Surgical Oncology 39 (2021) 101659

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Surgical Oncology
journal homepage: www.elsevier.com/locate/suronc

Characterization of inflammatory changes in the breast cancer associated

adipose tissue and comparison to the unaffected contralateral breast
Alecia M. Blaszczak a, 1, Dionisia Quiroga b, c, 1, Anahita Jalilvand a, Gina S. Torres Matias a,
Valerie P. Wright a, Joey Liu a, Lianbo Yu d, David Bradley a, Willa A. Hsueh a,
William E. Carson III b, e, *
a
Diabetes and Metabolism Research Center, Division of Endocrinology, Diabetes & Metabolism, Department of Internal Medicine, The Ohio State University Wexner
Medical Center, Columbus, OH, 43210, USA
b
The Ohio State University Comprehensive Cancer Center, The Ohio State University, 410 W 12th Avenue, Columbus, OH, 43210, USA
c
Department of Internal Medicine, Division of Medical Oncology, The Ohio State University, Starling Loving Hall, 320 W10th Ave, Columbus, OH, 43210, USA
d
Center for Biostatistics, The Ohio State University, 2012 Kenny Rd, Columbus, OH, 43221, USA
e
Department of Surgery, The Ohio State University, 410 W 10th Ave, N911 Doan Hall, Columbus, OH, 43210, USA

A R T I C L E I N F O A B S T R A C T

Keywords: Background: Adipose tissue has emerged as an important window into cancer pathophysiology, revealing po­
Breast cancer tential targets for novel therapeutic interventions. The goal of this study was to compare the breast adipose tissue
Adipose tissue (BrAT) immune milieu surrounding breast carcinoma and contralateral unaffected breast tissue obtained from
Cancer immunology
the same patient.
Materials and methods: Patients undergoing bilateral mastectomy for unilateral breast cancer were enrolled for
bilateral BrAT collection at the time of operation. After BrAT was processed, adipocyte and stromal vascular
fraction (SVF) gene expression was quantified by PCR. SVF cells were also processed for flow cytometric immune
cell characterization.
Results: Twelve patients underwent bilateral mastectomy for unilateral ductal carcinoma. BrAT adipocyte CXCL2
gene expression trended higher in the tumor-affected breast as compared to the unaffected breast. Macrophage
MCP-1 and PPARγ gene expression also tended to be higher in the tumor-affected breasts. T cell gene expression
of FOXP3 (p = 0.0370) were significantly greater in tumor-affected breasts than unaffected breasts. Affected
BrAT contained higher numbers of Th2 CD4+ cells (p = 0.0165) and eosinophils (p = 0.0095) while trending
towards increased macrophage and lower Th1 CD4+ cells infiltration than tumor-affected BrAT.
Conclusion: This preliminary study aimed to identify the immunologic environment present within BrAT and is
the first to directly compare this in individual patients’ tumor-associated and unaffected BrAT. These findings
suggest that cancer-affected BrAT had increased levels of T cell specific FOXP3 and higher levels of anti-
inflammatory/regulatory cells compared to the contralateral BrAT.

include altered gut microbiome, chronic low-grade inflammation,

1. Introduction
The rise in obesity prevalence both nationally and globally has been
linked to increased cancer incidences, including ovarian, gastrointes­
tinal, and hematologic malignancies [1]. In addition, obesity as a co­
morbidity is associated with increased cancer-associated mortality,
accounting for 14% of male and 20% of female cancer-associated deaths
[2]. Suggested etiologies behind obesity-related cancer incidence
altered adipokine secretion, as well as increased circulating insulin,
insulin-like growth factor and androgen production [1]. Adipose tissue
(AT) is a key player in mediating the inflammatory complications
associated with obesity in humans and mice [3–5]. In the lean state, AT
inflammation is suppressed by the concerted interaction between im­
mune suppressive cells, primarily regulatory T cells (Tregs) [6–8], [6–8]
[6–8] alternatively-activated/anti-inflammatory M2-like macrophages
[9] and innate lymphoid type 2 cells [10]. During chronic

* Corresponding author. OSU Comprehensive Cancer Center The Ohio State University 690 Tzagournis Medical Research Facility 420 W 12th Ave, Columbus, OH,
43210, USA.
E-mail address: william.carson@osumc.edu (W.E. Carson).
1
Both authors contributed equally to this work.

https://doi.org/10.1016/j.suronc.2021.101659
Received 25 August 2021; Accepted 6 September 2021
Available online 9 September 2021
0960-7404/© 2021 Elsevier Ltd. All rights reserved.
A.M. Blaszczak et al.
Abbreviations
AJCC American Joint Committee on Cancer
AT adipose tissue
BrAT breast adipose tissue
CLS crown-like structure
DCIS ductal carcinoma in situ
ER estrogen receptor
FBS fetal bovine serum
HER2 human epidermal growth factor receptor 2
PR progesterone receptor
PCR polymerase chain reaction
RPM revolutions per minute
SVF stromal vascular fraction
Treg regulatory T cells
over-nutrition, the inflammatory profile of AT shifts with a loss of
anti-inflammatory immune cells and an increase in pro-inflammatory
immune cell migration/infiltration including CD8+ cytotoxic T cells
[11], CD4+ Th1 cells [12], M1-like macrophages [9,13], and neutrophils
[14,15]. Not only are these cellular changes linked to the development
of obesity-related comorbidities (e.g., non-alcoholic fatty liver disease,
peripheral vascular disease, insulin resistance), but when these immune
networks are disrupted, murine models exhibit significant reductions in
these complications despite a high-fat diet [12,16–18]. These findings
highlight the importance of AT not only as a storage site for excess
caloric intake, but also as an important immunologic organ.
Although the existence of immunologic changes in cancer biology is
well-established, the interplay between AT immunology and tumori­
genesis is poorly understood and represents an important emerging
field. This is particularly relevant for breast cancer, as these tumors
develop uniquely within the AT milieu. Prior studies have suggested that
breast adipocytes which surround breast tumors may play a role in
tumor growth and metastasis [19,20]. Furthermore, others have sug­
gested that the BrAT surrounding breast tumors has altered levels of
leptin and adiponectin, known key regulators of lipid and glucose
metabolism [21]. The accumulation of excess lipids and glucose may
promote a pro-tumorigenic environment through the alteration of in­
flammatory pathways. Additionally, AT from breast cancer patients has
altered amounts of cytokine/chemokine secretion and aromatase
expression compared to AT from healthy donors which likely impacts
tumor development, growth and progression [22–25]. In particular,
adipocyte hypertrophy leads to the accumulation and proliferation of
macrophages surrounding dying adipocytes as part of the crown-like
structure (CLS). BrAT CLS are associated not only with a higher BMI,
but also an increased risk of breast cancer, metastases and death
[26–28]. These tumor associated macrophages tend to express greater
levels of both CD11c and MHCII molecules differentiating them from
typical BrAT macrophages and are thought to contribute to T cell
exhaustion [29].
To our knowledge, no prior studies have evaluated changes in im­
mune cell populations of BrAT in the setting of unilateral breast carci­
noma in comparison to the contralateral, unaffected BrAT. This self-
controlled study design helped to better elucidate localized immune
changes at the level of each individual patient. The primary objective of
this study was to characterize changes in immune cell composition and
gene expression patterns in BrAT adjacent to malignant breast tumors.
We hypothesized that tumor-associated BrAT would demonstrate pro-
inflammatory immune cell infiltration and gene expression changes
which was tested in a prospective paired study presented herein.
2
Surgical Oncology 39 (2021) 101659
2. Materials and Methods
2.1. Subject recruitment
Adult patients scheduled to undergo bilateral mastectomy with
biopsy-proven unilateral breast ductal carcinoma at The Ohio State
University Comprehensive Cancer Center were recruited and subse­
quently consented for study inclusion. Patients were excluded for
ongoing chronic steroid or anti-inflammatory medication usage and
end-stage kidney or liver disease. Twelve patients were included in this
study from which demographic information was collected, including
age, race, body mass index, breast cancer laterality, tumor pathology (e.
g. receptor status), and AJCC anatomic stage during their pre-operative
visit. Ethical approval of this study was obtained from The Ohio State
University Comprehensive Cancer Center institutional review board
(IRB protocol No. 2014H0471). Consent was obtained from all patients
to participate in this study.
2.2. Breast adipose tissue processing
BrAT was collected at the time of surgery from the cancer affected
and unaffected breasts. Adipose tissue was collected from the surface of
the mastectomy specimen in the quadrant of the breast containing the
tumor and from the mirror image region of the unaffected contralateral
breast. Samples were immediately placed in sterile fat media (RPMI with
3% FBS), placed on ice and taken to the laboratory for immediate pro­
cessing as previously described [30,31]. A small amount of the AT
sample was processed for histology. The tissue for histologic evaluation
was placed immediately in 10% formalin for 48 h before being stored in
70% ethanol at 4 ◦ C prior to processing. The remaining AT was minced
with scissors and subjected to a 50 min 1:1 collagenase II digestion. After
digestion, samples were diluted 1:1 with fat media and filtered through
serial 500 and 300 μM strainers. Samples were then centrifuged at 1100
RPM for 8 min to pellet the stromal vascular fraction (SVF) and to allow
the adipocytes to float to the top. Adipocytes were pipetted off, washed
with isolation buffer (PBS with 0.1% bovine serum albumin and 5 mM
ethylenediaminetetraacetic acid), re-centrifuged as stated above and
resuspended in 500 μL of TRI Reagent (Zymo Research, R2050-1-200).
The immune cell-containing SVF was further washed and subjected to
red blood cell lysis using Gibco™ ACK Lysing Buffer (Life Technologies,
A1049201) prior to designating aliquots for flow cytometry and mag­
netic bead isolation.
2.3. Quantification of adipocyte size
Tissue samples collected above for histology were paraffin-
embedded and then H&E stained. Images were taken at 4X for the
entire tissue section and five images were randomly selected for quan­
tification. Each image was split into quartiles and a random number
generator was used to select the quartile for quantification. The image
quartile area was traced along the tissue borders. Adipocyte and CLS
were subsequently counted, and the average adipocyte size was calcu­
lated. For each image, the adipocyte size was calculated as the number
of adipocytes divided by the traced quartile area. The five sections per
BrAT sample were then averaged to give an unaffected vs. affected
comparison.
2.4. Breast adipose tissue cell isolation and gene expression analyses
As stated above, whole BrAT was obtained and processed to separate
adipocytes and SVF. From the SVF, BrAT macrophages were isolated
using biotinylated anti-human CD14 (eBioscience, 13-0149-82, 1:100
dilution), followed by a biotin bead binder incubation and positive
magnetic stand separation. The remaining SVF was subsequently used
for T cell isolation using a biotinylated CD3 antibody (eBioscience, 13-
0038-82, 1:100 dilution), biotin bead binder incubation and positive
A.M. Blaszczak et al. Surgical Oncology 39 (2021) 101659

magnetic stand separation [32]. BrAT macrophages and T cells were Table 1
resuspended in TriReagent and used for quantitative polymerase chain Patient demographics.
reaction (qPCR) gene expression analysis. Characteristic N
Gene expression analyses were carried out on the isolated BrAT ad­
Gender Female 12
ipocytes, macrophages and T cells. RNA was isolated using the Direct-zol Male 0
RNA MiniPrep kit (Zymo Research, Irvine, CA). Sample concentration Age (years) Median 45
was quantified using the Nanodrop 2000. High-quality cDNA was Range 30–70
generated using the High-Capacity cDNA Reverse Transcription Kit Menopausal status Premenopausal 7
Postmenopausal 5
(Thermo Fisher Scientific, Waltham, MA) according to the manufac­ Family history of breast cancer (1st or 2nd Yes 8
turer’s instructions. SYBR Green primers and Taqman probes listed in degree relatives) No 4
Supplemental File 1 were ordered from Sigma for specific genes of in­ Germline genetic testing for pathologic BRCA Positive 2
terest along with the corresponding Bioline SensiFast No-ROX master gene variant Negative 9
Not tested/ 1
mix (Cat # BIO-98050 and BIO-86020 respectively). CT values of each
unknown
gene were normalized to sample PPIA control house-keeping gene BMI (kg/m2) Mean 32.2
expression and fold change was calculated in reference to the unaffected Range 23.3–43.3
BrAT samples [32]. Smoking history Never 9
Former 2
Current 1
2.5. Flow cytometry Breast tumor side Right 6
Left 6
For all 12 patients, SVF was collected from affected and unaffected Tumor receptor status ER+/PR+/ 5
breasts and samples were run independently. Flow cytometry analyses HER2neg
ER+/PR-/ 1
were carried out on all major immune cell types routinely identified in
HER2neg
AT including T cells, macrophages, neutrophils, eosinophils, innate ER+/PR-/HER2pos 1
lymphoid cells, innate lymphoid cells type 2, natural killer cells and ER-/PR-/HER2neg 5
myeloid-derived suppressor cells between the affected and unaffected AJCC anatomic stage 0 (DCIS) 1
breast of each patient (see gating structure in Supplemental File 2). After I 5
II 5
the isolation buffer wash, cells were resuspended in FACS buffer and IIIC 1
then blocked with Human TruStain FcX (Biolegend, Cat #422302 1:50 Neoadjuvant chemotherapy Yes 4
dilution) for 15 min before surface staining. Immediately following, cells No 8
were stained for 30 min in the dark with fluorochrome-conjugated an­
tibodies for cell surface identification listed in Supplemental File 3.
germline genetic testing, with only two being found to have known
Following surface staining and washing, live cells were stained with the
pathologic variants of genes associated with hereditary breast cancer
eBioscience Fixable Viability Dye (Cat# 65–0866) for 30 min. All cells,
(one with BRCA1 mutation, one with BRCA2 mutation). The median age
except T cells, were washed two times in FACS buffer and resuspended in
of the study population was 45 years with an average body mass index
a final volume of 100 μL. For T cell flow analysis, the eBioscience FOXP3
(BMI) of 32.2 kg/m2 58% (7/12) of the patients were premenopausal at
intracellular staining kit was utilized per manufacturer’s directions and
the time of bilateral mastectomy. Most of the patients had hormone
intracellular stains were used for further T cell characterization. Before
receptor-positive disease, including 42% (5/12) with ER+/PR+/
analysis, all cells were washed twice with FACS buffer and were run on
HER2neg. carcinoma, one patient with ER+/PR-/HER2neg. carcinoma,
the BD LSRII Flow Cytometer. All flow cytometry analyses were carried
and one patient with ER+/PR-/HER2pos. carcinoma. Triple-negative
out using FlowJo software (BD, Ashland, OR). For all samples, cells were
(ER-/PR-/HER2neg.) disease was also common, making up 42% (5/
first gated for all single cells (FSC-A vs SSC-A followed by FSC-H vs FSC-
12) of the patients. The most common anatomic stages were I and II,
A) followed by live immune cell gating (Fixable Viability Dye+ CD45+).
each comprising 42% (5/12) of the studied cohort. Additionally, there
Further cell type phenotyping is described in Supplemental File 4.
was one stage 0 ductal carcinoma in situ (DCIS), which measured 1.7 cm
in greatest length, and one stage III patient. 33% (4/12) of the enrolled
2.6. Statistical analyses
patients received neoadjuvant chemotherapy prior to surgery, including
three of the patients with triple-negative breast cancer and the one pa­
For all graphs, values are expressed as mean ± standard error of the
tient with HER2pos. disease. The patients with triple-negative breast
mean. Graphs were created using Prism 7.0 software (GraphPad, San
cancer all received anthracycline-containing regimens followed by a
Diego, CA) and all statistical analyses were completed using SAS version
taxane. The patient with ER+/PR-/HER2pos. breast cancer received a
24 (SAS, Cary, NC). Data distribution comparing BrAT from tumor-
taxane and platinum-based chemotherapy in addition to dual HER2pos.
affected and tumor-unaffected sides were determined by linear mixed
targeting monoclonal antibody therapy. Patients typically did not un­
modeling to allow for multiple testing corrections. In data comparing
dergo surgery until at least 4 weeks had passed since the last dose of
BrAT differences according to obese and non-obese BMI, unpaired Stu­
chemotherapy. All women had routine bilateral breast imaging and/or
dent’s T-tests were used. The associations between BMI, adipocyte size,
biopsy completed prior to surgery and were found to have no evidence of
and crown-like structures were tested with Spearman’s rank correlation.
tumor in the unaffected breast.
Significance was set at p < 0.05 for all analyses.

3. Results 3.2. Adipocyte size and gene expression

3.1. Patient characteristics
The study population consisted of 12 female patients with unilateral
breast ductal carcinoma undergoing elective bilateral mastectomy
(Table 1). Most of the patients were young (less than 50 years old),
premenopausal women who had a known 1st or 2nd degree relative with
a history of breast cancer. All but one of the patients had received
Increased adipocyte size is an important indicator of obesity and has
also been shown to influence leptin expression and angiogenesis [33].
Therefore, this cell measurement may have important implications in
cancer pathogenesis. Adipocytes from the affected and unaffected
breasts were measured and found to be similar in size with an average
area of 4.63 ± 0.15 μm2 and 4.62 ± 0.14 μm2 (p = 0.414, Fig. 1a),
respectively. The amount of crown-like structures (CLS) were also

3
A.M. Blaszczak et al. Surgical Oncology 39 (2021) 101659

Fig. 1. BrAT adipocyte characteristics and gene expression in cancer-affected and unaffected breasts. (A) Breast adipose tissue adipocyte size and (B) CLS
were measured in unaffected and affected breast samples with representative histologic slides (10X). Number of CLS in the unaffected breast samples were correlated
to patient BMI (C) and adipocyte size (D). (E) RNA was isolated from adipocytes and evaluated by qPCR. The amount of RNA expression of each gene is demonstrated
as a fold change as compared to the unaffected breast adipose tissue. (F) Fold changes of RNA expression of each gene were compared in patients who were non-obese
and obese in their tumor-affected breasts. For all graphs, bars represent the mean size of each group, error bars represent standard error of the mean, and * denotes p
< 0.05. Associations between BMI, adipocyte size, and crown-like structures were tested with Spearman’s rank correlation.

4
A.M. Blaszczak et al. Surgical Oncology 39 (2021) 101659

measured, which act as a gauge of adipose tissue inflammation as they
represent collections of immune cells surrounding adipocytes [3,28].
The number of CLS in each group were similar (Fig. 1b; 0.37 ± 0.16 mm2
vs. 0.32 ± 0.12 mm2, p = 0.404, respectively). In tumor unaffected
BrAT, the patients’ BMI appeared to positively correlate with the num­
ber of CLS present (Fig. 1c; p = 0.009), however, this relationship was
not found in the tumor affected BrAT (Supplemental File 5a; p = 0.967).
Moreover, adipocyte size and the number of CLS in the tumor unaffected
BrAT was positively correlated (Fig. 1d; p = 0.032), but this association
was not seen in tumor affected BrAT (Supplemental File 5b; p = 0.497).
An analysis of adipocyte transcript expression by PCR was informa­
tive. In contrast to the contralateral side, adipocytes from the cancer-
affected breast had no changes in the expression of other key inflam­
matory markers including IL-1β, IL-6, NLRP3, TNF, and adipokines
(including leptin and adiponectin). There were also no differences in
gene expression involved in the major histocompatibility complex II
(MHC II) pathway such as CIITA, HLA-DPA1, CD74, CD80, and CD86,
which are key mediators of adipocyte adaptive immune function.
Alternatively, consistent with prior studies, comparions of the tumor-
affected BrAT from non-obese BMI patients to those with an obese
BMI (30 kg/m2 or greater) revealed that the pro-inflammatory genes IL-
1β, IL-8, and TNF were expressed at higher levels in obese patients
(Fig. 1f) [4].
3.3. Altered macrophage and T cell expression of pro-inflammatory genes
found in ductal carcinoma-containing breasts
Using biotinylated beads, the BrAT macrophage fraction was isolated
from the SVF, processed for RNA, and evaluated for gene expression by
PCR. There was a trend towards increased MCP-1 (macrophage che­
moattractant) expression in cancer-associated BrAT over the contralat­
eral BrAT (1.67 ± 0.58 vs. 1.00 ± 0.40, p = 0.0680) (Fig. 2a). However,
there was also a trend towards increased expression of PPARγ (1.30 ±
0.25 vs. 1.00 ± 0.16, p = 0.1689), a regulator of M2-like macrophage
differentiation, thought to be anti-inflammatory in nature [34]. There
were no differences noted in macrophage expression of several inflam­
matory cytokines and MHC class II genes including IL-1β, NLRP3, TNF,
IL6, CIITA, CD74, CD80, CD86 and HLA-DPA1.
In addition to macrophage bead isolation, T cells were isolated from
BrAT SVF cells using CD3+ biotinylated beads. In the CD3+ isolated T
cell fraction, there was significantly increased expression of FOXP3
(1.52 ± 0.19 vs. 1.00 ± 0.13, p = 0.0370), (marker of regulatory T cells)
and a trend for increased GATA3 (1.33 ± 0.15 vs. 1.00 ± 0.08, p =
0.0710) (marker of T helper type 2 cells) in the cancer-associated BrAT
(Fig. 2b). There was no difference in the prevalence of the CD4+ and
CD8+ T cell populations when comparing the cancer-affected and un­
affected BrAT. Overall, this gene expression data suggests less inflam­
matory BrAT T cells in the setting of ductal carcinoma with increased

Fig. 2. BrAT SVF immune gene expression in cancer-affected and unaffected breasts. (A) Macrophages and (B) T cells were isolated using biotinylated beads
and processed to obtain RNA for qPCR analysis. The amount of RNA expression of each gene is demonstrated as a fold change as compared to the unaffected breast
adipose tissue. For all graphs, bars represent the mean size of each group, error bars represent standard error of the mean, and * denotes p < 0.05.

5
A.M. Blaszczak et al. Surgical Oncology 39 (2021) 101659

expression of Th2 and Treg T cell markers.
3.4. Altered profile of immune cells present in BrAT adjacent to ductal
carcinoma
BrAT SVF was analyzed for immune cell components by flow
cytometry. SVF from the cancer-affected breast was found to have
significantly increased levels of T helper type 2 cells (18.6 ± 2.2% vs.
13.6 ± 2.6%, p = 0.0165) and eosinophils (7.8 ± 0.6% vs. 5.6 ± 0.8%, p
= 0.0095) and a trend towards decreased T helper type 1 cells (16.1 ±
3.1% vs. 23.3 ± 4.2%, p = 0.0802) as compared to the contralateral
unaffected BrAT (Fig. 3a). There was no difference in the overall per­
centage of BrAT-associated neutrophils, macrophages or myeloid-
derived suppressor cells (MDSC). Further macrophage characterization
based on receptor expression demonstrated a trend towards increases in
CD80 (1.21 ± 0.50 vs. 4.16 ± 1.74, p = 0.0543) and CD64 (27.48 ±
15.15 vs. 101.11 ± 72.89, p = 0.2531) expression within the macro­
phage cell population of tumor affected versus unaffected BrAT
(Fig. 3b). Expression of CD64 on macrophages is characteristic of an M2-
like anti-inflammatory profile, while CD80 is known to be expressed
more highly on M1-like pro-inflammatory macrophages [9,35]. Overall,
these flow cytomertric findings are suggestive of a mainly
anti-inflammatory response in the cancer-associated BrAT, but show
some complexity with regard to macrophage populations.
4. Discussion
While many studies have evaluated the role of inflammation in
tumorigenesis [36], relatively few have focused on the inflammatory
state of cancer-associated AT. Given the importance of AT in mediating
chronic diseases, including obesity and diabetes, it was hypothesized
that cancer-associated AT would provide an important window into
breast cancer biology. In this study, ipsilateral BrAT from patients with
unilateral breast ductal carcinomas was compared to the contralateral,
unaffected BrAT. While many BrAT genes and cell populations were
similar bilaterally, several unique findings were identified. Importantly,

Fig. 3. Flow cytometric immune cell characterization of BrAT SVF in cancer-affected and unaffected breasts. SVF cells were separated from adipocytes,
stained, and analyzed by flow cytometry for the presence of immune cell subsets. (A) Complete SVF cell analysis is compared between the cancer-affected and
unaffected breast tissue. (B) Macrophage receptor expression of key M1-like and M2-like markers compared between the cancer-affected and unaffected breast tissue.
Bars represent the mean size of each group; error bars represent standard error of the mean. For all graphs, bars represent the mean size of each group, error bars
represent standard error of the mean, * denotes p < 0.05, and ** denotes p < 0.001.

6
A.M. Blaszczak et al.
this study identified an increased anti-inflammatory profile of
cancer-associated BrAT immune cells in the affected breasts, which is
interpreted to mean that the immunomodulatory effects of breast cancer
extend to the surrounding adipose tissue. However, existing data suggest
that obesity in of itself may generate an inflammatory profile. These two
observations are not mutual exclusive as the present study was focused
on evaluating the differences between the adipose tissue surrounding an
established breast cancer and the adipose of the contralateral unaffected
breast.
This study found that cancer-associated BrAT had increased anti-
inflammatory cells as compared to the unaffected breast as indicated
by a significant increase in T helper type 2 cells and eosinophils, and a
trend towards increased M1- and M2-like macrophages. Conversely, the
contralateral BrAT was characterized by a trend towards increased Th1
cells. The flow cytometric data was further supported by gene expression
data demonstrating a trend toward increased expression of anti-
inflammatory markers in the affected breast including macrophage
expression of PPARg and increased T cell expression of Treg marker
FOXP3. Overall, the increased anti-inflammatory profile of cancer-
associated BrAT immune cells suggests that the immunomodulatory
role of breast tumors extends beyond the tumor itself and may have an
impact on the surrounding AT. Breast cancer studies have shown that
increased Th2 cell signaling may promote the metastatic spread of
hormone-receptor-positive tumors [37], suggesting that inflammatory
signatures may also play a role in the progression of pre-existing tumors.
The analysis of gene expression in adipocytes did not reveal a pro­
nounced difference between the affected and the unaffected breast. The
discordant inflammatory findings between adipocyte gene expression
and the immune cell makeup (increased Th2, decreased Th1 cells near
the cancer site) in this study may be evidence of pro-tumorigenic/pro-
angiogenic signaling adipocytes with a lack of recruitment of Th1
cellular response to the surrounding tumor site, creating an ideal envi­
ronment for tumor development. Of note, pre-clinical mouse studies
have also found increased adipocyte pro-inflammatory gene expression
and a higher levels of tumor suppressor cells in breast cancer containing
mice [38].
Macrophages have been implicated in angiogenesis and neo­
vascularization of breast cancer, primarily through the secretion of pro-
angiogenic factors, such as VEGF-A. Previous studies in invasive breast
tumor microenvironments have not only shown a positive correlation
between vascularization and macrophage infiltration but also have
demonstrated a link between increased levels of macrophage infiltration
with decreased progression-free and overall survival rates [39]. Given
that M2-like macrophages function primarily to repair tissue injury and
increase angiogenesis, the present results (more M2 macrophages on the
cancer affected side) are in-line with these findings [40]. Taken
together, this data suggest that both the tumor microenvironment and
the associated AT may contribute to tumor vascularization and pro­
gression. The results for the patients with DCIS were similar to those
obtaianed for patients with invasive cancer which suggests that these
changes may develop early in the process of tumorigenesis.
Only a few other studies have examined the role of BrAT in breast
cancer. Lapeire et al., examined the cancer-associated AT SVF (cellular
fraction of AT) and secretome (proteins secreted from tissue) in patients
with breast cancer [41]. Comparing the AT SVF isolated from breast
cancer patients to control media in an in vitro co-culture model, increases
in oncostatin M expression and activation of JAK/STAT3 signaling in
CD45+ immune cells promoted tumor neovascularization. Targeted in­
hibition of the JAK/STAT3 pathway through the use of tofacitinib, an
anti-oncostatin M antibody, decreased tumor neovascularization in
orthotopic xenografts, suggesting that the cellular component of BrAT
promotes tumor cell scattering and vascularization [42]. Although
contralateral normal breast tissue was not available for comparison, this
data suggested that breast cancer-associated AT could exert its effects on
the tumor microenvironment through paracrine signaling, allowing for
increased tumor growth and vascularization. In the present study,
7
Surgical Oncology 39 (2021) 101659
evidence is presented supporting the concept that the immune cells in
the AT further contribute to the tumor microenvironment by promoting
an anti-inflammatory mileu that could allow for evasion of immune cell
surveillance.
In addition to comparing the affected BrAT to the unaffected BrAT, it
may also be important to study the affected BrAT qualities of certain
patient/tumor characteristics. For example, it is known that the tumor
microenvironment of breast cancers with different molecular subtypes
or different levels of invasion/grade can have unique levels of immune
activity (e.g. greater levels of macrophages and Treg cells in IDC than
DCIS) [43]. Our study has included a patient with DCIS as well as pa­
tients with invasive IDCs with all different levels of ER, PR, and HER2
expression. However, in future larger studies it would be greatly valu­
able to differentiate these into separate groups to determine if the BrAT
environment varies by breast pathology as well.
It was noted that the affected BrAT had significantly greater levels of
pro-inflammatory IL-1β, IL-8, and TNF gene expression in obese patients
compared to non-obese patients, in line with previous studies [24,44].
The positive correlation between BMI and adipocyte size as well as BMI
and number of CLS in unaffected BrAT is suggestive of increased
inflammation in the setting of obesity; this association has also been
verified in visceral and subcutaneous AT [45]. Interestingly, there is no
such relationship between BMI and adipocyte size or CLS number in
tumor affected BrAT, suggesting a significant alteration of the tumor
surrounding AT environment. Certain immune subsets also appeared to
be greater (e.g. MDSC) in obese patients affected BrAT as compared to
those who were non-obese at time of surgery.
Previous work has also demonstrated how neoadjuvant chemo­
therapy and breast tumor receptor status may affect the breast tumor
immune milieu [46,47]. Analysis of circulating T cells prior to and
following neoadjuvant chemotherapy in patients with breast cancer
have suggested that CD4+ T cell immune checkpoint receptor expression
decreases after chemotherapy, while CD8+ T cell immune checkpoint
receptor expression increases [46]. A study of hormone
receptor-positive breast cancers also revealed that tumor-infiltrating
lymphocyte and CD8+ T cell quantities were significantly lower after
chemotherapy, while there appeared to be a more predominant myeloid
RNA expression signature in the tumor [47]. As these changes have been
seen in circulating peripheral blood and intratumorally in breast cancer
patients following chemotherapy, it is plausible that
chemotherapy-induced changes may also occur in BrAT. It was noted in
this study that there may be trends in the affected BrAT according to
these parameters, for example, triple-negative breast cancers were found
to have lower amounts of BrAT T cell IFNg gene expression. Notably, the
patients who received neoadjuvant chemotherapy had profiles that were
quite similar to the other study participants; the primary difference was
that patients had greater macrophage IL-8 gene expression. Due to the
small size of this preliminary study, it would not be possible to stratify
data by menopausal status, BMI, or tumor/disease specific characteris­
tics. Therefore, differences in affected BrAT due to these factors would
be important to elucidate in future, larger studies.
5. Conclusion
The purpose of this study was to compare the local immune cell
composition of BrAT associated with ductal carcinoma to the contra­
lateral, unaffected BrAT. We report that BrAT has increased regulatory T
cell gene expression and a suppressive SVF immune cell environment
within the ipsilateral, cancer-affected breast characterized by increased
T helper type 2 cells and eosinophils. These preliminary findings not
only highlight a potentially important role for AT in breast cancer
biology, but may also help identify novel therapeutic approaches for
combatting breast cancer.
A.M. Blaszczak et al.
Ethics approval and consent to participate
Ethical approval of this study was obtained from The Ohio State
University Comprehensive Cancer Center institutional review board
(IRB protocol No. 2014H0471). Consent was obtained from all patients
to participate in this study.
Funding
This study was supported by grants from the American Diabetes
Association (1-16-ICTS-049) to WAH and from the National Cancer
Institute Center Core Grant (P30 CA016058). DQ was supported on an
National Cancer Institute T32 training grant (1T32CA247815-01).
Authors’ contributions
AMB: study concepts/design, literature research, experimental
studies, data acquisition, data/statistical analysis, manuscript prepara­
tion/editing. DQ: literature research, data/statistical analysis, manu­
script preparation/editing. AJ: experimental studies, data acquisition.
GSTM: experimental studies, data acquisition. VPW: experimental
studies, data acquisition. JL: experimental studies, data acquisition. DB:
study concepts/design, manuscript review. WAH: study concepts/
design, manuscript editing/review, funding. WEC: guarantor of the
integrity of the study, study concepts/design, clinical studies - patient
accrual, manuscript editing/review, funding.
Declaration of competing interest
WH serves on a Scientific Advisory Board Regeneron. WEC has
served on the Advisory Board for Dragonfly Therapeutics, Inc. All other
authors have no conflicts of interest to disclose.
Acknowledgements
Technical support was graciously provided by the Ohio State Uni­
versity Comprehensive Cancer Flow Cytometry Center Shared Resources
staff & faculty.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.suronc.2021.101659.
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