Download Harvesting of human material.

Tartrate resistant acid phosphatase: A marker for tumor infiltrating
immune cells in human colon cancer.
Cecilia Bengtssona, Gunnar Nylundb , Amir Khorram-Maneshb, Petra Wievegb,
Svante Nordgrena, and Dick S. Delbroa,c*
a
Department of Surgery, Sahlgrenska University Hospital, SE-413 45 Göteborg, Sweden
Department of Surgery, Kungälv Hospital, SE-442 83 Kungälv, Sweden
c
School of Pure and Applied Natural Sciences, University of Kalmar, SE-391 82 Kalmar,
Sweden
b
*
Corresponding author.: Tel.: +46 31 342 1235; fax: +46 31 4138 92.
E-mail address: [email protected].
Abstract
Tartrate resistant acid phosphatase (TRAP) is a marker of a subpopulation of activated
macrophages which are expressed in a variety of tissues. Clinically, serum levels of TRAP are
used in order to monitor the progression of diseases that engage bone tissue, like e.g. breast
cancer metastases. It was recently established that TRAP is expressed also breast cancer cells
which was suggested to contribute to their metastatic-osteolytic properties. In the current
study, we investigated whether TRAP expression may be noted also in human colon cancer
cells. We found that such was more or less absent from the HT-29 cell line. In
macroscopically tumor free colon tissue, TRAP expression was found in sub-epithelial
immune cells of the lamina propria. In colon cancer tissue, TRAP expression was restricted to
a subpopulation of infiltrating immune cells. To judge from the current findings, TRAP
appears not to be an important factor for tumor cell properties.
Introduction
Acid phosphatases, enzymes which catalyze the hydrolysis of orthophosphate
monoesters under acidic conditions, constitute a family of isoezymes of which its fifth
member, the tartrate resistant acid phosphatase (TRAP; EC 3.1.3.2), is of particular clinical
interest. TRAP is since many years a diagnostic feature of hairy-cell leukaemia (Hoyer et al.,
1997). Moreover, TRAP has been found to serve as a marker of bone engagement in various
diseases including cancer, since increases in osteoclast activity leads to the synthesis and
secretion of TRAP to the circulation and thereby increased serum TRAP. Elevated TRAP
concentrations can be demonstrated when malignant tumors metastazise to the skeleton.
Furthermore, TRAP expression on the mRNA, and protein levels have been demonstrated in
tumor cells from human breast, and ovarian cancer as well as melanoma, and also in
established cell lines from these kinds of tumors (Honig et al., 2006). In a very recent
immunohistochemical study, Adams et al. (2007) noted that while TRAP was expressed in human
breast cancer cell lines and tumor cells of human breast cancer, there was no expression in benign
breast tissue.
TRAP appears also to be a marker for a subpopulation of activated macrophages and dendritic cells
(Janckila et al., 2006) and has recently been noted to increase markedly in number in the lamina
propria of rat colon mucosa in the early phase of acute, experimental colitis (Lång, Lange, Delbro and
Andersson, in preparation). TRAP is expressed on the mRNA and protein levels in human colon but
its specific localization is insufficiently described (Hayman et al., 2001). It is, moreover, unknown
whether TRAP is expressed also in human colon cancer. In the current study, we have addressed
whether TRAP protein may be demonstrated by immunocytochemistry in the HT-29 human colon
cancer cell line, as well as by immunohistochemistry in colon cancer tissue and in adjacent,
macroscopically tumor free colon tissue.
Materials and methods
This study was approved by the local Ethics Committee of the Göteborg University.
Cell culture
The HT-29 (ATCC HTB 38; a kind gift from the laboratory of Prof. K. Lundholm,
Department of Surgery, Sahlgrenska University Hospital, Göteborg, Sweden) is a human
colon cancer cell line derived from an adenocarcinoma. The cells were maintained in culture
in McCoy’s 5a medium (Invitrogen, Stockholm, Sweden) supplemented with 1%L-glutamine
(Bio Whittaker Europe, Verviers, Belgium) and 1% penicillin–streptomycin (Invitrogen) in
the presence of 4% fetal calf serum (FCS, Invitrogen). A split ratio of 1:8 once weekly was
chosen, with a change of medium in between (McCoy’s 5a plus 2% FCS). Cells were renewed
after approximately 22 passages. For the experiments, the cells were seeded in chamber slides
(2 ml; 100,000 cells/ml). On day 5 following seeding, the cells were prepared for
immunocytochemistry. The experiments were repeated at least three times, unless specified
otherwise.
Immunocytochemistry
Before fixing the cells with phosphate-buffered formaldehyde, pH 7.4 (Substratavdelningen,
Sahlgrenska University Hospital, Göteborg, Sweden) for 25 min, the cells were washed twice
with PBS. Fixation was followed by a rinse (3-5 min) in TBS. Endogenous peroxidase was
blocked with 0.3% hydrogen peroxidase (VWR International, Stockholm, Sweden) in
methanol (Merck, Stockholm, Sweden) for 30 min followed by the blocking of unspecific
protein binding with 2% normal horse serum PK6200 (ImmunKemi, Järfälla, Sweden) for 1 h
in a moist chamber. The primary antibody: monoclonal mouse antibody to human TRAP (Lab
Vision, Freemont, Calif. USA; 1:20) diluted in horse serum was added and incubated in a
moist chamber at 4C over-night. The following day, the slides were washed in TBS (2 x 5
min), incubated with the secondary antibody for 30 min (also in a moist chamber), followed
by a second wash in TBS (2 x 5 min). For staining, the slides were incubated with the ABCreagents (Vectastain Elite ABC; Vector Laboratories, Burlingame, CA) for 30 min in a moist
chamber, again rinsed with TBS (2 x 5 min). Positive immunoreactivity was visualised with
3-diaminobenzidine tetrahydrochloride (DAB, DakoCytomation), resulting in brown staining.
The color reaction was stopped after about 8 min by a rinse in water for 10 min. The cells
were then counterstained with Mayer’s hematoxylin (Histolab, Göteborg, Sweden) for about
1–2 min, rinsed in water for 5 min before being mounted with Faramount Aqueous Mounting
Medium (DakoCytomation); they were then photographed under a light microscope (Nikon
Eclipse E400 & Nikon Digital Camera DXM 1200; Upplands Väsby, Sweden). Negative
controls were performed either by excluding the primary antibody and incubating the cells
instead with horse serum, resulting in no immunoreactivity.
Harvesting of human material.
Fourteen patients, 6 women (mean age: 74, range 65-82 years, 3 Dukes B and C, respectively)
and 8 men (mean age: 73, range 43-92 years, 4 Dukes B and C, respectively) underwent
surgical resection for colon tumours. All patients included in this study had their surgical
procedures performed at Kungälv Hospital, Kungälv, Sweden. They were treated on an
elective basis. For all patients, full-thickness samples of both neoplastic and macroscopically
tumour-free bowel wall, the latter resected 10 cm from the tumour, were dissected
immediately upon colonic resection. The portion of the tumour analysed was obtained from
the edge of the tumour, representing a viable area devoid of necrosis. The specimens were
immersed in 4% phosphate-buffered formaldehyde (pH 7.0, Apoteket AB, Sweden) for 24 h
and were then embedded in paraffin and were processed for immunohistochemistry.
Immunohistochemistry. Sections of 4 m thickness were cut. After de-paraffination, the tissue
sections were immersed in 10mM citrate buffer pH 6, and were placed in a microwave oven at
max power (2x5min). Next, endogenous peroxidase was blocked with 0.3% hydrogen
peroxide (VWR) in methanol (Merck) for 30 min. Non-specific protein binding was blocked
with 2% normal horse serum PK6200 (Immunokemi) for 60 min. The sections were incubated
with the monpclonal, mouse anti-TRAP antibody (see above; 1:20) over-night in a moist
chamber. Next, DAKO-Envision (DakoCytomation, Stockholm, Sweden) was applied for 30
min (also in a moist chamber), followed by a second wash in TBS (2-5 min). Positive
immunoreactivity was visualised with DAB (DakoCytomation), as above. The tissues were
counterstained with Mayer’s hematoxylin (Histolab). The tissue sections were mounted with
Pertex Mounting Medium (Histolab) and were then photographed under a light microscope as
above. Controls were performed as reported for immunocytochemistry (above).
Results
TRAP is very weakly expressed in HT-29 cells.
The antibody to TRAP produced very weak immunoreactivity in the HT-29 cells, seemingly
in the cytoplasm (Fig. 1b).
Fig. 1a. Control. HT-29 human colon Fig. 1b. Demonstration of TRAP immunocancer cells treated with secondary
reactivity. Antibody concentration: 1:20.
antibody only. Bar is 25m.
Bar is 25m.
TRAP is expressed in macroscopically tumor free, human colon tissue.
In Fig. 2 is may be seen that TRAP immunoreactivity is localized in immune cells of the
upper portion of the lamina propria.
Fig. 2. Immunohistochemical demonstration
of TRAP immunoreactivity in macroscopically
tumor free colon tissue. Bar is 50 m.
TRAP is expressed in the tumor stroma, in human colon cancer.
Fig. 3 demonstrates that TRAP is expressed in a subset of aggregations of tumor
infiltrating immune cells, while the expression in tumor cells is absent.
Fig. 3. Demonstration of TRAP immunoreactivity
in human colon cancer tissue. Bar is 50 m.
Discussion
This is the first demonstration of TRAP expression in human colon cancer. Somewhat
surprising, the expression in the HT-29 cells was very weak which is a contrast to the findings
in the human breast cancer cell lines as investigated by Honig et al. (2006) and Adams et al.
(2007). These authors found that in addition to the well-known finding that osteoclasts
produce TRAP, this enzyme is synthesized also by breast cancer cells, seemingly related to
the aggressiveness of the tumor. In a still unknown manner, TRAP may contribute to the
metastatic and osteolytic action of the breast cancer cells (Adams et al., 2007). It should be
noted, that the antibody used by Adams et al. (2007) is the same as in the current study.
Skeletal metastases of colorectal cancer are uncommon (Kanthan et al., 1999). It is tempting
to speculate that the difference between the two cancer types with regard to TRAP expression
may influence their respective tendency to metastasise to bone tissue.
In keeping with the very weak TRAP expression in the HT-29 cells, the tumor cells of the
colon cancer tissue did not exhibit TRAP immunoreactivity, while such was observed in a
subpopulation of tumor infiltrating mononuclear cells. To judge from previous reports
(Janckila et al., 2007), it may appear likely that these TRAP immunoreactive cells constitute
activated macrophages. The role for such cells for carcinogenesis and cancer progression is
obscure. Thus, one the one hand, tumor associated macrophages of the type 2 phenotype
participate in cancer progression by e.g. assisting angiogenesis and down-regulation of the
immune response to the tumor (Mantovani et al., 2006). On the other hand, in some tumors,
notably including colorectal cancer, the occurrence of tumor infiltrating macrophages are
linked to a favourable prognosis (Shunyakov et al., 2004).
In conclusion, we have demonstrated that TRAP is expressed in human colon cancer in a
subpopulation of immune cells of the tumor stroma, but the biological significance of this
finding awaits further studies in order to be elucidated.
Acknowledgements
The present study was supported by the Anna-Lisa and Bror Björnsson Foundation (D.
Delbro), the Assar Gabrielsson Foundation (D. Delbro), the Göteborg Medical Society (A.
Khorram-Manesh, G. Nylund), the King Gustaf V Jubilee Clinic Foundation (D. Delbro), the
LUA-ALF agreement (S. Nordgren), the University of Kalmar (D. Delbro) and the Västra
Götalandregionen (G. Nylund). The authors are grateful to Dr. F. Aldenborg for invaluable
advice and suggestions, to Prof. K. Lundholm for the gift of the HT-29 cells, and to Ms.
Lilian Karlsson for expert technical assistance.
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