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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

- i -

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),

- ii -

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

- iii -

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

- iv -

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

- 1 -

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

- 2 -

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

- 3 -

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

- 4 -

(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.

- 5 -

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

- 6 -

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

- 7 -

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.

- 8 -

- 9 -

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).

- 10 -

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

- 11 -

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.

- 12 -

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.

- 13 -

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.

- 14 -

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.

- 15 -

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.

- 16 -

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.

- 17 -

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).

- 18 -

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.

- 19 -

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.

- 20 -

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

- 21 -

Systems, Minnesota, USA) following the manufacturer’s instructions.

- 22 -

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).

- 23 -

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.

- 24 -

- 25 -

- 26 -

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.

- 27 -

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.

- 28 -

- 29 -

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