2. 124 I. Nurlaila et al. / Immunology Letters 156 (2013) 123–126
tumour cells within tumour nests, in TMC and AMC as a paired
model for studying tumour immunity.
2. Materials and methods
2.1. Tumour specimens
TMC and AMC samples were obtained from the Department of
Pathology’s archive at the Raja Isteri Pengiran Anak Saleha (RIPAS)
Hospital Brunei Darussalam in the form of formalin-fixed paraffin
embedded (FFPE) blocks from surgical resections performed in the
period 2004–2011 [10]. The FFPE blocks were cut into 4 m thick
sections using a microtome, affixed to silanized slides (Electron
Microscopy Science, USA) and prepared for immunohistochemical
staining as described [10].
2.2. Detection of CD8+ cells
Haematoxylin and eosin was used as a counter-stain for
immunohistochemical staining and to examine the morphological
features of TMC and AMC as previously described [10]. CD8+ cells
were identified by immunohistochemical staining using a mon-
oclonal mouse anti-human CD8 antibody clone C8/144B (Dako,
Germany) followed by a secondary goat anti-mouse IgG conju-
gated to peroxidase-labelled polymer solution (Dako, Denmark).
Diaminobenzidine was then used as a peroxidase substrate. The
TMC and AMC specimens were derived from six and eight dif-
ferent tumours respectively. Between five and seven fields were
examined per tumour under a light microscope (Olympus DP25,
Japan) at 600× magnification. Positively stained lymphocytes were
enumerated as a proportion of all lymphocytes in the same fields.
2.3. Detection of apoptotic cells
Apoptotic cells were detected by immunohistochemical stain-
ing with a rabbit monoclonal antibody to cleaved caspase-3
(Cell Signaling Technology, UK), followed by a secondary F(ab )2
peroxidase-conjugated goat antibody to rabbit IgG (Santa Cruz
Biotechnology, USA). The primary antibody detected the large frag-
ment of activated caspase-3 resulting from cleavage adjacent to
Asp175. A blocking peptide of cleaved caspase-3 (Cell Signaling
Technology, UK) was pre-mixed with the primary antibody before
application in order to establish the specificity of the primary anti-
body. Etoposide-treated Jurkat cell lines (Cell Signaling Technology,
UK) were employed as positive controls.
Apoptotic cells were enumerated by light microscopy (Olympus
DP25, Japan) at 600× magnification in six different TMC and five
different AMC samples. Between five to seven randomly selected
fields containing tumour nests were examined in each sample and
the percentage of apoptotic lymphocytes in contact with tumour
cells or apoptotic tumour cells in contact with lymphocytes in the
respective total lymphocyte or tumour cell populations in the fields
determined.
2.4. Statistical analysis
A two-tailed non-parametric Mann Whitney U-tailed test was
performed to determine the significance of differences between the
proportions of CD8+ and apoptotic cells in TMC and AMC.
3. Results
3.1. CD8+ lymphocytes in tumour nests and lymphocyte tracts
The CD8+ cells were abundant in both TMC and AMC (Fig. 1).
However, instead of being evenly distributed within the lympho-
cyte tract, CD8+ cells tend to accumulate in the periphery of the
tracts adjoining tumour nests (Fig. 1).
The mean percentages of CD8+ cells in the lymphocyte tracts
of TMC and AMC were 33.4% ± 6.6 (mean ± standard deviation)
and 25.1% ± 11.3 respectively. The results were not significantly
different (z = −1.48, p = 0.14). However the percentage of CD8+ lym-
phocytes within tumour nests of TMC (66.4% ± 14.3) was higher
than AMC (45.0% ± 16.2) with the difference being statistically sig-
nificant (z = −2.26, p = 0.02).
3.2. Apoptosis in tumour nests
Etoposide-treated Jurkat cell lines show prominent apoptotic
cells, indicated by dense brown staining (Fig. 2A). Fig. 2C and D
show apoptotic cells in representative experimental samples of
TMC and AMC respectively. In an AMC sample used as a negative
control, where cleaved caspase-3 blocking peptide was preincu-
bated with the primary antibody, some cells show characteristic
hyperchromicity, nuclear shrinkage and the presence of apoptotic
bodies (arrowed in Fig. 2B) but no brown staining (Fig. 2B). This
result confirms the specificity of detection of activated caspase-3.
The results also provide evidence to suggest that direct con-
tact between tumour cells and lymphocytes within the tumour
nest may lead to apoptosis. Fig. 3A shows an apoptotic tumour
cell (arrowed) in direct contact with an adjacent lymphocyte. Its
nucleus is condensed and hyperchromatic with a shrunken shape.
Fig. 3B shows apoptosis in two adjacent lymphocytes (arrowed)
that are in contact with a tumour cell. The upper lymphocyte has
Fig. 1. Localization of CD8+ lymphocytes in TMC and AMC. Panels show the location of CD8+ lymphocytes stained brown in (A) TMC and (B) AMC (100×). The full and dotted
lines delineate lymphocyte tracts (lt) and tumour nests (tn).
3. I. Nurlaila et al. / Immunology Letters 156 (2013) 123–126 125
Fig. 2. Apoptotic cells in TMC and AMC. Apoptotic cells stained brown were detected using a primary rabbit monoclonal antibody to cleaved caspase-3 in (A) Etoposide-
treated Jurkat cells as a positive control (200×), (B) AMC sample reacted with primary antibody that was pre-treated with cleaved caspase-3 blocking peptide as a negative
control (200×), (C) TMC (400×) and (D) AMC (400×). In the AMC sample used as a negative control (B), apoptotic tumour cells are morphologically observed (black arrows)
but no staining for cleaved caspase-3 is seen in them.
Fig. 3. Contact-mediated apoptosis of tumour cells and lymphocytes in TMC and AMC. The panels show apoptosis in a tumour cell in contact with a lymphocyte (A) and two
lymphocytes in contact with a tumour cell (B) at 600× magnification. The apoptotic cells are indicated with white arrowheads.
undergone nuclear shrinkage and condensation and a decrease in
size. The other lymphocyte has already formed an apoptotic body
since its organelles, especially the nucleus, has degraded into small
granules.
Apoptotic tumour cells within tumour nests that were in direct
contact with lymphocytes as a percentage of all tumour cells in
the fields examined were 0.8% ± 0.6 (mean ± standard deviation)
and 0.6% ± 0.5 in TMC and AMC respectively. The difference was
not statistically significant (p > 0.05). Apoptotic lymphocytes that
were in direct contact with tumour cells as a percentage of all lym-
phocytes in the fields examined were 7.4% ± 5.9 and 12.5% ± 21.7
in TMC and AMC respectively. The difference was not statistically
significant (p > 0.05).
4. Discussion
Despite the anaplastic cytological feature and high mitotic rate,
the prognosis for patients diagnosed with MBC, particularly TMC,
is better than for other types of DIC [1–8]. A positive correlation
between the intensity of lymphoid infiltration and patient survival
suggests that the immune system may be involved in restraining
the spread of this type of breast cancer [4].
4. 126 I. Nurlaila et al. / Immunology Letters 156 (2013) 123–126
CD8+ CTLs are a component of the adaptive immune system.
The capacity for rapid expansion and the ability of a single CD8+
CTL to destroy more than one target cell, while sparing ‘innocent’
bystanders, make CD8+ CTLs efficient antigen-specific effector cells.
Destruction of target cells by CD8+ CTLs typically requires cell con-
tact mediated by the recognition of peptide antigen presented on
MHC Class 1 molecules on the target cell by the T cell antigen recep-
tor, followed by target cell killing through the release of cytotoxic
granules or Fas/FasL interaction [11]. In a previous study on MBC in
Brunei, a higher proportion of CD8+ lymphocytes and a lower pro-
portion of CD20+ B-lineage cells were observed to be characteristic
of TMC in comparison to AMC [5]. Observations in the present and
previous studies [5,7] are consistent with close contact between
CD8+ lymphocytes and tumour cells in TMC. They suggest that the
better tumour control in TMC might be mediated by CD8+ CTLs.
However, the comparative distribution of CD8+ cells within tumour
nests and lymphocytic tracts of TMC and AMC had previously not
been reported. The present findings suggests that CD8+ lympho-
cytes in lymphocyte tracts tend to localize close to tumour nests
in MBC and that tumour nests of TMC had a significantly greater
percentage of CD8+ lymphocytes among the infiltrating lympho-
cytes than AMC. The lymphocyte tracts in TMC also tended to have
a greater proportion of CD8+ lymphocytes than AMC but statisti-
cal significance could not be established possibly due to the small
number of samples studied. These findings are however consistent
with CD8+ CTLs being responsible for the better tumour control in
TMC compared to AMC.
Apoptosis is a form of programmed cell death that plays an
essential role in many biological processes including normal cell
turnover, immune response, embryonic development and hor-
mone dependent atrophy [12,13]. Apoptosis in tumour cells has
been associated with the better prognosis in TMC [6,7]. A study
of 50 cases of MBC and 50 cases of non-medullary DIC utilizing
the terminal deoxynucleotidyl transferase-mediated dUTP-biotin
nick end-labelling (TUNEL) method for detecting apoptotic tumour
cells and immunohistochemistry for detecting p53, bcl-2, and Ki-
67 showed that tumour cells in MBC had higher apoptotic and
proliferative indices and significantly lower positivity for the anti-
apoptotic protein Bcl-2 than matched DIC controls [6]. A similar
independent study confirmed that tumour cells in both TMC and
AMC had higher apoptotic indices than matched cases of DIC [7].
No study to date has definitively established the cause of increased
apoptosis in MBC.
Taking into account previous observations of close contacts
between CD8+ lymphocytes and tumour cells in TMC [5,7], it was
expected that the better prognosis for TMC compared with AMC,
might be reflected in more tumour cells and the fewer lymphocytes
in TMC than AMC undergoing contact mediated-apoptosis within
tumour nests. However, while apoptosis in lymphocytes in contact
with tumour cells and in tumour cells in contact with lymphocytes
was observed within the tumour nests in both TMC and AMC, the
proportions of apoptotic cells were small and significant differences
could not be established between the two MBC types.
At least two strategies used by tumours to evade rejection by the
immune system are related to apoptosis [9,14]. Firstly, malignant
cells can alter the expression of molecules involved in apopto-
sis signalling, resulting in resistance to immune cell mediated
killing mechanisms. Secondly, tumours may adopt a mechanism
to delete attacking anti-tumour lymphocytes for example through
the expression of Fas ligand (CD95L) [9,14]. Tumour cells can
resist apoptosis at the membrane receptor and intracellular levels
[9,14]. Tumour cells down-regulate membrane Fas receptor (CD95)
expression thus inhibiting engagement with Fas ligand on immune
effector cells that can cause apoptosis in Fas-expressing tumour
cells. At the intracellular level, apoptosis resistance can be caused
by up-regulation of anti-apoptotic molecules or down-regulation
or loss of pro-apoptotic molecules. Higher levels of anti-apoptotic
Bcl-2 for example protects tumour cells against an immune attack
and promotes tumour survival and proliferation [6,13,14]. Details
of such possible mechanisms need to be investigated in parallel in
TMC and AMC. Additional roles for CD4+ T cells [15], other effector
cells such as NK cells [5], and antibodies in tumour immunity also
warrant further comparative investigation in TMC and AMC.
The present study underscores the previously proposed impor-
tance of TMC and AMC as a paired model system for studying
immune mechanisms in cancer [5]. Because Brunei has a popula-
tion of approximately 400,000 persons [10], and therefore only a
limited number of MBC cases, a larger number of samples from a
multi-centre investigation will be helpful in improving the statis-
tical power of such studies.
Conflict of interest statement
The authors declare no conflict of interest.
Acknowledgements
This study was supported by a graduate research scholarship to
IN from the Universiti Brunei Darussalam.
Author contributions: IN performed experiments; PUT provided
tumour specimens; RR and PUT conceived, designed and coordi-
nated the study; IN, RR and PUT drafted the manuscript. All authors
read and approved the final manuscript.
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