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CCL21 is an Effective Surgical Neoadjuvant for Treatment of Mammary Tumors
Abdelkader Ashour1,2, Xuede Lin1, Xiaojian Wang1, Hēth Turnquist1,3, Nicole Burns1,2, Anguraj Sadanandam3, Amit Tuli1,2, Rakesh Singh3, James Talmadge3, and Joyce Solheim1,2,3
1Eppley
Institute for Research in Cancer and Allied Diseases, 2Department of Biochemistry and Molecular Biology, and 3Department of Pathology and Microbiology
University of Nebraska Medical Center, Omaha, NE, USA
CCL21 Effect on Orthotopic Mammary Tumor Growth
In previous studies, the chemokine CCL21 has shown biological
activities that include T cell, natural killer (NK) cell, and dendritic
cell (DC) chemoattraction. The goal of this study was to determine
the effects of administering CCL21 to orthotopic mammary tumors
in terms of impact on tumor growth rate, immune cell infiltration of
the primary tumor, and survival. We found that a single intratumoral
administration of CCL21 in a slow release matrix (Hydron®) slowed
the growth of orthotopic mammary tumors and increased
intratumoral infiltration by T cells, NK cells, and DCs. CCL21Hydron® also prolonged survival of tumor-bearing mice, relative to
administration of PBS-Hydron®. Furthermore, mice that received
intratumoral neoadjuvant CCL21-Hydron®, prior to surgical
resection of tumors, survived significantly longer than control mice
(e.g., mice that received PBS-Hydron® before surgery, or CCL21Hydron® without resection). The surviving neoadjuvant CCL21Hydron®-treated mice, when challenged again with cl-66, had a
significantly slower rate of tumor growth than control mice. Overall,
our data indicate that CCL21-Hydron® treatment prior to mammary
tumor resection can significantly prolong survival and increase
resistance to subsequent tumor challenge, which suggests that it
generates anti-tumor protective immunity. Thus, intratumoral
administration of CCL21 in a slow release matrix has potential as a
neoadjuvant immunotherapy for breast cancer.
BALB/c mice were injected in the fourth inguinal mammary fat pad with 1X105 Cl-66
cells.
Once tumors were palpable, the mice received intratumoral implantation of 6 μg CCL21
in Hydron, or PBS- Hydron
Tumor growth and survival were monitored
Hydron is a commercially available hydrogel polymer
-sustained release drug delivery system
-utilized in several ongoing FDA-approved clinical trials, but has not been
examined as
a means to deliver CCL21 intratumorally
surgical resection
immunosuppression created by the tumor
protective immunity
necrosis and inflammation at the surgery site
+
attraction of immune cells into the region of the primary tumor by CCL21
Fig.2 CCL21 Effectively Inhibited Orthotopic Mammary Tumor Growth
*
2800
intensified local immune response that removes residual and metastatic disease
2600
*
2400
2200
Tumor Size (mm3)
Abstract
CCL21 and Surgery Against Mammary Carcinoma
Testing Surgery Accompanied by Surgery (Hy-CCL21
Neoadjuvant) Against Mammary Carcinoma
No Treatment
2000
PBS-Hydron
*
1800
CCL21-Hydron
1600
*
1400
1200
1000
800
*
600
*
*
400
200
0
Day 0
Day 4
Day 7
Day 10
Day 15
Day 19
Day 22
Day 25
Orthotopic mammary tumors were established by the injection of 1 X 105 cl-66 cells
into the fourth inguinal mammary fatpad of 180 female BALB/c mice
Once tumors reached 60 mm3 (Day 0), Hy-PBS or Hy-CCL21 (6 g of CCL21/mouse)
was implanted i.t.
Four days following initial treatment, surgical resection of the tumors was carried out.
 Survival was monitored.
On Day 100, surviving mice, as well as 20 naïve BALB/c mice, were re-challenged
with 1 X 105 cl-66 cells injected into the fourth inguinal mammary fatpad on the opposite
side
Tumor growth was monitored
Days Post Start of Therapy
Fig.5 Mice that received neoadjuvant CCL21-Hydron+tumor resection
survived significantly longer than mice that received control treatments
Introduction
Flow Cytometric Analysis of Tumor-infiltrating Cells
Fig.3 Intratumoral administration CCL21-Hydron significantly increased
the absolute number of CD8+ T cells and NK cells, relative to PBS-Hydron,
in cl-66 mouse mammary tumors
4500
4000
3500
3000
2500
2000
1500
1000
500
0
90
80
References
70
60
No Treatment
50
Surgery Only
40
PBS-Hydron (No Surgery)
30
CCL21-Hydron (No Surgery)
20
PBS-Hydron (Neoadjuv ant)
10
CCL21-Hydron (Neoadjuv ant)
0
0
10 20 30 40 50 60 70 80 90 100 110
Days Post Start of Therapy
*
CCL21-Hydron
PBS-Hydron
Fig.6 CCL21-Hydron neoadjuvant reduces lung metastases and inhibits
recurrence of tumors to primary or remote sites
*
CD8+ T cells
(CD3+ CD8+)
NK Cells
(DX5+)
DC cells
(CD11c+)
Immune Cell
Subset
Fig. 1 CCL21
Expressed in T-
cell zones of spleen
and lymph nodes
Multiple functions
to facilitate T cell
responses
Strong
chemoattractant for
DCs, NK cells, and
T cells
Induces antiapoptotic signaling
in DCs
Stimulates DC
phagocytosis
Confocal Analysis of Tumor-infiltrating Cells
CCL21 gradient
On day 0, 1, 3, or 7 after treatment with either CCL21-Hydron (black bars) or PBSHydron (white bars), mice (n=3 per time point and treatment type) were euthanized.
Tumors and marginal tissues were resected and frozen in Tissue Freezing Media
Frozen tumor samples were cryosectioned and fixed in ice cold 1:1 acetone: methanol.
Non-specific binding was blocked with 10% normal goat serum.
Rat anti-mouse CD8 and hamster anti-mouse CD11c were used as primary antibodies.
Cy™5-conjugated AffiniPure Goat Anti-Armenian Hamster IgG and Alexa Fluor 488
goat anti-rat IgG were used as secondary antibodies.
The antibodies were sequentially applied to the frozen, acetone/methanol fixed sections
and incubated according to the manufacturer’s instructions.
Fluorescence images were obtained with an LSM 410 confocal laser scanning
microscope
Fig.4 Lymphoid DCs (CD11c+CD8+) were more readily apparent in tumors from
the CCL21-Hydron-treated group, relative to the PBS-Hydron treated group
Fig.6 Tumor growth was significantly slower in re-challenged mice that
had previously received neoadjuvant CCL21-Hydron+tumor resection
*
130
120
Cell Line
110
Tumor Size (mm3)
100
Cl-66: aggressive mammary adenocarcinoma cell line of BALB/c
origin
-derived from a spontaneously arising mammary tumor
-consistently produces metastases to the bone marrow and lung
-The metastatic pattern produced in this model closely emulates
the progression of human metastatic breast cancer
-This maks Cl-66 an appropriated system for immunotherapeutic
drug development.
90
80
Naive
*
70
Surgery Only
60
PBS-Hydron (Neoadjuvant)
50
CCL21-Hydron (Neoadjuvant)
40
30
*
20
10
0
Day 0
CCL21-Hydron
PBS-Hydron
Multiple intratumoral injections of CCL21 (3 g daily for 3 days) have been
shown previously to slow the growth of MT-901 mammary tumors implanted
subcutaneously in the flank.16 Our study has extended these earlier findings in
several respects. In our study, mammary tumors in the orthotopic site (the
mammary fat pad) were used. The cellular infiltrates in the treated mammary
tumors were characterized, and found to include increased numbers of T cells
and NK cells by day 1 and lymphoid DCs by day 3 post-treatment. In addition,
our work combined the use of CCL21 with primary tumor resection, a model
that is relevant to a potential treatment protocol for many breast cancer patients.
Finally, we found that mice surviving after CCL21 neoadjuvant and tumor
resection had a significantly slower rate of tumor growth after cl-66 challenge
(relative to control treated mice or naïve mice), indicating that immunological
memory for the cl66 tumor had been developed.
In our study, the number of infiltrating T cells and lymphoid DCs was
increased by CCL21-Hydron treatment of cl-66 tumors. Consistent with our
results, the delivery of CCL21 via a herpes simplex virus-derived vector
directly into lymphomas or colon carcinomas resulted in tumor eradication or
slowed growth (respectively), associated with T cell and DC tumor
infiltration.23 These findings suggest that CCL21 may have anti-tumor
effectiveness mediated by DCs and T cells. However, the therapeutic efficacy in
our model may be at least partially due to the attraction of large numbers of NK
cells to the cl-66 tumors by CCL21-Hydron. NK cells have previously been
shown to be effective against breast tumors.2,4 In addition, the chemokine
CCL19 (which binds to the same receptor as CCL21) transduced into murine
mammary tumor cells has been shown to induce tumor rejection via a
mechanism involving NK cells as well as CD4+ cells.24 The relative importance
of T cell subsets and NK cells to the effectiveness of intratumoral CCL21Hydron as a surgical neoadjuvant against cl-66 will be analyzed in future
experiments using antibody depletion of cellular subsets.
100
Percentage Surviving Mice
Orthotopic cl-66 mammary tumors in BALB/c mice were generated and treated as
described above with CCL21-Hydron or PBS-Hydron (n = 4 mice per cohort)
Tumors were resected 24 hours after the treatment
Non-necrotic tumor tissue from each resected tumor was minced and digested with
collagenase and deoxyribonuclease I
Mononuclear cells were isolated with Lympholyte-M and analyzed by flow
cytometry for cell surface markers.
For NK cells, DX5-FITC antibody was used. For CD8+ T cells, PE-labeled anti-CD3
antibody and cychrome-labeled anti-CD8 antibody were used
Cells/mm3
Adjuvant or neoadjuvant immunotherapies can potentially be used
regionally or systemically against tumors, with minimal side effects.
Immunotherapy effects can be mediated by several cell types,
including antigen-presenting cells (such as DCs), T cells, and NK
cells. Specific immune responses against tumors .are initiated by DCs
via presentation of antigens from phagocytosed tumor cells to T
lymphocytes.1 NK cells provide innate immunity, and have been
shown to have therapeutic activity against several types of tumors,
including breast and pancreatic tumors.2-5
The trafficking of immune cells is regulated, in part, by
chemokines. For example, DCs, naïve T cells, and NK cells, due to
their expression of the CCR7 receptor, are attracted by the chemokine
CCL21 (also known as 6Ckine and secondary lymphoid tissue
chemokine).6-9 CCL21 is naturally expressed by high endothelial
venules and in T cell zones of spleen and lymph nodes.9 In addition
to its chemotactic function, CCL21 induces anti-apoptotic signaling in
DCs and mesangial cells, and stimulates phagocytosis by DCs.10-12
Ectopic expression of CCL21 can induce the formation of lymph
node-like structures composed of lymphocytes and DCs.13 In murine
models, CCL21 has been demonstrated to have therapeutic effects
against colon cancer, melanoma, and lung cancer.14-20 In addition, one
report showed that three daily intratumoral injections of CCL21
slowed the growth of a subcutaneously implanted mammary tumor.16
Discussions and Conclusions
Day 9
Day 13
Days Post Re-challenge with Cl-66
Day 16
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