Leukocyte composition of human breast cancer
Brian Ruffella, Alfred Aua,b, Hope S. Rugob,c, Laura J. Essermanb,d, E. Shelley Hwangb,d, and Lisa M. Coussensa,b,1
aDepartment of Pathology andbHelen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA 94143; and Departments
cMedicine anddSurgery, University of California, San Francisco, CA 94115
Edited by Kornelia Polyak, Dana–Farber Cancer Institute, Boston, MA, and accepted by the Editorial Board July 13, 2011 (received for review March 17, 2011)
Retrospective clinical studies have used immune-based biomarkers,
alone or in combination, to predict survival outcomes for women
with breast cancer (BC); however, the limitations inherent to
immunohistochemical analyses prevent comprehensive descrip-
state of leukocytes in BC stroma. To more fully evaluate this com-
plexity, and to gain insight into immune responses after chemo-
therapy (CTX), we prospectively evaluated tumor and nonadjacent
normal breast tissue from women with BC, who either had or had
not received neoadjuvant CTX before surgery. Tissues were evalu-
immunofluorescence and immunohistochemical analysis of tissue
sections. These studies revealed that activated T lymphocytes pre-
dominate in tumor tissue, whereas myeloid lineage cells are more
prominant in “normal” breast tissue. Notably, residual tumors from
an unselected group of BC patients treated with neoadjuvant CTX
contained increased percentages of infiltrating myeloid cells, ac-
of granzyme B-expressing cells, compared with tumors removed
from patients treated primarily by surgery alone. These data pro-
vide an initial evaluation of differences in the immune microenvi-
the degree to which CTX may alter the complexity and presence of
CD20+B cells, and multiple myeloid-lineage cells including
tumor-associated macrophages (TAMs) that are often identified
by immunohistochemical (IHC) detection of CD68 (1). High
lymphocyte infiltration is associated with increased survival in
patients <40 y of age (2) and with a favorable prognosis in
subsets of patients whose tumors are also heavily infiltrated by
TAMs (3). More specifically, large cohort studies of patients with
BC have revealed that the presence of CD68+cells in tumor
tissue correlates with poor prognostic features (4–6), higher tu-
mor grade (7–9), increased angiogenesis (10–13), decreased
disease-free survival (6, 11, 14, 15), and increased risk for sys-
temic metastasis when assessed in conjunction with endothelial
and carcinoma cell markers (16).
The functional significance of specific leukocytes in BC de-
velopment has been implied based on experimental studies using
homozygous null mutations in genes specifying leukocyte de-
expressing the polyoma virus middle T antigen regulated by the
mouse mammary tumor virus promoter (MMTV-PyMT mice),
progression of mammary carcinomas and metastases to lungs are
a cytokine critical for macrophage maturation and recruitment
vasculature (19), where their production of VEGFA fosters an-
giogenic programming of tissue (20, 21), and their production of
EGF promotes invasive tumor growth and subsequent metastases
(22, 23). Moreover, TAMs regulated by epithelial CSF1 express
that, in turn, mediate suppression of anti-tumor immunity by
everal subtypes of CD45-expressing leukocytes infiltrate
breast cancer (BC), including CD4+and CD8+T cells,
TAM presence and bioactivity within mammary tumors corre-
spond to their clinical activity, further indicating the importance
of TAMs, not only in promoting tumor development, but also in
suppression of anti-tumor immunity.
CD4+T cells isolated from human BC produce high levels of
type II helper (TH2) cytokines including IL-4 and IL-13 (25, 26),
which are significant in light of studies demonstrating that several
protumor activities of TAMs are regulated by IL-4 derived from
that infiltration by CD68+, CD4+, and CD8+immune cells in
human BC is predictive of overall survival, and that the ratio of
CD68 to CD8a mRNA in tumor tissue correlates with complete
pathologic response (pCR) in patients undergoing neoadjuvant
chemotherapy (CTX) for early stage BC (6). Despite the clear
correlation between these specific immune cell types and BC
clinical outcome, leukocyte complexity within tumor tissue
remains poorly described, with most studies relying on single-
marker IHC detection. Furthermore, although some studies have
examined the effects of CTX on the presence and function of
circulating peripheral blood leukocytes (28), data regarding the
effect of CTX on tumor-infiltrating immune cells are limited (29).
Herein, we evaluated leukocytic infiltrates in breast tissue
from predominantly hormone receptor positive patients who
had, or had not, received CTX before definitive surgery. In CTX-
naïve patients, we found that activated T lymphocytes comprised
the majority of immune cells within tumors, whereas myeloid-
lineage cells predominate in nonadjacent normal breast tissue. In
contrast, tumors from patients with residual disease after neo-
adjuvant CTX contained higher levels of infiltrating myeloid
cells, with a simultaneous shift away from a TH2 dominated
Increased Presence of T Cells in Tumor Tissue. To evaluate the
composition of tumor-infiltrating leukocytes in human BC,
tumors from 20 patients were evaluated by polychromatic flow
cytometry and IHC detection of leukocyte lineages in tissue
sections as described in Materials and Methods. Nine invasive
ductal carcinomas (IDC) and five invasive lobular carcinomas
(ILC)—mostly histological grade two or three—were obtained
from patients with no prior exposure to CTX (CTX-naïve) at the
time of primary surgery for early stage BC, although one patient
had received neoadjuvant tamoxifen. Six tumor samples were
obtained from patients previously treated with neoadjuvant CTX
before resection (CTX-treated), consisting entirely of grade two
or three IDC. Notably, three of six CTX-treated tumors were
HER2/neu-positive, compared with only 1 of 14 CTX-naïve
tumors, whereas both groups contained roughly equivalent per-
centages of tumors negative for estrogen, progesterone, and
HER2 receptors (triple negative). Details of tumor pathology
are outlined in Table S1. Ipsilateral nonadjacent tissue was also
obtained from seven CTX-naïve and four CTX-treated patients
Author contributions: B.R. and L.M.C. designed research; B.R. performed research; A.A.,
H.S.R., L.J.E., and E.S.H. contributed new reagents/analytic tools; B.R. and L.M.C. analyzed
data; and B.R. and L.M.C. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. K.P. is a guest editor invited by the Editorial
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
www.pnas.org/cgi/doi/10.1073/pnas.1104303108 PNAS Early Edition
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BREAST CANCER SPECIAL
for use as “normal” tissue, in addition to tissues from two con-
tralateral prophylactic mastectomies from patients with ipsilat-
eral ductal carcinoma in situ (DCIS).
were present in both normal and tumor tissue, but with substantially
by using a combination of lineage markers to identify specific sub-
populations (Figs. S1 and S2), with the complexity of these pop-
ulations shown in Fig. 1B as a percentage of the total number of
CD45+cells in each sample. BC tissues from CTX-naïve patients
contained infiltrates dominated by T lymphocytes (CD3ε+), with
minor populations of natural killer cells (CD3ε−CD56+NKG2D+)
and B lymphocytes (CD19/20+HLA-DR+CD3−). In comparison,
DR+), mast cells (FcεR1α+CD117+CD11b−CD49d+) and neu-
tissuefromthesepatients.A similar immuneprofile wasobservedin
breast tissues obtained from the two prophylactic mastectomies
Increased Presence of Myeloid-Lineage Cells in Residual Tumors from
Patients Exposed to Neoadjuvant CTX. Comparative analysis of
residual BC tissue removed from patients after neoadjuvant CTX
revealed an obvious difference in the percentages of myeloid-
lineage cells compared with the CTX-naïve group. With some
exceptions, this difference included an increased presence of mac-
rophages as a percent of total leukocytes (Fig. 2A), as well as by
densityevaluationofCSF1 receptor(CSF1R)-positivecells intissue
by IHC (Fig. 2B). Increased percentages of mast cells (Fig. 2C) and
neutrophils (Fig. 2D) were also evident in most CTX-treated
patients, with an ≈14-fold increase in CTX-treated versus CTX-
naïve groups. Basophils (FcεR1α+CD117−CD11b−CD49d+; Fig.
2E) were highly increased in only one of six CTX-treated samples,
whereas the percentage of myeloid dendritic cells (CD11c+HLA-
DR+CD14lo/-; Fig. 2F) was unchanged. Evaluation of plasmacytoid
dendritic cells expressing CD85g/ILT7 detected an insufficient
number of events for analysis. Thus, with the exception of baso-
phils, dendritic cells, and CD15+CD11b+CD49d+eosinophils—
which were present just at a detectable level in the tissues ex-
amined—increased presence of myeloid-lineage cells typified
residual tumors of women treated with neoadjuvant CTX.
CD68 Is Not a Macrophage-Specific Marker in Human BC. Macro-
phages are well established as regulators of murine mammary
tumorigenesis (30), where they can represent up to 80% of
leukocytes present within late stage mammary carcinomas (1). In
human BC, immunoreactivity for CD68 has been used exten-
sively for identification of macrophages, with CD68+cell density
associated with reduced overall survival (6, 11, 14, 15).
The high number of CD68+cells reported in the literature, and
shown in Fig. 3A, was in contrast to the limited number of
cytometry in the BC suspensions examined (Figs. 1B and 2A). To
BC tissue sections, compared with CD163 (a hemoglobin scav-
engerreceptor alsocommonlyused asa markerformacrophages)
correlation in cell density among the three markers. We next
evaluated frozen BC tissue sections by confocal microscopy after
immunofluorescent detection of CD68 in combination with
CSF1R or CD45 (Fig. 3B). Although all cells expressing high lev-
els of the CSF1R also expressed CD68, there was a distinct pop-
ulation of CD68+cells that expressed neither CSF1R nor CD45.
CD31+endothelial cells, or smooth muscle actin α-expressing
mural cells surrounding vasculature (Fig. 3C). This expression
contrasted with murine mammary tumors isolated from MMTV-
PyMT transgenic mice (17), where CD68+cells coexpressed
both CSF1R and the murine macrophage marker F4/80 (Fig.
S3). In agreement with historic literature (31, 32), these results
thus indicate that CD68 is not a macrophage-specific marker in
Tumor-Infiltrating T Cells Display an Activated Phenotype. To reveal
the phenotype of T cells infiltrating BCs, we examined surface
marker and chemokine receptor expression of tissue-infiltrating
CD4+and CD8+T cells (Fig. 4 A and B). Specifically, both
CD4+and CD8+T cells displayed increased expression of ac-
tivation markers CD69 and HLA-DR compared with peripheral
eosin (H&E) staining of tissue sections (Left) with representative immunohis-
tochemistry for CD45 (Right) shown for each. (B) Flow cytometric analysis of
leukocyte populations within human breast tumors. Results are shown as
a percent of total CD45+cells with markers used to define specific lineages
Leukocyte infiltration of human breast tumors. (A) Hematoxylin and
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blood T cells, with a corresponding loss of markers for naïve T
cells, CD45RA, and CCR7. Furthermore, although all T cells
constitutively expressed the costimulatory receptor CD28 (Fig.
S4A), expression of CD27, another costimulatory receptor, was
reduced in a large proportion of tissue-infiltrating cells, in-
dicative of shedding after interaction with its ligand CD70 (33)
and potential acquisition of effector functions (34, 35). CD4+
and CD8+T cells also displayed substantially up-regulated ex-
pression of chemokine receptors CCR4 and CCR5 (Fig. 4B), and
although CD8+T cells constitutively expressed CXCR3, tissue-
infiltrating CD4+T cells exhibited higher CXCR3 expression
than their counterparts in peripheral blood. Surface marker ex-
pression by tissue-infiltrating CD4+and CD8+T cells was subtly
different between tumor and benign tissue in some samples;
however, these changes were not consistent across patients, or
between CTX-naïve and CTX-treated groups (Fig. S4B).
Altered Lymphocyte Balance in Residual Tumors After Neoadjuvant
CTX. Although we observed no difference in the percent of
CD3e−CD56+NKG2D+natural killer (NK) cells (Fig. 5A),
higher levels of CD19/CD20+HLA-DR+B cells were evident in
several CTX-naïve tumors compared with both normal tissue
and CTX-treated tumors. As has been reported (36), B cells
were clustered together in association with T cells (Fig. 5C).
Notably, CD4+T cells as a percent of the total CD45+pop-
ulation were also increased in CTX-naïve tumors compared with
both normal tissue and residual postneoadjuvant tumors (Fig.
5D). As the percent of CD8+T cells was unchanged (Fig. 5E),
the lower percentage of CD4+T cells within the CTX-treated
group resulted in an increased CD8 to CD4 ratio (Fig. 5F). Al-
though it was unclear whether the density of CD8+cells in CTX-
treated residual tumors was increased (Fig. 5G), the number of
cells expressing granzyme B was strikingly evident in two of six
CTX-treated tumors (Fig. 5H), whereas minimal granzyme B
staining was observed in CTX-naïve tumors, even in areas with
high numbers of CD8+T cells (Fig. 5I).
Despite the reduced percentage of CD4+T cells in tumors
from CTX-treated patients, there was no change in the density
of IHC detected regulatory T cells expressing FoxP3 (Fig. S5A),
which was specifically expressed by CD3+CD4+cells in the
tumor (Fig. S5B). Gating on CD25hicells, consisting of >80%
FoxP3+cells in all samples tested, also revealed that the relative
percentage of these cells was invariant between groups (Fig.
S5C). Phenotypically, CD4+FoxP3+cells displayed an activated
phenotype with equivalent surface levels of CD45RO and CD69
patients. (A) CD14hiCD11b+HLA-DR+macrophages shown as a percent of
total CD45+cells as determined by flow cytometry. (B) Representative im-
munohistochemistry for CSF1R in tumors from either CTX-naïve (Upper) or
CTX-treated (Lower) patients. Red arrows indicate cells displayed in enlarged
insets. FcεR1α+CD117+CD11b−CD49d+mast cells (C), CD15+CD11b+CD49d−
neutrophils (D), FcεR1α+CD117−CD11b−CD49d+basophils (E), and CD11c+HLA-
DR+CD14lo/−(F) DCs shown as a percent of total CD45+cells. N, nonadjacent
normal; T, tumor.
Increased myeloid-lineage leukocyte infiltration within CTX-treated
tissue. (A) Representative immunohistochemistry within tumors for CD68
(Left), CSF1R (Center), and CD163 (Right) in serial sections from a CTX-trea-
ted patient. Red arrows indicate cells displayed in enlarged insets. (B) Im-
munofluorescent staining of human breast tumors for CD68 (red) in
conjunction with CSF1R (i and ii) or CD45 (iii and iv). (C) Immunofluorescent
staining for CD68 (red) in conjunction with pan-keratin (green; i), or CD31
(green) and smooth muscle actin-α (SMA; purple; ii).
CD68 is not a specific macrophage marker in human breast tumor
Ruffell et al. PNAS Early Edition
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BREAST CANCER SPECIAL
to CD4+FoxP3−cells and, as has been reported for cells in pe-
ripheral blood (37), expressed lower levels of CD127 (Fig. S5D).
Interestingly, although not all FoxP3+cells expressed HLA-DR,
they did comprise the majority of HLA-DR–expressing CD4+T
cells, in addition to coexpressing high levels of CD25.
These data collectively reveal a shift within tumors toward
a TH2-type response in BC characterized by increasedpresence of
B cells and CD4+T cells, in comparison with nonadjacent normal
breast tissue. This shift is reversed in tumors obtained from CTX-
treated patients, with additional evidence of a cytotoxic T-cell
response through a more favorable CD8/CD4 T-cell ratio and
increased presence of granyzme B-expressing lymphocytes; thus,
even residual tumors from patients with a poor response to CTX
may contain immune microenvironments that are more favorably
skewed towards an anti-tumor, TH1-type immune response.
Herein, we present a detailed description of leukocyte com-
plexity in BC as evaluated in a cohort of CTX-naïve patients with
stage 2/3 tumors, compared with patients with significant residual
disease after neoadjuvant CTX. T lymphocytes were the major
population within both CTX-naïve and CTX-treated tumors,
found almost exclusively in an activated state as determined by
increased expression of CD69 and chemokine receptors, with
simultaneous loss of naïve markers CCR7 and CD45RA. The
presence of activation markers, however, does not definitively
demonstrate that intratumoral T cells are functionally active. In
fact, granzyme B expression was minimal within tumors from
CTX-naïve patients, suggesting negligible cytotoxic activity by
infiltrating CD8+T cells. In comparison, granzyme B was highly
expressed in one-third of the CTX-treated tumors, suggestive of
a more cytotoxic T-cell response within some tumors after ex-
posure to CTX.
Importantly, residual tumors from CTX-treated patients also
contained reduced percentages of B cells and CD4+T cells.
Tumor-infiltrating CD4+T cells in BC are known to express the
TH2 cytokines IL-4 and IL-13 concomitantly with the production
of IFN-γ (25, 26), consistent with coexpression of CXCR3 and
CCR4 (38, 39) as we observed herein. It remains to be de-
termined whether cytokine production by CD4+T cells is altered
by neoadjuvant CTX; however, the combined reduction in both
CD4+T cells and B cells is indicative of a favorable shift away
from a TH2 microenvironment. This shift could be relevant for
TAM function, as has been described in the MMTV-PyMT
model where TAMs are programmed by IL-4 toward a TH2
phenotype (1), and more recently in pancreatic ductal adeno-
carcinoma during treatment where an agonist CD40 monoclonal
antibody fostered cytolytic macrophage activities (40).
Although the extent of lymphocyte infiltration has been as-
sociated with improved prognosis in subsets of patients (2, 3),
and with pCR after CTX (41, 42), information regarding the
relationship between individual lymphocyte subsets to survival is
limited. High FoxP3 counts correlate with reduced overall and
relapse-free survival in estrogen receptor (ER)-positive tumors
(43), and pCR to neoadjuvant CTX is associated with reduced
FoxP3 grading (44, 45). Although two studies examining T-cell
infiltration by flow cytometry found conflicting results regarding
the CD8:CD4 ratio and lymph node metastasis (46, 47), the
number of CD8+T cells within tissue has been associated with
improved patient survival (48). We have also reported a CD68/
CD4/CD8 immune signature predicting overall and relapse-free
survival, with inverse correlations evident for CD4 when used in
conjunction with other markers (6). There is thus an urgent need
for additional prospective investigations where multiple param-
eters of lymphocytic infiltration and functionality are evaluated
to determine the most significant biomarker comparisons that
predict outcome and guide specific therapy.
type. (A and B) Representative histograms of CD3+CD4+(Upper)
or CD3+CD8+(Lower) T cells isolated from a single CTX-treated
patient with both normal (blue) and tumor (red) tissue. Ex-
pression of activation markers CD69 (Left), HLA-DR (Center
Left), CD45RA (Center Right), and CD27 (Right) are shown in A,
and expression of chemokine receptors CCR4 (Left), CCR5
(Center Left), CCR7 (Center Right) and CXCR3 (Right) are shown
Tissue-infiltrating T cells display an activated pheno-
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| www.pnas.org/cgi/doi/10.1073/pnas.1104303108 Ruffell et al.
Although used successfully in multiple studies to relate TAM
infiltration with clinically relevant outcomes, our results indicate
in human breast tissue given that multiple stromal cells express it
and that a subset of these are CSF1R- and CD45-negative. We
observed that the nonleukocytic CD68+cells were predominantly
located within tumor stroma and, thus, based on this localization
and morphology, we speculate that CSF1R−CD68+cells likely
reflect tumor-associated fibroblasts or monocyte-derived fibro-
do not invalidate CD68 as a clinically relevant marker and, im-
portantly, CSF1-response gene signatures have been identified in
breast adenocarcinomas that are predictive of recurrence risk and
metastasis (53, 54). However, given the important role that
fibroblasts (and perhaps fibrocytes) play in fostering aspects of
tumorigenesis (55–57), differentiating among macrophages,
fibroblasts, and other stromal populations within tumors has the
potential to improve diagnostic information currently generated
by immunodetection of CD68.
As we have reported for expression of csf1 mRNA (6), mul-
tiple genes encoding myeloid cell chemoattractants are differ-
entially expressed by human BC cell lines, with variable
induction of these genes in response to CTX (Fig. S6). Although
differential expression between cell lines corresponding to par-
ticular subtypes of BC is evident, it is doubtful these cell lines
accurately represent the response of BC tumor tissue; thus, we
are investigating whether differences in myeloid cell infiltrates
reflect distinct molecular subtypes of BC and to what extent
these differ in residual tumors from CTX-treated patients.
It is important to acknowledge that leukocyte composition
within tumors responding to CTX likely differs substantially from
residual or recurrent tumors from patients that have received
CTX, given what is known regarding immune responses to CTX-
induced cell death (28). However, we recently reported that in
mammary carcinomas of MMTV-PyMT mice, blockade of the
CSF1-CSF1R pathway critical for TAM recruitment improved
response to CTX through a CD8+T-cell–dependent effect (6).
Thus, even though the findings presented herein are based on
a small dataset of heterogeneous tumor subtypes, and our results
may be biased because of sample selection favoring large and/or
less CTX-responsive tumors among the CTX-treated group, the
clear distinctions in the myeloid profiles between CTX-naive and
CTX-treated tumors is provocative and indicates that a CSF1-
targeted strategy may be a promising approach to enhance
therapeutic efficacy of cytotoxic CTX, particularly for treatment
of refractory BC. Moreover, given the increase in granulocytic
populations within tumors resistant to CTX, and the involvement
of these cells in regulating immune responses in chronic in-
flammatory diseases (58–62), these populations may also be
functionally relevant, and targeting common pathways of im-
mune suppression within the tumor microenvironment may
provide additional therapeutic opportunities to increase efficacy
of neoadjuvant CTX.
a percent of total CD45+cells as determined by flow cytometry. (C) Immunofluorescent staining of tumors for CD20 (green) and CD3 (red). CD3ε+CD4+T cells
(D) and CD3ε+CD8+T cells (E) are shown as a percent of total CD45+cells. (F) Ratio of CD8+to CD4+T cells within CTX-naive versus CTX-treated tumors.
Number of CD8-positive (G) and granzyme B-positive (H) cells per area as determined by automated counting. (I) Representative sections stained with CD8 or
granzyme B from CTX-naive (Left) or CTX-treated (Right) tumors. Red arrows indicate cells displayed in enlarged insets. N, nonadjacent normal; T, tumor.
Improved cytotoxic T-cell response in CTX-treated tumors. CD3ε−CD56+NKG2D+natural killer cells (A) and CD3ε−CD19/20+HLA-DR+B cells (B) shown as
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BREAST CANCER SPECIAL
Materials and Methods Download full-text
Tissues were collected at the time of surgery from consenting patients at the
University of California, San Francisco under approval from the institutional
by a certified pathologist (A.A.) and were prepared for analysis on the day of
resection. The percent of macrophages and CD8+T cells hasbeen reported for
a subset of the patients described here (6). Flow cytometry, immunohisto-
chemistry, and immunofluorescence were performed as described (6), with
detailed methods contained in SI Materials and Methods, and a list of anti-
bodies available in Tables S2 and S3. Statistical differences between two in-
dependent groups were determined by using Student’s t test via Prism 4.0
software (GraphPad Software).
work was supported by a Department of Defense Breast Cancer Research
Program Fellowship (to B.R.); a grant from the Breast Cancer Research
Foundation (to H.S.R.); National Institutes of Health/National Cancer Institute
Grants R01CA130980, R01CA132566, R01CA140943, and P50CA58207; a Dr.
Susan Love Research Foundation Instructional grant; and Department of
Defense Grants W81XWH-06-1-0416 and PR080717 (to L.M.C.).
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