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PUBLISHED 17 March 2023
DOI 10.3389/fonc.2023.1140738
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EDITED BY
Xiuwei Yang,
University of Kentucky, United States
REVIEWED BY
Yingjun Ding,
University of Oklahoma Health Sciences
Center, United States
•CORRESPONDENCE
Jan Bouchal
jan.bouchal@upol.cz
'Deceased
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Frontiers in Oncology
RECEIVED 09 January 2023
ACCEPTED 27 February 2023
PUBLISHED 17 March 2023
CITATION
Ondruššek R, Kvokačková B, Kryštofova' K,
Brychtová S, Souček K and Bouchal J
(2023) Prognostic value and multifaceted
roles of tetraspanin CD9 in cancer.
Front Oncol. 13:1140738.
doi: 10.3389/fonc.2023.U40738
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© 2023 Ondruššek, Kvokačková', Kryštofova',
Brychtová, Souček and Bouchal. This is an
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Prognostic value and
multifaceted roles of
tetraspanin CD9 in cancer
Robert Ondruššek12
, Barbora Kvokačková'3 4 5
,
Karolína Kryštofova'67
, Světlana Brychtova'11
, Karel Souček3 4 5
and Jan Bouchal18
*
'Department of Clinical and Molecular Pathology, Institute of Molecular and Translational Medicine,
Faculty of Medicine and Dentistry, Palacký University, Olomouc, Czechia,2
Department of Pathology,
EUC Laboratoře CGB a.s., Ostrava, Czechia, department of Cytokinetics, Institute of Biophysics of
the Czech Academy of Sciences, Brno, Czechia, international Clinical Research Center, St. Anne's
University Hospital, Brno, Czechia, department of Experimental Biology, Faculty of Science, Masaryk
University, Brno, Czechia, 6
Proteomics Core Facility Central European Institute of Technology,
Masaryk University, Brno, Czechia, 'National Centre for Biomolecular Research, Faculty of Science,
Masaryk University, Brno, Czechia, department of Clinical and Molecular Pathology, University
Hospital Olomouc, Olomouc, Czechia
CD9 is a crucial regulator of cell adhesion in the immune system and plays
important physiological roles in hematopoiesis, blood coagulation or viral and
bacterial infections. It is involved in the transendothelial migration of leukocytes
which might also be hijacked by cancer cells during their invasion and metastasis.
CD9 is found at the cell surface and the membrane of exosomes affecting cancer
progression and therapy resistance. High expression of CD9 is mostly associated
with good patients outcome, with a few exceptions. Discordant findings have
been reported for breast, ovarian, melanoma, pancreatic and esophageal cancer,
which might be related to using different antibodies or inherent cancer
heterogeneity. According to in vitro and in vivo studies, tetraspanin CD9 is not
clearly associated with either tumor suppression or promotion. Further
mechanistic experiments will elucidate the role of CD9 in particular cancer
types and specific conditions.
KEYWORDS
CD9, cancer, immunohistochemistry, prognosis, exosomes
1 Introduction
Tetraspanin CD9, also known as TSPAN29 or motility-related protein 1, is a member
of the transmembrane 4 superfamily proteins, which are characterized by four
transmembrane domains, two extracellular loops, and short intracellular N-and Cterminal
tails (Figure 1A) (1, 2). Like other tetraspanins, C D 9 can undergo
palmitoylation on each of its membrane-proximal cysteines which affects its interactions
with other partners (3). Tetraspanins generally form tetraspanin-enriched microdomains
(TEMs) i n cell membranes. Within these domains, they interact with various
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FIGURE 1 (Continued)
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Ondrussek et al. 10.3389/fonc.2023.1140738
FIGURE 1 (Continued)
Structure of tetraspanin CD9 and its role in cancer, including exosome trafficking. (A) The CD9 protein consists of four transmembrane domains (1-
4), short (EC1, SEL) and long (EC2, LEL) extracellular loop, short intracellular loop and short intracellular N- and C-termini. There are several possible
palmitoylation sites made up of membrane-proximal cysteines and a possible N-glycosylation site in the SEL. In the LEL, there are two disulfide
bridges, each containing one cysteine of the CCG motif (152-154), a typical feature of the tetraspanin family. Based on UniProt (AC: P21926, cited
1.8.2022). (B) CD9 was implicated both in tumor promoting and suppressing mechanisms. Several studies have described its role in cell migration
and invasion, e.g. by affecting actin-polymerization and reorganisation at the cell protrusions (5, 6) or by increasing the production of the proteinase
MMP-2 which cleaves ECM components during cell invasion (6). Increased CD9 expression was also linked to increased signalling in the
protumorigenic NF-KB pathway (7). However, increased CD9 expression was shown to attenuate EGFR signalling and thus suppress cell
proliferation (8, 9). Another study described higher metastatic rate in cells with decreased CD9 expression (10). CD9 downregulation was also
observed in cells which underwent EMT (11). CD9 can also affect tumor neoangiogenesis by promoting VEGFR3 signalling in endothelial cells (12).
Last but not least, transendothelial migration of tumor cells is supported by CD9 reorganisation at points of contact between endothelial and tumor
cells (13). (C) Exosomes can transport cargo between cells in the tumor microenvironment (other tumor cells, stromal cells, immune cells) and thus
enable mutual communication (14, 15). They also help establish the premetastatic niche in the target organ before colonization (16-20). Exosomes
can also promote drug resistance via several mechanisms, e.g. by transporting drugs out of the tumor cells (21, 22) or by neutralisation of antibodyconjugated
drugs (23).
transmembrane and intracellular partners, including other
tetraspanins, integrins, proteases, immunoglobulins, and
intracellular signaling proteins (4). Therefore, the biological
effects of CD9 depend on these dynamic interactions within the
context of TEMs (1, 24). Recently, a "concatenation model" for
forming CD9/EWI-F assemblies has been suggested, which may
explain the occurrence of these TEMs (25). Besides tetraspanins
(e.g. CD63, CD81, CD151 and TSPAN4), CD9 interacts with
numerous single-span transmembrane proteins, such as integrins
(e.g. CD49c/ITGA3 and CD29 (1, 26), immunoglobulin
superfamily proteins (e.g. EWI-F/PTGFRN and EWI-2/IGSF8) (1,
27), heparin-binding EGF-like growth factor (28), and
metalloprotease A D A M 17 (A Disintegrin And Metalloproteinase
17) (29). Previously, CD9 has also shown the ability to interact with
other proteins such as CD19, CD46 and CD117 (30-32).
An equally important role is played by the interaction of
tetraspanins with intracellular signaling molecules, although
significantly fewer of them have been identified compared to
transmembrane partners (4). In the context of CD9, these are
mainly interactions with small GTPases of the Rho family (Rac
and RhoA) that affect the actin cytoskeleton (33, 34), ERM proteins
(ezrin-radixin-moesin) that mediate binding with the cytoskeleton,
and PKC (35), which regulates the function of a wide range of
proteins and intracellular signaling. Obviously, tetraspanin CD9
plays a complex role both in physiological conditions as well as in
many diseases including cancer (Figure IB).
2 Physiological roles of CD9
CD9 is a key regulator of cell adhesion in the immune system
and plays an important role in the physiology of leukocytes and
endothelial cells as well as in hematopoiesis and blood coagulation.
Other physiological processes with the important role of CD9
include sperm-egg fusion (36), neurite outgrowth (37) or
myotube formation (38). Recently, CD9 and tetraspanin 4 were
revealed as membrane curvature sensors which play an essential
role in the formation of migrasome and fertilization (39). In this
sense, it was also shown that the reversed cone-like molecular shape
of CD9 generates membrane curvature in the crystalline lipid layers,
which explains the CD9 localization in regions with high membrane
curvature and its implications in membrane remodeling (40). CD9
is also an exosomal marker and may affect the cellular exosomal
transport and interactions between tumor cells and the stromal
microenvironment (see chapter below).
One of the important functions of CD9 is the regulation of
hematopoietic stem cell differentiation in the bone marrow and
critical hematopoiesis events. CD9 is essential in megakaryocyte
(41), lymphoid and myeloid differentiation (42). In vitro work
proved that immature CD34+ bone marrow cells express high
levels of CD9, while differentiated cells lose their expression (41).
Also, CD9-expressing stromal cells of bone marrow affect
hematopoietic cells and may be one factor that determines the
degree of stem cell differentiation (43). Next, CD9 is involved in the
blood clotting process, because it is a component of integral
membrane proteins expressed on the cell surface and granular
membranes of thrombocytes, which play an essential role in the
coagulation process. It is part of an alpha 2b/beta 3 - CD9 - CD63
integrin - tetraspanin complex in activated platelets (44, 45).
The role of CD9 in different immune cells and its relevance to
inflammation has recently been reviewed by Brosseau et al. (2). For
example, the tetraspanin CD9 plays an important role in the
immune synapse in two ways:/l/Through its association with
LFA-1 on the T cell, CD9 controls the state of aggregation and
adhesive capacity of this integrin./2/On the surface of the antigenpresenting
cells, CD9 recruits ICAM-1 into TEMs, thus increasing
its adhesive capacity (1). Its importance was also shown for the
endothelial receptors such as integrin ligands ICAM-1 and V C A M -
1, which facilitate leukocyte adhesion to the endothelium and their
subsequent transmigration. Another important CD9 function is the
inhibition of A D A M 10 and A D A M 17 sheddase activity, which
enhances cell-cell adhesion and costimulatory capacity (1).
CD9 also affects viral (46) and bacterial infections (47, 48).
Within TEMs, CD9 modulates various virus-induced processes at
the membrane, including membrane fusion, viral budding and viral
release. Sims et al. demonstrated that exosomes could enhance
HIV-1 entry into human T and monocytic cell lines via exosomal
tetraspanin proteins CD81 and CD9 (46). Infection by enveloped
coronaviruses initiates with viral spike proteins binding to cellular
receptors and is followed by proteolytic cleavage which prompts
virus-cell membrane fusion. Infection, therefore, requires the
proximity of receptors and proteases which ensures that virus-cell
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Ondrussek et al. 10.3389/fonc.2023.1140738
entry occurs at the appropriate time and place. Earnest et al. showed
that CD9 is crucial for condensing these receptors and proteases
(DPP4 and TMPRSS2, respectively) which allows viruses to enter
cells efficiently and rapidly (49, 50). Although it might be
reasonable to think that SARS-CoV-2 virulence relies on CD9
activity by clustering and scaffolding receptor and protease (i.e.
ACE2 and TMPRSS2, respectively) for efficient cell entry, this
hypothesis has not been validated yet in current literature (50, 51).
3 CD9 in exosomes and
cell-to-cell communication
There is a growing interest in cell-cell communication mediated
by secreted vesicles termed exosomes (Figure 1C) (52). Exosomes
are nanoscaled extracellular vesicles (EVs) (generally, their sizes
range from 30 to 150 nm) released by almost all cell types.
Tetraspanins are an abundant component of exosome
membranes, with CD9 being one of the most frequently found
along with CD63, CD81, CD82 and TSPAN8 (53, 54). Functionally
it has been demonstrated that CD9 knock-down in extracellular
vesicles from breast cancer cells or recipient cells reduced
endocytosis (55). Importantly, tetraspanins can influence the
composition of exosomes through interactions with their binding
partners. For example, a decrease in CD9 expression led to a
significant reduction in the metalloprotease CD 10 content in
exosomes of pre-B-lymphocytes (56). CD10 can serve as a
positive (57) and negative (58) prognostic factor in some cancers,
but its role in exosomes has not yet been described. It has also been
shown that the alteration of CD9 and CD 151 on prostate cells alters
the proteome of their resultant EVs and that these EVs can enhance
the migratory and invasive capabilities of a non-tumorigenic
prostate cellular population (59). The cargo of exosomes reflects
the state of tumor cells from which they are derived. They can be
explored as minimally invasive biomarkers for the early detection,
diagnosis and prognosis of various cancers (16, 44, 60). Currently,
there are many methods for exosome isolation and detection,
ranging from classical ultracentrifugation or filtration to
immunoaffinity, flow cytometry and acoustics-based microfluidic
techniques (61, 62).
The mechanisms of exosome biogenesis are highly regulated
through several distinct pathways, including ESCRT (endosomal
sorting complexes required for transport) - dependent and ESCRTindependent
pathways (17). Although the exosome release is a
physiological process, its increased rate and specific cargo are
favorable for oncogenic progression and metastases (16).
Regarding CD9, its alterations affect extracellular vesicle secretion
and mitophagy in melanoma cells (63). The exosome-mediated
communication is not limited to the cancer cells, it has also been
shown in different cell types within the tumor microenvironment
locally and distantly. Bioactive molecules, including CD9 in
exosomes derived from cancer and stromal cells, provide the
essential signals for the re-education of various cells and
remodeling the tumor architecture (14, 15). For instance, in
pancreatic ductal adenocarcinoma, CD9 mediated E V uptake
from cancer-associated fibroblasts that promoted tumor
development (15). In the model of colon cancer blocking EVderived
C D 9 by antibody prevented the morphological
transformation and migratory phenotype of cancer cells that
uptake EVs (64). The CD9-positive EVs were higher in patients
with prostate cancer compared to ones with benign prostate
hyperplasia, and its secretion can be modulated in response to
dihydrotestosterone. Importantly, siRNA knockdown of
endogenous CD9 reduced cellular proliferation and expression of
AR and prostate-specific antigen. However, knockdown of AR did
not alter CD9 expression, implicating CD9 as an upstream regulator
of AR (65). The exosomes may also determine organotropism and
prepare the pre-metastatic niche in the sense of Stephen Paget's
seed and soil hypothesis (17, 66). Cancer cells from primary tumors
release oncogenic biomolecules to the distant site before the cell
invasion occurs, forming a pre-metastatic niche in the target organ
that promotes successful metastatic outgrowth (16-20).
Therapy resistance in cancer can also occur via exosomes in
several ways (17). Corcoran et al. reported the transfer of MDR1 by
exosomes which enhanced a docetaxel efflux out of the recipient
cells (67). Exosomes from drug-resistant breast cancer cells could
also transmit chemoresistance do adriamycin and docetaxel by a
horizontal transfer of microRNAs (68). Aung et al. have shown that
tumor-derived exosomes can protect cancer cells by transporting an
abundance of proteins targeted by drugs, hence neutralising the
therapy effects (23). Similarly, cells of the tumor microenvironment
also release exosomes that can enhance drug resistance in cancer
cells. For example, fibroblast-derived exosomes have been shown to
decrease the efficiency of chemotherapy and radiation in cancer by
activating STAT1 and NOTCH3 signaling, which resulted in the
expansion of therapy-resistant tumor-initiating cells (69).
Chemotherapeutic drugs may also be excreted from cancer cells
via exosomes (21, 22). Together, these studies described various
mechanisms of exosome-mediated drug resistance either through
pumping anticancer drugs out of cells or transferring molecular
cargo between cells.
4 CD9 in cancer cells: Dr. Jekyll
and Mr. Hyde
CD9 expression is deregulated in a number of pathologies,
including cancer, but the precise mechanism underlying these
changes and the associated consequences are not fully understood
(4). Bioinformatic analysis of binding sites in the promoter/
enhancer region of the CD9 gene identified E2F, NFkB, SP1 and
STAT3 as top transcription factors often associated with both the
process of carcinogenesis and disease prognosis (70). Nevertheless,
the CD9 protein plays a dual role in cancer progression, exhibiting
both tumor-supportive and tumor-suppressive properties that are
context-dependent.
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Ondrussek et al. 10.3389/fonc.2023.1140738
4.1 Tumor-promoting properties
The plasma membrane protrusions enable the spreading of
neoplastic cells, helping them to move between and invade
surrounding stromal cells. In addition to passing through
intercellular gap junctions, the neoplastic cell can also use a
transcellular route for intra/extravasation, which are the essential
steps of the metastatic process (71). Overexpression of CD9 has
been shown to enhance FAK phosphorylation and reorganisation of
the cortical actin cytoskeleton in fibrosarcoma HT1080 cells plated
on laminin (5). This was also associated with the induction of
MMP2 and PI3K-dependent signaling (6). Aggressive triplenegative
breast cancer MDA-MB-231 cells displayed significant
alterations of their plasma membrane protrusions after CD9
knockdown and had reduced tumorigenic and metastatic capacity
in mouse xenografts (72). The potential role of CD9 in metastasis
was indicated in a different study, describing increased expression of
CD9 in breast cancer bone metastasis compared to primary tumors,
where CD9 antibody treatment in vivo moderately inhibited the
progression of bone lesions (73). In this sense, CD9 expression and
migration were induced by native type IV collagen through a
DDR1-dependent pathway in the breast cancer model, but not in
non-tumorigenic MCF10A and MCF12A cells (66). Surprisingly,
along with the plasma membrane, CD9 can also localize in nuclei
and its depletion led to polynucleation and multipolar mitosis (74).
CD9 has also been shown to interact with VEGFR3 signaling.
After intrathoracic implantation of lung cancer cells, metastasis to
lymph nodes was diminished and accompanied by decreased
neoangiogenesis and lymphangiogenesis in CD9 knock-out mice
(12). Knocking down CD9 in human lymphatic endothelial cells
also decreased their migration, proliferation and tube formation
which was associated with attenuated VEGFR3 signaling.
Importantly, active redistribution of endothelial CD9 was also
observed during interactions between melanoma and endothelial
cells in an intravasation assay (13). Anti-CD9 monoclonal
antibodies specifically inhibited the transendothelial migration of
melanoma cells. Association of CD9 with transendothelial invasion
has also been observed by immunohistochemistry in cervical cancer
as well as in melanomas (75, 76). Similarly, Hori et al. found CD9
expression at severe vessel invasion in gastric cancer (77). CD9
upregulation was also detected in ovarian carcinomas by expression
profiling and immunohistochemistry (7). The CD9 upregulation
associated with enhanced expression of TNF-alpha and NFkB
signaling and treatment with CD9 blocking antibody ALB6
resulted in reduced tumor growth in-vivo (7). CD9 can also
attenuate EGF signaling pathways in gastrointestinal cancer cells
by colocalizing with EGFR (8).
One of the key drivers of tumor progression are cancer stem
cells (CSCs) capable to self-differentiate, self-renew and fueling
tumor growth. It has been reported that CD9 identifies a
subpopulation of pancreatic cancer stem cells (CSCs) able to
initiate and sustain pancreatic cancer growth as demonstrated in
CD9 deficient organoid and mice models (78). Mechanistically,
CD9 promoted the plasma membrane localisation of the glutamine
transporter ASCT2, enhancing glutamine uptake in cancer cells.
CD9 has been identified as a marker of CSCs also i n the
glioblastoma model, where its disruption led to decreased cell
proliferation, invasion, and inhibition of tumor growth (79, 80).
Decreased cell migration was also reported in highly metastatic
hepatocellular carcinoma cells upon CD9 silencing (81) or in breast
cancer cells using CD9-binding peptide (82). The same group
successfully reduced melanoma lung metastasis after peptide
binding to tetraspanin CD9. The CD9-binding peptide impeded
tetraspanin web formation, cancer cell invasion and significantly
reduced secretion and uptake of cancer cell exosomes (83).
Antibody targeting of CD9 in pancreatic cancer disrupted CD9/
A D A M interactions and led to decreased proliferation, migration
and colony formation (84).
Regarding small-cell lung cancer (SCLC), CD9 was expressed
preferentially in SCLC tumors and metastases from three of seven
relapsed patients, whereas chemonarve primary tumors from 16
patients were CD9 negative with only one exception (85).
Mechanistically, CD9 was upregulated in chemoresistant cell
lines, which adhered more tightly to fibronectin via (31 integrin,
but they were less motile than the respective chemosensitive
parental lines, implying a potential role of CD9 molecule in the
cell adhesion-mediated drug resistance (85).
4.2 Tumor-inhibiting properties
Regarding breast cancer, Remsik et al. observed the
downregulation of CD9 in cancer cells that underwent epithelialmesenchymal
transition (EMT) both in vitro and in vivo (11). High
CD9 expression is associated with epithelial phenotype and
favorable prognosis regarding recurrence-free survival (11).
However, further mechanistic studies will be needed to clarify the
role of CD9 in EMT and breast cancer progression. In ovarian
cancer, the downregulation of CD9 attenuated the expression of
several integrins and rearranged junctional and cytoskeletal
molecules which was associated with weaker adhesion to the
extracellular matrix (10). Enhanced peritoneal dissemination was
observed for subclones with low CD9 expression (10), consistent
with a previous report of inverse correlation of CD9 and ovarian
cancer tumor stage (86). Decreased CD9 clustering may reflect the
tendency of malignant cells to have less organized cell-cell junctions
where tetraspanins are typically known to be clustered (3). Low
affinity anti-CD9 antibody, C9BB, which binds preferentially to
CD9 homodimer was used in experiments documenting a shift to
heterodimers in cancer cells. This may be associated with decreased
CD9 palmitoylation or altered expression of CD9 partners (3).
Takeda et al. observed a decreased lymph node metastasis of
lung cancer cells transduced with CD9 without impact on the
primary tumor growth (87). Similarly, ectopic expression of CD9
in fibrosarcoma cell line HT1080 reduced their lung metastatic
ability by forming a complex with podoplanin, suppressing
podoplanin-induced platelet aggregation (88). In line with these
studies, genetic ablation of CD9 in a model of mouse prostate
adenocarcinoma did not affect primary tumor development. Still, it
increased the incidence of metastases to the liver but not the lungs,
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Ondruššek et al. 10.3389/fonc.2023.1140738
suggesting a possible tissue-specific manner of CD9 interactions in
this model (89).
The antiproliferative effect of CD9 was also observed in in vitro
model of human glioblastoma executed via inhibition of EGFR
phosphorylation (9). In contrast, the downregulation of CD9
promoted cancer growth and metastasis through the upregulation
of EGF in pancreatic cancer (90). In the context of SCLC that
develops distant metastases extremely early, Funakoshi et al.
observed the downregulation of tetraspanin CD9 in all cell lines.
CD9 recovery suppressed cell motility of SCLC cells, suggesting that
low expression of CD9 affects cell motility and may contribute to
the highly invasive and metastatic phenotype of SCLC (91).
Likewise CD9 overexpression in hepatocellular carcinoma
inhibited proliferation in vitro and in vivo while CD9 knockdown
enhanced in vivo growth (92).
5 Prognostic value of CD9 in
solid tumors and a problem
of different antibodies
Besides studies dealing with the molecular function of CD9,
expression of this gene was also monitored in large cancer patient
cohorts concerning tumor aggressiveness and survival. Relevant
articles since 1993 are summarized in Table 1 and some are briefly
commented on below. High expression of CD9 is mostly associated
with good patient outcomes, with a few exceptions. These might be
attributed to special cancer subtypes and different antibodies used
for CD9 staining.
Discordant findings have been reported for breast, ovarian,
melanoma, pancreatic and esophageal cancer. The good prognostic
value of high CD9 expression was described in studies using the inhouse
mouse monoclonal antibody m31-15 (93, 100) and a
monoclonal antibody from Dako (94). Of note, studies using the
antibody clone m31-15 consistently report the good prognostic
value of high CD9 in all cancer types (see Tables 1A, IB). On the
other hand, other groups reported poor outcomes for breast and
ovarian cancer patients with high CD9 expression using Abeam
monoclonal antibody EPR2949 or an antibody from Millipore (7,
113,114). Kwon et al. also evaluated stromal immune cells and their
CD9 expression was associated with good patient outcome (113).
The same group used the EPR2949 antibody also for colorectal
cancer where the high CD9 expression in tumors was associated
with a good prognosis (103). Regarding stroma, high stromal CD9
evaluated with antibody clone C-4 was also associated with better
survival of patients with pancreatic ductal adenocarcinoma, while
positive tumor CD9 showed opposite results (115).
Another frequently used antibody is clone 72F6 from
Novocastra. High levels of CD9 evaluated with this antibody were
associated with poor prognosis in gastric cancer (111), while the
association with good prognosis was found for mesothelioma,
cervical and prostate cancer (75, 105, 116). Several other studies
used an antibody from Novocastra without closer specification,
complicating their findings' interpretation. One of these studies
TABLE 1 (A) Summary of immunohistochemical studies - high CD9 expression associates with good clinical outcome.
Authors Tumor Type Patients Detection method
Antibody
clone
Source Therapy
Clinical outcome
with high CD9
Miyake et al., 1995
(93)
breast ca 143
IHC, RT-PCR, western
blotting
m31-15 in-house s less aggressive disease
Arihiro et al., 1998
(94)
breast ca 93 IHC, western blotting n.s. Dako s
good prognosis, low risk of
L N metastases
Houle et al., 2002
(86)
ovarian ca 40 IHC m31-15
in-house, Dr.
Miyake
S/AC less aggressive disease
Sauer et al., 2003
(75)
cervical ca 44 IHC 72F6 Novocastra S good prognosis
Miyamoto et al.,
2001 (95)
endometrial ca 71 IHC TP82
Temecula,
C A
S good prognosis
Wang et al., 2007
(27)
prostatic ca 167 IHC 72F6 Novocastra S less aggressive disease
Mhawech et al.,
2003 (96)
urothelial
blader ca
320 IHC n.s. Novocastra TURBT good prognosis
Ai et al., 2007 (97)
urothelial
blader ca
52 IHC n.s.
Jingmei
Biotech
TURBT good prognosis
Blüm et al., 2010
(98)
oral squamous
ca
179 IHC, RT-PCR n.s. Neomarkers S good prognosis
Kusukawa et al.,
2001 (99)
oral squamous
ca
78 IHC, western blotting clone 007 in-house S good prognosis
(Continued)
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Ondruššek et al. 10.3389/fonc.2023.1140738
TABLE 1 Continued
Authors Tumor Type Patients Detection method
Antibody
clone
Source Therapy
Clinical outcome
with high CD9
Uchida et al., 1999
(100)
esophageal
squamous ca
108 IHC m31-15 in-house s good prognosis
Zou et al., 2012
(101)
ca of gallblader 108 IHC n.s. Dako s good prognosis
Khushman et al.,
2019 (102)
pancreatic ca 29 IHC n.s. Abeam s less aggressive disease
Kim et al., 2016
(103)
colorectal ca 305 IHC EPR2949 Abeam S/AC
good prognosis in left-side
tumors only
Hashida et al. 2003
(104)
colorectal ca 146 IHC, RT-PCR m31-15 in-house S good prognosis
Amatya et al., 2013
(105)
mesothelioma 112 IHC 72F6
Novus
Biologicals
S/AC/RT good prognosis
Si et Hersey 1993
(106)
melanoma 55 IHC FMC56
in-house, Dr.
Zola
S less aggressive disease
Woegerbauer et al.,
2010 (107)
Merkell cell ca 25
IHC, RT-PCR, western
blotting
RDI-MCD9 Fitzgerald S/AC/RT good prognosis
Kohmo et al., 2010
(85)
small cell lung
ca
24
IHC, RT-PCR, western
blotting, flow cytometry
72F6 Novocastra A C good prognosis
Adachi et al., 1998
(108)
non small lung
ca
172 IHC, RT-PCR m31-15 in house S good prognosis
Higashiyama et al.,
1995 (109)
adenoca of lung 132 IHC, RT-PCR m31-15 in house S good prognosis
AC, adjuvant chemotherapy; ca, carcinoma; IHC, immunohistochemitry; LN, lymph node; NAC, neoadjuvant chemotherapy; n.s. not specified; RT, radiotherapy; S, surgery; TURBT; transuretral
resection of bladder tumor.
TABLE 1 (B) Summary of immunohistochemical studies - high CD9 expression associates with poor clinical outcome.
Authors
Tumor
Type
Patients Detection method
Antibody
clone
Source Therapy
Clinical outcome with
high CD9
Kim et al.,
2019 (110)
papillary
thyroid ca
553 IHC n.s. Novocastra s associated with L N metastases
Hori et al.,
2004 (77)
gastric ca 78
IHC, nothern and western
blotting
72F6 and
007
Novocastra and Dr.
Mekada, resp.
S/AC
associated with advanced
disease
Soyuer et al.,
2010 (111)
gastric ca 49 IHC 72F6 Novocastra S/AC poor prognosis
Miki et al.,
2018 (14)
gastric ca 619 IHC n.s. Life Technologies S
poor prognosis, in particular
of the scirrhous type
Huan et al.,
2015 (112)
esophageal
squamous ca
104 IHC, western blotting n.s. Santa Cruz S
poor prognosis, associated
with advanced disease
Kwon et al.,
2017 (113)
breast ca 1349 IHC EPR2949 Abeam S/AC
poor prognosis in luminal A
subtype
Baek et al.,
2019 (114)
lobular breast
ca
113 IHC EPR2949 Abeam S/AC/RT poor prognosis
Hwang
et al.2012 (7)
ovarian ca 30
IHC, microarray, RT-PCR,
western blotting
n.s. Millipore S
associated with advanced
disease
Lucarini et al.,
2022 (76)
cutaneous
melanoma
120 IHC 72F6 Novocastra S
poor prognosis, associated
with L N metastases
Han et al.,
2022 (115)
pancreatic ca,
N A C
179 IHC C-4 Santa Cruz NAC/S poor prognosis
AC, adjuvant chemotherapy; ca, carcinoma; IHC, immunohistochemitry; LN, lymph node; NAC, neoadjuvant chemotherapy; n.s. not specified; RT, radiotherapy; S, surgery; TURBT; transuretral
resection of bladder tumor.
Frontiers in Oncology 07 frontiersin.org
Ondruššek et al. 10.3389/fonc.2023.1140738
investigated breast cancer samples and found no prognostic value of
CD9 (117).
Overall, the interpretation of immunohistochemistry studies is
problematic due to the use of different antibodies that are not
thoroughly validated and essential details are missing. For example,
epitopes for the most frequently used monoclonal antibodies m31-
15, 72F6 and EPR2949 are unavailable. Two recent meta-analyses
concluded that low CD9 expression is significantly associated with
poor prognosis of cancer patients (118, 119) however, these results
are oversimplified and problematic with respect to the
abovementioned issues.
As described in the previous chapter, CD9 is not clearly
associated with either tumor suppression or promotion.
Additional mechanistic studies have recently been thoroughly
reviewed by Lorico et al. They also summarized CD9 targeting
with therapeutic antibodies and drew attention to the potential side
effects of this strategy. This approach might further be complicated
by the presence of CD9 in exosomes which may neutralize the
therapeutic antibodies or enhance the pro-metastatic effects of
exosomes by their enhanced endocytosis (53).
6 Conclusion
A growing body of evidence points to the important role of CD9
in various physiological processes and cancer. As highlighted in the
review, CD9 is not unequivocally associated with either tumor
suppression or promotion, and the antibodies used to detect CD9
might be problematic. Despite conflicting results in different types
of cancer, the clinical relevance of CD9 has been highlighted by
several immunohistochemical and, more importantly, mechanistic
studies. Therapeutic targeting of CD9 is emerging, however, this
approach may be complicated by the presence of CD9 in exosomes,
which may neutralize therapeutic antibodies or enhance the prometastatic
effects of exosomes by their increased endocytosis. In
conclusion, fully validated antibodies and well-designed functional
studies may help to elucidate further the role of CD9 in cancer
progression and patient clinical outcome.
Author contributions
JB, SB, KS - conceptualization. RO - writing, preparation of
original draft. BK, KK, KS - writing, reviewing and editing. KK graphical
figures preparation. JB - supervision, writing, reviewing
and editing. All authors contributed to the article and approved the
submitted version.
Funding
This work was supported by funds from the Czech Health
Research Council (grant NU21-08-00023) and the Czech Science
Foundation (grant nr. 20-229845S). JB was supported by DRO
(FNOL, 00098892) from the Czech Ministry of Health and by the
project of National Institute for Cancer Research (Programme
EXCELES, ID Project No. LX22NPO5102) - Funded by the
European Union - Next Generation EU.
Conflict of interest
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
Publisher's note
All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher.
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