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공학석사학위논문
M1 macrophage-derived
nanovesicles repolarize M2
macrophages for cancer
treatment
암 치료를 위한 M1 대식세포에서 추출한
나노베지클에 의한 M2 대식세포의 재분화 연구
2018년 8월
서울대학교 대학원
화학생물공학부
추 연 웅
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Abstract
M1 macrophage-derived
nanovesicles repolarize M2
macrophages for cancer
treatment
Yeon Woong Choo
School of Chemical and Biological Engineering
The Graduate School
Seoul National University
Cancer immunotherapy is a treatment that activate immune
cells to induce anti-tumor immune response. Since macrophages are
common immune cells in tumor microenvironment (TME),
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tumor-associated macrophages (TAM) are studied as a attractive
target for cancer immunotherapy. Macrophages are known to have
two different phenotypes; M2 macrophages release anti-inflammatory
cytokines and angiogenesis factor that potentiate tumor growth. M1
macrophages release pro-inflammatory cytokines and induce
anti-tumor immune response. Cancer cells release cytokines that
affect TAM to polariae to M2 macrophages. Thus, repolarazation of
M2 TAM to M1 macrophages may be promising immunotherapy.
Exosomes are known as nanocarrier that can induce
phenotype change in recipient cells. In this study, we used
exosome-like nanovesicles derived from M1 macrophages (M1NV) to
repolarize M2 macrophages to M1 macrophages. M1NV treatment to
M2 macrophage showed successful upregulation of M1 marker mRNA
expression, protein expression, and cytokines expression in M2
macrophages. Thus, our study indicates M1NV treatment repolarize
M2 TAM to M1 macrophages for cancer immunotherapy.
Keywords: cancer immunotherapy, macrophage repolarization,
nanovesicle, tumor-associated macrophages
Student Number: 2016-21059
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Contents
Abstract ················································································ i
List of Figures ·································································· 1
1. Introduction ··································································· 5
2. Experimental Section ·············································· 10
2.1 Cell Culture ···························································· 10
2.2 Preparation of M1NV ········································ 12
2.3 Preparations of Exosome ··································· 13
2.4 Physicochemical Characterization of
M1NV ······································································· 14
2.5 mRNA Quantification of NV and Cells ········· 15
2.6 MicroRNA Profiling Assay ······························· 16
2.7 In vitro Cellular Uptake of M1NV ················· 17
2.8 Viability of Cells after Treatment with
M1NV ···········································································18
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2.9 Transwell Assay ················································ 19
2.10 In vitro Analyses of Macrophage
Polarization ································································20
3. Results and Discussion ·········································· 22
3.1 Characterization of M1NV and Exosomes ···· 22
3.2 In vitro Cellular Uptake and Cytotoxicity of
M1NV ······································································· 27
3.3 Comparison of the Therapeutic Effects between
M1NV and M1 Macrophages ···························· 30
3.4 M1NV Polarize M2 Macrophages to
M1 Type. ·····································································34
4. Conclusion ··································································· 39
5. Reference ····································································· 40
요약 (국문초록) ································································ 46
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List of Figures
Figure 1. Schematic of the preparation of M1NV and the therapeutic
effects of M1NV in TME. M2 TAM secret anti-inflammatory cytokines
that promote tumor growth (Left). When M1NV polarize M2 TAM to
M1 type, M1 macrophages secret pro-inflammatory cytokines that
attacks cancer cells (Right).
Figure 2. Characterization of M1NV. (A) Size distributions and zeta
potentials of M1NV and M1 macrophage-derived exosomes, as
analyzed by nanoparticle tracking (n=3) and electrophoretic light
scattering (n=3), respectively. (B) Transmission electron microscopic
images of M1NV and M1 macrophage-derived exosomes. (C)
Comparison of the number and protein amounts of M1NV and
exosomes, both of which were produced from the same number of
M1 macrophages (*p<0.05 vs. exosomes). (D) Relative mRNA
amounts of pro-inflammatory M1 macrophage markers (CD86, IL–6,
iNOS, and TNF–α) in M0 macrophages (M0 cell), M1 macrophages
(M1 cell), M0NV and M1NV. The amounts of mRNA from genes of
interest were normalized to the GAPDH amount. (E) Microarray
analysis of M1, M2 macrophage-related miRNA expressions in M1NV
compared to M0NV. Red and green indicate upregulation and
downregulation, respectively, in the miRNA expressions of M1NV as
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compared to those of M0NV. The number indicates log2 ratio of
M1NV miRNA to M0NV miRNA. Only miRNAs whose difference in
the amount between M1NV and M0NV is over 1.5 fold are
represented.
Figure 3. Cellular uptake and cytotoxicity of M1NV in vitro. (A)
Fluorescent image analysis and (B) FACS analysis for M1NV uptake
by M2 macrophages and cancer cells. M1NV were labeled with a red
fluorescent dye (DiI) prior to the M1NV (50 µg/mL) treatments for
fluorescent imaging (4 h) and FACS analyses (1 h) (*p<0.05 vs.
cancer cells). NS: no staining. (C) Relative viable cell number of
cancer cells and M2 macrophages treated with various doses of
M1NV for 1 day, as evaluated by the CCK-8 assay (n=3) (*p<0.05
vs. 0 µg/mL). (D) Effects of M0NV or M1NV treatments for 24 h on
the angiogenic gene expressions in cancer cells. Amounts of mRNA
from genes of interest were normalized to the GAPDH amount (n=3,
ns: not significant).
Figure 4. Comparison of the therapeutic effects and macrophage
polarization induction effects between M1 macrophages and M1NV.
(A) Relative viable cancer cell number co-cultured with M1
macrophage-co-cultured-M2 macrophages (Group 2) or with M1NV
treated-M2 macrophages (Group 3) for 1 day was examined by
CCK-8 assay (n=3) compared to control group (co-cultured with
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untreated M2 macrophage; Group 1). n=4-6, ns: not significant,
*p<0.05 vs. Control. (B) Relative mRNA expressions of M1 and M2
markers in M1 macrophages co-cultured with either M2 macrophages
(group 2) or cancer cells (group 3) for 24 h, as compared to the
control group (M1 macrophages only, group 1). (C) Relative mRNA
expressions of M1 and M2 markers in M2 macrophages after 24 h of
co-culture with M1 macrophages (group 2) or after 24 h of M1NV
treatment (group 3), as compared to the control group (M2
macrophages only, group 1). (B)–(C) n=3, *p<0.05 vs. control, and
†p<0.05 vs. group 2. The mRNA expression from genes of interest
was normalized to GAPDH and expressed as a relative change.
Figure 5. Polarization of M2 macrophages to M1 macrophages in
vitro induced by the M1NV treatment. M2 macrophages were treated
with either M0NV or M1NV for 24 h. (A) Relative expressions
mRNA in M1 (iNOS, TNF–α, and IL–6) and M2 markers (IL–4,
IL–10, and Fizz–1), as evaluated by qRT–PCR. (B) Angiogenic
gene (VEGF), metastatic gene (CCL18) expressions in M2
macrophages after M0NV or M1NV treatments for 24 h. The mRNA
expressions from genes of interest were normalized to GAPDH. NT
denotes no treatment. (C) Immunofluorescence staining for CD86 (red,
M1 marker) and CD206 (red, M2 marker) of M2 macrophages treated
with either M0NV or M1NV for 24 h. Nuclei were stained with DAPI
(blue). (D) ELISA assay assessment for secretion of
pro-inflammatory cytokine (IL–6) and anti-inflammatory cytokine
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(IL–4) from M2 macrophages treated with either M0NV or M1NV
for 24 h. ND denotes no detection. (A)–(D) n=3 per group, *p<0.05
vs. NT, †p<0.05 vs. M0NV.
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1. Introduction
Cancer immunotherapy is a treatment activating immune cells
such as T cells, B cells, and macrophages to induce immune response
against cancer (1). Traditionally, cancer immunotherapy was focused
on T cells activation, since activated T cells directly kill cancer cells,
and secret cytokines that induce immune response against cancer (2).
However, recently, macrophages are also focused as new target for
cancer immunotherapy, since macrophages are the most common
immune cells and major immune regulators in tumor
microenvironment (TME) (3). TME is a combination of multiple
components including immune cells, fibroblasts, cytokines, extracellular
matrix, and cancer cells (4). Cancer cells release signaling cytokines
and extracellular vesicles in TME to suppress immune cell activation
and promote its own growth. Macrophages in TME, tumor-associated
macrohages (TAM) are affected by exposure to cytokines including
IL-4, IL-10, TGF-β1 and lactic acid and tumor extracellular vesicles
in TME and polarized to alternatively activated M2 type (5, 6). M2
TAM secret molecules that suppress anti-tumor immune response
and promote angiogenesis in TME so that promote tumor growth (7,
8, 9). In contrast, classically activated M1 macrophages suppress
tumor growth by releasing pro-inflammatory factors to support Th1
immunity and kill tumor cells by stromal destruction and normalizing
tumor vascular (10, 11, 12). Thus, there have been efforts to induce
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repolarization of M2 TAM to M1 TAM for tumor suppression with
anti-CD40 antibody (13), anti-MARCO antibody (14), BTK inhibitor
(15), iron oxide nanoparticles (11) and expression regulation of
immunomodulatory histidine-rich glycoprotein (16).
Exosomes are subpopulations of extracellular vesicles that
have a diameter of 40-150 nm and serve as molecule carriers for
intracellular communication (17, 18). Exosomes contain RNAs and
proteins originated from their parent cells and deliver these contents
to recipient cells, which induce phenotype changes within recipient
cells (19) or modulate immune responses (20, 21, 22). Thus, using
exosomes as nanocarriers that induce phenotype change in target
cells has proven to be successful method for cell therapy (23, 24). In
this context, we postulate that exosomes derived from M1
macrophages may repolarize M2 macrophages to M1 macrophages.
However, the amount of exosomes naturally released from cells is
low, and the method of exosome separation is inefficient (18). Given
the current limitation of low exosome production yield, several studies
have suggested nanovesicles (NV) as a substitute for exosomes (18,
25). NV are prepared by a serial extrusion of cells (18), have cell
membrane and size similar to those of exosomes, and are more
enriched in proteins and RNAs than exosomes. Thus, it is expected
that NV have similar but more intense effects on target cells than
exosomes (15, 18). Here, we propose M1 macrophage-derived NV
(M1NV) can be used to repolarize M2 TAM to M1 TAM for cancer
immunotherapy as M2 TAM suppress immune response to tumor and
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destabilize T cell function (7). In this study, we investigated whether
M1NV could be used as an immune regulator that repolarize M2
macrophage to M1 type in vitro.
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Figure 1. Schematic of the preparation of M1NV and the therapeutic
effects of M1NV in TME. M2 TAM secret anti-inflammatory cytokines
that promote tumor growth (Left). When M1NV polarize M2 TAM to
M1 type, M1 macrophages secret pro-inflammatory cytokines that
attacks cancer cells (Right).
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2. Experimental Section
2.1 Cell Culture
RAW264.7 macrophage cell line and CT26 colon carcinoma
cell line were purchased from Korean Cell Line Bank (Seoul, Korea)
and cultured in high-glucose Dulbecco’s Modified Eagle’s Medium
(DMEM, Gibco, NY, USA) supplemented with 10% (v/v) fetal bovine
serum (FBS, Gibco) and 1% (v/v) penicillin/streptomycin (Gibco). The
cells were incubated at 37℃ with 5% CO2 saturation. To obtain
mouse bone marrow-derived macrophages (BMDM), bone marrow
was collected from the femurs of 6-week-old female BALB/c mice as
described previously (26). In brief, hind legs were removed from mice,
and we removed muscle tissue from bone of legs. Then, we flushed
the bones with phosphate-buffered saline (PBS) using syringe to get
primary cells. Primary macrophages were differentiated for 7 days in
a macrophage differentiation medium, which is high-glucose DMEM
supplemented with 10% (v/v) FBS, 1% (v/v) penicillin/streptomycin,
and 10% (v/v) L929 cell-conditioned medium. L929 cell-conditioned
medium was prepared by growing L929 cells in high-glucose DMEM
containing 10% (v/v) FBS, 1% (v/v) penicillin/streptomycin for 10
days. The medium containing macrophage colony stimulating factor
secreted by L929 cells was harvested. At day 3, additional
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macrophage differentiation medium was added to the BMDM culture.
BMDM were collected at day 7 for further in vitro analysis. M1
classically activated macrophages were prepared by addition of 100
ng/mL lipopolysaccharide (LPS) to RAW264.7 cells. M2 alternatively
activated macrophages were induced by addition of 20 ng/mL IL-4
(Invitrogen, CA, USA) to BMDM.
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2.2 Preparation of M1NV
NV were prepared from M1 macrophages as described
previously (18). In brief, LPS-pretreated RAW264.7 cells (M1
macrophages) were suspended at a concentration of 5×106 cells/mL in
PBS, and sequentially extruded 11 times through polycarbonate
membrane filters (Whatman plc, Maidstone, Kent, UK) with pore size
of 1 µm, 400 nm, and 200 nm using a mini-extruder (Avanti Polar
Lipids, Alabaster, Alabama, USA) to form homogeneous nanosized
extracellular vesicles. The NV were then ultracentrifuged in a density
gradient formed by 50 and 10% OptiPrep layers at 100,000 g for 2 h
at 4℃. M1NV obtained from the interface of the layers were further
ultracentrifuged at 100,000 g for 2 h at 4℃. The protein concentration
of the isolated M1NV were quantified using Bradford reagent
(Sigma-Aldrich, St. Louis, Missouri, USA) according to the
manufacturer’s protocol.
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2.3 Preparations of Exosome
Exosomes were prepared from M1 macrophages as described
previously with modification (27). Exosome-depleted FBS was
prepared by ultracentrifugation at 100,000 g for 16 h at 4℃. M1
macrophages were incubated in DMEM medium containing 10% (v/v)
of exosome-depleted FBS and 1% (v/v) penicillin/streptomycin for 24
h. The culture medium was collected, and cells and debris were
eliminated by serial centrifugation at 500 g for 10 min, and 3,000 g
for 15 min, at 4℃. Exosomes were collected by ultracentrifugation at
100,000 g for 2 h at 4℃. The protein concentration of the isolated M1
macrophage-derived exosomes were quantified using the Bradford
reagent according to the manufacturer’s protocol.
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2.4 Physicochemical Characterization of M1NV
The size and particle density of M1NV and exosomes were
measured by nanoparticle tracking analysis using the Nanosight LM10
system (Malvern, Worcestershire, UK). M1NV and exosomes were
dispersed in PBS at 500 ng total proteins/mL, the NV particle size
and density were determined measured with CDD camera at level 14,
a slide shutter of 1,500, and a slider gain of 420. The chamber
temperature was maintained at 23℃. The measurements were
obtained in triplicate, and each individual measurement duration was 1
min. the data were analyzed using nanoparticle tracking analysis
software version with a detection threshold of 5 (multiple) with
autosetting of max jump distance and blur. ELS Z-1000 (Otsuka
Electronics, Osaka, Japan) was used to take electrophoretic light
scattering (ELS) measurements for particle zeta potential of M1NV
and exosomes. The structure of the M1NV and exosomes were
examined using a transmission electron microscope (LIBRA 120, Carl
Zeiss, Germany). A drop of M1NV or exosomes at a concentration of
4 µg∕mL was deposited onto a glow-discharged carbon-coated grid.
A drop of 1% uranyl acetate stain was added to the grid after three
minutes when the sample was deposited. The grid was rinsed with 5
drops of distilled water. The grid was subsequently dried and
visualized using a LIBRA 120, 120KV Energy-Filtering Transmission
Electron Microscope.
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2.5 mRNA Quantification of NV and Cells
Total RNA were extracted from non-treated RAW264.7 cells
(M0 macrophages), M1 macrophages, M0NV, and M1NV using 1 mL
of Trizol (Qiagen Valencia, CA, USA). The total RNA concentration
was determined using a NanoDrop spectrometer (ND-2000, NanoDrop
Technologies, USA). Six hundred nanograms of total RNA from each
sample were reverse-transcribed into cDNAs, and SYBR green-based
quantitative real time polymerase chain reaction (qRT–PCR) was
performed by a Step One Plus real-time PCR system (Applied
Biosystems, Waltham, Massachusetts, USA) with TOPreal qPCR 2X
PreMIX (Enzynomics, Daejeon, Korea). Cycling conditions were the
following: initial denaturation at 95℃ for 15 min, followed by 45
cycles at 95℃ for 10 s, 60℃ for 15 s, and 72℃ for 30 s. All of the
data were analyzed using the comparative Ct method (28). Three
samples were analyzed per group.
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2.6 MicroRNA Profiling Assay
M0NV and M1NV are prepared, as mentioned earlier. Total
RNA were prepared from M1NV and M0NV, as mentioned earlier and
quantified by NanoDrop spectrometer. microRNA (miRNA) expression
profiling with Affymetrix GeneChip® miRNA 4.0 assay are conducted
by a commercial service (BioCore, Seoul, Korea). All the experimental
results were saved as Microsoft Excel files.
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2.7 I n Vitro Cellular Uptake of M1NV
IL–4 treated BMDM (M2 macrophages) and CT26 cells were
allowed to attach to culture plates containing 10% (v/v)
FBS-containing medium for 24 h. Subsequently, DiI-labeled M1NV
were added into the culture at a concentration of 50 µg/mL and
incubated for 4 h. Cellular uptake of M1NV was evaluated using
fluorescence microscopy. Additionally, 1 h after the treatment with DiI
–labeled M1NV, M2 macrophages and CT26 cells were washed with
PBS and analyzed with Fluorescence-activated cell sorting (FACS).
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2.8 Viability of Cells after Treatment with M1NV
Various concentrations (10, 50, and 100 µg/mL) of M1NV
were added to cultures of M2 macrophages and CT26 cells, and the
cells were incubated for 24 h. The number of live cells was
determined with cell counting kit-8 (CCK-8) assay (DoGenBio, Seoul,
Korea) after 24 h, according to the manufacturer’s protocol (n=3 per
group). After replenishing with fresh medium, CCK-8 solution was
added into each well of 24 well plate, and the cells were incubated
for 2 h. Absorbance (= relative viable cell number) was measured at
450 nm using Powerwave X340 (Bio-Tek Instruments, Winooski,
Vermont, USA). The relative viable cell number was expressed
relatively to the viable cell number of the no treatment group. qRT–
PCR was performed to determine the mRNA expression of angiogenic
factors (FGF–2, VEGF, and PDGFβ). Three samples were analyzed
per group.
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2.9 Transwell Assay
M2 macrophages were co-cultured with M1 macrophages
using Transwell, or treated with 50 ng/mL M1NV for 24 h. CT26
cells were co-cultured with either untreated M2 macrophages, M2
macrophages co-cultured with M1 macrophages, or M2 macrophages
treated with M1NV for 24 h. The CCK-8 assay was performed on
cancer cells to evaluate cancer cell growth, as mentioned earlier. M1
macrophages were co-cultured with either M2 macrophages or CT26
cells for 24 h. M2 macrophages were co-cultured with M1
macrophages or treated with M1NV. Moreover, qRT–PCR was
performed to determine the expression levels of the M1 (CD86 and IL
–6) and M2 marker genes (CD206 and Fizz–1) in M1 macrophages
and M2 macrophages. Three samples were analyzed per group.
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2.10 I n Vitro Analyses of Macrophage Polarization
M2 macrophages were induced, as mentioned earlier. M2
macrophages are treated with 50 ng/mL of either M0NV or M1NV
for 24 h for repolarization. qRT–PCR was performed to determine
the expressions of M1 (IL–6, iNOS, and TNF–α) and M2-associated
genes (Fizz–1, IL–4, and IL–10), as mentioned earlier. Three
samples were analyzed per group. The protein expressions of the M1
macrophage marker (CD86) and M2 macrophage marker (CD206)
were evaluated by an immunocytochemistry assay. Cells were fixed
with 4% paraformaldehyde for 10 min at RT and washed in PBS.
Primary antibodies against CD86 (Santa Cruz Biotechnology, CA,
USA) and CD206 (Abcam, Cambridge, UK) were used for staining.
The samples were then incubated in PBS containing
Rhodamine-conjugated secondary antibodies (Jackson-Immunoresearch,
West Grove, Pennsylvania, USA) for 1 h at RT. All samples were
mounted with mounting solution containing
4',6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame,
CA, USA) to stain the nuclei, and were imaged using a fluorescent
microscope (Olympus, Tokyo, Japan). To confirm the production of
the M1 marker cytokine IL–6 and suppression of the M2 marker
cytokine IL–4 in M2 macrophages treated with 50 µg/mL M0NV or
M1NV for 24 h, cytokine secretion was analyzed using mouse IL–6
and IL–4 enzyme-linked immunosorbent assay (ELISA) kits (R&D
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Systems, Minnesota, USA) following the manufacturer’s instructions.
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3. Results and Discussion
3.1 Characterization of M1NV and Exosomes
Nanoparticle tracking analysis of M1NV and exosomes
showed size distributions with a mean diameter of 189.7 ± 2.5 nm,
146.0 ± 5.9 nm, respectively (Figure 2A). Electrophoretic light
scattering (ELS) of M1NV and exosomes revealed that M1NV and
exosomes have similar zeta potential (Figure 2A). The closed
vesicular morphology of M1NV and exosomes were analyzed with
Energy-Filtering Transmission Electron Microscopy (Figure 2B).
Particle number and protein amounts of M1NV are higher compared
to that of exosomes produced from the same number of macrophages
(Figure 2C). As M1NV were prepared by serially extruding M1
macrophages, we evaluated pro-inflammation and M1 macrophage
markers mRNAs such as CD86, IL-6, TNF-α, and iNOS in cells and
NV. We compared the mRNA expression of M0NV and M1NV to
that of respective cells via qRT-PCR (Figure 2D). This analysis
showed that both M1 macrophage and M1NV show higher mRNA
expression of pro-inflammation-relative and M1 macrophage marker
mRNAs compared to M0NV and M0 macrophages. Thus, we
concluded that NV maintains contents including RNAs of M1
macrophage markers of their parent cells (24).
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Since certain types of miRNAs induce macrophages to
polarize to certain type (29). we evaluated the difference in the
miRNA amount between M1NV and M0NV via microarray assay.
The M1 and M2 macrophage-related miRNA expressions of M1NV
are shown as compared to those of M0NV (Figure 2E). Substantially,
M1 macrophage-related miRNAs are upregulated and M2
macrophage-related miRNAs are downregulated in M1NV (30, 31, 32,
33). Importanly, upregulated expressions of miR-155, miR-125, and
miR-21 are known to polarize macrophage to M1 type, and
downregulated expressions of miR-34a, let-7c, and let-7f are known
to polarize macrophage to M2 type (29, 32, 34). Thus, the miRNA
profiles of M1NV confirm that M1NV have potential to polarize M2
TAM to M1 macrophages.
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Figure 2. Characterization of M1NV. (A) Size distributions and zeta
potentials of M1NV and M1 macrophage-derived exosomes, as
analyzed by nanoparticle tracking (n=3) and electrophoretic light
scattering (n=3), respectively. (B) Transmission electron microscopic
images of M1NV and M1 macrophage-derived exosomes. (C)
Comparison of the number and protein amounts of M1NV and
exosomes, both of which were produced from the same number of
M1 macrophages (*p<0.05 vs. exosomes). (D) Relative mRNA
amounts of pro-inflammatory M1 macrophage markers (CD86, IL–6,
iNOS, and TNF–α) in M0 macrophages (M0 cell), M1 macrophages
(M1 cell), M0NV and M1NV. The amounts of mRNA from genes of
interest were normalized to the GAPDH amount. (E) Microarray
analysis of M1, M2 macrophage-related miRNA expressions in M1NV
compared to M0NV. Red and green indicate upregulation and
downregulation, respectively, in the miRNA expressions of M1NV as
compared to those of M0NV. The number indicates log2 ratio of
M1NV miRNA to M0NV miRNA. Only miRNAs whose difference in
the amount between M1NV and M0NV is over 1.5 fold are
represented.
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3.2 I n vitro Cellular Uptake and Cytotoxicity of
M1NV
In vitro uptake of M1NV was evaluated to determine whether
M1NV are better taken up by TAM compared to cancer cells in
TME, where macrophages and cancer cells are abundant. M2
macrophages were obtained as substitute of M2 TAM. Then, obtained
M2 macrophages and CT26 cells were treated with DiI-conjugated
M1NV. fluorescence images and FACS analysis revealed that M1NV
were taken up by 80.4% of M2 macrophage, and were taken up by
12.0% of cancer cells (Figure 4A and B). The exact mechanism for
the higher cellular uptake of M1NV by M2 macrophages than cancer
cells is not clear, but phagocytic activity of macrophages may be
responsible for the higher cellular uptake.
To determine whether M1NV affect viability of cells, a cell
viability assay using CCK-8 assay was performed on M2
macrophages and CT26 cells cultured in vitro for 24 h in the
presence of 0, 10, 50 and 100 µg/mL M1NV. Figure 4C showed that
M1NV exhibited no cellular toxicity to both M2 macrophages and
cancer cells. In addition, M1NV had no effects on angiogenic gene
expression of CT26 (Figure 4D). This data reveals that M1NV
treatment itself does not induce apoptosis in macrophages or cancer
cells.
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Figure 3. Cellular uptake and cytotoxicity of M1NV in vitro. (A)
Fluorescent image analysis and (B) FACS analysis for M1NV uptake
by M2 macrophages and cancer cells. M1NV were labeled with a red
fluorescent dye (DiI) prior to the M1NV (50 µg/mL) treatments for
fluorescent imaging (4 h) and FACS analyses (1 h) (*p<0.05 vs.
cancer cells). NS: no staining. (C) Relative viable cell number of
cancer cells and M2 macrophages treated with various doses of
M1NV for 1 day, as evaluated by the CCK-8 assay (n=3) (*p<0.05
vs. 0 µg/mL). (D) Effects of M0NV or M1NV treatments for 24 h on
the angiogenic gene expressions in cancer cells. Amounts of mRNA
from genes of interest were normalized to the GAPDH amount (n=3,
ns: not significant).
- 30 -
3.3 Comparison of the Therapeutic Effects
between M1NV and M1 Macrophages
To determine whether M2 macrophages repolarized to M1
type affect cancer cell growth, a CCK-8 assay was performed on
CT26 cells co-cultured with M2 macrophages. We postulate that M2
macrophages were repolarized by either co-culturing with M1
macrophages or M1NV treatment. M2 macrophages treated with
M1NV (group 3 in Figure 4A) suppressed the cancer cell growth,
compared to M2 macrophage only (group 1 in Figure 4A) and M2
macrophages co-cultured with M1 macrophages (group 2 in Figure
4A). The data of Figure 4A suggest that repolarization of M2
macrophages to M1 type suppresses cancer cell proliferation, since
M1 macrophages are known to suppress tumor growth.
To examine how M1 macrophages are affected by M2
macrophages and cancer cells, both of which are abundant cell types
in tumors, M1 macrophages were co-cultured with M2 macrophages
or CT26 cells for 24 h. Subsequently, the mRNA expressions of the
M1 marker (CD86) and M2 markers (CD206 and Fizz–1) in the M1
macrophages were evaluated by qRT–PCR (Figure 4B). It is shown
that mRNA expression of the M1 marker was decreased and that of
the M2 markers were increased in M1 macrophages co-cultured with
M2 macrophages or CT26 cells. The data indicate that M1
macrophages can be polarized to M2 macrophages through
- 31 -
interactions with M2 macrophages and cancer cells. Thus,
implantation of M1 macrophages to TME for cancer therapy may
result in polarization of the implanted M1 macrophages to M2 TAM
and ultimate stimulation of tumor growth by increased M2 TAM (35).
Additionally, the mRNA expressions of the M1 marker (IL–6) and
M2 marker (Fizz–1) in the M2 macrophages co-cultured with M1
macrophages or treated with M1NV were evaluated by qRT–PCR
(Figure 4C). The mRNA expression of the M2 marker was shown to
be decreased in both groups, and that of the M1 marker was
significantly increased only in the M1NV treatment group.
Importantly, the data revealed that M1NV treatment is more effective
in the repolarization of M2 macrophages to M1 macrophages than
direct treatment with M1 macrophages. Thus, we concluded that
using M1NV is more effective way for repolarize M2 TAM to M1
macrophages than using M1 macrophages.
- 32 -
- 33 -
Figure 4. Comparison of the therapeutic effects and macrophage
polarization induction effects between M1 macrophages and M1NV.
(A) Relative viable cancer cell number co-cultured with M1
macrophage-co-cultured-M2 macrophages (Group 2) or with M1NV
treated-M2 macrophages (Group 3) for 1 day was examined by
CCK-8 assay (n=3) compared to control group (co-cultured with
untreated M2 macrophage; Group 1). n=4-6, ns: not significant,
*p<0.05 vs. Control. (B) Relative mRNA expressions of M1 and M2
markers in M1 macrophages co-cultured with either M2 macrophages
(group 2) or cancer cells (group 3) for 24 h, as compared to the
control group (M1 macrophages only, group 1). (C) Relative mRNA
expressions of M1 and M2 markers in M2 macrophages after 24 h of
co-culture with M1 macrophages (group 2) or after 24 h of M1NV
treatment (group 3), as compared to the control group (M2
macrophages only, group 1). (B)–(C) n=3, *p<0.05 vs. control, and
†p<0.05 vs. group 2. The mRNA expression from genes of interest
was normalized to GAPDH and expressed as a relative change.
- 34 -
3.4 M1NV Polarize M2 macrophages to M1 type.
To determine whether M1NV repolarize M2 macrophages to M1
type, we evaluated the mRNA expression changes of M1 (iNOS,
TNF-α, and IL-6) and M2 (IL-4, IL-10, and Fizz-1) type markers in
M2 macrophages after 24 h treatment with M1NV (Figure 5A). To
exclude the possibility that NV that do not contain pro-inflammatory
factors could repolarize M2 macrophages to M1 type, M0NV
treatment was used as a comparison group. The data showed that
mRNA levels of M1 markers were significantly higher in the M1NV
treatment group, whereas those of some M2 markers were lower
compared to the no treatment group and the M0NV treatment group.
The mRNA expressions of the angiogenesis factor VEGF and
metastatic factor CCL–18 were also evaluated in M2 macrophages
after 24 h treatment with M1NV. no significant difference was
observed in mRNA expression of VEGF among the three groups and
significant decrease was observed in mRNA expression of CCL–18
of M1NV-treatment group compared to other groups. (Figure 5B).
Similarly, immunofluorescence analysis of M2 macrophages obtained
after 24 h of treatment with M1NV showed increased number of
CD86+ cells and decreased number of CD206+ cells compared with
the no treatment group and the M0NV treatment group (Figure 5C).
In addition, the repolarization of M2 macrophages to M1 type resulted
in the enhanced pro-inflammatory cytokine (IL-6) expression and the
reduced anti-inflammatory cytokines (IL-4) expression (Figure 5D).
- 35 -
This suggest that M1NV effectively repolarize M2 marcophages to
M1 type. Also, this data revealed that the contents in NV were the
major facts in repolarizing M2 macrophages, and just treatment of
NV itself did not affect repolarization much, since repolarization by
M0NV were not stronger than those of M1NV. Thus, we postulate
that M1NV may repolarize M2 TAM to M1 type and inhibit the
growth of tumor.
- 36 -
- 37 -
- 38 -
Figure 5. Polarization of M2 macrophages to M1 macrophages in
vitro induced by the M1NV treatment. M2 macrophages were treated
with either M0NV or M1NV for 24 h. (A) Relative expressions
mRNA in M1 (iNOS, TNF–α, and IL–6) and M2 markers (IL–4,
IL–10, and Fizz–1), as evaluated by qRT–PCR. (B) Angiogenic
gene (VEGF), metastatic gene (CCL18) expressions in M2
macrophages after M0NV or M1NV treatments for 24 h. The mRNA
expressions from genes of interest were normalized to GAPDH. NT
denotes no treatment. (C) Immunofluorescence staining for CD86 (red,
M1 marker) and CD206 (red, M2 marker) of M2 macrophages treated
with either M0NV or M1NV for 24 h. Nuclei were stained with DAPI
(blue). (D) ELISA assay assessment for secretion of
pro-inflammatory cytokine (IL–6) and anti-inflammatory cytokine
(IL–4) from M2 macrophages treated with either M0NV or M1NV
for 24 h. ND denotes no detection. (A)–(D) n=3 per group, *p<0.05
vs. NT, †p<0.05 vs. M0NV.
- 39 -
4. Conclusion
Our data demonstrated that M1NV treatment repolarizes
macrophages from pro-tumor M2 type to anti-tumor M1 type, which
suppress tumor tissue growth. M1NV have higher yield than naturally
produced exosomes and maintain RNAs and proteins originated from M1
macrophages. M1NV also have miRNAs known to participate in M1
polarization. M1NV are more taken up by M2 macrophages than CT26
cells. In vitro treatment of M2 macrophages with M1NV induced
repolarization toward M1 type. The repolarization was proved by
increased M1 markers and decreased M2 markers in RNAs, membrane
proteins, cytokines. M1NV treatment needs to prove its therapeutic
efficiency for in vivo tumor model in the further study.
- 40 -
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요약 (국문초록)
암 치료를 위한 M1 대식세포에서 추출한
나노베지클에 의한 M2 대식세포의 재분화 연구
암 면역 치료는 몸 안의 면역세포를 활성화하여 암에 대항하는
면역작용을 일으켜 암을 치료하는 방법이다. 대식세포가 종양의 미세환
경에서 가장 많은 면역세포이므로, 종양 관련 대식세포는 암 면역 치료
를 위해서 연구되고 있다. 대식세포는 두 가지 다른 표현형을 가지고 있
는데, 하나는 염증을 감소시키고, 혈관 형성 등을 하여 암의 성장을 돕
는 M2형, 다른 하나는 염증을 일으키고, Th1 면역작용을 일으켜 암의 성
장을 막는 M1형이다. 암 주변에는 암세포가 내뿜는 여러 가지 물질들에
의하여 M2 대식세포로 변한 종양 관련 대식세포가 많이 존재한다. 이
M2 종양 관련 대식세포가 면역 억제, 혈관 생성 등을 통해 암 성장을
도우므로, 이 M2 종양 관련 대식세포를 암의 성장을 억제하는 M1형으로
재분화하는 것이 일종의 암 면역 치료가 될 수 있다.
이번 연구에서는 M2 종양 관련 대식세포를 M1 형으로 재분화할
물질로서, M1 대식세포에서 추출한 엑소좀 모사 M1 나노베지클을 이용
하였다. 엑소좀은 모세포에서 유래한 유전물질과 단백질을 수용 세포에
전달하여 표현형의 변화를 일으킬 수 있다고 알려져 있다. 단 자연적으
- 47 -
로 만들어진 엑소좀은 수득률이 낮으므로, 이 연구에서는 엑소좀을 대신
하여 세포를 압축시켜 나노베지클을 만들어 사용하였다.
M1 나노베지클은 엑소좀과 비슷한 크기를 가지고 있고, 더 많은
단백질을 포함하고 있다. 또한 M1 대식세포의 RNAs, miRNAs 등을 포함
하고 있고, 암세포보다 M2 대식세포에 더 많이 흡수되므로, 암 미세환경
에서 M1 대식세포 유래의 물질들을 M2 대식세포에 더 많이 전달할 수
있을 것으로 기대할 수 있다. 또한, M1 대식세포를 그대로 처리하면 처
리해 준 M1 대식세포가 M2 대식세포와 암세포의 영향으로 M2형으로 재
분화되어 M2 대식세포를 늘리는 결과가 예상되는 것에 비해, M1 나노베
지클은 그런 위험이 없고 M2 대식세포를 재분화하는 효과가 M1 대식세
포에 비해 높으므로 M1 나노베지클이 재분화에 더 적합한 물질이라 판
단되었다. M1 나노베지클을 M2 대식세포에 처리하였을 때, 대식세포 내
에서 M2 마커 RNAs, 단백질, 사이토카인의 발현량이 감소하고, M1 마커
RNAs, 단백질, 사이토카인의 발현량이 증가하므로 M1 나노베지클의 처
리가 M2 대식세포를 M1형으로 재분화시킨다는 결론을 내렸다.
향후에는 실제 암 동물모델에서 암 성장 억제 효과를 가지고 있
는지 확인하여 M1 나노베지클의 유용성을 확인하고, 암뿐만이 아니라
M2 대식세포과 관련하여 생기는 면역질환 등에 쓰일 수 있을 것으로 보
인다.
주요어: 암 면역 치료, 대식세포 분화, 나노베지클, 종양 관련 대식세포
학번: 2016-21059