Transfer of miRNA in Macrophage-Derived Exosomes Induces Drug Resistance in Pancreatic Adenocarcinoma

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is known for its resistance to gemcitabine, which acts to inhibit cell growth by termination of DNA replication. Tumor-associated macrophages (TAM) were recently shown to contribute to gemcitabine resistance; however, the exact mechanism of this process is still unclear. Using a genetic mouse model of PDAC and electron microscopy analysis, we show that TAM communicate with the tumor microenvironment via secretion of approximately 90 nm vesicles, which are selectively internalized by cancer cells. Transfection of artificial dsDNA (barcode fragment) to murine peritoneal macrophages and injection to mice bearing PDAC tumors revealed a 4-log higher concentration of the barcode fragment in primary tumors and in liver metastasis than in normal tissue. These macrophage-derived exosomes (MDE) significantly decreased the sensitivity of PDAC cells to gemcitabine, in vitro and in vivo. This effect was mediated by the transfer of miR-365 in MDE. miR-365 impaired activation of gemcitabine by upregulation of the triphospho-nucleotide pool in cancer cells and the induction of the enzyme cytidine deaminase; the latter inactivates gemcitabine. Adoptive transfer of miR-365 in TAM induced gemcitabine resistance in PDAC-bearing mice, whereas immune transfer of the miR-365 antagonist recovered the sensitivity to gemcitabine. Mice deficient of Rab27 a/b genes, which lack exosomal secretion, responded significantly better to gemcitabine than did wildtype. These results identify MDE as key regulators of gemcitabine resistance in PDAC and demonstrate that blocking miR-365 can potentiate gemcitabine response.
Significance: Harnessing macrophage-derived exosomes as conveyers of antagomiRs augments the effect of chemotherapy against cancer, opening new therapeutic options against malignancies where resistance to nucleotide analogs remains an obstacle to overcome. Cancer Res; 78(18); 5287–99. ©2018 AACR.

Full text

Tumor Biology and Immunology
Transfer of miRNA in Macrophage-Derived
Exosomes Induces Drug Resistance in
Pancreatic Adenocarcinoma
Yoav Binenbaum
1
, Eran Fridman
1
, Zvi Yaari
2
, Neta Milman
1
, Avi Schroeder
2
,
Gil Ben David
1
, Tomer Shlomi
3
, and Ziv Gil
1,4
Abstract
Pancreatic ductal adenocarcinoma (PDAC) is known for its
resistance to gemcitabine, which acts to inhibit cell growth by
termination of DNA replication. Tumor-associated macro-
phages (TAM) were recently shown to contribute to gemcita-
bine resistance; however, the exact mechanism of this process
is still unclear. Using a genetic mouse model of PDAC and
electron microscopy analysis, we show that TAM communi-
cate with the tumor microenvironment via secretion of
approximately 90 nm vesicles, which are selectively internal-
ized by cancer cells. Transfection of artificial dsDNA (barcode
fragment) to murine peritoneal macrophages and injection to
mice bearing PDAC tumors revealed a 4-log higher concen-
tration of the barcode fragment in primary tumors and in liver
metastasis than in normal tissue. These macrophage-derived
exosomes (MDE) significantly decreased the sensitivity of
PDAC cells to gemcitabine, in vitro and in vivo. This effect was
mediated by the transfer of miR-365 in MDE. miR-365
impaired activation of gemcitabine by upregulation of the
triphospho-nucleotide pool in cancer cells and the induction
of the enzyme cytidine deaminase; the latter inactivates
gemcitabine. Adoptive transfer of miR-365 in TAM induced
gemcitabine resistance in PDAC-bearing mice, whereas
immune transfer of the miR-365 antagonist recovered the
sensitivity to gemcitabine. Mice deficient of Rab27 a/b genes,
which lack exosomal secretion, responded significantly better
to gemcitabine than did wildtype. These results identify
MDE as key regulators of gemcitabine resistance in PDAC and
demonstrate that blocking miR-365 can potentiate gemcita-
bine response.
Significance: Harnessing macrophage-derived exosomes
as conveyers of antagomiRs augments the effect of chemo-
therapy against cancer, opening new therapeutic options
against malignancies where resistance to nucleotide analogs
remains an obstacle to overcome. Cancer Res; 78(18); 5287–99.
2018 AACR.
Introduction
Pancreatic ductal adenocarcinoma (PDAC) ranks fourth
among cancer-related deaths. Despite decades of research, the
cure rate of the disease remains disappointingly low (<5%; ref. 1).
This dismal prognosis is due to late detection and to resistance of
tumors to all known systemic therapies.
Gemcitabine, the first-line drug for the treatment of PDAC, is a
cytidine analog that acts to inhibit cell growth by arrest of DNA
replication. Resistance to gemcitabine develops within weeks of
initiation of therapy, as a result of intrinsic resistance and envi-
ronmental factors (2). Gemcitabine is metabolized intracellularly
by deoxycytidine kinase (dCK), to active phospho-nucleosides;
the incorporation of these nucleosides into DNA and RNA leads to
replication arrest. Among the mechanisms known to cause gem-
citabine resistance are loss of membranal transporters, deficiency
of dCK, competition with de novo CTP, and upregulation of
cytidine deaminase (CDA), the enzyme that metabolizes gemci-
tabine to its inactive form. Treatment with nab-paclitaxel was
shown to reduce CDA expression and potentiate gemcitabine
efficacy; this highlights the importance of CDA in mediating drug
resistance (3).
Macrophages are associated with poor prognosis in PDAC
(4) and were shown to secrete soluble factors that induce gemci-
tabine resistance of PDAC cells (5). We hypothesized that tumor-
associated macrophages (TAM) secrete vesicles that transfer
molecular signals to cancer cells, thus inducing drug resistance.
Here, we demonstrate a mechanism by which resistance to
chemotherapy is mediated through shuttling of miRNAs between
TAM and cancer cells, via exosomes.
Materials and Methods
Animals
All animal experiments were approved by The Institutional
Animal Care and Use Committee at the Technion, approval#
IL-086-07-2013 and IL-124-12-2012. Wild-type (WT) C57/bl
1
The Laboratory for Applied Cancer Research, Department of Otolaryngology
Head and Neck Surgery, Clinical Research Institute at Rambam Healthcare
Campus, Haifa, Israel.
2
Laboratory for Targeted Drug Delivery and Personalized
Medicine Technologies, Technion, Israel Institute of Technology, Haifa, Israel.
3
Departments of Computer Science and Biology, Technion, Israel Institute of
Technology, Haifa, Israel.
4
Technion Integrated Cancer Center, Rappaport
Institute of Medicine and Research, Technion, Israel Institute of Technology,
Haifa, Israel.
Note: Supplementary data for this article are available at Cancer Research
Online (http://cancerres.aacrjournals.org/).
Corresponding Author: Ziv Gil, Rambam Medical Center, Haifa 3525408, Israel.
Phone: 972-4-7772480; E-mail: G_Ziv@rambam.health.gov.il
doi: 10.1158/0008-5472.CAN-18-0124
2018 American Association for Cancer Research.
Cancer
Research
www.aacrjournals.org 5287
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mice were purchased from Harlan. Rab27a
/
b
/
were a kind
gift from Miguel C. Seabra of the National Heart and Lung Insti-
tute, Imperial College, London, United Kingdom. Rab27a
/
b
/
mice were bred and genotyped as described (6, 7).
Tissue culture
PDAC K989 cell line is an in-house line, generated from an
explant culture of a pancreatic tumor from a KPC mouse (8). Cells
were authenticated by sequencing of Kras G12D and TP53 R172H
mutations, and pdx-1 CRE insertion (primers are detailed in
Supplementary Table S1). The following early passage ATCC cell
lines were used in our experiments: NIH-3T3 (ATCC CRL-1658
passage 8–12), Mia PaCa 2 (ATCC CRL-1420 passage 3–7), and
THP-1 (ATCC TIB-202, passage 3–6). All cells were tested for
Mycoplasma on a regular basis. Murine peritoneal macrophages
(mpMacrophages) were isolated from the peritoneal lavage of
WT C57/Bl mice or Rab27a
/
b
/
mice. mpMacrophages were
incubated for 24 hours with 100 ngr/mL LPS and 20 ngr/mL g-IFN
for M1 polarization, and with 20 ngr/ml IL4 (Peprotech) for
M2 polarization. Polarization was verified by RT-PCR for the
transcripts Arginase-1, CD206, CD86, and iNOS, as described
(9). THP-1 monocytes were differentiated to macrophages as
described (10), except that 100 ng/mL of phorbol 12-myristate
13-acetate (Sigma; P8139) was used. Macrophages were polarized
toward the M2 phenotype as described above, using human IL4
(Peprotech).
Exosome purification and characterization
Exosomes were isolated from macrophage conditioned media
as described previously (11). One dose corresponded to exosomes
from 30 mL media. Unless otherwise stated, one dose of exosomes
was used. Exosome size distribution and concentrations were
assessed with the Nanoparticle Tracking Analysis (NTA) system
(Nanosight NS300). One dose contained approximately 2 mgof
protein, and 5 10
11
exosomes, as measured by Nanosight. RNA
was extracted by the Hybrid-R miRNA purification Kit (GeneAll).
Cryo-TEM sample preparation and acquisition, and NTA sample
acquisition and analysis are described in the Supplementary Data
Section.
Exosome internalization experiments
Exosomes were labeled with PKH67 (Sigma) or CFSE (Molec-
ular Probes) according to the manufacturer's instructions. Excess
dye was removed using exosome spin columns (Thermo Fisher
Scientific). PKH67-stained exosomes were applied on cells for 75
minutes, and cells were then vigorously washed thrice. Cells were
fixed by 4% PFA and counterstained by PKH26 (Sigma). Slides
were viewed on a LSM-550 confocal microscope (Ziess), with a
X63 objective, through a pinhole of 120 to 134 mm, with 23–30
Z-stacks per field of view. Images were analyzed using Imaris
software with an Imaris-Cell module (Bitplane) that identified the
nucleus and the cell membrane.
Gene expression RT-qPCR, Western blot analysis, and immu-
nofluorescence were previously described (12–15). Western blot-
ting and immunoprecipitation are described in Supplementary
Materials. Supplementary Tables S2 and S3 provide lists of anti-
bodies and qPCR primers used in the study. For miRNA detection,
RT-qPCR was performed with commercial TaqMan MicroRNA
Assays (ABI, Thermo Fisher Scientific) using 5 ngr of small RNA
per reaction. U6 was used as an internal control. For these
experiments, primers designed for the homo sapiens miRNAs
were used, due to complete sequence homology. Assay IDs are
detailed in Supplementary Table S4.
More methods are described in the Supplementary Methods
section in the Supplementary Materials.
Results
Secretion of exosomes from mpMacrophages
The accumulation of TAM (bearing M2 markers) in sections
from patients with PDAC, together with our previous findings that
M2-polarized macrophages are involved in gemcitabine resistance,
led us to investigate the mechanism of M2 macrophage–induced
drug resistance (5). M2-polarized murine mpMacrophages were
generated by adding cytokine IL4 to the media (Supplementary
Fig. S1A; refs. 9, 16). The media were purified by differential cen-
trifugations (11) and examined by a cryogenic transmission elec-
tron microscope. The electron microscope imaging revealed the
presence of nanovesicles of variant sizes (Fig. 1A; Supplementary
Fig. S1B; ref. 17). The diameter distribution of these nanovesicles
revealed a mean size of 90 nm for cryopreserved spheres (Fig. 1B),
or a mean diameter of 135 nm in room temperature (Supple-
mentary Fig. S1C). This variation in size can be explained by
expansion of vesicle volume at room temperature, relative to the
cryo-TEM environment (17, 18), or by inherent differences
between measurement techniques (electron microscope vs. NTA;
ref. 19). Immunoblotting of lysates from purified nanovesicles,
using known exosomal markers (11, 20, 21), demonstrated that
these nanovesicles expressed CD63 and LAMP2 (Fig. 1C). This
observation, together with the size distribution, suggested that
these were MDE.
MDE and gemcitabine resistance
As M2 mpMacrophages can secrete soluble signals that induce
chemotherapy resistance, we conjectured that MDE may also play
a role in this process. We evaluated the effect of gemcitabine on
PDAC K989 cells in the presence of MDE. MDE significantly
decreased the sensitivity of K989 cells to gemcitabine (Fig. 1D,
P<0.01 at 5–50 mmol/L of gemcitabine). At a gemcitabine
concentration of 50 mmol/L, the survival of K989 cells was
increased by 100% after adding MDE compared with control
(P¼0.001). MDE affected the response of K989 cells to gemci-
tabine (5 mmol/L) in a dose-dependent manner (P¼0.02, Fig.
1E). Similar to the KPC cell line, the MiaPaCa-2 human pancreatic
cell line incubated with MDEs from THP-1 cells also demon-
strated reduction of sensitivity to gemcitabine (Fig. 1F, P<0.05).
Selectivity of MDE
To further investigate the mode of interaction between MDE
and cancer cells, MDE were stained by the lipophilic dye, PKH67
(green), and incubated with nonfixed K989 cells for 75 minutes,
followed by vigorous washing. After fixation, the cell membranes
were stained with PKH26 (red). MDE could be detected in the
cytoplasm of cancer cells (Fig. 2A). Analysis of the intracellular
architecture revealed that cytoplasmic spheres culminated close to
the plasma membrane (Fig. 2B and C; Supplementary Fig. S1D).
These data suggest that exosomes secreted by M2 mpMacrophages
are readily internalized by K989 cells.
Next, we evaluated the selectivity of MDE, by comparing their
uptake by cancer cells and stromal cell monocultures. We first
incubated K989 cells and fibroblasts (NIH-3T3) in the presence of
PKH67-labeled MDE for 75 minutes and evaluated the MDE
Binenbaum et al.
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uptake levels by measuring the green PKH67 signal in each cell
line. Supplementary Fig. S2A shows that 26.6% of the K989 cells
were positive for PKH67, whereas none of the fibroblasts showed
positive MDE uptake (P<0.001, Supplementary Fig. S2A).
Despite the detection of a robust exosome signal in the cytoplasm
of K989 cells, immunofluorescence microscopy detected only low
signal levels in NIH-3T3 cells (Supplementary Fig. S2B). To
further evaluate exosomal distribution in PDAC tumors ex vivo,
tumors from 5-month-old KPC mice were dissociated to a single-
cell suspension and plated to adhere to a tissue culture dish. The
cells were incubated with PKH67-labeled MDE and analyzed by
flow cytometry. Supplementary Fig. S2C demonstrates that anti–
cytokeratin antibody-stained K989 cells, but not NIH-3T3 fibro-
blast or mpMacrophages. In 21.5% of the CK
þ
cancer cells,
intracellular PKH67-labeled exosomal staining was observed,
compared with 0.38% of the CK-negative cells (Fig. 2D; Supple-
mentary Fig. S2D).
To investigate whether macrophages transfer exosomes to
pancreatic cancer cells in vivo, we synthesized a unique 75-nt-long
dsDNA "barcode fragment," which was transfected to mpMacro-
phages. Following verification of the barcode in MDE (Supple-
mentary Fig. S2E), barcode-transfected mpMacrophages were
injected i.p. to mice carrying K989-PDAC tumors. Mice were
sacrificed after 48 hours, and their organs were separately disso-
ciated to single-cell suspensions. Tumor cells (CK-positive) and
mpMacrophages (F4/80 positive) were sorted by FACS, and the
abundance of the barcode in these populations was assessed by
qRT-PCR. Figure 2E shows that in both the primary tumors and
the liver metastases, the DNA barcode accumulated predominantly
in CK
þ
PDAC cells compared with the CK-negative stromal cells
(P<0.001). Uptake in the normal pancreas, spleen, and liver was
4–5 log less than in the primary tumor or in metastases. Overall,
these results suggest a selective transfer of exosomes from mpMa-
crophages to cancer cells, both ex vivo and in vivo.
Exosomal transfer of miRNA from macrophages to PDAC cells
Growing evidence indicates that exosomes are enriched in
miRNAs (22, 23). Analysis of the content of MDE using the
Agilent Bioanalyzer RNA LabChip revealed abundant short RNAs
measuring 18–22 nt, the size of miRNAs (Supplementary Fig.
S3A), whereas RNA fragments longer than 200 nt were not evident
in MDE (Supplementary Fig. S3B). A literature search revealed
0
0.2
0.4
0.6
0.8
1
1.2
No exo Exo
0
0.3
0.6
0.9
1.2
1.5
1.8
Relave cell number
P < 0.001
P = 0.02
BA
D
E
C
CD63
LAMP-2
M2 MDEs
0400200 600
40
20
0
Number of vesicles
0
0.2
0.4
0.6
0.8
1
1.2
50301510510.10
Relave cell number
Relave cell number
μmol/L Gemcitabine
μmol/L Gemcitabine
NoExo Exo
F
0 0.01 10 1000.1
P < 0.05
P < 0.01
60
Vesicle diameter (nm)
Figure 1.
MDE and gemcitabine resistance.
A, Cryo-TEM image of exosomes in mouse
peritoneal macrophage media (bar, 200
nm). B, Distribution of exosome diameters
measured in the cryo-TEM images. C,
Western blot analysis of exosomes from
M2-polarized mpMacrophages. D,
Proliferation of K989 cells, pretreated with
exosomes (empty circles) or control (filled
triangles), incubated with escalating doses
of gemcitabine (P<0.001). Samples were
normalized to proliferation without
gemcitabine. E, Proliferation of K989
cells treated with escalating doses of
MDE 5mmol/L gemcitabine (P¼0.02).
Samples were normalized to proliferation
without gemcitabine. F, Proliferation of
MiaPaCa-2 cells, pretreated with
THP-1–derived exosomes (empty circles
bars) or control (filled triangles), incubated
with escalating doses of gemcitabine
(P<0.001). Samples were normalized to
proliferation with gemcitabine only.
Exosomes Induce Gemcitabine Resistance in Pancreatic Cancer
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Phase Membrane DAPIMDEs Overlay
B
A
C
0
20
40
60
80
100
120
-3 -1 1 3 5 7 9 11131517192123252729
Number of vesicles
Distance (μm)
Distance from plasma membrane
Distance from nucleus's membrane
75 min
Incubaon
PDAC
Dissociaon
Stained MDEs Cytokeran staining
FACS
DE
MDEs
+
-
0.01
0.1
1
10
100
1,000
10,000
100,000
1000,000
Normal
panc
Tumor Liver
met
Liver Spleen Lung
Relave quanty
Tissue
CK+ Negave F4/80+
*
*
*
Distance to nucleus membrane
PKH67
CK- CK+
CK- CK+
Cell count
-MDEs
+MDEs
% PKH67-Posive cells
99.6% 0.38%
69.32% 21.57%
0
5
10
15
20
25
CK- CK+
Figure 2.
Selectivity of MDE. A, Confocal images of K989 cells with or without stained exosomes (green). Cell membranes are shown in red (bar, 10 mm). B, 3D cell image of
exosome distribution inside a K989 cell. C, Analysis of the distance of internalized exosomes from the plasma membrane or nucleus. Negative values denote
exosomes inside the nucleus. D, Exosome internalization by K989 cells. Top, experiment design. Bottom, FACS analysis of exosome (PKH67) uptake by K989 cells
(cytokeratin-positive) or stromal cells (cytokeratin-negative). The bar graph shows percentages of PKH67-positive in indicated cell populations. E, qPCR for
detection of the ds-DNA barcode, recovered from indicated tissue: PDAC cells (CK
þ
), macrophages (F4/80
þ
), and stromal cells (negative). ,P<0.001. panc,
pancreas; met, metastasis.
Binenbaum et al.
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that miRNAs 21,181b, 320, 365, and Let-7a were previously
implicated in the induction of chemotherapy resistance
(24–26, 27). We profiled the miRNA content of M1 and M2
mpMacrophages using nCounter Mouse miRNA Expression
Assay (Nanostring). We found miR-365 to rank among the
most differentially upregulated miRNAs in M2 compared with
M1 mpMacrophages (Supplementary Table S5). The relative
abundance of miRNAs 21,181b, 320, 365, and Let-7a was
compared by real-time PCR in M1 and M2 mpMacrophage
MDE. Figure 3A shows that MDE from M2 mpMacrophages
were rich in miRNAs 181b, 320, and 365, relative to exosomes
from M1 (3.82-, 3.39-, and 10.25-fold, respectively; P¼0.04,
P¼0.01, and P¼0.002, respectively). miRNAs 320, 181b, and
21, but not miR-365 and Let-7a, were also enriched in exo-
somes secreted from M2 macrophages compared with exo-
somes secreted from na€
ve (M0) macrophages (Supplementary
Fig. S3C). Incubation of K989 cells with gemcitabine further
induced miR-181b and miR-365 expression (Fig. 3B). Most
importantly, incubation of K989 cell MDE and gemcitabine
had a profound synergistic effect on miR-365 expression com-
pared with other miRNAs. The finding that miR-365 is marked-
ly upregulated in K989 cells treated with gemcitabine and MDE
suggests its potential role in gemcitabine resistance.
Exosomal transfer of miR-365 induces gemcitabine resistance
We next investigated the role of miR-365 transfer by MDE in
gemcitabine resistance. To this end, we performed a series of miR-
365 perturbations in K989 cells and evaluated the contribution of
M2 mpMacrophages to gemcitabine resistance. Transfection of
miR-365 mimic to K989 cells significantly increased the miR-365
levels compared with controls (P¼0.01), whereas antagomiR-
365 transfection significantly reduced miR-365 expression in the
cancer cells (P¼0.03). Incubation with MDE increased the
expression of miR-365 in K989 cells, whereas transfection of
antagomiR-365 to K989 cells significantly reduced the effect of
MDE on miR-365 levels (Fig. 3C, P<0.01). We incubated K989
cells with MDE alone, or transfected K989 cells with 50 nmol/L or
100 nmol/L of antagomiR-365, and then incubated them with
MDE. Comparing the amounts of miR-365 in these cells, we
found that antagomiR transfection reduced the increase observed
by incubation with MDE in a dose-dependent manner (Supple-
mentary Fig. S3D). Congruent with these results, Fig. 3D shows
that transfection of miR-365 to K989 cells induced gemcitabine
resistance relative to miR-control, whereas transfection of antag-
omiR-365 to K989 cells restored the effect of gemcitabine (P¼
0.01). Previous studies have demonstrated that overexpression of
miR-365 can inhibit proliferation (28). K989 cells treated with
M2 MDE or miR-365 without gemcitabine showed increased
proliferation levels, indicating that gemcitabine resistance did
not result from reduced proliferation (Supplementary Fig. S3E).
To further assess the effect of miR-365 transfer by exo-
somes, we cocultured K989 cells and M2 mpMacrophages in a
transwell system. AntagomiR-365 transfection to M2 mpMacro-
phages resulted in a dramatic reduction in miR-365 expression
in mpMacrophages (Supplementary Fig. S3F). mpMacrophages
transfected with antagomiR-365 or miR-control were plated in
inserts with 220-nm pore size and incubated with K989 cells for
48 hours (Fig. 3E). K989 cells were then harvested and analyzed
by qRT-PCR and FACS for miR-365 expression and apoptosis.
Figure 3F shows that K989 cells incubated with mpMacrophages
transfected with miR-control had significantly higher miR-365
levels than cells incubated with M2 mpMacrophages transfected
with antagomiR-365 (P¼0.01).
Figure 3G shows that M2 mpMacrophages transfected with
miR-control induced a significantly lower level of cell death and
apoptosis (30.1% and 20.23%, respectively) in K989 cells than
did M2 mpMacrophages transfected with antagomiR-365 when
incubated with gemcitabine (73.2% and 55.5%, respectively).
Taken together, these data show that exosomal transfer of miR-
365 via exosomes induced gemcitabine resistance, and that
antagomiR-365 treatment of the mpMacrophages can restore the
sensitivity of cancer cells to gemcitabine.
Exosomal modulation of pyrimidine metabolism and CDA
expression in PDAC
To further explore the mechanism by which MDE and miR-365
induce gemcitabine resistance, we analyzed, by liquid chroma-
tography–mass spectrometry (LC/MS), cell lysates of K989 cells
incubated with MDE or transfected with miR-365 mimic. Heat
maps of the top 50 metabolites of K989 cells treated with MDE
and miR-365 mimic are presented in Fig. 4A and B and Supple-
mentary Table S6, respectively. The analysis revealed a significant
increase in pyrimidine metabolism of K989 by MDE or miR-365
(Fig. 4C and D, respectively, P<0.001), and a significant increase
in triphosphate-nucleotide (NTP) concentration in both miR-
365–transfected and MDE-treated K989 cells compared with
controls (Fig. 4E and F). Metabolomic analysis of M2-derived
exosomes did not detect high levels of nucleotides that could
account for the observed increase in nucleotide pools in K989 cells
after treatment with MDE or miR-365 (Supplementary Metabo-
lomics Data MDE).
High levels of NTPs upregulate CDA, the enzyme that controls
the cellular pyrimidine pool, by catalyzing cytidine to uridine
(29). CDA inactivates gemcitabine by converting dFdCytidine to
dFdUridine (2). Figure 4G shows that increasing intracellular
NTPs upregulates CDA expression in K989 cells. To examine the
possibility that miR-365 and MDE upregulate CDA expression, we
transfected miR-365 mimic to K989 cells and evaluated CDA
expression by four methods. A qRT-PCR analysis demonstrated
that transfection of miR-365 mimic increased CDA transcript
levels in a dose-response manner (P<0.05), whereas antago-
miR-365 significantly reduced the relative expression of CDA (P¼
0.04; Fig. 4H). The expression of the gemcitabine transporter
hENT1 did not change significantly after treatment of K989 cells
with MDE or transfection with miR-365 mimic compared with
controls (MDEþ/MDE–,RQ¼1.9, P>0.05; miR-control/miR-
365, RQ ¼0.9, P>0.1). Similarly, increased CDA protein levels
were observed when K989 cells were incubated with MDE or
transfected with miR-365 mimic (Fig. 4I). LC/MS analysis of K989
cells, incubated for 48 hours with MDE, had a 2.6-fold higher
concentration of dFdUridine in their media than did controls (P¼
0.01, Fig. 4J). This supports the hypothesis that increased CDA
expression is a component of the mechanism by which MDE and
miR-365 reduce sensitivity to gemcitabine. In agreement, LC/MS
analysis revealed a significant increase in dFdUridine in the media
of K989 cells transfected with miR-365 mimic compared with
miR-control, 16 hours after initiation of the experiment (Fig. 4K,
P¼0.02). Immune precipitation did not reveal the presence of
CDA protein in MDE, ruling out the possibility that direct CDA
transport occurs via exosomes (Fig. 4L).
The above results show that MDE and miR-365 modulate
pyrimidine metabolism in PDAC cells. Increasing NTP
Exosomes Induce Gemcitabine Resistance in Pancreatic Cancer
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P = 0.6
P = 1
P = 0.01
P = 0.04
0
1
2
3
4
5
Let-7a miR-21 miR-181b miR-320 miR-365
Relave quanty
Normal media GEM GEM+MDEs
0
2
4
6
8
10
12
14
Let-7a miR-21 miR-181b miR-320 miR-365
Relave quanty
M1 M2
CD
EF
AB
M2
macrophages
Transfecon
antagomiR-365 or miR-
control
IL-4 Polarizaon
PDAC Cells
0
0.5
1
1.5
2
2.5
Viability index (rao of cells of control)
With GEM (5 μmol/L)
P = 0.01
P = 0.09
P = 0.019
P < 0.01
0
0.2
0.4
0.6
0.8
1
1.2
Relave quanty miR-365 in PDAC
P = 0.01
P = 0.002
P = 0.4
P = 0.1
P = 0.02
P = 0.8
P = 0.06 P = 0.9
P < 0.01
P = 0.03
P < 0.01
P < 0.01
0.1
1
10
100
Relave quanty of miR-365 in PDAC
P = 0.01
P = 0.03
P < 0.01
P < 0.01
P = 0.03
AntagomiR-365miR control
0
20
40
60
80
miR control AntagomiR-365
Apoptoc Dead
% of Total PDAC cells
G
PI
AnnexinV
Figure 3.
Exosomal transfer of miR-365 induces gemcitabine resistance. A, Relative enrichment of indicated miRNAs in M1- and M2-derived exosomes, measured by qRT-P CR.
miRNA levels in M1 NDEs were used for normalization. B, Modulation of miRNA abundance in K989 cells by gemcitabine (GEM), gemcitabine with exosomes
(GEMþMDE), and control (normal media), as evaluated by qRT-PCR. miRNA levels in K989 cells grown in control media were used for normalization.
C, miR-365 perturbation in K989 cells. Black bars, K989 cells transfected with miR-control, miR-365 mimic, or antagomiR-365. Gray bars, K989 cells treated with
MDEs with or without antagomiR-365 transfection in the presence of 5 mmol/L gemcitabine. miRNA levels in cells transfected with miR-control were used for
normalization. D, The effect of perturbations described in Con K989 cell proliferation in the presence of gemcitabine (5 mmol/L). Proliferation of K989 cells
transfected with miR-control was used for normalization. E, Experimental design. K989 cells cocultured with M2 mpMacrophages transfected with miR-control or
antagomiR-365. F, miR-365 expression in K989 cells. miR-365 levels in K989 cells cocultured with mpMacrophages transfected with miR-control were used for
normalization. G, FACS analysis of K989 cells cocultured with M2 mpMacrophages transfected with antagomiR-365/miR-control (gemcitabine 5 mmol/L). Bar
graph, Apoptosis and cell death levels in K989 cells from M2 mpMacrophagesþantagomiR-365 and miR-M2 mpMacrophagesþmiR-control groups.
Binenbaum et al.
Cancer Res; 78(18) September 15, 2018 Cancer Research5292
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CEF
0
0.5
1
1.5
2
2.5
3
3.5
4
Relave expression CDA
P = 0.6
P = 0.01
P = 0.04
P = 0.04
0
5
10
dGTP dATP dTTP dCTP
Relave concentraon
No MDEs MDEs
*
*
*
*
CDA 16 kDa
Acn 48 kDa
G
0.0
0.2
0.4
0.6
0.8
1.0
00.51 2 4 16
Relave concentraion dFdUridine
Time (h)
365-Mimic miR-Control
K
CDA
16 kDa
Acn 48 kDa
H
0
0.2
0.4
0.6
0.8
1
1.2
24 48
Relave concentraon dFdUridine
Time (h)
*
*
*
MDEs
No MDEs
*
Pyrimidine
metabolism
L
CDA 16 kDa
IgG control CDA anbody
No MDESMDEs
AB
miR-365 mimic miR-control
0
2
4
6
8
10
dGTP dATP dTTP dCTP
Relave concentraon
miR Control Mimic-365
*
*
*
*
Pyrimidine
metabolism
Pathway impact
–Log (P)
–Log (P)
Pathway impact
D
IJ
Control
miR-Control
AntagomiR
Mimic 10 nmol/L
Mimic 20 nmol/L
Mimic 30 nmol/L
dGTP 20 µmol/L
dATP 20 µmol/L
dATP 50 µmol/L
dGTP 50 µmol/L
Figure 4.
Macrophage-derived exosomes and miR-365 regulate pyrimidine synthesis and CDA expression. A, Heat map of LC-MS metabolomics. Abundant metabolites
for K989 cells pretreated with MDE or control and incubated with gemcitabine (5 mmol/L). B, Heat map of LC-MS metabolomics of K989 cells transfected with
miR-365 or miR-control and incubated as in A.C, "Metabolome view" for pathway enrichment cells treated as in A.D, Concentration of dNTPs, measured by
LC/MS analysis in K989 cells, with or without MDE (,P<0.05). E, "Metabolome view" for pathway enrichment cells treated as in B.F, Concentration of
dNTPs, measured by LC/MS analysis in K989 cells, transfected with miR-365 or miR-control (,P<0.05). G, Western blot of CDA in K989 cells loaded with
increased concentrations of NTPs (dGTP/dATP). H, qPCR of CDA expression in K989 cells transfected with indicated oligonucleotides (gemcitabine 5 mmol/L).
CDA levels in K989 cells transfected with miR-control were used for normalization. I, Western blot of CDA in K989 cells treated as indicated. J, LC/MS
analysis of dFdUridine excreted by K989 cells, with/without MDE (,P¼0.01). K, MS analysis of dFdUridine excreted by K989 cells treated as indicated (,P¼0.02).
L, Immunoprecipitation of CDA from lysates prepared from K989 cells and MDE as indicated.
Exosomes Induce Gemcitabine Resistance in Pancreatic Cancer
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concentration induced miR-365–upregulated CDA expression.
CDA inactivates gemcitabine by its conversion to dFdUridine
(2). Increased concentration of NTPs in PDAC cells also promoted
gemcitabine resistance, as dCTP competes with dFdCTP for DNA
incorporation.
Modulation of gemcitabine resistance in vivo
To investigate the contribution of MDE to gemcitabine resis-
tance in vivo, we used the Rab27a
/
b
/
(Rab27KO) mouse
model. Rab27KO mice have impaired exosomal packaging and
secretion due to hampered intracellular trafficking (7, 30).
Rab27KO mpMacrophages had significantly lower exosome
secretion than did WT controls (P¼0.05, Supplementary
Fig. S4A and S4B; ref. 24).
We implanted the pancreata of WT and Rab27KO mice with
PDAC K989 cells and followed tumor size by small animal
sonography (n¼8 per group). The tumor kinetics were similar
between Rab27KO and WT mice 7 weeks after tumor implanta-
tion (Supplementary Fig. S4C, P¼0.49).
At 2 weeks after implantation, the mean tumor volume was
similar in both groups. Next, both groups were treated with
gemcitabine for 5 weeks (Fig. 5A). Figure 5B shows the tumor
growth kinetics in each animal separately. As shown in Fig. 5C, 5
weeks after gemcitabine treatment, tumors in the Rab27KO group
responded significantly better to chemotherapy than did those in
the WT group (274 223 mm
3
and 865 545 mm
3
, respectively,
P¼0.003). Immunofluorescence analysis of sections from the
animal described above, stained with anti-F4/80 and anti-CDA
(Fig. 5D and E), demonstrated that although the distribution of
mpMacrophages was similar in both groups (105 vs. 122; F4/80þ
cells/field, P¼0.51, Fig. 5F), CDA expression in PDAC tumors was
significantly lower in Rab27KO mice than in WTs (mean fluo-
rescence intensity of 2.6 and 9.1, respectively, P<0.05, Fig. 5G).
CDA staining intensity was similar in 5-week postimplantation
tumors induced in WT and Rab27KO without gemcitabine treat-
ment (Supplementary Fig. S4D). Supplementary Fig. S4E demon-
strates CDA expression in cytokeratin-positive ductal cells in the
tumor. Cytokeratin-positive cells had more intense CDA staining
than cytokeratin-negative cells in WT and Rab27KO tumors
(Supplementary Fig. S4E and S4F).
Immune transfer of antagomiR-365 augments gemcitabine
response
To overcome the effect of miR-365 on gemcitabine resistance,
we used mpMacrophages as carriers of antagomiR-365. We
implanted K989 PDAC tumors in Rab27KO mice to minimize
endogenous MDE secretion. This enabled manipulation of exo-
somes predominantly in the immune-transferred mpMacro-
phages. Tumors were grown in the pancreata of Rab27KO mice
for 2 weeks. We then performed immune transfer of mpMacro-
phages to the PDAC-bearing Rab27KO mice (5 million mpMa-
crophages/dose, i.p. injection, twice weekly), with three distinct
macrophage populations: WT donor mpMacrophages transfected
with antagomiR-365 (n¼6), WT donor mpMacrophages trans-
fected with miR-control (n¼7), and mpMacrophages from
Rab27KO donors transfected with miR-control (n¼6). Mice
were then treated with gemcitabine and followed for 10 weeks
(Fig. 6A). Immunofluorescence of pancreatic specimens demon-
strated similar macrophage density in tumors from all groups
(T TEST WTþmiR-control, Rab27KOþmiR-control, P¼0.6;
T TEST WTþmiR-control, WTþantagomiR-365, P¼0.2). Our
staining cannot distinguish between endogenous and immune-
transferred mpMacrophages (Fig. 6B). CDA expression was sig-
nificantly higher in the group treated with WT mpMacrophages
transfected with miR-control than in those treated with Rab27KO
mpMacrophages transfected with miR-control, or with WT
mpMacrophages transfected with antagomiR-365 (n¼6–7 mice,
P<0.05, Fig. 6B and C). Kaplan–Meier graphs showed that mice
injected with WT mpMacrophages transfected with miR-control
had significantly shorter survival than mice treated with WT
mpMacrophages transfected with antagomiR-365 (P¼0.03,
Fig. 6D). Similarly, mice injected with WT mpMacrophages
transfected with miR-control had shorter survival than mice
injected with Rab27KO mpMacrophages transfected with
miR-control (P¼0.01). Mice injected with WT mpMacrophages
transfected with miR-control had larger tumors than mice treat-
ed with WT mpMacrophages transfected with antagomiR-365,
and than mice injected with Rab27KO mpMacrophages trans-
fected with miR-control (Supplementary Fig. S4G). Overall,
the data show that immune transfer of antagomiR-365 via
mpMacrophages can restore sensitivity to gemcitabine in vivo.
Discussion
In this work, we uncovered a mechanism by which macro-
phages communicate with PDAC cells to induce chemotherapy
resistance. We showed that miRNAs containing MDE are trans-
ferred from macrophages to PDAC cells, altering their gene
expression and metabolism. The latter results in excretion of
gemcitabine out of cells and chemotherapy resistance.
Accumulating evidence suggests that the tumor microenviron-
ment plays a pivotal role in the development of drug resistance
(2, 31). M2 macrophages are a prominent constituent in the
pancreatic cancer microenvironment and have been associated
with poor prognosis (32, 33), neural invasion (4, 34), and poor
response to treatment (35). However, the mechanism for inter-
cellular communication between macrophages and PDAC cells
is poorly understood. Here, we demonstrated that macrophages
transmit molecular signals to cancer cells by shuttling exosomes
that are selectively internalized by PDAC cells. We found most of
the internalized MDE to be cytosolic and close to the plasma
membrane; and a minority of the signals was perinuclear. Exo-
somes appear to selectively enter cancer cells ex vivo, but rarely to
enter their noncancerous stromal counterparts.
In vivo, the dsDNA barcode that was delivered from TAM- to
PDAC-bearing mice was recovered almost exclusively from
cancer cells in primary tumors and in distant metastases.
Selective uptake of exosomes by cancer cells can be explained
by protein–protein interactions, by specific lipid properties, or
by macropinocytosis.
Both receptor-mediated endocytosis, requiring the recognition
of a specific ligand by a receptor on the host cell, and raft-mediated
endocytosis, requiring the presence of cholesterol and sphingo-
lipid-rich microdomains, were implicated in exosome internali-
zation (28). Ras-transformed PDAC cells were reported to display
enhanced micropinocytosis (36). This could explain the prefer-
ential uptake of exosomes by PDAC cells compared with other
stromal cells.
MDE-mediated transfer of miR-365 plays a pivotal role in
chemotherapy resistance, as MDE-treated K989 cells display
increased survival in response to gemcitabine, relative to untreat-
ed controls. We found that in the transfer of miR-365, MDE
Binenbaum et al.
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0
200
400
600
800
1,000
1,200
1,400
1,600
257257
Tumor volume (mm
3
)
Week
0
500
1,000
1,500
2,000
2,500
3,000
3,500
4,000
4,500
Tumor volume (mm
3
)
A
D
B
DAPI
CDA Overlay
Rab27KO WT
DAPI
F4/80 Overlay
Rab27KO WT
P = 0.003
C
E
Tumor
inducon
Day 0 Day 14
Study
ends
Week 7
5 weeks
GG G
WT Mice
RAB27 KO Mice
GEM 80 mg/kg
WT Rab27KO
Average volume
Week
F
0
5
10
15
20
25
Rab27KO WT
CDA Mean fluorescence intensity
P < 0.05
0
50
100
150
200
250
Rab27KO WT
F4/80 Cells/field
P = 0.51
G
Week 2 Week 5 Week 7
Average tumor volume (mm3) Rab 27 KO 60.29±20 107.32±241 191.5±315
Average tumor volume (mm3) WT 136.12±9 169.78±287 919.36±264
t test (P value) 0.15 0.78 0.03
Figure 5.
MDE and gemcitabine resistance in vivo.A, Experiment layout. Pancreata of mice were implanted with K989 cells. Gemcitabine was administered after 14 days
for 5 weeks. B, Tumor volumes in WT and Rab27KO mice at weeks 2–7, measured by ultrasound (gray line, average volume). C, Tumor volumes in WT and
Rab27KO mice, measured at autopsy (P¼0.003). D, Immunofluorescence of macrophages (F4/80-red) in WT and Rab27KO mice tumors (bar, 500 mm).
E, Immunofluorescence of CDA in WT tumors and Rab27KO mice tumors (bar, 500 mm). F, Quantification of F4/80 cells/field. G, Quantification of mean
fluorescence intensity of CDA signal in E.
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Exosomes Induce Gemcitabine Resistance in Pancreatic Cancer
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inhibit the effect of gemcitabine. However, transfection of antag-
omiR-365 to K989 cells partially blocked the effect of MDE on
gemcitabine (P¼0.019). Our observation that antagomiR-365
only partially blocked the effect mediated by MDE raises the
possibility that other miRNA delivered by MDE may be involved
in the process. Previous works suggested that miR-365 down-
regulates BCL2, hence hastening apoptosis (37), or participates in
signal transduction during mitogenic assault (38). In cutaneous
squamous cell carcinoma, miR-365 is considered an oncomiR
(39) that acts by targeting nuclear factor I/B (27). Interestingly,
miR-193b-mir-365 appears to be involved in metabolic regula-
tion, being essential to brown fat cell differentiation (40), and
abundant in the mitochondria (41). Our mass spectroscopy
analysis concurred with these data and revealed that miR-365
upregulates pyrimidine metabolism and increases NTP levels in
cancer cells. Increased levels of NTP upregulate CDA, one of
several deaminases responsible for maintaining the cellular
pyrimidine pool (42), and the enzyme responsible for gemcita-
bine inactivation in humans. CDA expression in PDAC tumors
was significantly lower in Rab27KO mice than in WT. Neverthe-
less, we cannot rule out the possibility that some of the signals we
detected in the tumor originate from spillover of CDA from
macrophages to cancer cells. CDA deaminates gemcitabine to
dFdU (43, 44), which is passively excreted out of the cell. Indeed,
we observed increased excretion of dFdU from cancer cells fol-
lowing miR-365 transfection. Alternatively, increased nucleotide
pools can affect resistance to gemcitabine by molecular compe-
tition (45). Therefore, dCTP upregulated by miR-365 could com-
pete directly with gemcitabine for incorporation into the DNA
chain, further potentiating resistance (46, 47). Figure 7 sum-
marizes the proposed mechanism by which macrophages transfer
exosomes loaded with miR-365 to PDAC cells and modulate
gemcitabine metabolism.
One implication of our study is a possible strategy to overcome
gemcitabine resistance by the immune transfer of antagomiR-365
to primary tumors via macrophages. This approach resulted in
significant improvement in the effect of gemcitabine on survival
of tumor-bearing mice.
0
0.5
1
1.5
2
2.5
3
Tumor
inducon
Day 0
G
Day 14
Study
ends
Week 10
MGMG
X7 weeks
Harvesng of
Macrophages
Transfecon with
miR-Control
Rab27KO Mice
Transfecon with
AntagomiR-365
Transfecon with
miR-Control
WT Mice
Recipient
Rab27 KO Mice
A
D
B
WT
Macrophages
miR-Control
Rab27KO
Macrophages
miR-Control
WT
Macrophages
AntagomiR-365
F4/80
CDA
Type of immune transferred macrophages
C
Mean fluorescence intensity
P = 0.6
P = 0.2
F4/80
WT
Macrophages
AntagomiR-365
Rab27KO
Macrophages
miR-Control
WT
Macrophages
miR-Control
CDA
Surviving
0.0
0.2
0.4
0.6
0.8
1.0
21 28 35 42 49 56 63
Day
Rab27KO donors+ miR-
Control
WTdonors + miR-Control
WT donors + AntagomiR-365
P = 0.03
P = 0.01
Figure 6.
Immune transfer of mpMacrophages carrying antagomiR-365 in vivo.A, Two weeks after implantation of K989 tumors in Rab27KO mice, WT and Rab27KO-
derived mpMacrophages (transfected with miR-control or antagomiR-365) were injected into the mice. Gemcitabine was administered until week 10. B,
Immunofluorescence with anti-F4/80 and anti-CDA Abs in PDAC tumors (bar, 500 mm). C, Quantification of the pixel area of the CDA and F4/80 signal
of the images in B. Black bars, F4/80; gray bars, CDA. D, Kaplan–Meier graph of the experimental groups in A.
Binenbaum et al.
Cancer Res; 78(18) September 15, 2018 Cancer Research5296
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Gemcitabine is the cornerstone of treatment of patients with
PDAC, despite its modest efficacy. Our findings suggest a new
avenue for the development of interventions aimed to potentiate
the effect of gemcitabine. Treatments directed to block the pro-
tective effect of macrophages on cancer could prolong survival
and reduce morbidity. The knowledge gained from this study is
anticipated to be applicable to other cancers for which gemcita-
bine and other nucleoside analogues are the treatment of choice.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: Y. Binenbaum, E. Fridman, Z. Gil
Development of methodology: Y. Binenbaum, Z. Yaari, N. Milman,
A. Schroeder
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): Y. Binenbaum, E. Fridman, Z. Yaari, N. Milman,
A. Schroeder, G.B. David, T. Shlomi
Analysis and interpretation of data (e.g., statistical analysis, biostatistics,
computational analysis): Y. Binenbaum, E. Fridman, N. Milman, A. Schroeder,
G.B. David, Z. Gil
Writing, review, and/or revision of the manuscript: Y. Binenbaum, N. Milman,
A. Schroeder, Z. Gil
Gemcitabine
(dFdCydine) dFdC
dCK
dFdCMP
5’NT
CDA
dFdU
Extracellular
space
Cytoplasm
NMPK
dFdCDP
RR
CDP dCDP dCTP
NDPK
dFdCTP
DNA
dFdU
miR-365
miR-365 miR-365
Pancreac cancer cells
Tumor-associated macrophages
miR-365
Macrophage-derived
exosome
Compeve
inhibion
Figure 7.
Summary of the mechanism by which TAM induce gemcitabine resistance. MDE transmit miR-365 selectively to PDAC cells. Gemcitabine (dFdC) is
transported into PDAC cells and is phosphorylated by dCK to produce dFdCTP, or deaminated by CDA to dFdU, which is excreted out of the cells. miR-365
is upregulated in PDAC cells and increases the concentration of intracellular NTP, which competes with dFdCTP for DNA incorporation. An increase in
NTPs also upregulates CDA expression, further contributing to dFdC deamination. Enzymes and metabolites that regulate gemcitabine resistance are
in bold. dCK, deoxycytidine-kinase; CDA, cytidine-deaminase; NMPK, nucleoside monophosphate kinase; NDPK, nucleoside diphosphate kinase; 50NT,
50-nucleotidase; RR, ribonucleotide-reductase.
Exosomes Induce Gemcitabine Resistance in Pancreatic Cancer
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Administrative, technical, or material support (i.e., reporting or organizing
data, constructing databases): Y. Binenbaum, G.B. David
Study supervision: N. Milman, T. Shlomi, Z. Gil
Acknowledgments
We thank Lana Ginene and Yotam de la Zerda for tech nical assistance, Cindy
Cohen for her editorial assistance, Naama Koifman and Yeshayahu Talmon and
the staff of the imaging unit at the Biomedical Core Facility, and The Ruth and
Bruce Rappaport Faculty of Medicine at the Technion for their support. Z. Gil
was supported by the Israel Science Foundation. N. Milman was supported by
Israel Cancer Research Fund, The Barbara S. Goodman Endowed RCDA for
Pancreatic Cancer, and The Israel Cancer Association.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
Received January 13, 2018; revised May 17, 2018; accepted July 13, 2018;
published first July 24, 2018.
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