Rom J Morphol Embryol 2015, 56(2):481490

RJME
ORIGINAL PAPER Romanian Journal of
Morphology & Embryology
http://www.rjme.ro/

Interrelations between hepatic stellate cells and immune

system cells in patients with hepatocellular carcinoma
ALIN GABRIEL IONESCU1), SERGIU MARIAN CAZACU1), COSTIN TEODOR STREBA1),
MIRCEA CTLIN FOROFOIU2), MARIUS EUGEN CIUREA3), MIHAELA IONESCU4), OTILIA ROGOVEANU5),
VIOLETA COMNESCU6), CRISTIN CONSTANTIN VERE1)
1)
Department of Internal Medicine and Gastroenterology, University of Medicine and Pharmacy of Craiova, Romania
2)
Department of Medical Semiology, University of Medicine and Pharmacy of Craiova, Romania
3)
Department of Reconstructive Surgery, University of Medicine and Pharmacy of Craiova, Romania
4)
Department of Medical Informatics, University of Medicine and Pharmacy of Craiova, Romania
5)
Department of Physical Medicine and Rehabilitation, University of Medicine and Pharmacy of Craiova, Romania
6)
Department of Pathology, Emergency County Hospital, Craiova, Romania

Abstract
Objective: Our aim was to identify potential correlations between activated hepatic stellate cells (HSCs) and immune systems cells in
patients with viral C hepatocellular carcinoma, by quantifying the percentage of activated HSCs, T-lymphocytes, natural killer cells and
B-lymphocytes, in three distinct regions: tumor, transition area and the vicinity tissue (25 mm). Patients and Methods: We prospectively
included 20 samples prelevated at necropsy from patients with HCC and C viral infection. We assessed the percentage of alpha-smooth
muscle actin (-SMA), CD45RO, NK1 and CD20 expression using immunohistochemistry and a semi-quantitative scoring method. Results:
We found an inverse correlation between the number of -SMA-positive HSCs and the number of NK1-positive cells in tumor (p=0.0007),
in the transition area/tumor capsule (p=0.024) and in the vicinity tissue (p=0.038). Regarding T-lymphocytes, we have also identified an
inverse correlation with the number of -SMA-positive HSCs in tumor (p=0.0036), in the transition area/tumor capsule (p=0.034) and in the
vicinity tissue (p=0.047). We found no correlation between the number of activated HSCs and the number of CD20-positive cells in all three
examined areas. Conclusions: The analysis of HSCs activity within specified areas of tumoral liver tissue may lead to new perspectives in
early diagnosis of relapses and in the development of future neoadjuvant therapies.
Keywords: hepatocellular carcinoma, hepatic stellate cells, alpha-smooth muscle actin, immune system cells, immunohisto-
chemical assessment.

Introduction
According to World Health Organization (WHO), C
viral hepatitis represents a major affection, with a global
incidence of 3% [1], in Romania its prevalence being
3.5% within the adult population [2]. An average of 20%
to 30% of all patients infected with hepatitis C virus will
progress towards liver cirrhosis, with an annual rate of
3% to 5% developing hepatocellular carcinoma (HCC).
Thus, we scan predict that, in a 10 years period, around
a third of all patients with C viral liver cirrhosis will
acquire HCC.
Liver fibrosis and cirrhosis emerge because of liver
aggression generated by the hepatitic C virus. The increa-
sed extracellular matrix (ECM) production and accumu-
lation is the main feature of chronic liver disease. Within
the injured hepatic tissue, activated hepatic stellate cells
(HSCs) are the principal collagen producing cells. Follo-
wing hepatic injury, quiescent HSCs activate and trans-
differentiate into myofibroblast-like proliferative, fibro-
genic and contractile cells [36].
WHO states that 600 000 new HCC cases are diag-
nosed every year, this value constantly increasing in both
Europe and United States of America. Worldwide, HCC is
the third cause of mortality and morbidity due to malign
diseases [7], being the fifth most common type of cancer
in men and seventh in women [8]. HCC remains a disease
with a highly lethal prognosis, despite recent treatment
strategies like chemotherapy and tyrosine kinase inhibitor
drugs, hepatic resection and liver transplantation [8, 9].
Viral hepatic cirrhosis is a major risk factor in the deve-
lopment of HCC. Carcinogenesis is favored, on one hand,
by the chronic inflammation of the liver, due to a non-
specific and ineffective activation of the immune system
[10, 11] and, on the other hand, by the interactions between
tumor cells and the surrounding microenvironment [9].
Liver carcinogenesis is initiated by the combined effect
of a series of growth factors synthesized by activated
HSCs. These cells, by amplifying the activity of signaling
pathways mediated by nuclear factor kappaB (NF-kB) and
extracellular signal-regulated kinase (ERK), intervene in
HCC progression, on one hand by stimulating tumoral
cells proliferation and, on the other hand, by inhibiting
their apoptosis [12]. Several inflammatory cytokines are
secreted into the tumor microenvironment by HCC cells
and the infiltrating immune cells, which can influence
tumor progression and impair immune systems anti-
tumor response [1315]. Previous in vitro and in vivo
studies have demonstrated that HSCs may be activated by
malignant cells. Following their activation, they become

ISSN (print) 12200522 ISSN (on-line) 20668279

482 Alin Gabriel Ionescu et al.
involved through growth factors in the development of
intratumoral and peritumoral HCC stroma, thus contri-
buting to HCC progression and its increased aggressi-
veness [16, 17].
Several previous immunohistochemical studies have
reported an increased number of activated HSCs in tumoral
sinusoids, fibrous septae, as well as in tumor capsule
[17]. Recent studies have emphasized the major role
played by activated HSCs in inhibiting hepatic immune
response, as well as in stimulating neoangiogenesis in
patients with chronic viral infection [18, 19].
Aim
The aim of this study was to identify potential corre-
lations between activated HSCs and immune systems
cells in patients with viral C HCC. We have done this by
quantifying the percentage of activated HSCs, T-lympho-
cytes, natural killer cells and B-lymphocytes, immuno-
stained with specific markers, in three distinct regions:
tumor, transition area or tumor capsule (for encapsulated
tumors), and the vicinity tissue (25 mm).
Materials and Methods
Study group
In order to assess the number of activated HSCs, T-
lymphocytes, B-lymphocytes and natural killer cells in
patients with HCC and C viral infection, we conducted
a two-year prospective study, between 20102012, on
samples prelevated at necropsy from 20 patients, all
previously under investigation within the 1st Medical
Clinic, Emergency County Hospital, Craiova, Romania,
with ages varying between 39 and 78 years. HCC diagnosis
was defined based on increased seric value of alpha-feto-
protein, corroborated with imagistic diagnosis following
contrast enhanced abdominal ultrasound and computer
tomography. Viral C etiology was previously established
in these patients by detecting the presence of HCV anti-
bodies in their serum. We have obtained the consent to
prelevate samples from all patients included in this study.
All necessary approvals were obtained from the Hospital
Ethical Commission.
HCC diagnosis was confirmed based on histopatho-
logical assessment performed in the Laboratory of
Histological, Histopathological and Immunohistochemical
Techniques within the Research Centre for Microscopic
Morphology and Immunology, University of Medicine
and Pharmacy of Craiova.
Histopathology and immunohistochemistry
Histopathological study was performed on hepatic
tissue samples obtained after necropsy, which were
prelevated, formalin fixed and paraffin embedded. The
assessment of histopathological aspects was conducted
by an experienced pathologist (M.C.), according to the
work protocol used in the Laboratory of Histological,
Histopathological and Immunohistochemical Techniques
within the Research Centre for Microscopic Morphology
and Immunology, University of Medicine and Pharmacy
of Craiova [20].
Quiescent HSCs contain cytoplasmic lipid droplets;
after activation, they lose their droplets and form multiple
microfilaments mainly consisting in alpha-smooth muscle
actin (-SMA). Several studies recommend -SMA as a
trustworthy marker in immunostaining activated HSCs
[2123].
In order to evaluate the number of activated HSCs
from the tumoral tissue prelevated during necropsy, slices
cut from paraffin blocks that were previously used for
histological evaluation, were subsequently displayed on
glass slides pre-treated with poly-L-Lysine for immuno-
histochemical staining. The primary antibody was the
HHF35 clone, anti-human mouse, diluted 1:200, an anti-
-SMA antibody, and the second antibody was IgG horse
anti-mouse, diluted 1:500. The immunostaining obtained
with the help of DAB (3,3-diaminobenzidine) chromogen
was counterstained with Hematoxylin, and emphasized at
cytoplasmic and membranar levels. The antibodies and
clones used for B-lymphocytes (CD20), T-lymphocytes
(CD45RO) and natural killer cells (NK1) immunostaining
are summarized in Table 1.
Table 1 Antibodies, clones and dilutions used for
immunohistochemical analysis
Antibody Clone Dilution Manufacturer
-SMA HHF35 1:200 DAKO
CD20 L26 1:100 DAKO
CD45RO UCHL1 1:100 DAKO
NK1 NK1 1:50 DAKO
In order to evaluate the number of activated HSCs,
B-lymphocytes, T-lymphocytes and natural killer cells,
we used a semi-quantitative method that determined the
percentage of immunostained cells from specific regions:
tumor, transition area/tumor capsule, and the vicinity tissue
(25 mm). The percentage of immunostained cells was
categorized as following: absent, up to 3% (marked as 0);
slight, 333% (marked as 1); moderate, 3466% (marked
as 2); severe, more than 66% (marked as 3) (Table 2).
Table 2 Protocol of semi-quantitative assessment
of hepatic stellate cells immunostained with -SMA,
CD45RO, NK1 and CD20, in studied areas
Percentage of cells immunostained
Areas with -SMA, CD45RO, NK1 and CD20
<3% 333% 3466% >66%
Tumor
Transition area/tumor
capsule Absent Slight Moderate Severe
Vicinity tissue
(25 mm)
Glass slides were examined with a Nikon Eclipse E200
microscope, with 10, 20, and 40 magnification
objectives. Most relevant images were captured using
a Nikon DS-Fi1 digital camera and the LUCIA NET
software application version 1.16.5.
Statistical analysis was performed using XLSTAT
suite for Microsoft Excel. The correlations between the
number of -SMA immunostained HSCs from all three
studied regions (tumor, transition area/tumor capsule, and
the vicinity tissue) with the number of CD45RO-positive
T-lymphocytes, CD20-positive B-lymphocytes and NK1-
positive natural killer cells, were assessed using Kendal
correlation test with a p-value <0.05 considered statisti-
cally significant.
 Interrelations between hepatic stellate cells and immune system cells in patients with hepatocellular carcinoma
Results
Our study group consisted of 20 patients: 14 males and
six females (gender ratio M/F = 2.33/1), with a mean
age of 64.959.89.
Histopathological aspects
The macroscopic examination revealed that three
patients had encapsulated tumors, while the rest had non-
encapsulated tumors.
In most patients, we found nodular HCC, characterized
by the presence of several nodules, some with regular
form, others with irregular aspect, having various sizes,
disseminated within the entire hepatic parenchyma, and
which compressed the surrounding tissue. For the three
patients with encapsulated HCC, the tumor was well
delimited from the surrounding parenchyma, having
several small sized satellite nodules.
The microscopic examination of the sections revealed
three histological types of HCC: well, moderately, and
poorly differentiated.
In the well-differentiated HCC, we found, within the
same tumor, trabecular and acinar (pseudo glandular)
varieties. The trabecular form of HCC was characterized
by a proliferation of malign hepatocytes, which formed
irregular anastomotic plaques, often separated by poorly
distinguished sinusoids, covered in flat cells similar to
Kupffer cells. Trabeculae resembled those in the normal
adult liver, but often presented a thickened aspect, being
composed by several cellular layers. We have noticed few
collagen fibers, with an adjacent disposition to sinusoids
walls. Malign hepatocytes had polygonal shapes, with an
abundant, slightly granular cytoplasm, which presented
less eosinophilic staining than normal hepatocytes. Their
nuclei were large and hyperchromatic, with prominent
nucleoli (Figures 1 and 2).
A characteristic of HCC, regardless of its type, is
represented by the bile synthesis. In the acinar form of
HCC, we have encountered structures that had a glandular
aspect. They consisted of layers of malign hepatocytes
that surrounded the lumen of a bile canaliculus, which
contained a concentrated bile secretion. The tubular or
pseudopapillary aspect resulted after degeneration and
cytolysis, or following the formation of cystic spaces in
an otherwise solid trabecula. Compared to trabecular form
of HCC, cells specific to acinar form are cylindrical and
more elongated.
In the moderately differentiated HCC, we have found
solid, scirrhous and with clear cells types. The solid
type was characterized by small cells, with considerably
varying shapes. Occasionally, we have also encountered
pleomorphic multinucleated giant cells. The tumor deve-
lops in solid formations or in cellular groups. There is a
reduced bile secretion, and diminished connective tissue.
Large tumors frequently had ischemic central necrosis
(Figure 3).
The scirrhous type was defined by malign hepatocytes
that proliferated as narrow fascicules, surrounded by
abundant fibrous stromae. Occasionally, we have noticed
duct-like structures. For most tumoral formations, the
neoplasic cells presented a hepatocyte-like aspect.
483
For the clear cells type, observed malign hepatocytes
displayed a predominantly clear cells appearance. It is
known that tumors frequently have clear cells areas, due
to an increased content of glycogen or, in some cases, of
lipids.
For poorly differentiated HCC, the examined cells
had pleomorphic aspects, with different shapes and sizes,
as well as significant variations of nuclei. We have
identified a large number of giant cells with particular
aspects, with fusiform shapes, similar to sarcoma cells
(Figures 4 and 5).
In all HCC types, we have noticed globular hyaline
structures that reflected the presence of alpha-fetoprotein,
alpha1-antitripsin or other proteins, occasionally finding
hyaline Mallory bodies.
After the histological examination of all samples
prelevated from patients included in our study group,
we have noticed the following distribution, according to
the degree of tumor differentiation: nine patients had
poorly differentiated HCC, five patients presented mode-
rately differentiated HCC, and six patients well differ-
entiated HCC. Among patients with poorly differentiated
HCC, there were three patients with encapsulated tumors.
Immunohistochemical aspects
The semi-quantitative analysis of HSCs, natural killer
cells, as well as B- and T-lymphocytes, from the three
examined regions (tumor, transition area/tumor capsule,
and the vicinity tissue), revealed different results.
In the tumor, -SMA immunostaining was moderate
and severe for most patients (Figures 6 and 7). The semi-
quantitative analysis of T-lymphocytes immunostained
with CD45RO was predominantly slight and moderate
(Figure 8). For natural killer cells, NK1 immunostaining
was especially slight and absent (Figure 9).
The semi-quantitative analysis of B-lymphocytes
indicated a predominantly slight immunostaining with
CD20 in most patients (Figure 10). We have summarized
the results in Table 3, and the corresponding chart in
Figure 11.
Table 3 Assessment of -SMA, CD45RO, NK1 and
CD20 immunostained cells in the tumor
Tumor
Antibodies
<3% 333% 3466% >66%
-SMA 0 4 9 7
CD45RO 2 10 8 0
NK1 5 12 3 0
CD20 6 14 0 0
In the transition area (tumor capsule for the three
patients with encapsulated HCC), we have noticed a severe
immunostaining of -SMA-positive HSCs in most patients,
including those with encapsulated tumors (Figure 12). The
immunostaining with CD45RO, CD20 and NK1 was
mostly slight for all three types of studied lymphocytes:
T (Figure 13), natural killer (Figure 14), and B (Figure 15).
The results of our semi-quantitative analysis are presented
in Table 4 and Figure 16.
484 Alin Gabriel Ionescu et al.
Table 4 Assessment of -SMA, CD45RO, NK1 and assessment of CD20 immunostained cells was predo-
CD20 immunostained cells in the transition area minantly slight (Figure 20). The results of immuno-
(tumor capsule) histochemical analysis were centralized in Table 5, and
Transition area (tumor capsule) graphically illustrated in Figure 21.
Antibodies
<3% 333% 3466% >66% Table 5 Assessment of -SMA, CD45RO, NK1 and
CD20 immunostained cells in the vicinity tissue (2
-SMA 0 2 6 12
5 mm)
CD45RO 3 14 3 0
Vicinity tissue (25 mm)
NK1 8 10 2 0 Antibodies
<3% 333% 3466% >66%
CD20 4 11 5 0
-SMA 0 10 10 0
In the vicinity tissue (25 mm), the semi-quantitative CD45RO 2 3 10 5
analysis of immunostained HSCs was moderate and slight, NK1 0 8 10 2
in an even distribution (Figure 17). The immunostaining
CD20 1 12 7 0
of T-lymphocytes and natural killer cells was moderate
in the majority of patients (Figures 18 and 19). The

Figure 1 Well-differentiated microtrabecular hepato- Figure 2 Hepatocellular carcinoma with an acinar

cellular carcinoma, with formed pseudorosettes resulted
following bile canaliculae dilation, surrounded by hepa-
tocytes; biliar thrombosis is also noticed. Dilaated sinusoids
are present intratrabecular. HE staining, 200.
growth pattern, tumoral cells display pseudorosette aspect
in some areas. Some tumoral cells contain lipidic deposits
(steatosis). We can notice tumor emboli and inflamma-
tory infiltrate. Tumoral nodules are surrounded by layers
of fibrocollagenous tissue. HE staining, 100.

Figure 3 Moderately differentiated hepatocellular car-
cinoma. HE staining, 100.
Figure 4 Solid poorly differentiated hepatocellular car-
cinoma; hepatic architecture is modified, lacking portal
spaces. We can notice polygonal tumor cells, with un-
specificity and mitosis, with a reduced collagen stroma
delimiting tumoral cells formations. HE staining, 100.
 Interrelations between hepatic stellate cells and immune system cells in patients with hepatocellular carcinoma 485

Figure 5 Anaplastic poorly differentiated macrotra- Figure 6 Hepatocellular carcinoma, -SMA present
becular hepatocellular carcinoma, with giant multi- in hepatic stellate cells from tumoral stroma. IHC for
nucleate tumoral cells, with cerebriform nuclei and -SMA, 100.
numerous unspecificities and mitosis, with macrovesi-
cular steatosis, desmoplastic stroma with dilated vessels,
which create a focal aspect of peliosis. HE staining, 100.

Figure 7 Hepatocellular carcinoma, -SMA present Figure 8 T-lymphocytes diffusely distributed in the
in hepatic stellate cells from tumoral stroma. IHC for tumor. IHC for CD45RO, 40.
-SMA, 20.

Figure 9 Intratumoral groups of natural killer cells. Figure 10 Rare B-lymphocytes located inside the tumor.
IHC for NK1, 100. Moderate immunostaining in the transition area and in
the vicinity tissue. IHC for CD20, 40.
486 Alin Gabriel Ionescu et al.

Figure 11 Graphical representation of -SMA, CD45RO, Figure 12 Hepatocellular carcinoma, -SMA present
NK1 and CD20 immunostained cells in the tumor. in hepatic stellate cells from tumoral stroma and tran-
sition area. IHC for -SMA, 100.

Figure 13 T-lymphocytes diffusely distributed in the Figure 14 Groups of natural killer cells present inside
tumor and in the transition area, and numerous in the the tumor and in the transition area. IHC for NK1, 40.
vicinity tissue. IHC for CD45RO, 100.

Figure 15 Groups of B-lymphocytes with a nodular Figure 16 Graphical representation of -SMA, CD45RO,
disposition in the vicinity tissue. Rare B-lymphocytes in NK1 and CD20 immunostained cells in the transition
the transition area and absent inside the tumor. IHC area (tumoral capsule).
for CD20, 40.
 Interrelations between hepatic stellate cells and immune system cells in patients with hepatocellular carcinoma
Figure 17 Encapsulated tumor, -SMA present in
hepatic stellate cells from tumoral stroma, transition
area and vicinity tissue. IHC for -SMA, 100.
Figure 19 Groups of natural killer cells present inside
the tumor and in the transition area, more abundant in
the vicinity tissue. IHC for NK1, 20.
Figure 21 Graphical representation of -SMA, CD45RO,
NK1 and CD20 immunostained cells in the vicinity tissue
(25 mm).
Statistical analysis
The statistical analysis in tumor revealed an inverse
487
Figure 18 T-lymphocytes diffusely distributed in the
tumor and in the transition area, and numerous in the
vicinity tissue. IHC for CD45RO, 100.
Figure 20 Groups of B-lymphocytes with a nodular
disposition in the vicinity tissue. Rare B-lymphocytes in
the tumor and transition area. IHC for CD20, 20.
correlation between the number of -SMA-positive HSCs
and the number of CD45RO-positive cells (p=0.0036)
and NK1-positive cells (p=0.0007).
We have found no correlation between the number
of activated HSCs and the number of CD20-positive
cells.
In the transition area/tumor capsule, we have found
an inverse correlation between the number of activated
HSCs and the number of natural killer cells (p=0.024)
and T-lymphocytes (p=0.034). There was no correlation
between the number of activated HSCs and the number
of B-lymphocytes.
The statistical analysis in the vicinity tissue (25 mm)
indicated an inverse correlation between the number of
activated HSCs and the number of natural killer cells
(p=0.038) and T-lymphocytes (p=0.047). We have found
no correlation between the number of activated HSCs
and the number of B-lymphocytes present in the vicinity
tissue.
Our results are summarized in Table 6.
488 Alin Gabriel Ionescu et al.
Table 6 Overview of all statistic correlations we have identified in the present study
Tumor Transition area (tumor capsule)
Antibodies -SMA immunostained HSCs -SMA immunostained HSCs
P P
CD45RO (UCHL1) 0.0036
NK1 0.0007
CD20 (L26) 0.1682
Discussion
Our results confirm the higher prevalence of HCC in
men compared to women and its development mainly in
the sixth decade of life [8].
Because of hepatic injury, lymphocytes migrate to
Disse space, where they interact with activated HSCs
[24]. Natural killer and Kupffer cells situated in the liver
initially release -Interferon and interleukins, leading to
an increase of TNF- concentration. These cytokines
interfere with the synthesis of cellular adhesion molecules
of the sinusoid endothelial cells, which allows recruitment
and sinusoidal transmigration of inflammatory cells, pre-
ceding hepatocytary apoptosis [25]. Previous studies have
reported that increased serum levels of IL-6 are associated
with a more severe prognosis for HCC patients, while
high intratumoral overexpression of IL-8 was correlated
with an amplified frequency of invasion and metastasis
[13, 2628].
There is a direct interaction, through adhesion, between
activated HSCs and lymphocytes populations that infiltrate
the sub-endothelial space [29].
HSCs activation begins in the portal spaces, with a
subsequent expansion along the fibrous septae, pheno-
menon followed by the recruitment, in the same direction,
of lymphocytes populations. Hepatic inflammation is
initiated by hepatic cells apoptosis and recruitment of
extrahepatic inflammatory cells [30]. Although leukocytes
reach the liver through portal tracts, sinusoids and centri-
lobular vein, the distribution of inflammatory infiltrate
depends especially upon the histopathological localization
of the lesion. The inflammatory infiltrate may contain
T-lymphocytes with a tendency to peripheral distribution,
B-lymphocytes with a mainly central distribution, plas-
matic cells, histiocytes, eosinophils, neutrophils, macro-
phages, natural killer cells and mast cells. Following
liver injury, perisinusoidal HSCs and interstitial fibro-
blasts are activated both playing a major role in the
formation of fibrous septae [31].
Previous studies have reported that T-lymphocytes
intervene in fibrosis mediation, while natural killer cells
generate an antifibrotic effect [24].
T-lymphocytes response inhibition by the intratumoral
activated HSCs contribute to HCCs invasion and metas-
tasis. Vinas et al. demonstrated a new role played by
HSCs as intrahepatic specialized antigen-presenting cells
that activate and stimulate T-lymphocytes proliferation,
also triggering a series of specific responses from T-
lymphocytes that concern proteic and lipid antigens [32].
Based on an in vitro study, Xia et al. reported that tumoral
HSCs induce activated T-lymphocytes apoptosis, thus
contributing to HCCs invasion and metastasis [33].
Tumoral HSCs are characterized by specific structural
changes, as well as an increased collagen synthesis.
Vicinity tissue (25 mm)
-SMA immunostained HSCs
P
0.0345 0.0473
0.0247 0.0388
0.2031 0.8760
Although active chronic viral hepatitis or autoimmune
liver diseases are both associated with significantly in-
creased numbers of activated T-lymphocytes and deve-
lopment of liver fibrosis, immunosuppression is still
present. A main cause of hepatic immunosuppression is
represented by HSCs activation, which induces activated
T-lymphocytes apoptosis, through the signaling pathway
mediated by apoptosis ligand 1 (B7-H1) [34]. Quiescent
HSCs synthesize molecules with an important role in
immunoregulation. In HCC, tumoral activated HSCs inhibit
T-lymphocytes response, thus demonstrating the apparition
of a new immunoregulatory activity, subsequent to their
activation. HCC cells synthesize molecules and cytokine
that trigger HSCs activation.
HSCs activation is a consequence of the following
phenomena: inhibition of membranar receptors MHC
class I, class II, CD86 and CD54; stimulation of apoptosis
inhibiting receptors located on cells surface, through
B7-H1 ligands; amplification of inflammatory cytokines
synthesis (IL-6, IL-1) and inhibitors (TNF-a, TGF-b3).
Tumoral HSCs role as co-stimulator and antigen-pre-
senting cells is diminished by blocking MHC class I,
class II, CD86 and CD54 receptors. Tumoral HSCs
immunosuppressing activity is generated through B7-H1
signaling pathway that leads to T-lymphocytes apoptosis
[34].
B7-H1 identification on tumoral HSCs surface suggests
the interaction between proinflammatory response and
immune tolerance of HCCs microenvironment. A recent
in vitro study demonstrated that the inhibition of T-
lymphocytes by tumoral HSCs favors HCCs invasion
and metastasis [33].
Natural killer cells represent the majority of lympho-
cytes present in the liver, being disposed in hepatic
sinusoid endothelium. Unlike natural killer cells situated
in periphery or spleen, those from the liver have immuno-
phenotypic, morphological and functionally unique
features. The main characteristic of liver natural killer
cells is increased cytotoxicity, due to a high level of cyto-
toxic effectors. In normal liver, natural killer cells play a
major role in the first line of defense of the innate immune
system, also intervening in constant immune monitoring
of the liver, by eliminating invading pathogens, toxins
and circulating tumoral cells [35].
In patients infected with hepatitic C virus, natural
killer cells have a dual behavior, on one hand they induce
tissular injuries and block hepatocytary regeneration,
and on the other hand, they protect the liver from C
virus aggression and tumoral cells proliferation. Despite
these antagonist effects, natural killer cells inhibit liver
fibrosis by directly inducing activated HSCs apoptosis.
They induce the apoptosis of HSCs in the early stages of
activation or senescent, but not of HSCs already activate
or quiescent, which are immune to their stimulation [36].
 Interrelations between hepatic stellate cells and immune system cells in patients with hepatocellular carcinoma
Due to fenestrations present in the hepatic sinusoidal
endothelium, natural killer cells directly interact with
parenchymatous cells, like hepatocytes, as well as with
non-parenchymatous cells present in Disse space, like
HSCs. Also, they may migrate to the edge of cicatricial
fibrotic tissue, in the vicinity of activated HSCs that
synthesize all the elements needed for their interaction.
Natural killer cells activation is controlled by a fragile
equilibrium between anti- and pro-activation signals
mediated through the presence of activating or inhibiting
receptors, with their corresponding ligands, located on
the membrane of target cells. Moretta et al. proved that
strong immune anti-tumor responses are a consequence
of NK-cells infiltrated in tumor sites [37]. A new study
has demonstrated that high intratumoral levels of NK-
cells are correlated with an increased survival in several
other types of cancer [38]. Recently, a positive correlation
was identified between the density of intratumor NK-
cells and tumor apoptosis, and a negative correlation
with tumor proliferation [10], knowing that activated
HSCs induce tumor proliferation [12]. Also, Zhao et al.
have found a positive correlation between the number of
tumor infiltrating natural killer cells and the intratumor
expression of IL-37, significantly associated with better
survival rates of HCC patients [39].
In our study, we have found an inverse correlation
between the number of -SMA positive cells and CD45
and NK1-positive cells, which implies the inhibiting effect
generated by activated HSCs on natural killer cells and
T-lymphocytes. Knowing that natural killer cells induce
apoptosis of HSCs either in early stages of activation or
senescent, we can conclude that, in our examined areas,
there is a population of fully activated HSCs which are
resistant to apoptotic signals generated by natural killer
cells and which, in turn, generates an inhibiting effect
on these cells [36]. Regarding the interaction between
activated HSCs and B-lymphocytes in patients with HCC,
we have not found a correlation in any of the examined
areas.
In the layers of fibrous tissue that surround the tumor,
we have observed a proliferation of activated HSCs.
Previous studies have demonstrated that these myofibro-
blast transformed cells are also present in tissular spaces
between tumoral cells trabeculae and endothelial cells
[17], presenting cytoplasmic extensions that occasionally
surround neoplasic cells trabeculae. Thus, we consider
that HSCs activated by peritumoral hepatocytes injuries,
may be involved in the formation of capsules around
hepatic tumors, being also suggested by the presence
of a severe immunostaining in all three patients with
encapsulated tumors. A major limitation of our study
was the small number of patients included in our study
group. Further studies are necessary in order to better
understand the interrelations between HCC cells, activated
HSCs and immune systems cells.
HSCs present in non-necrotic tumoral tissue produce
ECM, therefore maintaining the structure of neoplasic
trabeculae [17]. Activated HSCs are the main source of
matrix metalloproteinases (MMP)-2 and type I collagen
that stimulates MMP-2 activation, a very important
enzyme involved in the ECM remodeling process asso-
ciated with tumoral invasion and metastasis. In tumoral
489
progression, activated HSCs intervene, on one hand by
altering ECM remodeling process and, on the other hand,
by synthesizing angiopoietin and vascular endothelial
growth factor, both responsible of tumoral angiogenesis
[40].
Even if chronic viral hepatitis is characterized by an
increased number of activated T-lymphocytes, immuno-
suppression is present in the liver, mainly due to HSCs
activation which, by triggering activated T-lymphocytes
apoptosis, favors both HCC invasion and metastasis.
Intratumoral, activated HSCs inhibit T-lymphocytes
response.
Conclusions
A better understanding of physiopathological mecha-
nisms through which activated HSCs are involved in liver
carcinogenesis, could help in elaborating new therapeutic
targets that would aim HSCs inhibition. Thus, a neoadju-
vant post-surgical therapy may prevent the development
of relapses in the peritumoral tissue. Also, by inducing the
apoptosis of peritumoral activated HSCs or their reversion
to quiescent stages, it would be possible to decrease the
incidence of HCC relapses, leading to a greater survival
rate.
Conflict of interests
The authors declare that they have no conflict of
interests.
Author contribution
All authors had an equal contribution in preparing
this manuscript and thus share first authorship.
Acknowledgments
This paper is supported by the Sectorial Operational
Programme Human Resources Development (SOP HRD),
financed from the European Social Fund and by the
Romanian Government under the contract number
POSDRU/159/1.5/S/132395.
References
[1] Wasley A, Grytdal S, Gallagher K; Centers for Disease
Control and Prevention (CDC). Surveillance for acute viral
hepatitis United States, 2006. MMWR Surveill Summ, 2008,
57(2):124.
[2] Gheorghe L, Iacob S, Csiki IE. Prevalence of hepatitis C in
Romania: different from European rates? J Hepatol, 2008,
49(4):661662; author reply 663.
[3] Mengshol JA, Golden-Mason L, Rosen HR. Mechanisms of
disease: HCV-induced liver injury. Nat Clin Pract Gastro-
enterol Hepatol, 2007, 4(11):622634.
[4] Spengler U, Nattermann J. Immunopathogenesis in hepatitis
C virus cirrhosis. Clin Sci (Lond), 2007, 112(3):141155.
[5] Ionescu AG, Streba LA, Vere CC, Ciurea ME, Streba CT,
Ionescu M, Comnescu M, Irimia E, Rogoveanu O. Histo-
pathological and immunohistochemical study of hepatic
stellate cells in patients with viral C chronic liver disease.
Rom J Morphol Embryol, 2013, 54(4):983991.
[6] Irimia E, Mogoant L, Predescu IO, Efrem IC, Stnescu C,
Streba LA, Georgescu AM. Liver steatosis associated with
chronic hepatitis C. Rom J Morphol Embryol, 2014, 55(2):351
356.
[7] Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer
statistics, 2002. CA Cancer J Clin, 2005, 55(2):74108.
[8] Guo CL, Yang HC, Yang XH, Cheng W, Dong TX, Zhu WJ,
Xu Z, Zhao L. Associations between infiltrating lymphocyte
subsets and hepatocellular carcinoma. Asian Pac J Cancer
Prev, 2012, 13(11):59095913.
490 Alin Gabriel Ionescu et al.
[9] Yang JD, Nakamura I, Roberts LR. The tumor microenvi-
ronment in hepatocellular carcinoma: current status and
therapeutic targets. Semin Cancer Biol, 2011, 21(1):3543.
[10] Chew V, Tow C, Teo M, Wong HL, Chan J, Gehring A, Loh M,
Bolze A, Quek R, Lee VK, Lee KH, Abastado JP, Toh HC,
Nardin A. Inflammatory tumour microenvironment is asso-
ciated with superior survival in hepatocellular carcinoma
patients. J Hepatol, 2010, 52(3):370379.
[11] Nakamoto Y, Guidotti LG, Kuhlen CV, Fowler P, Chisari FV.
Immune pathogenesis of hepatocellular carcinoma. J Exp
Med, 1998, 188(2):341350.
[12] Amann T, Bataille F, Spruss T, Mhlbauer M, Gbele E,
Schlmerich J, Kiefer P, Bosserhoff AK, Hellerbrand C.
Activated hepatic stellate cells promote tumorigenicity of
hepatocellular carcinoma. Cancer Sci, 2009, 100(4):646653.
[13] Akiba J, Yano H, Ogasawara S, Higaki K, Kojiro M.
Expression and function of interleukin-8 in human hepato-
cellular carcinoma. Int J Oncol, 2001, 18(2):257264.
[14] Hu P, Hu HD, Chen M, Peng ML, Tang L, Tang KF, Matsui M,
Belladonna ML, Yoshimoto T, Zhang DZ, Xiang R, Ren H.
Expression of interleukins-23 and 27 leads to successful
gene therapy of hepatocellular carcinoma. Mol Immunol,
2009, 46(89):16541662.
[15] Ikeguchi M, Hirooka Y. Interleukin-2 gene expression is a
new biological prognostic marker in hepatocellular carcinomas.
Onkologie, 2005, 28(5):255259.
[16] Mikula M, Proell V, Fischer AN, Mikulits W. Activated hepatic
stellate cells induce tumor progression of neoplastic hepato-
cytes in a TGF-beta dependent fashion. J Cell Physiol, 2006,
209(2):560567.
[17] Enzan H, Himeno H, Iwamura S, Onishi S, Saibara T,
Yamamoto Y, Hara H. Alpha-smooth muscle actin-positive
perisinusoidal stromal cells in human hepatocellular carci-
noma. Hepatology, 1994, 19(4):895903.
[18] Friedman SL. Hepatic stellate cells: protean, multifunctional,
and enigmatic cells of the liver. Physiol Rev, 2008, 88(1):
125172.
[19] Zhao W, Zhang L, Yin Z, Su W, Ren G, Zhou C, You J, Fan J,
Wang X. Activated hepatic stellate cells promote hepato-
cellular carcinoma development in immunocompetent mice.
Int J Cancer, 2011, 129(11):26512661.
[20] Mogoant L, Popescu CF, Georgescu CV. Ghid de tehnici
de histologie, citologie i imunohistochimie. Ed. Medical
Universitar, Craiova, 2007.
[21] Cassiman D, Libbrecht L, Desmet V, Denef C, Roskams T.
Hepatic stellate cell/myofibroblast subpopulations in fibrotic
human and rat livers. J Hepatol, 2002, 36(2):200209.
[22] Buniatian G, Hamprecht B, Gebhardt R. Glial fibrillary acidic
protein as a marker of perisinusoidal stellate cells that can
distinguish between the normal and myofibroblast-like pheno-
types. Biol Cell, 1996, 87(12):6573.
[23] Guyot C, Lepreux S, Combe C, Doudnikoff E, Bioulac-Sage P,
Balabaud C, Desmoulire A. Hepatic fibrosis and cirrhosis:
the (myo)fibroblastic cell subpopulations involved. Int J Bio-
chem Cell Biol, 2006, 38(2):135151.
[24] Melhem A, Muhanna N, Bishara A, Alvarez CE, Ilan Y,
Bishara T, Horani A, Nassar M, Friedman SL, Safadi R. Anti-
fibrotic activity of NK cells in experimental liver injury through
killing of activated HSC. J Hepatol, 2006, 45(1):6071.
[25] Batusic DS, Armbrust T, Saile B, Ramadori G. Induction of
Mx-2 in rat liver by toxic injury. J Hepatol, 2004, 40(3):446453.
[26] Cressman DE, Greenbaum LE, DeAngelis RA, Ciliberto G,
Furth EE, Poli V, Taub R. Liver failure and defective hepa-
Corresponding author
Sergiu Marian Cazacu, Lecturer, MD, PhD, Department of Internal Medicine and Gastroenterology, University of
Medicine and Pharmacy of Craiova, 2 Petru Rare Street, 200349 Craiova, Romania; Phone +40351443 500,
e-mail: cc.vere.umf@gmail.com
Received: January 19, 2015
tocyte regeneration in interleukin-6-deficient mice. Science,
1996, 274(5291):13791383.
[27] Nakagawa H, Maeda S, Yoshida H, Tateishi R, Masuzaki R,
Ohki T, Hayakawa Y, Kinoshita H, Yamakado M, Kato N,
Shiina S, Omata M. Serum IL-6 levels and the risk for
hepatocarcinogenesis in chronic hepatitis C patients: an
analysis based on gender differences. Int J Cancer, 2009,
125(10):22642269.
[28] Wong VW, Yu J, Cheng AS, Wong GL, Chan HY, Chu ES,
Ng EK, Chan FK, Sung JJ, Chan HL. High serum inter-
leukin-6 level predicts future hepatocellular carcinoma deve-
lopment in patients with chronic hepatitis B. Int J Cancer,
2009, 124(12):27662770.
[29] Muhanna N1, Horani A, Doron S, Safadi R. Lymphocyte
hepatic stellate cell proximity suggests a direct interaction.
Clin Exp Immunol, 2007, 148(2):338347.
[30] Lee JI, Lee KS, Paik YH, Nyun Park Y, Han KH, Chon CY,
Moon YM. Apoptosis of hepatic stellate cells in carbon
tetrachloride induced acute liver injury of the rat: analysis of
isolated hepatic stellate cells. J Hepatol, 2003, 39(6):960
966.
[31] Salmi M, Adams D, Jalkanen S. Cell adhesion and migration.
IV. Lymphocyte trafficking in the intestine and liver. Am J
Physiol, 1998, 274(1 Pt 1):G1G6.
[32] Vias O, Bataller R, Sancho-Bru P, Gins P, Berenguer C,
Enrich C, Nicols JM, Ercilla G, Gallart T, Vives J, Arroyo V,
Rods J. Human hepatic stellate cells show features of anti-
gen-presenting cells and stimulate lymphocyte proliferation.
Hepatology, 2003, 38(4):919929.
[33] Xia Y, Chen R, Ye SL, Sun R, Chen J, Zhao Y. Inhibition of
T-cell responses by intratumoral hepatic stellate cells contri-
bute to migration and invasion of hepatocellular carcinoma.
Clin Exp Metastasis, 2011, 28(7):661674.
[34] Yu MC, Chen CH, Liang X, Wang L, Gandhi CR, Fung JJ,
Lu L, Qian S. Inhibition of T-cell responses by hepatic stellate
cells via B7-H1-mediated T-cell apoptosis in mice. Hepato-
logy, 2004, 40(6):13121321.
[35] Gao B, Radaeva S, Park O. Liver natural killer and natural
killer T cells: immunobiology and emerging roles in liver
diseases. J Leukoc Biol, 2009, 86(3):513528.
[36] Radaeva S, Sun R, Jaruga B, Nguyen VT, Tian Z, Gao B.
Natural killer cells ameliorate liver fibrosis by killing activated
stellate cells in NKG2D-dependent and tumor necrosis factor-
related apoptosis-inducing ligand-dependent manners. Gastro-
enterology, 2006, 130(2):435452.
[37] Moretta L, Pietra G, Montaldo E, Vacca P, Pende D, Falco M,
Del Zotto G, Locatelli F, Moretta A, Mingari MC. Human NK
cells: from surface receptors to the therapy of leukemias
and solid tumors. Front Immunol, 2014, 5:87.
[38] Senovilla L, Vacchelli E, Galon J, Adjemian S, Eggermont A,
Fridman WH, Sauts-Fridman C, Ma Y, Tartour E, Zitvogel L,
Kroemer G, Galluzzi L. Trial watch: Prognostic and predictive
value of the immune infiltrate in cancer. Oncoimmunology,
2012, 1(8):13231343.
[39] Zhao JJ, Pan QZ, Pan K, Weng DS, Wang QJ, Li JJ, Lv L,
Wang DD, Zheng HX, Jiang SS, Zhang XF, Xia JC. Inter-
leukin-37 mediates the antitumor activity in hepatocellular
carcinoma: role for CD57+ NK cells. Sci Rep, 2014, 4:5177.
[40] Wirz W, Antoine M, Tag CG, Gressner AM, Korff T,
Hellerbrand C, Kiefer P. Hepatic stellate cells display a
functional vascular smooth muscle cell phenotype in a three-
dimensional co-culture model with endothelial cells. Different-
iation, 2008, 76(7):784794.
Accepted: July 10, 2015