Article

Endosialin expression in relation to clinicopathological and biological variables in rectal cancers with a Swedish clinical trial of preoperative radiotherapy

Department of Oncology, Institute of Clinical and Experimental Medicine, Linköping University, Linköping, S-581 85, Sweden.
BMC Cancer (Impact Factor: 3.36). 03/2011; 11(1):89. DOI: 10.1186/1471-2407-11-89
Source: PubMed
ABSTRACT
The importance of changes in tumour-associated stroma for tumour initiation and progression has been established. Endosialin is expressed in fibroblasts and pericytes of blood vessels in several types of tumours, and is involved in the progression of colorectal cancer. In order to see whether endosialin was related to radiotherapy (RT) response, and clinicopathological and biological variables, we investigated endosialin expression in rectal cancers from the patients who participated in a Swedish clinical trial of preoperative RT.
Endosialin was immunohistochemically examined in normal mucosa, including distant (n = 72) and adjacent (n = 112) normal mucosa, and primary tumours (n = 135). Seventy-three of 135 patients received surgery alone and 62 received additional preoperative RT.
Endosialin expression in the stroma increased from normal mucosa to tumour (p < 0.0001) both in RT and non-RT group. In the RT group, endosialin expression in the stroma was positively associated with expression of cyclooxygenase-2 (Cox-2) (p = 0.03), p73 (p = 0.01) and phosphates of regenerating liver (PRL) (p = 0.002). Endosialin expression in the tumour cells of both in the RT group (p = 0.01) and the non-RT group (p = 0.06) was observed more often in tumours with an infiltrative growth pattern than in tumours with an expansive growth pattern. In the RT group, endosialin expression in tumour cells was positively related to PRL expression (p = 0.02), whereas in the non-RT group, endosialin expression in tumour cells was positively related to p73 expression (p = 0.01).
Endosialin expression may be involved in the progression of rectal cancers, and was related to Cox-2, p73 and PRL expression. However, a direct relationship between endosialin expression and RT responses in patients was not found.

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Available from: Zhi-Yong Zhang, Jan 19, 2016
RESEARCH ARTIC LE Open Access
Endosialin expression in relation to
clinicopathological and biological variables in
rectal cancers with a Swedish clinical trial of
preoperative radiotherapy
Zhi-Yong Zhang
1,2,3
, Hong Zhang
4
, Gunnar Adell
5
, Xiao-Feng Sun
1*
Abstract
Background: The importance of changes in tumour-associated stroma for tumour initiation and progression has
been established. Endosialin is expressed in fibroblasts and pericytes of blood vessels in several types of tumours,
and is involved in the progression of colorectal cancer. In order to see whether endosialin was related to
radiotherapy (RT) response, and clinicopathological and biologi cal variables, we investigated endosialin expression
in rectal cancers from the patients who participated in a Swedish clinical trial of preoperative RT.
Methods: Endosialin was immunohistochemically examined in normal mucosa, including distant (n = 72) and
adjacent (n = 112) normal mucosa, and primar y tumours (n = 135). Seventy-three of 135 patients received surgery
alone and 62 received additional preoperative RT.
Results: Endosialin expression in the stroma increased from normal mucosa to tumour (p < 0.0001) both in RT and
non-RT group. In the RT group, endosialin expression in the stroma was positively associated with expression of
cyclooxygenase-2 (Cox-2) (p = 0.03), p73 (p = 0.01) and phosphates of regenerating liver (PRL) (p = 0.002).
Endosialin expression in the tumour cells of both in the RT group (p = 0.01) and the non-RT group (p = 0.06) was
observed more often in tumours with an infiltrative growth pattern than in tumours with an expansive growth
pattern. In the RT group, endosialin expression in tumour cells was positively related to PRL expression (p = 0.02),
whereas in the non-RT group, endosialin expression in tumour cells was positively related to p73 expression (p =
0.01).
Conclusions: Endosialin expression may be involved in the progression of rectal cancers, and was related to Cox-2,
p73 and PRL expression. However, a direct relationship between endosialin expression and RT responses in patients
was not found.
Background
Colorectal cancer is one of the most common malignant
diseases in western countries. Rectal cancer is a frequent
presentation, with an estimated 35% of cases found situ-
ated in the rectum [1]. New surgical techniques [2] and
preoperative radiotherapy (RT) [3] have improved the
local control and disease-free survival of patients with
rectal c ancer. However the incidence of recurrence and
mortality are still high, even following RT. T herefore, it
is important to gain a better understanding of the
changes induced in tumours following RT of rectal
cancer patients and search for new biological markers in
order to evaluate their therapeutic effects.
The initiatio n and progression of tumours are influ-
enced by the behaviour of the tumour microenviron-
ment, consisting of the extracellular matrix (ECM), the
newly formed vasculature, inflammatory cells and fibr o-
blasts [4,5]. Tumour-associated fibroblasts (activated
fibroblasts and myofibroblasts) have a well-recognized
role as a source of paracrine (cell-to-cell) growth factors
* Correspondence: xiao-feng.sun@liu.se
1
Department of Oncology, Institute of Clinical and Experimental Medicine,
Linköping University, Linköping, S-581 85, Sweden
Full list of author information is available at the end of the article
Zhang et al. BMC Cancer 2011, 11:89
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© 2011 Zhang et al; licensee BioMed Cent ral Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
Page 1
that influence the growth, migration and invasion of
cancer cells during the carcinogenic process. Activated
fibroblasts are also responsible for the synthesis, deposi-
tion and remodelling of the ECM in tumour-associated
stroma [4]. Angiogenesis is a multistep process in
tumour progression that involves both endothelial cells
and pericytes. Alternative potential targets fo r inhibiting
tumours may be invol ved in the tumour-associated
stroma that contains newly for med blood vessels, active
fibroblasts and ECM proteins.
One of these ECM proteins is endosialin. The gene for
this protein is located in chromosome 11q13 [6], and its
product is a type I transmembrane protein, which is a
highly sialylated cell surface receptor found conserved in
humans and in mice. Its extracellular portion consists of
five globular domains, which are N-terminal C-type l ec-
tin domain, a sushi-like domain, and three epidermal
growth factor (EGF)-like repeats, followed by a mucin-
like region [7,8]. Endosialin was first reported to be
selectively expressed in tumour-associate d endo thel ium,
which results in an alternate designation of tumour
endothelial marker 1 (TEM1) [9]. Recently, this designa-
tion was challenged by a series of studies in which
endosialin was shown to be expressed in pericytes (peri-
endothelial mural cells) and activated fibroblasts [10-14].
Thus, endosialin plays an important role in overall
tumour vasculature [15]. Targeting on endosialin or its
related pathways may therefore offer an attractive thera-
peutic opportunity for cancer patients [12].
In the present study, we examined endosialin expres-
sion in distant and adjacent normal mucosa, as well as
in primary tumours, from rectal cance r pa tients, with or
without preoperative RT. We aimed to investigate the
relationships of endo sialin expression with RT
responses, and clinicopathological and biological vari-
ables associated with rectal cancers.
Methods
Patients
Endosialin was immunohistochemically examined in dis-
tant mucosa samples (n = 72, in which 65 cases were
matched with primary tumours), adjacent normal
mucosa sample s (n = 112) and primary tumours (n =
135) from the patients with rectal adenocarcinoma. The
patients were from the Southeast Swedish Health Care
region and part icipated in a Swedish clinical trial of pre-
operative RT between 1987 and 1990 [3]. The distant
normal mucosal samples were taken from a resected
distant margin that was histologically free from tumours,
and adjacent normal mucosa was adjacent to the pri-
mary tumo ur on the same histologic sectio n. The study
was approved by the et hical committee of the Faculty of
Health Sciences, Universities of Linköping and Uppsala,
Sweden. All participants gave informed consents.
Among 135 patients, 73 patients received surgery alone
and 62 received additional preoperative RT. A total of
25 Gy of radiation was a dministered in five fractions
before surgery, over a median of 8 days (range, 6-
15 days). Surgery was performed at a median of 3 days
(range, 0-8 days) after RT. None of the patients received
adjuvant chemotherapy before or after surgery. The
mean age of the patients was 67 years (range, 36-
85 years; median, 69 years). All patients were included in
the follow-up, with mean and median follow-up periods
of 86 and 75 months (range, 0-193 mo nths), respectively.
Follow-up sessions were scheduled at the end of 2004, by
which time 49 patients had died from rectal cancer.
The growth pattern of the tumours was classified (by
two pathologists) as either expansive or infiltrative pat-
tern, based on their patterns of growth and invasiveness.
Tumours were graded as well, moderately or poorly dif-
ferentiated. Other patient and tumour characteristics are
presented in Table 1. No statistically significant
Table 1 Characteristics of patients and rectal cancers
Characteristics Non-
Radiotherapy
Radiotherapy p
-value
n. (%) n. (%)
Gender 0.64
Male 43 (59) 39 (63)
Female 30 (41) 23 (37)
Age (years) 0.65
67 29 (40) 27 (44)
>67 44 (60) 35 (56)
TNM stage 0.26
I 21 (51) 20 (49)
II 18 (47) 20 (53)
III 29 (66) 15 (34)
IV 5 (42) 7 (58)
Differentiation 0.87
Well 2 (3) 2 (3)
Moderately 57 (78) 46 (74)
Poorly 14 (19) 14 (23)
Numbers of other
tumours*
0.07
Single 63 (86) 47 (76)
Multiple 8 (11) 14 (22)
Surgical type 0.16
Rectal amputation 37 (51) 24 (39)
Anterior resection 36 (49) 38 (61)
Resection margin 0.25
Tumour free 71 (97) 57 (92)
Tumour 2 (3) 5 (8)
To anal verge (cm) 0.14
#
Mean 7.67 8.67
* Other colorectal cancer and/or other type of tumour besides the present
rectal cancer.
#
For T-te st, and the other p values for X
2
test.
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differences between the non-RT and RT groups regard-
ing gende r, age, TNM stage, grade of differentiation,
and number of other tumours, surgical type, resection
marg in and mean distance to the anal ver ge were fo und
(p > 0.05).
The data for expre ssion of cyclooxygenase-2 (Cox-2)
[16], p73 [17] and phosphates of regenerating liver
(PRL) [18] determined by immunohistochemistry, were
obtained from the previous studies carried out at our
laboratory.
Immunohistochemical staining
The mouse monoclonal antibody (B1/35), which is
directed against human endosialin, was provided by
Prof. Clare Isacke (Institute of Cancer Research, Sutton,
UK), and described previously [11,12].
Five micrometer sections obtained from paraffin-
embedded tissue blocks were incubated overnight at
60 °C, deparaffinized in xylene, and rehydrated in graded
ethanol and distilled water. Sections were boi led in
0.01M citrate buffer (pH 6.0) in a high-pressure cooker
for 1 min at 120 °C and then kept at room temperature
for 30 min, followed by w ashing in phosphate-buffer ed
saline (PBS; pH 7.4) buffe r. Th e sections were th en
incubated overnight at 4°C with the primary antibody
diluted to 1:250 in PBS. Sections were rinsed with PBS
and incubated with polymerized horseradish peroxidise
(HRP) -anti mouse/rabb it IgG for 30 min at room tem-
perature (Real Envision HRP (rabbit/mouse) kit,
Dako). After w ashing with PBS, the peroxidase reaction
was run for 8 min w ith 3, 3 - diaminobenzididine
(DAB). Sections were then counterstained with hema-
toxylin and mounted for microscopic examination.
The normal mucosa and primary tumour samples
were stained in the same immunostaining run to avoid
biases in the staining pattern and intensity. Sections
known to stain positively were included as negative and
positive co ntrols. For negati ve controls, sections incu-
bated with universal mouse IgG (Dako), instead of the
primary antibody, were not stained, whereas, the positive
controls were stained with the primary antibody.
The slides were examined with a microscope and scored
independently by two pathologists who were given no clin-
ical or pathological information. To avoid artifacts, areas
with poor morphology, section margins, and any necrotic
regions were not considered. The staining intensity in the
entire stroma was scored as either weak (including nega-
tive) or strong. The staining intensity of tumour cells over
the entire s lide area was scored as negative or positive (if
positive cells >5% of tumour cells).
Statistical analysis
The significance of the difference in the intensity of
endosialin expression between normal mucosa and
primary tumours, as well as between stroma and tumour
cells, was examined by X
2
or McNemars test. The rela-
tionship between endosialin expression and clinico-
pathological/biological factors was examined by the X
2
method. The relationship between endosialin expression
and survival was tested by using Cox s proportional
hazard model. All p values were two sided, and values
of p < 0.05 were considered as statistically significant.
Results
Endosialin expression in the stroma of distant normal
mucosa, adjacent normal mucosa and tumour
Endosialin was expressed in fibroblasts and blood vessels
in the stroma of normal mucosa, including distant and
adj acent normal mucosa, a nd tumour (Figure 1A-D) . In
the fibroblasts, endosialin expression of intratumoural or
peritumoural areas ranged from a prominent to a diffuse
pattern. In the blood vessels (mainly mircrovessels), on
the other han d, endosialin pr esented in the cells around
vessels, either in the stroma of normal mucosa or
tumour.
Endosialin expression in the stroma significantly
increased from distant or adjac ent normal mucosa to
the tum our (p < 0.0001) in both the non-RT group (3%,
5%, an d 63%, respectively) and the RT groups (12%, 5%,
and 65%, respectively, Figure 2). No significant differ-
ence was observed between distant and adja cent normal
mucosa in the two subgroups (p > 0.05; Figure 2).
Endosialin expression in the stroma in relation to
clinicopathological and biological variables
As shown in Table 2, in the RT group, the frequencies
of strong endosialin expression in the stroma in TNM
stages I to IV were 45%, 70%, 87%, and 57%, respectively
(p = 0.07). I f TNM stage I (45%) was compared with
other stages (II+III+IV, 74%) the difference was statisti-
cally significant (p = 0.03). Endosialin expression was
positively associated with expression of Cox-2 (p =
0.03), p73 (p = 0.01), a nd PRL (p = 0.002), whereas
there was no such relationship in the non-RT group
(p > 0.05; Table 2). No significant associations of endo-
sialin expressio n in the strom a with other clinicopatho-
logical variables, including gender, age, t umour location,
differentiation, complication, local/distant recurrence,
overall survival and disease-free survival, in either the
RT g roup o r the non-RT group ( p >0.05;datanot
shown) were also observed.
Endosialin expression in the epithelial cells of distant
normal mucosa, adjacent normal mucosa and carcinoma
cells of tumours
There was no positive expression in epithelial cells of dis-
tant and adjacent normal mucosa in either the non-RT
group or the RT group (Figure 1A-C). While in 25 cases
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of non-RT group and 21 cases of RT group, some tumour
cells had positive endosialin expression (Figure 1C-D).
We further analyzed the relationship between endosia-
lin expression in the stroma and that in the tumour
cells in non-RT and RT group (Table 3). The expression
in the s troma and in the tumour cells was c oncordant
in 24 weak/negative cases (33%) and 22 strong/positive
cases (30%) in non-RT group. Only 27 cases (37%)
showed different expression in the two locations (3
tumours (4%) showed positive expression in tumour
cells but weak expression in stroma, and 24 tumours
(33%) s howed strong expression in stroma but negative
expression in tumour cells). In the RT group, the con-
cordance of endosialin expression in stroma and in
tumour cells was 20 weak/negative cases (32%) and 19
strong/positive cases (31%). The discordance was 23
cases (37 %), which showed diff erent expression in the
two locations (2 tumours (3%) showed positive e xpres-
sion in tumour cells but weak expression in stroma, and
21 tumours (34%) showed strong expression in stroma
but negative expression in tumour cells). There was a
significant difference between the expression in the
Figure 1 Endosialin expression in rectal normal muc osa and in rectal cancer (representative images from different groups). (A and B)
Weak expression in the fibroblasts and capillaries in the stroma, and lack of expression in epithelial cells of normal mucosa ((A) ×100 and (B)
×400). (C) Strong expression in fibroblasts of tumour-associated stroma (black arrow) and weak expression in fibroblasts of normal mucosa
(green arrow), normal epithelial cells are negative (yellow arrow), and a few tumour cells are positive (red arrow) and negative (pink arrow)
(×100). (D) Strong expression in fibroblasts of tumour-associated stroma (black arrow) and tumour cells (red arrow), whereas some tumour cells
are negative (pink arrow) (×400).
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stroma and in the tumour cells in the non-RT (p =
0.0001) and RT groups (p = 0.0003).
Endosialin expression in tumour cells in relation to
clinicopathological and biological variables
In the non-RT group, endosialin expr ession was more
frequent in tumours with infiltrative growt h patterns
than those with expansive growth patt erns (p =0.01;
Table 4) and was positively related to p73 expression
(p =0.01;Table4).IntheRTgroup,asimilartrendof
endosialin expression was observed in tumours with
infiltrative growth patterns w hen compared to those
with expansive growth patterns (p = 0.06; Table 4).
Endosialin expression was positively related to PRL
expression (p = 0.02; Table 4).
No significant correlation between endosialin expres-
sion in tumour cells and survival, r ecurrence and o ther
pathological/biological variables were found in either the
non-RT group or in the RT group (p >0.05; data not
shown).
Discussion
Endosialin, also referred to as TEM1, was origina lly dis-
covered as a human embryonic fibroblast-specific anti-
gen and later reported to be expressed in the
endothelium. Endosialin is ba rely detectable in normal
tissues other than its moderate expression in the smooth
muscle of colon and prostate [13]. Therefore, TEM1 was
once considered as an important can didate as a vascular
target [6,9,19]. However, recent studies have demon-
strated that endosialin is expre ssed in pericytes of breas t
tumours and b rain gliomas, and not selectively in
tumour endothelium [10-12]. In the present study,
endosialin was prominently expressed in the tumour-
associated stroma, especially in fibroblasts and blood
vessels, and only weakly expressed in the stroma of dis-
tant or adjacent normal mucosa in both the non-RT
group and in the RT groups. When compared to the
normal mucosa, endosialin expressio n was much higher
in tumour, thus agreeing with other re ports on co lon
Figure 2 Endosialin expression in the stroma in distant normal m ucosa, adjacent norma l mucosa and tumour in no n-RT and RT
groups. A significant increase was observed from the distant normal mucosa or adjacent normal mucosa to the tumour (p < 0.0001).
Table 2 Endosialin expression in tumour-associated
stroma in relation to clinicopathological and biological
variables in rectal cancer patients
Variables Non-
Radiotherapy
p -value Radiotherapy p -value
Weak
(%)
Strong
(%)
Weak
(%)
Strong
(%)
TNM stage 0.92 0.07
I 9 (43) 12 (57) 11 (55) 9 (45)
II 6 (33) 12 (67) 6 (30) 14 (70)
III 10 (34) 19 (66) 2 (13) 13 (87)
IV 2 (40) 3 (60) 3 (43) 4 (57)
Cox-2 0.89 0.03
Weak 14 (37) 24 (63) 13 (50) 13 (50)
Strong 12 (35) 22 (65) 7 (22) 25 (78)
p73 0.11 0.01
Weak 13 (46) 15 (54) 11 (55) 9 (45)
Strong 12 (28) 31 (72) 9 (19) 26 (81)
PRL 0.47 0.002
Weak 4 (44) 5 (56) 8 (73) 3 (27)
Strong 18 (32) 38 (68) 10 (24) 32 (76)
p values for X
2
test; Cox-2: cyclooxygenase-2; PRL: phosphates of regenerat ing
liver.
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and breast cancers [20,21]. Up-regulated endosialin in
the t umour-associated stromamayplayaroleinthe
tumorigenesis of rectal cancers.
In the present study, endosialin expression in the
stroma was more frequently observed in advanced TNM
stages, and positively related to the tumour cellular
expression of Cox-2, p73, and PRL in the RT group,
whereas th ere were no such associations in the non-RT
group. The level of endosialin expression in the tumour-
associated stroma was significantly higher in breast
cancers with nodal involvement compared to those with
negative nodes [21]. Endosialin has also be en found in
glioblastoma multiforme, anaplastic astrocytomas, and
metastatic carcinomas that are of highly invasive activity
[12,22]. Endosialin is also more abundant in melanoma
metastases than in the primary tumours [13]. In colorec-
tal cancer, endosialin was up-regulated in Dukes B
compared to Dukes A [20]. Cox-2 is an inducible isoen-
zyme of cyclooxygenase that is undetectable in normal
colonic mucosa but is overexpressed in 80% of colonic
tumours [23]. Cox-2 is involved in a multistep process
of colorectal tumorigenesis, such as apoptosis inhibition
of c ellular proliferation a nd angiogenesis enhanc ement,
tumour cell invasion and differentiation. One of our
previous studies has shown that Cox-2 expression is
higher in more advanced tumours [24]. It is interesting
to see, in the further study, whether or not endosialin
and C ox-2 have interactions in the tumour develop-
ment, especially in enhancing angiogenesis. We have
also found that p73 expression is increased during the
development of colorectal cancers and its overexpres-
sion is further associated with poor prognosis in patients
[25]. PRL, which stimul ates the Rho signalling pathway
to promote cell motility a nd invasion [26], is up-
regulated in colorectal cancer and associated with
tumour invasion and metastasis [18]. In the same series
of the patients, our previous studies have demonstrated
that tumours with p53-negative expression (wild t ype
p53), or p73-negative expression, or weak Cox-2 expres-
sion had less local recurrence after RT [16,17,27]. PRL
expression is related to distant recurrence and poor sur-
vival after RT [18]. In further studies of colon cancer
cell lines, we found that, af ter radiation, the antiapopto-
tic ΔNp73 and mitosis factor PRL-3 increase [28] and
the overexpression of ΔNp73b increases the viability of
cell lines and cisplatin induces the degradation of
ΔNp73b in a dose-dependent manner (unpublished
data). All these results indicate that certain biological
factors may be involved in response to therapy in rectal
cancer patients. If we could further confirm the data
obtained from studies of these biological factors in rela-
tion to the clinicopathological issues above as well as
Table 3 Relationship of endosialin expression in stroma and tumour cells of rectal cancer patients
Non-Radiotherapy Radiotherapy
Variables Stroma Weak (%) Stroma Strong (%) *p-value Stroma Weak (%) Stroma Strong (%) *p-value
Tumour cells 0.0001 0.0002
Negative (%) 24 (33) 24 (33) 20 (32) 21 (34)
Positive (%) 3 (4) 22 (30) 2 (3) 19 (31)
Total (%) 27 (37) 46 (63) 22 (35) 40 (65)
* For McNemars test.
Table 4 Endosialin expression in tumour cells in relation to clinicopathological and biological variables in rectal
cancer patients
Variables Non-Radiotherapy p-value Radiotherapy p-value
Negative (%) Positive (%) Negative (%) Positive (%)
Growth pattern 0.01 0.06
Expansive 35 (71) 14 (29) 21 (75) 7 (25)
Infiltrative 7 (39) 11 (61) 12 (50) 12 (50)
Cox-2 0.16 0.10
Weak 22 (58) 16 (42) 20 (77) 6 (23)
Strong 25 (74) 9 (26) 18 (56) 14 (44)
p73 0.01 0.44
Weak 23 (82) 5 (18) 14 (70) 6 (30)
Strong 23 (53) 20 (47) 19 (59) 13 (41)
PRL 0.25 0.02
Weak 14 (74) 5 (26) 16 (84) 3 (16)
Strong 27 (59) 19 (41) 18 (53) 16 (47)
p values for X
2
test; Cox-2: cyclooxygenase-2; PRL: phosphates of regenerat ing liver.
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their pathways in therapies, we may be able to apply
them to clinical practices where rectal cancer patients
may receive individual therapies based on their biologi-
cal profile. For example, the targeting of mu ltipl e biolo-
gical factors instead of only one in certain therapies may
yield greater responses to them.
Radiated stromal fibroblasts in the carcinogenic pro-
cess have been shown to induce sub-lethal DNA
damage. Radiation-induced alterations of the stroma
have been found to produce more mammary carcinomas
compared to non-radiated stroma [29]. All of these
results hinted that the patients with up-r egulated
expression of endosialin in a tumour-associated stroma
may be linked to other biological variables that are
related to aggressive c haracterization after radiation.
However, we did not find that endosialin expression was
further r elated to survival in either the whole group or
subgroups (with or without RT) of patients. This may
be partly due to the limited number of the patients and/
or the hypothesis that endosialin may play a survival
role in certain groups o f the patients. It would be inter-
esting to determine the survival significance of endosia-
lin in subgroups where the endosialin is relat ed to the
clinicopathological variables,suchasTNM,Cox-2,p73
and PRL. We did find that endosialin presented different
results in each subgroup, but firm conclusions are diffi-
cult to draw at this point due to the limited number.
In the present study, we also observed endosialin
expression in tumour cells. Po sitive endosialin expres-
sion in tumour cells was more frequently observed in
tumours with infiltrative growth pat tern compared to
expansive growth p attern, regardless of RT. Endosialin
was positively related to p73 expression in the non-RT
group, and to PRL expression in the RT group. Why
was stromal endosialin positively related to p73, PRL
and Cox-2 in the RT-group, but not in the non-RT
group?Onespeculationmaybethatradiationinflu-
enced biological factors by up-regulating or down-
regulating expression. After radiation, bad factors,
such as Cox-2, p73, and PRL-3 increased their expres-
sion. This indicates that there may be some mech anism
by which tumors try to protect themselves by increasing
the expression of bad factors against the damage of
radiation. Furthermore, why did stro mal endos ialin and
tumour cellular endosialin have different relationships
with biological factors (p73 and PRL)? For example,
stromal endosialin was related to p73 expression in the
RT group, whereas tumour cellular endosialin was
related to p73 in the non-RT group. One possible expla-
nation may be that the effects of radiation on stroma
differ on different types of tumour cells, resulting in dif-
ferent expression and relat ionships to the biological fac-
tors investigated. In addition, the p73 gene contains two
promoter regions, giving rise to a p53-like protein
named TAp73, and the N-terminally t runcat ed ΔNp73,
which lacks the transact ivation domain and p53 homo l-
ogy. ΔNp73isthoughttohavearegulatoryfunction,
down-regulating both TAp73 by protein-protein interac-
tion and p53 by competitive binding with DNA. In this
autoregulatory l oop, ΔN p73 protein is also up-regulated
by both TAp73 and p53. The functional cooperation
among these family members seems to vary depending
on cell types, stimuli and p53 status [30]. In other
words, the roles of the two isoforms may depend on
their locations in the stroma or tumour cells.
Recent studies have raised th e concept of the coevolu-
tion of t umour cells with tumour-associated stroma.
The stromal environment of tumour s appears to be a
leading factor, and not just a supporting one in the
initiation of tumours [31]. The tumour microenviron-
ment and interactions between tumour and stromal
cells have a reciprocal relationship in tumour develop-
ment and progression. Another interesting c oncept is
epithelial-mesenchymal transition (EMT), a process by
which cells l ose their polarized epithelial structures and
concomitantly acquire a migratory or mesenchymal phe-
notype. EMT is essential for normal em bryonic develop-
ment and progression of non-invasive adenomas into
malignant, metastatic carcinomas. Alterations in cell-cell
adhesion, cell-substrate interaction, extracellular matrix
degradation and cytoskeleton organization are the major
events that occur during EMT [32]. Overexpressed thy-
mosin b4(Tb4) induces EMT in colorectal carcinoma
by increasing integrin-linked kinase (ILK) complex for-
mation with particularly interesting new cysteine-
histidine rich protein (PINCH) [32]. We have studied
PINCH expression in colorectal carcinomas and found
that PINCH overexpressed on fibroblasts in the tumour-
associated stroma compared to its expression in normal
mucosa [33], similar to e ndosialin expression in tumour
versus normal mucosa. In the present study, endosialin
was expressed in the both tumour-associated stroma
and tumour cells. Furthermore, endosialin expression in
the tumour-associated stroma was positively correlated
with that in tumour cells, giving more information
regarding the interactions of endosialin between tumour
microenvironments and tumours. Changes in signal
conduction related to endosialin appear to play an
important role in enhancing tumour pr ogression after
radiation. There was evidence that antiangiogenic thera-
pies targeting both endothelial cells and pericytes were
more effec tive than single-agent t herapies [34 ]. In the
present study, endosialin was not only obviously up-
regulated in stroma but also up-regulated in tumour
cells when compared to normal mucosa. If the therapies
target endosialin in both stroma and tumour cells, t hey
may provide more efficient strategies of therapy for the
patients with rectal cancers.
Zhang et al. BMC Cancer 2011, 11:89
http://www.biomedcentral.com/1471-2407/11/89
Page 7 of 9
Page 7
Regarding the discrepancies of endosialin localization,
in stroma and/or in tumour cells found in different stu-
dies, several factors may be responsible, including the
number and clinicopathological characteristics of the
patients included in such studies, as well as the methods
used in the s ame. For example, if tissue arrays were
used for staining endosialin [13], it is possible that a
limited sample of tissue was obtained from the tumour
blocks and the selected arrays may not be representative
of the complete characteristics of the tumour because of
tumour heterogeneity. In the present study, we used
ordinary sections for staining endosialin and observed
that only 34% of the cases showed positive endosialin
expression in tumour ce lls. In fact, in some positive
cases, only a few po sitive tumour cells were determined
in the entire tumour sections. In addition, s ome studies
which em ployed real-time PCR or quantitative real-ti me
PCR methods could not determine the location of the
expression [20,21]. In the present study, we used immu-
nohistochemical staining, which is one of the best meth-
ods of identifying the location of endosialin expression.
Conclusions
Endosialin expression may be involved in the progres-
sion of rectal cancers. It is also r elated to Cox-2, p73,
and PRL expression. Ho wever, a direct relationship
between endosialin expression and RT responses in
patients was not found.
Acknowledgements
The authors are grateful to Prof. Clare M. Isacke and Dr. Nicole Simonavicius
for providing the endosialin antibody. The study was supported by grants
from the Service Centre for Experts and Scholars of Hebei Province of China
to ZYZ and the Swedish Cancer Foundation and the Health Research
Council of the South-East of Sweden to XFS.
Author details
1
Department of Oncology, Institute of Clinical and Experimental Medicine,
Linköping University, Linköping, S-581 85, Sweden.
2
Department of
Pathology, Tangshan Gongren Hospital, Tangshan, PR China.
3
Laboratory
Centre, First Hospital of Hebei Medical University, Shijiazhuang, PR China.
4
Division of Biomedicine, School of Life Science, University of Skövde,
Skövde, Sweden.
5
Department of Oncology, Karolinska University Hospital,
Stockholm, Sweden.
Authors contributions
ZYZ conducted the experiments, analyzed the results with XFS, and wrote
the drafts of the manuscript. ZYZ and HZ read the immunohistochemical
slides. AG provided the samples for the experiments and information
regarding the therapy. XFS designed the experiments and helped write the
manuscript. All authors have read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 29 October 2009 Accepted: 1 March 2011
Published: 1 March 2011
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Pre-publication history
The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1471-2407/11/89/prepub
doi:10.1186/1471-2407-11-89
Cite this article as: Zhang et al.: Endosialin expression in relation to
clinicopathological and biological variables in rectal cancers with a
Swedish clinical trial of preoperative radiotherapy. BMC Cancer 2011
11:89.
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Zhang et al. BMC Cancer 2011, 11:89
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  • Source
    • "Colorectal cancer (CRC) is the fourth most common cancer in men and the third most common in women[1]. Rectal cancer is a frequent presentation, with an estimated 35% of cases found situated in the rectum [2]. Different factors like environmental factors, genetic factors such as aberrant DNA methylation, behavioral and metabolic risk factors have been suspected in etiology and outcome of this tumor[3]. "
    Full-text · Article · Jan 2016
  • Source
    • "Since the discovery of CD248 [45] , clinical and genetic evidence has pointed to it as a promoter of tumor growth and inflammation (reviewed in [2]). Increased expression of CD248 is detected in stromal cells surrounding most tumors, and high levels often correlate with a poor prog- nosis [20,23]. Means of interfering with the tumorigenic effects of CD248 have eluded investigators due to a lack of knowledge surrounding the regulation of CD248. "
    [Show abstract] [Hide abstract] ABSTRACT: CD248 is a cell surface glycoprotein, highly expressed by stromal cells and fibroblasts of tumors and inflammatory lesions, but virtually undetectable in healthy adult tissues. CD248 promotes tumorigenesis, while lack of CD248 in mice confers resistance to tumor growth. Mechanisms by which CD248 is downregulated are poorly understood, hindering the development of anti-cancer therapies. We sought to characterize the molecular mechanisms by which CD248 is downregulated by surveying its expression in different cells in response to cytokines and growth factors. Only transforming growth factor (TGFbeta) suppressed CD248 protein and mRNA levels in cultured fibroblasts and vascular smooth muscle cells in a concentration- and time-dependent manner. TGFbeta transcriptionally downregulated CD248 by signaling through canonical Smad2/3-dependent pathways, but not via mitogen activated protein kinases p38 or ERK1/2. Notably, cancer associated fibroblasts (CAF) and cancer cells were resistant to TGFbeta mediated suppression of CD248. The findings indicate that decoupling of CD248 regulation by TGFbeta may contribute to its tumor-promoting properties, and underline the importance of exploring the TGFbeta-CD248 signaling pathway as a potential therapeutic target for early prevention of cancer and proliferative disorders.
    Full-text · Article · Feb 2014 · BMC Cancer
  • [Show abstract] [Hide abstract] ABSTRACT: Particularly interesting new cysteine-histidine-rich protein (PINCH) was found to be up-regulated in the stroma of colorectal carcinomas (CRCs) in our previous studies and was involved in angiogenesis through activation of fibroblasts in extracellular matrix (ECM) in response to tumors. Here, we examined PINCH mRNA expression in colorectal cancer and investigated its relationship with the clinicopathological features and proliferation cell nuclear antigen (PCNA), vascular endothelial growth factor (VEGF) and FAS. The primary cancer tissues, adjacent noncancerous tissues and the proximal and distant margins of normal mucosa were collected from 81 colorectal cancer patients during surgery. PINCH, PCNA, VEGF and FAS mRNA expression was examined by reverse transcriptional PCR (RT-PCR). PINCH mRNA expression was significantly increased in primary tumors compared with that in adjacent noncancerous tissues, and the proximal and distant margins of normal mucosa (p<0.0001). Expression of PINCH mRNA in colon cancer tended to be higher than expression in rectal cancer (p=0.051). Tumors which had infiltrated through the wall of the colorectum trended to have higher PINCH mRNA expression (p=0.073). PINCH mRNA expression in primary tumors was positively related to the expression of PCNA mRNA (r=0.534, p=0.010), VEGF mRNA (r=0.431, p=0.022) and FAS mRNA (r=0.542, p=0.012). PINCH mRNA was overexpressed in colorectal cancer and associated with PCNA mRNA, VEGF mRNA and FAS mRNA expression. PINCH mRNA was involved in the development of colorectal cancer and might play a role in the epithelial mesenchymal transition in the rectum differently than in the colon, through the adenomatous polyposis coli (APC)/catenin pathway.
    No preview · Article · Dec 2011 · Anticancer research
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