgR-EV
+Akt Inh IV
XP-pIgR-EV
+PRI-724
6
** 6
***
4.17% 5.57% 4.46% 3.35% ** ** *** ***
CD90

5 5

CD133 (%)
4 4
CD90 (%)

3 3

2 2 DMSO
2.53% 5.40% 3.19% 2.75%
CD133

1 1 Akt Inh IV
PRI -724
0 0
XP-CTL XP-pIgR XP-CTL XP-pIgR
-EV -EV -EV -EV

50 ***
G XP-CTL-EV XP-pIgR-EV pg
XP-pIgR-EV XP-pIgR-EV ** ***
Apoptotic cells (%)

40
+DMSO +DMSO +Akt Inh IV +PRI-724
30

20
DMSO
40.4% 36.8% 41.8% 45.8% 10 Akt Inh IV
PI

PRI -724
0
XP-CTL XP-pIgR
Annexin
Anne in V -EV -EV

Fig. 7. Inhibitors of Akt and b-catenin obstruct the promoting effect of pIgR-enriched EVs on cancer and cancer stemness properties. (A) Immunoblot
showing Akt, phospho-Akt (S473), GSK3b, and p-GSK3b (S9) expression in PLC/PRF/5 cells treated with XP-CTL-EV and XP-pIgR-EV and cotreated with either
vehicle, Akt inhibitor IV (Akt Inh IV) or b-catenin inhibitor PRI-724. Anti-b-actin was used as the loading standard. Treated cells were subjected to colony for-
mation (B), migration (C) and invasion (D) assays. (E) Spheroid formation assays of the cells treated as described above were performed for 2 passages, P1 and P2,
each with 1,000 cells. Flow cytometry analysis of CD90+ and CD133+ cells (F) and apoptotic cells (G) was performed. The percentages of CD90+ and CD133+ cells
and apoptotic cells were plotted. Data are presented as the mean ± SEM. Student’s t test was used for 2 groups. *p <0.05; **p <0.01; ***p <0.001. EV, extracellular
vesicle; XP, expression vector.

promote cancer and cancer stemness properties through the (Fig. 8B). Remarkably, cotreatment with sorafenib and anti-pIgR
PDK1/Akt/GSK3b/b-catenin axis. antibody exerted the most potent inhibitory effect on tumor
formation. We did not observe signs of distress or significant
changes in the body weights of mice in any treatment group
Targeting EV-pIgR with a pIgR neutralizing antibody is a
(Fig. 8C). The weight and dimensions of dissected tumors were
potential therapeutic option for HCC
significantly reduced in mice subjected to combined treatment
The therapeutic effect of the anti-pIgR antibody and sorafenib
with sorafenib and anti-pIgR antibody (Fig. 8D). Immunohisto-
was tested in mice implanted with PDTXs that expressed pIgR
chemistry analysis revealed no change in cellular pIgR expres-
(Fig. 8A). Administration of anti-pIgR antibody or sorafenib
sion in tumors from different treatment groups, suggesting that
suppressed tumor growth compared to vehicle treatment

892 Journal of Hepatology 2022 vol. 76 j 883–895

A B 1,200 PBS/IgG C 25
Sorafenib
1,000

Tumor volume (mm3)

Subcutaneous pIgR Ab

Body weight (g)

injection Sorafenib+pIgR Ab
800 20
* ** ***
600
0 3 6 9 12 15 Days
400 * 15 PBS/IgG
Tumor size Sorafenib Sorafenib
reached 200 pIgR Ab
about 90 mm3 pIgR Ab
Sorafenib+pIgR Ab
Sorafenib+pIgR Ab 0 10
0 3 6 9 12 15 0 3 6 9 12 15
Day Day

D 0.8 ** 20 ***

Max. tumor dimension (mm)

n.s. *
* *** n.s.

Tumor weight (g)

PBS/IgG 0.6 15 n.s.
*
Sorafenib *
0.4 n.s. 10
**
pIgR Ab
0.2 5
Sorafenib/pIgR Ab

0 0
IgG nib Ab Ab IgG nib Ab Ab
BS/
or afe pI gR pI gR BS/
or afe pI gR pI gR
P S ib/ P S ib/
en en
oraf oraf
S S
Fig. 8. Treatment with sorafenib and pIgR neutralizing antibody effectively inhibited the development of patient-derived tumor xenografts. (A) Schematic
diagram of the treatment regimen applied to mice subcutaneously implanted with patient-derived tumor xenografts. Mice were administered vehicle (DMSO in
PBS and IgG), sorafenib, anti-pIgR neutralizing antibody or a combination of sorafenib and anti-pIgR neutralizing antibody. DMSO/PBS and sorafenib were
administered daily via oral gavage. Control IgG and anti-pIgR neutralizing antibody were injected peritoneally every 3 days (n = 8). (B) Tumor growth was
monitored and recorded every 3 days until the end of the experiment. Tumor volume was estimated and plotted against time. (C) The body weight of the mice
was examined every 3 days until the experimental endpoint. The tumors were excised from the mice. (D) The weight and maximum dimension of tumors were
measured and plotted. Data are presented as the mean ± SEM. Two-way ANOVA with Turkey’s test was used for multiple groups, or Student’s t test was used for 2
groups. *p <0.05; **p <0.01; ***p <0.001; n.s., not significant. Ab, antibody; pIgR, polymeric immunoglobulin receptor.

the pIgR neutralizing antibody acts by targeting EV-pIgR but not that pIgR is highly expressed in hepatocarcinoma cells and plays
endogenous pIgR (data not shown). important oncogenic functions in HCC. More than 30 years ago,
the detection of high levels of secretory components, which
Discussion result from proteolytic cleavage of pIgR, in the sera of patients
Dysregulation of pIgR was initially reported 45 years ago in with HCC was reported.27 High levels of secretory pIgR have also
malignantly transformed epithelial cells.11–13 Since then, studies been detected in the sera of patients with bladder carcinoma,
have reported contrasting expression of pIgR in different human lung carcinoma and colonic carcinoma with liver metastasis.29–31
carcinomas,14–23 and its role in prognostication of carcinomas Previously, the existence of pIgR within EVs has been reported in
remains inconclusive. pIgR was previously implicated in metas- both CCA and HCC.32 In the present study, our data underscore
tasis by a study demonstrating its positive expression in hepatic the importance of EV-pIgR in hepatocarcinogenesis and the po-
metastatic tissues of colon carcinoma.24 pIgR has been reported tential of EV-pIgR as a diagnostic and prognostic marker in HCC.
to be mostly expressed in cholangiocytes, contributing to an Although alterations in pIgR expression have been detected in
increased secretory pIgR level in the serum.25–27 Immunohisto- different cancer types, the understanding of the mechanism
chemistry of pIgR revealed low to medium staining in hepato- leading to dysregulation of pIgR is limited. In HBV-related HCC,
cytes but no staining in cholangiocytes based on data retrieved pIgR interacts with and activates the Smad2/3 complex leading
from the Human Protein Atlas. Comparison of tissue expression to epithelial-mesenchymal transition (EMT), providing a missing
levels in cholangiocarcinoma (CCA) and HCC in the Cancer link between chronic inflammation and HCC metastasis.8 Of
Genome Atlas and the Genotype-Tissue Expression databases note, it is possible that HBV-related chronic inflammation might
revealed pIgR expression in both carcinoma types, although contribute to changes in the microenvironment, which result in
there was no significant difference between tumorous and non- increased pIgR expression and promote HCC metastasis. Addi-
tumorous tissue. In line with the reported overexpression of pIgR tionally, a previous study suggested EV-pIgR is a non-specific
in HCC,8,9,28 we observed increased transcript and protein levels biomarker related to inflammation in CCA and HCC.32 However,
of pIgR in tumorous tissue compared with their counterparts, as we did not observe an increased level of EV-pIgR in either pa-
well as in metastatic HCC cells compared with non-metastatic tients with HBV or cirrhosis. Thus, the involvement of EV-pIgR in
HCC cells and immortalized liver cells. Our findings suggest persistent inflammation and viral hepatitis requires further in-

Journal of Hepatology 2022 vol. 76 j 883–895 893

Research Article
depth investigation. The functional link between pIgR and EMT is
also substantiated by its inverse correlation with E-cadherin
expression in pancreatic cancer.33 In addition, pIgR activates the
Rac1/CDC42-MEK/ERK cascade to promote HCC tumor growth.9
Another study revealed that bufalin inhibits HCC cell growth
and motility by suppressing APOBEC3 and pIgR.34 pIgR has been
shown to exert oncogenic functions by activating ribosomal
proteins in HCC.28 However, these studies were either conducted
in a physiologically irrelevant canine kidney cell line MDCK or in
SMMC7721, BEL7404 and SKHEP1 cells which have been re-
ported to be either HeLa cell contaminated or of endothelial
origin.35 Therefore, it is not surprising that neither endogenous
pIgR nor EV-pIgR activated Smad2/3 signaling and induced EMT
in HCC cells (data not shown).8 Here, the EV-pIgR-induced PDK1/
Akt/GSK3b/b-catenin axis was observed in different HCC cell
lines, regardless of their b-catenin mutation or activation sta-
tus.36 This mechanistic finding demonstrates how an immuno-
globulin can be associated with tumorigenesis induced by EVs.
Intriguingly, during our establishment of stable clones with
enhanced pIgR expression, we noticed the discrete molecular
size of endogenous pIgR and EV-pIgR. Treatment of cells with the
N-glycosylation inhibitor tunicamycin blocks the glycosylation of
pIgR, revealing 2 distinct sizes of 110 kDa and 83 kDa.17,37
Functionally, the glycosylation of pIgR aids in transcytosis and
mediates the attachment of bacteria.37,38 To date, the functional
implications of non-glycosylated pIgR remain elusive. Our find-
ings suggest that pIgR residing within EVs is in a non-
glycosylated form. Importantly, our observation reveals an un-
reported oncogenic function of non-glycosylated EV-pIgR.
However, the detailed regulatory mechanism of deglycosylation
of pIgR during its packaging into EVs remains unanswered and
requires further investigation.
Sorafenib is the first-line systemic therapy for patients with
inoperable advanced HCC; unfortunately, the effect of sorafenib
is modest, and most patients are highly resistant to sorafenib.39
Other multikinase inhibitors, such as lenvatinib, regorafenib
and carbozantinib, are not superior to sorafenib in terms of
survival. In 2020, bevacizumab (anti-VEGF antibody) plus ate-
zolizumab (anti-PD-L1 antibody) treatment was approved as a
first-line treatment for unresectable HCC.40 The combination
strategy, which has a substantially better outcome than sorafenib
alone, acentuates the advantages of combination treatment over
monotherapy. Herein, our findings suggest that blockade of EV-
pIgR with neutralizing antibodies either alone or in combina-
tion with sorafenib or other therapeutic agents may serve as a
therapeutic option beyond the current limited treatments
for HCC.
In conclusion, our study provides compelling evidence
implicating patient-derived EVs with enhanced pIgR expression
as key mediators of HCC cancer stemness and tumorigenesis. We
also demonstrate that EV-pIgR exerts oncogenic functions via
activation of b-catenin and that blockade of EV-pIgR with a
neutralizing antibody is a potential treatment alternative.
Abbreviations
Akt, protein kinase B; ANOVA, analysis of variance; CCA, chol-
angiocarcinoma; CSC, cancer stem cell; E–HCC–EVs, EVs from
patients with early HCC; ELISA, enzyme-linked immunosorbent
assay; EMT, epithelial-mesenchymal transition; EpCAM,
894 Journal of Hepatology 2022 vol. 76 j 883–895
Hepatic and Biliary Cancer
epithelial cell adhesion molecular; EVs, extracellular vesicles;
GSK3b; glycogen synthase kinase 3b; HBV, hepatitis B virus; HCC,
hepatocellular carcinoma; L–HCC–EVs, EVs from patients with
late HCC; PBS, phosphate-buffered saline; PDK1, 3-
phosphoinositide dependent kinase 1; PDTX, patient-derived
tumor xenograft; pIgR, polymeric immunoglobulin receptor;
qPCR, quantitative PCR; SAM, synergistic activation mediators;
SEM, standard error of mean; XP, XPack expression vector.
Financial support
The work was supported by in part by the funding of Research
Grants Council Theme-Based Research Project on Liver Cancer
Stemness (Project No. T12-704/16-R); The University of Hong
Kong Seed Funding for Strategic Interdisciplinary Research
Scheme (Project no.: 102009863 and 007000142).
Conflict of interest
The authors have no conflict of interest to declare.
Please refer to the accompanying ICMJE disclosure forms for
further details.
Author’s contributions
SKT, SWKW contributed equally to this work. Conceptualization:
SKT, JWPY; Methodology: SKT, JWPY; Investigation: SKT, SWKW,
JYTC, XM, THN, CLSY, ZL, HLF, AHNT, SKYM, TKWL; Resources:
DKHW, LM, MY, CS, ION, PC, KZ, YG, JPY; Manuscript writing: SKT,
JWPY; Funding Acquisition: JWPY.
Data availability statement
We declare that all data involved in this study are available in the
article along with the supplementary materials.
Acknowledgment
We acknowledge the assistance of The University of Hong Kong,
Li Ka Shing Faculty of Medicine, Centre for PanorOmic Sciences
Imaging and Flow Cytometry Core for providing equipment
needed for animal imaging and confocal microscopy. Raw data
were acquired using Mass spectrometer maintained by CPOS
Proteomics and Metabolomics Core. We also thank Centre for
Comparative Medicine Research for providing animals and fa-
cility for animal experimentation and the Electron Microscope
Unit for providing service and support needed for experiments
involving electron microscope.
Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.jhep.2021.12.005.
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