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Büyüceketal. Biomarker Research (2024) 12:154
https://doi.org/10.1186/s40364-024-00702-w
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Biomarker Research
Prevalence andclinical signicance
ofClaudin-3 expression incancer: atissue
microarray study on14,966 tumor samples
Seyma Büyücek1, Nina Schraps2, Anne Menz1, Florian Lutz1, Viktoria Chirico1, Florian Viehweger1,
David Dum1, Ria Schlichter1, Andrea Hinsch1, Christoph Fraune1,3, Christian Bernreuther1, Martina Kluth1,
Claudia Hube‑Magg1, Katharina Möller1, Viktor Reiswich1, Andreas M. Luebke1, Patrick Lebok1,3,
Sören Weidemann1, Guido Sauter1, Maximilian Lennartz1, Frank Jacobsen1, Till S. Clauditz1, Andreas H. Marx1,4,
Ronald Simon1*, Stefan Steurer1, Eike Burandt1, Natalia Gorbokon1, Sarah Minner1, Till Krech1,3 and
Morton Freytag1
Abstract
Background Claudin‑3 (CLDN3) participates in the formation of the tight‑junctions (TJs) that regulate intercellular
permeability. Altered CLDN3 expression has been linked to tumor progression in multiple tumor types. Despite its
widespread expression in normal epithelial cells, CLDN3 is considered an attractive drug target candidate, since it may
be more accessible in cancer cells than in normal cells due to their less orchestrated cell growth.
Methods To comprehensively determine the prevalence of CLDN3 expression in cancer, a tissue microarray contain‑
ing 14,966 samples from 133 different tumor types and subtypes as well as 608 samples of 76 different normal tissue
types was analyzed by immunohistochemistry.
Results CLDN3 immunostaining was observed in 8,479 (68.9%) of 12,314 analyzable tumors, including 11.6%
with weak, 6.2% with moderate, and 51.1% with strong positivity. CLDN3 staining was found in 96 of 133 tumor cat‑
egories, 80 of which contained at least one strongly positive case. CLDN3 positivity was most seen in neuroendocrine
neoplasms (92–100%) and in adenocarcinomas (67–100%), tumors of the female genital tract, including various sub‑
types of ovarian and endometrial carcinoma (up to 100%), as well as different subtypes of breast cancer (95.3–100%).
CLDN3 positivity was less common in squamous cell carcinomas (0–43.2%) and mainly absent in melanoma, mesen‑
chymal, and hematolymphatic neoplasms. In clear cell renal cell carcinoma (ccRCC), low CLDN3 was strongly linked
to poor ISUP (p < 0.0001), Fuhrman (p < 0.0001), and Thoenes (p < 0.0001) grades, advanced pT category (p < 0.0001),
high UICC stage (p = 0.0006) and distant metastasis (p = 0.0011), as well as shortened overall (p = 0.0118) and recur‑
rence‑free (p < 0.0001) survival. In papillary RCC (pRCC), low CLDN3 was associated with poor grade (p < 0.05), high
pT (p = 0.0273) and distant metastasis (p = 0.0357). In urothelial carcinoma high CLDN3 was linked to high grade
(p < 0.0001) and nodal metastasis (p = 0.0111). The level of CLDN3 staining was unrelated to parameters of tumor
aggressiveness in pancreatic, gastric, and breast cancer.
*Correspondence:
Ronald Simon
R.Simon@uke.de
Full list of author information is available at the end of the article
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Büyüceketal. Biomarker Research (2024) 12:154
Conclusion In conclusion, our data demonstrate significant levels of CLDN3 expression in many different tumor enti‑
ties and identify reduced CLDN3 expression as a potential prognostic marker in RCC.
Keywords CLDN3, Tissue microarray, Cancer, Renal cell carcinoma, Biomarker
Introduction
Claudin-3 (CLDN3) is one of 27 known members of the
claudin family [1]. Together with occludin and other
junctional adhesion molecules, the claudins form the
tight-junctions (TJs) that regulate intercellular perme-
ability [2]. Claudins can be distinguished into paracellu-
lar barrier forming and pore forming claudins allowing
for controlled diffusion of ions and water through TJs [3].
TJs display characteristic individual compositions and
ratios of different claudins which define individual “pen-
etrability properties” in different tissues and cell types
[2, 4]. CLDN3 is a rather ubiquitously expressed barrier
forming claudin which occurs in the intestine and many
other epithelial tissues [5, 6].
Despite their widespread expression in normal cells,
TJ components are considered attractive drug target
candidates, since they may be more accessible in cancer
cells than in normal cells. In normal epithelia, the acces-
sibility of TJ proteins is limited by the orchestrated cell
growth, the protection of individual TJ proteins by intact
TJ structures, and the predominant expression of TJs
at apical surfaces [7–10]. e misorientation of the cell
division in cancerous tissues results in a markedly higher
exposure of TJ components [7, 10, 11]. e expression
of CLDN3 in cancer has been analyzed in more than 45
studies using immunohistochemistry (IHC). Aberra-
tions of CLDN3 expression have been reported to occur
in colorectal [12], breast [13–15], ovarian [16, 17], pro-
static [18, 19], gastric [20–22], hepatic [23] and pulmo-
nary cancers [24]. Several of these studies have found a
link between either elevated [14, 18, 25] or reduced [19,
23, 24] CLDN3 expression levels and poor prognosis of
cancer patients. It is of note that the reported rates of
CLDN3 positivity varied considerably between studies.
For example, the range of reported CLDN3 positive cases
ranged from 25 to 73.6% in gastric cancer [20, 21], from
32 to 95% in breast cancer of no special type [26, 27], and
from 41.4 to 97.0% in pulmonary adenocarcinoma [28,
29]. Such conflicting data between studies are typically
caused by the use of different antibodies, IHC protocols,
and criteria to define CLDN3 positivity.
To better understand the prevalence and potential clin-
ical significance of CLDN3 expression in cancer, a com-
prehensive study analyzing a large number of neoplastic
and non-neoplastic tissues under highly standardized
conditions is needed. erefore, CLDN3 expression was
analyzed in more than 14,500 tumor tissue samples from
133 different tumor types and subtypes as well as 76 non-
neoplastic tissue categories by IHC in a tissue microarray
(TMA) format in this study.
Material andmethods
Tissue Microarrays (TMAs)
e normal tissue TMA was composed of 8 samples
from 8 different donors for each of 76 different nor-
mal tissue types (608 samples on one slide). e cancer
TMAs contained a total of 14,966 primary tumors from
133 tumor types and subtypes. Detailed histopathological
data on grade, pathological tumor stage (pT) or patho-
logical lymph node status (pN) were available from breast
cancers (n = 600), urothelial carcinomas (n = 829), ovar-
ian cancers (n = 344), endometroid endometrial cancers
(n = 182), thyroid (n = 518), gastric (n = 327), and pancre-
atic carcinomas (n = 598) as well as clear cell (n = 1,224)
and papillary (n = 310) renal cell carcinomas (ccRCC,
pRCC). Clinical follow up data were available from 789
patients with ccRCC and from 177 patients with pRCC
with a median follow-up time of 48.0 and 50.5 months
(range 1–250 and 1–247). e composition of both
normal and cancer TMAs is described in detail in the
results section. All samples were from the archives of the
Institute of Pathology, University Medical Center Ham-
burg, Germany, the Institute of Patholgy, Clinical Center
Osnabrueck, Germany, and Department of Pathology,
Academic Hospital Fuerth, Germany. Tissues were fixed
in 4% buffered formalin and then embedded in paraffin.
e TMA manufacturing process was described ear-
lier in detail [30, 31]. In brief, one tissue spot (diameter:
0.6mm) per patient was used. e use of archived rem-
nants of diagnostic tissues for TMA manufacturing, their
analysis for research purposes, and the use of patient
data were according to local laws (HmbKHG, §12) and
analysis had been approved by the local ethics commit-
tee (Ethics commission Hamburg, WF-049/09). All work
has been carried out in compliance with the Helsinki
Declaration.
Immunohistochemistry (IHC)
Freshly cut TMA sections were immunostained on one
day and in one experiment. Slides were deparaffinized
with xylol, rehydrated through a graded alcohol series and
exposed to heat-induced antigen retrieval for 5min in an
autoclave at 121°C in pH 7.8 Tris–EDTA-Citrat (TEC)
puffer. Endogenous peroxidase activity was blocked with
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Büyüceketal. Biomarker Research (2024) 12:154
Dako REAL Peroxidase-Blocking Solution (Agilent Tech-
nologies, Santa Clara, CA, USA; #S2023) for 10 min.
Primary antibody specific against CLDN3 protein (rab-
bit recombinant monoclonal, HMV-309, ardoci GmbH,
Hamburg, Germany) was applied at 37°C for 60min at a
dilution of 1:150. For the purpose of antibody validation,
the normal tissue TMA was also analyzed by the rabbit
recombinant monoclonal CLDN3 antibody EPR19971
(Abcam Limited, Cambridge, GB) at a dilution of 1:40
and an otherwise identical protocol. Bound antibody was
then visualized using the Dako REAL EnVision Detection
System Peroxidase/DAB + , Rabbit/Mouse kit (Agilent
Technologies, Santa Clara, CA, USA; #K5007) according
to the manufacturer’s directions. e sections were coun-
terstained with hemalaun. IHC scoring was predefined
and has been used in multiple previous studies [32–34].
For tumor tissues, the percentage of positive neoplastic
cells was estimated, and the staining intensity was semi-
quantitatively recorded (0, 1 + , 2 + , 3 +) [35]. For statis-
tical analyses, the staining results were categorized into
four groups. Tumors without any staining were consid-
ered negative. Tumors with 1 + staining intensity in ≤ 70%
of tumor cells and 2 + intensity in ≤ 30% of tumor cells
were considered weakly positive. Tumors with 1 + stain-
ing intensity in > 70% of tumor cells, 2 + intensity in
31–70%, or 3 + intensity in ≤ 30% of tumor cells were
considered moderately positive. Tumors with 2 + inten-
sity in > 70% or 3 + intensity in > 30% of tumor cells were
considered strongly positive. e analysis by one pathol-
ogist enables the best possible consistency of interpreta-
tion within the study. A possible impact of interobserver
variation was excluded as much as possible by a four-tier
categorization of tumor staining. Although interobserver
variation is common in TMA studies between 1 + and
2 + there is little discrepancies between 0 + and 3 + .
Statistics
Statistical calculations were performed with JMP17®
software (SAS®, Cary, NC, USA). Contingency tables and
the chi2-test were performed to search for associations
between CLDN3 immunostaining and tumor phenotype.
Survival curves were calculated according to Kaplan–
Meier. e Log-Rank test was applied to detect signifi-
cant differences between groups.
Results
Technical issues
A total of 12,314 (82.3%) of 14,966 tumor samples were
interpretable in our TMA analysis. Non-interpretable
samples demonstrated lack of unequivocal tumor cells or
lack of entire tissue spots. A sufficient number of samples
(≥ 4) of each normal tissue type was evaluable.
CLDN3 immunostaining innormal tissues
CLDN3 immunostaining was predominantly membra-
nous. CLDN3 staining was particularly strong in luminal
cells of breast glands, prostate, and seminal vesicle, folli-
cular cells of the thyroid,respiratory epithelial cells,glan-
dular cells of salivary glands, a small subset of gastric
epithelial cells in the neck and in glandular pits,all epi-
thelial cells of the small intestine and the colorectum,
bile ducts in the liver and gallbladder epithelium, acinar
cells of the pancreas, collecting ducts of the kidney, most
epithelial cells in the cauda epididymis, epithelial cells of
endometrium glands, the fallopian tube, and the endocer-
vix (predominantly basolateral), megakaryocytes of the
bone marrow, subsets of high endothelial venules and of
monocytic cells in germinal centers of lymph nodes, as
well as in squamous epithelial cells of tonsil crypts and
corpuscles of Hassall’s in the thymus. A less intense, weak
to moderate membranous CLDN3 staining was observed
in the urothelium (predominantly in the upper half), epi-
thelial cells of the parathyroidal gland, few epithelial cells
of the adrenal gland, hepatocytes (predominantly at the
bile secreting apical membrane), excretory ducts of sali-
vary glands, islets cells of the pancreas,chief cells in the
corpus epididymis, some renal tubular cells,hepatocytes,
a large subset of corpus luteum cells of the ovary, pneu-
mocytes, a subset of cells in the white pulp of the spleen,
and the syncytiotrophoblast (surface membrane) of the
first trimenon placenta. CLDN3 staining was absent in
squamous epithelial cells of the epidermis, the ectocer-
vix, and the esophagus, amnion, chorion, all muscle cells,
and the brain. Representative images are shown in Fig.1.
All cell types identified as CLDN3 positive by HMV-309
were also positive by using EPR19971, although the signal
was less intense for EPR19971 even at a dilution of 1:40
(Supplementary Fig.1).
CLDN3 immunostaining inneoplastic tissues
CLDN3 staining was observed in 8,479 (68.9%) of 12,314
analyzable tumors, including 11.6% with weak, 6.2%
with moderate, and 51.1% with strong staining intensity.
CLDN3 staining varied both in intensity and in its pat-
tern between samples. Most CLDN3 positive tumors
showed a purely membranous staining pattern but some
tumors showed an additional cytoplasmic positivity. Rep-
resentative images are shown in Fig.2.
At least an occasional weak CLDN3 positivity was
detected in 96 of 133 tumor categories and 80 categories
included at least one case with strong CLDN3 positivity
(Table1).
CLDN3 positivity was most seen in adenocarcinomas
(67–100%) and neuroendocrine neoplasms (92–100%)
from various organs as well as in other tumors of the
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Büyüceketal. Biomarker Research (2024) 12:154
Fig. 1 CLDN3 immunostaining of normal tissues. The panels show a strong membranous CLDN3 immunostaining of the luminal cells of breast
glands (a) and of the prostate (b), epithelial cells of the fallopian tube (c), a small subset of gastric epithelial cells in the neck and in glandular pits
(d), epithelial cells of the colorectum (e), the upper half of urothelial cells (f), and in collecting ducts of the kidney medulla (g) while CLDN3 staining
is absent in squamous epithelial cells of the epidermis (h)
Fig. 2 CLDN3 immunostaining in cancer. CLDN3 immunostaining was purely membranous in most tumors, with some showing
an additional cytoplasmic positivity. The panels show a strong CLDN3 positivity in cancer cells of a neuroendocrine tumor of the appendix
(a), an adenocarcinoma of the prostate (b), an endometrioid endometrial carcinoma (c), an invasive breast cancer of no special type (d),
a muscle‑invasive urothelial carcinoma (e), and clear cell renal cell carcinoma (f). CLDN3 staining is lacking in another clear cell renal cell carcinoma
(g) and squamous cell carcinoma of the lung (h)
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Table 1 CLDN3 immunostaining in human tumors
CLDN3 immunostaining
Tumor entity on TMA (n) analyzable (n) negative (%) weak (%) moderate (%) strong (%)
Tumors of the skin Basal cell carcinoma
of the skin 41 13 100.0 0.0 0.0 0.0
Squamous cell carcinoma
of the skin 95 80 93.8 6.3 0.0 0.0
Malignant melanoma 19 19 100.0 0.0 0.0 0.0
Malignant melanoma lymph
node metastasis 86 82 98.8 1.2 0.0 0.0
Merkel cell carcinoma 2 2 50.0 0.0 50.0 0.0
Tumors of the head and
neck Squamous cell carcinoma
of the larynx 109 89 78.7 11.2 6.7 3.4
Squamous cell carcinoma
of the pharynx 60 41 65.9 24.4 0.0 9.8
Oral squamous cell carcinoma
(floor of the mouth) 130 102 84.3 4.9 4.9 5.9
Pleomorphic adenoma
of the parotid gland 50 45 37.8 31.1 20.0 11.1
Warthin tumor of the parotid
gland 49 48 2.1 31.3 43.8 22.9
Basal cell adenoma of the sali‑
vary gland 15 15 20.0 13.3 20.0 46.7
Tumors of the lung, pleura
and thymus Adenocarcinoma of the lung 196 130 3.8 3.8 4.6 87.7
Squamous cell carcinoma
of the lung 80 45 71.1 17.8 2.2 8.9
Mesothelioma, epithelioid 40 24 95.8 4.2 0.0 0.0
Mesothelioma, biphasic 29 17 100.0 0.0 0.0 0.0
Thymoma 29 29 69.0 6.9 13.8 10.3
Lung, neuroendocrine tumor
(NET) 29 25 8.0 0.0 8.0 84.0
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Table 1 (continued)
CLDN3 immunostaining
Tumor entity on TMA (n) analyzable (n) negative (%) weak (%) moderate (%) strong (%)
Tumors of the female geni-
tal tract Squamous cell carcinoma
of the vagina 30 26 76.9 11.5 3.8 7.7
Squamous cell carcinoma
of the vulva 107 87 90.8 3.4 2.3 3.4
Squamous cell carcinoma
of the cervix 88 74 56.8 31.1 5.4 6.8
Adenocarcinoma of the cervix 23 23 4.3 4.3 4.3 87.0
Endometrioid endometrial
carcinoma 288 261 0.4 5.7 10.3 83.5
Endometrial serous carcinoma 36 32 0.0 0.0 6.3 93.8
Carcinosarcoma of the uterus 57 46 21.7 17.4 4.3 56.5
Endometrial carcinoma, high
grade, G3 13 13 30.8 7.7 15.4 46.2
Endometrial clear cell carci‑
noma 9 8 0.0 12.5 0.0 87.5
Endometrioid carcinoma
of the ovary 93 71 0.0 4.2 4.2 91.5
Serous carcinoma of the ovary 530 445 0.2 1.3 2.7 95.7
Mucinous carcinoma
of the ovary 75 51 19.6 19.6 7.8 52.9
Clear cell carcinoma
of the ovary 51 40 0.0 5.0 0.0 95.0
Carcinosarcoma of the ovary 47 36 19.4 11.1 5.6 63.9
Granulosa cell tumor
of the ovary 44 42 90.5 4.8 2.4 2.4
Leydig cell tumor of the ovary 4 4 100.0 0.0 0.0 0.0
Sertoli cell tumor of the ovary 1 1 0.0 100.0 0.0 0.0
Sertoli Leydig cell tumor
of the ovary 3 3 66.7 0.0 33.3 0.0
Steroid cell tumor of the ovary 3 3 66.7 0.0 0.0 33.3
Brenner tumor 32 26 88.5 7.7 0.0 3.8
Tumors of the breast Invasive breast carcinoma
of no special type 499 345 0.3 13.9 9.6 76.2
Lobular carcinoma
of the breast 150 107 4.7 17.8 16.8 60.7
Medullary carcinoma
of the breast 8 7 0.0 14.3 0.0 85.7
Tubular carcinoma
of the breast 2 1 0.0 0.0 100.0 0.0
Mucinous carcinoma
of the breast 7 4 0.0 0.0 0.0 100.0
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Table 1 (continued)
CLDN3 immunostaining
Tumor entity on TMA (n) analyzable (n) negative (%) weak (%) moderate (%) strong (%)
Tumors of the digestive
system Adenomatous polyp, low‑
grade dysplasia 50 25 0.0 0.0 0.0 100.0
Adenomatous polyp, high‑
grade dysplasia 50 38 0.0 0.0 0.0 100.0
Adenocarcinoma of the colon 2483 2219 0.1 0.9 1.0 98.0
Gastric adenocarcinoma, dif‑
fuse type 215 189 23.3 9.5 2.6 64.6
Gastric adenocarcinoma,
intestinal type 215 184 9.8 8.7 10.9 70.7
Gastric adenocarcinoma,
mixed type 62 50 8.0 14.0 8.0 70.0
Adenocarcinoma
of the esophagus 83 76 6.6 7.9 10.5 75.0
Squamous cell carcinoma
of the esophagus 76 69 68.1 17.4 4.3 10.1
Squamous cell carcinoma
of the anal canal 91 65 83.1 10.8 0.0 6.2
Cholangiocarcinoma 58 46 15.2 21.7 23.9 39.1
Gallbladder adenocarcinoma 51 44 6.8 20.5 11.4 61.4
Gallbladder Klatskin tumor 42 27 22.2 33.3 25.9 18.5
Hepatocellular carcinoma 312 274 11.7 56.6 14.6 17.2
Ductal adenocarcinoma
of the pancreas 659 415 33.0 33.3 12.5 21.2
Pancreatic/Ampullary adeno‑
carcinoma 98 65 12.3 7.7 10.8 69.2
Acinar cell carcinoma
of the pancreas 18 17 5.9 17.6 11.8 64.7
Gastrointestinal stromal tumor
(GIST) 62 58 100.0 0.0 0.0 0.0
Appendix, neuroendocrine
tumor (NET) 25 14 0.0 0.0 0.0 100.0
Colorectal, neuroendocrine
tumor (NET) 12 9 0.0 0.0 0.0 100.0
Ileum, neuroendocrine tumor
(NET) 53 45 0.0 0.0 0.0 100.0
Pancreas, neuroendocrine
tumor (NET) 101 76 2.6 0.0 3.9 93.4
Colorectal, neuroendocrine
carcinoma (NEC) 14 12 8.3 8.3 8.3 75.0
Ileum, neuroendocrine carci‑
noma (NEC) 8 7 0.0 0.0 0.0 100.0
Gallbladder, neuroendocrine
carcinoma (NEC) 4 4 0.0 0.0 75.0 25.0
Pancreas, neuroendocrine
carcinoma (NEC) 14 9 0.0 22.2 11.1 66.7
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Table 1 (continued)
CLDN3 immunostaining
Tumor entity on TMA (n) analyzable (n) negative (%) weak (%) moderate (%) strong (%)
Tumors of the urinary
system Non‑invasive papillary
urothelial carcinoma, pTa G2
low grade
87 79 77.2 16.5 5.1 1.3
Non‑invasive papillary
urothelial carcinoma, pTa G2
high grade
80 73 63.0 26.0 8.2 2.7
Non‑invasive papillary urothe‑
lial carcinoma, pTa G3 126 116 36.2 35.3 6.9 21.6
Urothelial carcinoma, pT2‑4
G3 735 520 46.0 25.2 11.9 16.9
Squamous cell carcinoma
of the bladder 22 21 100.0 0.0 0.0 0.0
Small cell neuroendocrine
carcinoma of the bladder 5 5 0.0 20.0 0.0 80.0
Sarcomatoid urothelial
carcinoma 25 12 100.0 0.0 0.0 0.0
Urothelial carcinoma
of the kidney pelvis 62 55 65.5 25.5 1.8 7.3
Clear cell renal cell carcinoma 1287 1045 10.1 25.6 16.6 47.7
Papillary renal cell carcinoma 368 275 2.9 5.1 8.7 83.3
Clear cell (tubulo) papillary
renal cell carcinoma 26 18 5.6 5.6 11.1 77.8
Chromophobe renal cell
carcinoma 170 133 30.1 43.6 12.8 13.5
Oncocytoma of the kidney 257 191 22.0 61.3 13.6 3.1
Tumors of the male genital
organs Adenocarcinoma of the pros‑
tate, Gleason 3 + 3 83 83 0.0 1.2 2.4 96.4
Adenocarcinoma of the pros‑
tate, Gleason 4 + 4 80 80 0.0 0.0 1.3 98.8
Adenocarcinoma of the pros‑
tate, Gleason 5 + 5 85 85 0.0 0.0 1.2 98.8
Adenocarcinoma of the pros‑
tate (recurrence) 258 237 0.0 1.3 0.8 97.9
Small cell neuroendocrine
carcinoma of the prostate 2 2 0.0 0.0 0.0 100.0
Seminoma 682 640 95.5 3.9 0.6 0.0
Embryonal carcinoma
of the testis 54 41 82.9 14.6 2.4 0.0
Leydig cell tumor of the testis 31 30 96.7 0.0 3.3 0.0
Sertoli cell tumor of the testis 2 2 100.0 0.0 0.0 0.0
Spermatocytic tumor
of the testis 1 1 100.0 0.0 0.0 0.0
Yolk sac tumor 53 40 60.0 37.5 2.5 0.0
Teratoma 53 40 72.5 10.0 7.5 10.0
Squamous cell carcinoma
of the penis 92 67 95.5 3.0 1.5 0.0
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Table 1 (continued)
CLDN3 immunostaining
Tumor entity on TMA (n) analyzable (n) negative (%) weak (%) moderate (%) strong (%)
Tumors of endocrine organs Adenoma of the thyroid gland 63 63 0.0 4.8 12.7 82.5
Papillary thyroid carcinoma 341 329 0.0 4.6 6.7 88.8
Follicular thyroid carcinoma 109 106 0.0 7.5 8.5 84.0
Medullary thyroid carcinoma 57 57 3.5 0.0 1.8 94.7
Parathyroid gland adenoma 43 41 9.8 31.7 19.5 39.0
Anaplastic thyroid carcinoma 19 19 84.2 5.3 0.0 10.5
Adrenal cortical adenoma 48 43 95.3 2.3 2.3 0.0
Adrenal cortical carcinoma 27 23 60.9 17.4 4.3 17.4
Pheochromocytoma 51 48 97.9 2.1 0.0 0.0
Tumors of hematopoetic
and lymphoid tissues Hodgkin’s lymphoma 103 91 100.0 0.0 0.0 0.0
Small lymphocytic lymphoma,
B‑cell type (B‑SLL/B‑CLL) 50 39 100.0 0.0 0.0 0.0
Diffuse large B cell lymphoma
(DLBCL) 113 101 100.0 0.0 0.0 0.0
Follicular lymphoma 88 77 100.0 0.0 0.0 0.0
T‑cell non‑Hodgkin’s lym‑
phoma 25 19 100.0 0.0 0.0 0.0
Mantle cell lymphoma 18 15 100.0 0.0 0.0 0.0
Marginal zone lymphoma 16 12 100.0 0.0 0.0 0.0
Diffuse large B‑cell lymphoma
(DLBCL) in the testis 16 15 100.0 0.0 0.0 0.0
Burkitt lymphoma 5 2 100.0 0.0 0.0 0.0
Tumors of soft tissue and
bone Granular cell tumor 23 13 100.0 0.0 0.0 0.0
Leiomyoma 50 49 100.0 0.0 0.0 0.0
Leiomyosarcoma 94 87 100.0 0.0 0.0 0.0
Liposarcoma 96 82 100.0 0.0 0.0 0.0
Malignant peripheral nerve
sheath tumor (MPNST) 15 13 100.0 0.0 0.0 0.0
Myofibrosarcoma 26 25 100.0 0.0 0.0 0.0
Angiosarcoma 42 28 100.0 0.0 0.0 0.0
Angiomyolipoma 91 69 100.0 0.0 0.0 0.0
Dermatofibrosarcoma protu‑
berans 21 11 100.0 0.0 0.0 0.0
Ganglioneuroma 14 11 100.0 0.0 0.0 0.0
Kaposi sarcoma 8 2 100.0 0.0 0.0 0.0
Neurofibroma 117 84 100.0 0.0 0.0 0.0
Sarcoma, not otherwise speci‑
fied (NOS) 74 63 98.4 0.0 1.6 0.0
Paraganglioma 41 27 100.0 0.0 0.0 0.0
Ewing sarcoma 23 12 100.0 0.0 0.0 0.0
Rhabdomyosarcoma 7 6 83.3 16.7 0.0 0.0
Schwannoma 122 93 100.0 0.0 0.0 0.0
Synovial sarcoma 12 9 100.0 0.0 0.0 0.0
Osteosarcoma 19 10 100.0 0.0 0.0 0.0
Chondrosarcoma 15 8 100.0 0.0 0.0 0.0
Rhabdoid tumor 5 5 60.0 0.0 20.0 20.0
Solitary fibrous tumor 17 17 100.0 0.0 0.0 0.0
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Büyüceketal. Biomarker Research (2024) 12:154
female genital tract such as in various subtypes of ovar-
ian and endometrial carcinoma (up to 100%) and differ-
ent subtypes of breast cancer (95.3–100%). CLDN3 was
less common in squamous cell carcinomas (0–43.2%)
and mainly absent in melanoma, mesenchymal neopla-
sia, and in tumors of hematopoetic and lymphoid tissues.
A graphical representation of a ranking order of CLDN3
positive and strongly positive cancers is given in Fig.3.
In pRCC, low CLDN3 staining was associated with
poor ISUP (p = 0.0019), Fuhrman (p = 0.0064), and
oenes (p = 0.0315) grades, high pT (p = 0.0273), and
distant metastasis (p = 0.0357). In urothelial carcinoma
high CLDN3 staining was associated with high grade in
non-invasive carcinomas (p < 0.0001), tumor invasive-
ness (pTa vs. pT2-4; p < 0.0001) as well as with nodal
metastasis (p = 0.0111) and lymphovascular invasion
(L1 status; p = 0.0062) in the subset of muscle-invasive
carcinomas. e level of CLDN3 immunostaining was
unrelated to parameters of tumor aggressiveness in
ductal adenocarcinoma of the pancreas, gastric cancer
and breast cancer. Associations with tumor phenotype
are summarized in Table2.
Fig. 3 Ranking order of CLDN3 positive immunostaining in different human tumors.In ccRCC, low CLDN3 staining was strongly linked to poor ISUP
(p < 0.0001), Fuhrman (p < 0.0001), and Thoenes (p < 0.0001) grades, advanced pT stage (p < 0.0001), high UICC stage (p = 0.0006), distant metastasis
(p = 0.0011), as well as shortened overall (p = 0.0118; Fig. 4a) and recurrence‑free (p < 0.0001; Fig. 4b) survival
Fig. 4 CLDN3 immunostaining and recurrence‑free (A) and overall survival (B) in clear cell renal cell carcinoma
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Büyüceketal. Biomarker Research (2024) 12:154
Table 2 CLDN3 immunostaining and tumor phenotype
CLDN3 immunostaining
n negative (%) weak (%) moderate (%) strong (%) p
Invasive breast cancer of no special type All cancers 294 0.3 13.3 9.9 76.5
pT1 115 0.0 10.4 9.6 80.0 0.1192
pT2 147 0.7 11.6 11.6 76.2
pT3-4 29 0.0 31.0 3.4 65.5
G1 8 0.0 0.0 12.5 87.5 0.6289
G2 160 0.0 13.1 10.6 76.3
G3 126 0.8 14.3 8.7 76.2
pN0 148 0.7 12.8 9.5 77.0 0.6641
pN + 117 0.0 12.8 12.0 75.2
Endometrioid endometrial carcinoma pT1 105 0.0 6.7 4.8 88.6 0.2436
pT2 23 0.0 8.7 17.4 73.9
pT3-4 37 0.0 8.1 13.5 78.4
pN0 50 0.0 4.0 14.0 82.0 0.7811
pN + 29 0.0 6.9 10.3 82.8
Clear cell renal cell carcinoma all cancers 999 10.0 25.9 16.5 47.5
ISUP
1229 5.2 18.3 13.1 63.3 < 0.0001
2355 5.9 25.4 16.9 51.8
3221 10.4 34.4 17.6 37.6
464 48.4 23.4 14.1 14.1
Fuhrman
154 1.9 11.1 5.6 81.5 < 0.0001
2598 5.7 24.4 17.4 52.5
3252 11.9 32.1 18.7 37.3
478 43.6 28.2 11.5 16.7
Thoenes
1300 6.0 19.7 15.3 59.0 < 0.0001
2418 9.6 30.4 19.6 40.0
390 31.1 33.3 7.8 27.8
UICC
1261 6.9 24.9 14.6 53.6 0.0006
231 12.9 32.3 9.7 45.2
383 21.7 26.5 10.8 41.0
465 26.2 32.3 7.7 33.8
pT1 574 5.1 23.7 17.1 54.2 < 0.0001
pT2 111 6.3 22.5 20.7 50.5
pT3-4 303 20.8 31.0 14.2 34.0
pN0 151 15.2 27.2 11.9 45.7 0.1249
pN + 22 27.3 27.3 22.7 22.7
pM0 88 9.1 19.3 12.5 59.1 0.0011
pM + 83 25.3 31.3 10.8 32.5
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Table 2 (continued)
CLDN3 immunostaining
n negative (%) weak (%) moderate (%) strong (%) p
Papillary renal cell carcinoma All cancers 239 2.9 5.9 9.2 82.0
ISUP
129 0.0 3.4 13.8 82.8 0.0019
2113 0.9 4.4 3.5 91.2
362 6.5 8.1 17.7 67.7
44 25.0 25.0 25.0 25.0
Fuhrman
12 0.0 0.0 0.0 100 0.0064
2153 0.7 3.9 5.9 89.5
363 6.3 7.9 15.9 69.8
48 12.5 12.5 37.5 37.5
Thoenes
145 0.0 4.4 13.3 82.2 0.0315
2128 2.3 6.3 7.0 84.4
314 14.3 0.0 28.6 57.1
UICC
177 1.3 3.9 9.1 85.7 0.0625
29 0.0 22.2 22.2 55.6
33 0.0 0.0 0.0 100.0
49 22.2 22.2 11.1 44.4
pT1 169 1.8 5.3 5.3 87.6 0.0273
pT2 39 2.6 7.7 17.9 71.8
pT3-4 25 12.0 8.0 16.0 64.0
pN0 20 0.0 10.0 15.0 75.0 0.2338
pN + 12 16.7 8.3 16.7 58.3
pM0 23 0.0 4.3 17.4 78.3 0.0357
pM + 11 18.2 18.2 27.3 36.4
Ductal adenocarcinoma of the pancreas pT1 8 50 25 0 25 0.662
pT2 43 37.2 39.5 9.3 14
pT3 242 35.5 33.9 12.8 17.8
pT4 17 52.9 29.4 11.8 5.9
G1 10 50 20 0 30 0.092
G2 218 35.3 33 11.9 19.7
G3 68 41.2 38.2 13.2 7.4
pN0 65 40 29.2 12.3 18.5 0.8041
pN + 244 36.1 35.7 11.9 16.4
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Büyüceketal. Biomarker Research (2024) 12:154
Discussion
e results of our successful analysis of 14,966 tumors
from 133 different tumor categories provide a com-
prehensive overview of CLDN3 expression in cancer.
Although CLDN3 expression could be found in a wide
range of tumor entities, it showed that CLDN3 positiv-
ity was most seen in neuroendocrine neoplasms and ade-
nocarcinomas, as well as in tumors of the female genital
tract and various subtypes of breast cancer. CLDN3 posi-
tivity was less frequent in squamous cell carcinomas and,
as described by others [36], only rarely seen in hematol-
ymphoid and in most mesenchymal neoplasms. Although
previous studies on CLDN3 expression in cancer were
limited in number and had provided partially conflicting
data (summarized in Fig.5), several earlier results are in
agreement with our data. For example, CLDN3 positiv-
ity was described in 95% of 20 [37] and in 89% of 57 [38]
esophageal adenocarcinomas (our study: 93.4%), 100%
of 16 colorectal neuroendocrine tumors [39] (our study:
100%), and in 97% of 34 pulmonary adenocarcinomas
[29] (our study: 96.2%).
Claudins, which are essential for the formation of TJs
in human epithelial and endothelial cells, are altered in
a variety of tumors. Because both downregulation and
upregulation of CLDN3 have been found in different
tumor entities and both alterations have been associ-
ated with aggressive tumor characteristics, a tissue type
dependency of CLDN3 function has been assumed
[40]. e striking associations between a reduced
CLDN3 expression and unfavorable histopathological
tumor parameters and poor prognosis in ccRCC repre-
sents a key finding of our study. As adjuvant systemic
therapies are increasingly being administered in high
and intermediate risk RCC, there is a need for a bet-
ter assessment of the individual risk of progression in
these tumors [41, 42]. In the future, CLDN3 IHC could
evolve towards a clinically useful prognostic marker
in RCC, optimally in combination with other markers.
A link between reduced CLDN3 expression and poor
patient outcome or unfavorable tumor characteristics
was previously also found in other cancer types. Jung
et al. [21] described an association between reduced
CLDN3 expression and L1 status as well as advanced
T-stage in a study on 72 gastric adenocarcinomas. Che
et al. [24] reported low CLDN3 expression in squa-
mous cell carcinomas of the lung with high pT stage,
nodal metastasis, and disease recurrence. Orea et al.
[19] found lower disease-free survival and time to clini-
cal progression in prostatic adenocarcinomas with low
CLDN3 expression. Jiang etal. [23] found a shortened
Table 2 (continued)
CLDN3 immunostaining
n negative (%) weak (%) moderate (%) strong (%) p
Gastric adenocarcinoma All cancers 316 13.9 13.9 9.8 62.7
pT1-2 42 7.1 7.1 4.8 81.0 0.2596
pT3 103 15.5 11.7 12.6 60.2
pT4 105 15.2 14.3 10.5 60.0
pN0 61 13.1 8.2 8.2 70.5 0.5204
pN + 189 13.8 13.8 11.1 61.4
Urothelial carcinoma All cancers 619 50.7 27.6 10.3 11.3
pTa G2 low 79 77.2 16.5 5.1 1.3 < 0.0001
pTa G2 high 73 63.0 26.0 8.2 2.7
pTa G3 86 36.0 37.2 9.3 17.4
pT2 97 50.5 20.6 14.4 14.4 0.5546
pT3 183 46.4 31.1 10.4 12.0
pT4 92 43.5 28.3 12.0 16.3
G2 18 55.6 27.8 11.1 5.6 0.6878
G3 354 46.3 27.7 11.9 14.1
pN0 203 51.2 27.1 12.3 9.4 0.0111
pN + 143 38.5 29.4 11.2 21.0
L0 146 52.7 27.4 11.0 8.9 0.0062
L1 132 36.4 30.3 11.4 22.0
Abbreviations: G grade, pM pathologic status of distant metastasis, pN pathologic lymph node status, pT pathologic tumor stage, LLymphatic invasion status, ISUP
International Society of Urologic Pathologists, UICC Union for International Cancer Control
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Page 14 of 17
Büyüceketal. Biomarker Research (2024) 12:154
overall survival in hepatocellular carcinomas with
reduced CLDN3 mRNA expression. Functional stud-
ies on cell line models identified associations between
reduced CLDN3 expression and various cancer driv-
ing mechanisms such as a decrease in epithelial bar-
rier function [43], invasiveness [43], dedifferentiation
[43], proliferative potential [44], and reduced adhesion
[40]. Alternatively, it cannot be excluded that reduced
CLDN3 expression in tumors derived from CLDN3
expressing normal cells merely reflects tumor cell dedi-
fferentiation which always parallels cancer progression.
at not only downregulation but also upregulation
can be associated with tumor progression in a tumor type
dependent manner is demonstrated in our study by the
strong relationship between CLDN3 upregulation and
grade and stage progression in urothelial carcinomas.
is is in line with data from an earlier study by Nakani-
shi etal. showing a link between high CLDN3 expression
Fig. 5 CLDN3 protein expression in cancer (own findings vs. literature data). Graphical representation of CLDN3 data from this study (X) compared
to the previous literature. The colors of the dots represent the number of tumors analyzed in these studies: red: n ≤ 20; yellow: n = 21 to 100; green:
n > 100. For raw data and references, see suppl. Tab. 1
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Page 15 of 17
Büyüceketal. Biomarker Research (2024) 12:154
and advanced stage, high grade and poor overall survival
in a cohort of 129 urothelial cancers of the upper urinary
tract [45]. A significant association between CLDN3
upregulation and tumor progression had also been
reported for breast [25] and ovarian cancer [17]. Mech-
anisms that were suggested to explain a tumor promot-
ing role of CLDN3 in cancer include a regulatory impact
on cancer stemness [46] and increased chemoresistance
[46]. In ovarian cancer cell lines, Agarwal etal. [47] found
associations between CLDN3 upregulation and increased
cell survival, invasion and motility. Again, it cannot be
excluded that CLDN3 neo-expression can be caused by
random alterations occurring during dedifferentiation in
tumors cell derived from CLDN3 non-expressing normal
cells.
Claudins represent potential therapeutic cancer drug
targets for several cancer types due to their membra-
nous localization [48]. Initial evidence for druggability of
CLDN3 came from experiments with Clostridium per-
fringens enterotoxin (CPE), which causes food poisoning,
and selectively binds to the ECL2 motive of CLDN3 [49,
50]. Non-cytotoxic CPE fragments have therefore been
interrogated for their therapeutic potential in cancer.
ey showed anti-tumor efficacy in prostate [50], breast
[51], and ovarian cancer cells [52]. Moreover, C-terminal
fragment of CPE increased the efficacy of chemotherapy
in ovarian cancer [53] and were also successfully used
as a carrier to specifically deliver therapeutic drugs to
ovarian cancer cells [54]. While CPE is not specific for
CLDN3 but also binds to other claudins, specific anti-
bodies have been developed for treating cancer [7, 55,
56]. Human monoclonal antibodies such as KM390755,
IgGH6 [57], and h4G3 [7] been developed against the
CLDN3 ECL1 and ECL2 domains. ese antibodies were
shown to induce antibody-dependent cellular cytotoxic-
ity (ADCC) and in case of KM3907 also a complement-
dependent cytotoxicity (CDC) [48, 55].
Considering the large scale of our study, our assay was
extensively validated by comparing our IHC findings
in normal tissues with data obtained by another inde-
pendent anti-CLDN3 antibody and CLDN3 RNA data
derived from three different publicly accessible databases
[58–61]. is validation procedure was suggested by
the international working group of antibody validation
(IWGAV) [62]. To ensure an as broad as possible range
of proteins to be tested for cross-reactivity, 76 different
normal tissues categories were included in this analysis.
Validity of our assay was supported by the detection of
strongest claudin-3 immunostaining in tissues with high-
est RNA expression (intestine, thyroid, pancreas, and
the prostate). True CLDN3 expression in tissues and
cell types found to be CLDN3 positive by HMV-309 but
lacking documented RNA expression (germinal center
cells in lymphatic tissues, megakaryocytes in the bone
marrow, squamous epithelium positivity in the thymus
and the tonsil crypts, gallbladder, urothelium, placenta,
epididymis, gastric mucosa, adrenal gland, and the para-
thyroidal gland) as well as in tissues with only very low
CLDN3 RNA levels (endometrium) were confirmed by
identical stainings seen by the independent antibody
EPR19971 (Suppl. Figure 1). Given that these CLDN3
positive cell types constituted very small subpopulations
of the respective organs, we assume that CLDN3 RNA
had not been detected due to a massive dilution if RNAs
from total organs were analyzed. Overall, these data
document a high specificity of our IHC assay for CLDN3
detection.
Conclusion
Our data demonstrate significant levels of CLDN3
expression in many different tumor entities, and show
that both increased and decreased levels of CLDN3 can
occur during tumor progression in a cancer type depend-
ent manner. e strong association between low CLDN3
expression and unfavorable prognostic tumor features
may suggest a clinically useful role of CLDN3 expression
measurement in ccRCC.
Abbreviations
CLDN3 Claudin 3
TJ Tight junction
RCC Renal cell carcinoma
ccRCC Clear cell renal cell carcinoma
pRCC Papillary renal cell carcinoma
IHC Immunohistochemistry
TMA Tissue microarray
CPE Clostridium perfringes enterotoxin
ADCC Antibody‑dependent cellular cytotoxicity
CDC Complement‑dependent cytotoxicity
IWGAV International working group of antibody validation
G Grade
pM Pathologic status of distant metastasis
pN Pathologic lymph node status
pT Pathologic tumor stage
L Lymphatic invasion status
ISUP International Society of Urologic Pathologists
UICC Union for International Cancer Control
Supplementary Information
The online version contains supplementary material available at https:// doi.
org/ 10. 1186/ s40364‑ 024‑ 00702‑w.
Supplementary Material 1.
Supplementary Material 2.
Acknowledgements
We are grateful to Laura Behm, Melanie Steurer, Inge Brandt, Maren Eisenberg,
and Sünje Seekamp for excellent technical assistance.
Authors’ contributions
SB, NS, RS, GS, MF: contributed to conception, design, data collection, data
analysis and manuscript writing. AM, FL, VC, FV, DD, RS, AH, CF, CB, KM, VR,
AML, PL, SW, ML, FJ, TC, AHM, SS, EB, NG, SM, TK: participated in pathology
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 16 of 17
Büyüceketal. Biomarker Research (2024) 12:154
data analysis, data interpretation, and collection of samples RS, MK, CHM: data
analysis RS, GS, MF: study supervision All authors agree to be accountable for
the content of the work.
Funding
Open Access funding enabled and organized by Projekt DEAL.
Data availability
All data generated or analyzed during this study are included in this published
article.
Declarations
Ethics approval and consent to participate
The use of archived remnants of diagnostic tissues for manufacturing of TMAs
and their analysis for research purposes as well as patient data analysis has
been approved by local laws (HmbKHG, §12) and by the local ethics commit‑
tee (Ethics commission Hamburg, WF‑049/09). All work has been carried out in
compliance with the Helsinki Declaration. Patient consent was waived due to
local laws (HmbKHG, §12,1) that permit research with anonymized diagnostic
left‑over tissue samples.
Consent for publication
Not applicable.
Competing interests
Conflict of interests The CLDN3 antibody clone HMV‑309 was provided from
ardoci GmbH (owned by a family member of GS).
Author details
1 Institute of Pathology, University Medical Center Hamburg‑Eppendorf, Mar‑
tinistr. 52, Hamburg 20246, Germany. 2 General, Visceral and Thoracic Surgery
Department and Clinic, University Medical Center Hamburg‑Eppendorf,
Hamburg, Germany. 3 Institute of Pathology, Clinical Center Osnabrueck,
Osnabrueck, Germany. 4 Department of Pathology, Academic Hospital Fuerth,
Fuerth, Germany.
Received: 15 October 2024 Accepted: 30 November 2024
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