biomedicines

Article
MiR-506 Promotes Antitumor Immune Response in Pancreatic
Cancer by Reprogramming Tumor-Associated Macrophages
toward an M1 Phenotype
Tiantian Yang, Yitong Han, Junhang Chen, Xiaoyu Liang and Longhao Sun *

Department of General Surgery, Tianjin Medical University General Hospital, Tianjin 300052, China;
[email protected] (X.L.)
* Correspondence: [email protected]; Tel.: +86-22-6036-1727

Abstract: Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant cancer with a poor
prognosis, and effective treatments for PDAC are lacking. In this study, we hypothesized that miR-506
promotes antitumor immune response in PDAC by reprogramming tumor-associated macrophages
toward an M1 phenotype to reverse its immunosuppressive tumor microenvironment (TME). First,
the relationship between TME and the expression of miR-506 was assessed using clinical samples.
Our results provided evidence that lower expression of miR-506 was associated with poor prognosis
and immunosuppressive TME in PDAC patients. In addition, miR-506 inhibit the PDAC progression
and reversed its immunosuppressive microenvironment in a macrophage-dependent manner. Next,
we established a PDAC mouse model by orthotopic injection to further explore the role of miR-506
in vivo. Mechanistic investigations demonstrated that miR-506 could reprogram the polarization
of M2-like macrophages toward an M1-like phenotype through targeting STAT3. Meanwhile, miR-
506 could also sensitize PDAC to anti-PD-1 immunotherapy, because the tumor microenvironment
remodeling effects of miR-506 could reprogram macrophage polarization and subsequently promote
cytotoxic T lymphocyte (CTL) infiltration. These findings suggest a relationship between miR-506
and TME, especially M2-like macrophages, thus providing novel insights into mechanisms of tumor
Citation: Yang, T.; Han, Y.; Chen, J.;
progression and potential immunotherapeutic targets for further clinical treatment.
Liang, X.; Sun, L. MiR-506 Promotes
Antitumor Immune Response in
Keywords: miR-506; pancreatic cancer; reprogram; signal transducer and activator of transcription 3;
Pancreatic Cancer by
tumor-associated macrophage
Reprogramming Tumor-Associated
Macrophages toward an M1
Phenotype. Biomedicines 2023, 11,
2874. https://doi.org/10.3390/
biomedicines11112874 1. Introduction

Academic Editors: Satoshi Wada,

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy. Its
Cinzia Bagalà and Marta Chiaravalli
5-year survival rate is <10% [1,2]. Surgical resection with chemotherapy remains the most
effective therapeutic strategy [3,4], yet, considering that most patients present with late-
Received: 21 August 2023 stage cancer (tumor being either unresectable or metastatic), alternative treatment options
Revised: 3 October 2023 are considered. While immunotherapy and targeted therapy have been well investigated
Accepted: 10 October 2023 in past decades, no therapy has been found to be fully effective [5]. Therefore, exploring
Published: 24 October 2023
novel therapeutic targets and biological markers for early diagnosis and treatment is
urgently needed.
Unlike other types of cancers, the extensive desmoplastic stroma, which mainly refers
Copyright: © 2023 by the authors. to the TME rich in stroma cells and the deposition of extracellular matrix (ECM), is a
Licensee MDPI, Basel, Switzerland. unique hallmark of PDAC [6,7]. The TME of PDAC consists of fibroblasts, neurons, en-
This article is an open access article dothelial cells, EMC proteins, and immune infiltration cells [8]. Studies have shown that
distributed under the terms and immune cells have a vital role in the biology of TME in PDAC patients [9,10]. For example,
conditions of the Creative Commons macrophages, accounting for the highest proportion in the TME, including both resident
Attribution (CC BY) license (https:// and recruited circulating macrophages, have a critical role in the homeostasis of TME and
creativecommons.org/licenses/by/ tumor progression [11,12]. Furthermore, in the TME, macrophages can be transformed
4.0/).

Biomedicines 2023, 11, 2874. https://doi.org/10.3390/biomedicines11112874 https://www.mdpi.com/journal/biomedicines

Biomedicines 2023, 11, 2874 2 of 16

into an M1-like phenotype with anti-cancer immunity and M2-like phenotype with im-
munosuppressive function. These cells can repolarize and transform mutually due to
the alteration of the TME [13,14]. M2-like macrophages, also known as tumor-associated
macrophages (TAMs), possess potent immunosuppressive activity. Previous studies also
demonstrated that M2 TAMs are strongly associated with tumor progression, metastasis,
and drug resistance in PDAC patients [15,16], suggesting that TAMs could be potentially
used as immunotherapeutic targets for clinical treatment with PDAC patients.
Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that
regulates a series of biological processes [17,18]. Hyperactivated STAT3 has been found
in multiple cancers and has been associated with poor prognoses [19]. Activated STAT3
also affects immune cells in the TME by regulating the expression of several cytokines
(interleukin 6 (IL-6) and IL-10) and vascular endothelial growth factor (VEGF) [20]. More-
over, these tumor-derived cytokines and factors can activate and enhance STAT3 signaling
in the TME and tumor cells, which promotes tumor progression and impairs antitumor
immunity [21–23].
MicroRNAs (miRNAs) are 20- to 25-nucleotide-long noncoding single-stranded RNA
molecules. They can play an oncogenic or oncosuppressive role in various malignancies by
regulating the expression of oncogenic or tumor-suppressive target genes. Notably, several
miRNAs have been found to be upregulated or downregulated in various tumors, including
PDAC, which can break down the mRNA transcript or inhibition of the translation of the
mRNA to proteins implicated in cancer pathogenesis [24,25]. MiR-506 has been negatively
associated with tumor T stage and lymph node metastasis, and its deregulation has been
associated with pancreatic progression [26,27]. Moreover, another study proved that STAT3
is a direct target of miR-506 in PDAC [28].
In this study, we further explored the relationship between the expression of miR-506
and alteration of the TME in PDAC patients, revealing that miR-506 can promote antitumor
immune response by reprogramming M2 macrophages to M1-like phenotype through
targeting the STAT3 signaling pathway and enhancing the sensitivity of immunosuppres-
sive therapy.

2. Materials and Methods

2.1. Clinical Specimens
Paired human PDAC and adjacent nontumor pancreatic tissue samples were collected
from 126 patients with biopsy-proven PDAC at Tianjin Medical University General Hospital
(TMUGH) between 2018 and 2021. Fresh surgical samples were processed into single-cell
suspensions containing 2.5 U/mL hyaluronidase, 0.1 mg/mL DNase, 1 mg/mL collagenase.
Samples were then subjected to flow cytometry; antibodies against human CD8, CD4,
FOXP3, CD25, CD45, CD163, and CD68 (all BD Biosciences) were used as cell surface
markers to identify human TAMs (CD68+ /CD163+ ), M1 TAMs (CD68+ /CD163− ), Tregs
(CD4+ CD25+ FOXP3+ ), M2 TAMs (CD68+ /CD163+ ), cytotoxic T cells (CD8+ /CD45+ ), and
apoptotic T cells (annexin V+ /CD8+ ) (all from BD Biosciences, San Jose, CA, USA). Enzyme-
linked immunosorbent assay (ELISA) was used to analyze the IFN-γ in tumor tissue,
according to the manufacturer’s procedure (R&D Systems, Minneapolis, MN, USA). Mirror-
image tumor samples were collected and snap-frozen in liquid nitrogen and stored until
RNA extraction. Clinical and biological information was registered for each patient, and
follow-up data were recorded after informed consent was obtained. The institutional
research ethics committee at TMUGH approved this study.

2.2. Macrophage Analysis

Density gradient centrifugation with Histopaque-1077 (Sigma-Aldrich, St. Louis, MI,
USA) was used to obtain PBMCs isolated from healthy blood donors. CD14+ monocytes
were then purified using CD14 microbeads (Miltenyi Biotech, Miltenyi Biotech) and cul-
tured in RPMI 1640 medium (with 50 ng/mL GM-CSF (Peprotech, London, UK) and 20%
Biomedicines 2023, 11, 2874 3 of 16

heat-inactivated FBS (Sigma-Aldrich, St. Louis, MI, USA) for five days to differentiate into
nonpolarized M0 macrophages.

2.3. Macrophage Polarization

On day 5, M0 macrophages were differentiated using M1 medium (50 ng/mL IFN-γ,
50 ng/mL GM-CSF, 100 ng/mL lipopolysaccharide, and 20 ng/mL TNF-α) or M2 medium
(100 ng/mL M-CSF, 10 ng/mL IL-10, and 10 ng/mL TGF-β; Peprotech, London, UK), with
miR-506 or miR-ctrl for 48 h. On day 7, polarized macrophages were analyzed using flow
cytometry; antibodies against human MHC-II, CD86, CD163, CD204, CD80, and CD206 (all
from BD Biosciences, San Jose, CA, USA) were used for cell surface markers.

2.4. Phagocytosis Assay

FITC-labeled Escherichia coli BioParticles (Thermo Fisher Scientific, Waltham, MA,
USA) was mixed with polarized macrophages. Cells were then incubated with for 2 h
in a humidified atmosphere containing 5% CO2 /95% air at 37 ◦ C. After removing the
BioParticle loading suspension, trypan blue suspension was mixed with the solution. A
microplate spectrophotometer was then used to analyze the relative phagocytosis.

2.5. Nitric Oxide (NO) Production

The Griess Reagent System (Promega, Madison, WI, USA) was used to analyze NO
production. Briefly, a supernatant medium of different polarized macrophages was mixed
with Griess reagent. The mixture was then incubated for ten min at 20 ◦ C in the dark, after
which the absorbance at 540 nm was measured using a microplate spectrophotometer; NO
concentration was determined with sodium nitrite as a standard.

2.6. Arg1 Activity

Arg1 activity was analyzed as previously described [29]. Briefly, macrophages were
lysed. Then, the supernatant was heated at 56 ◦ C for 7 min to activate the Arg1. Then,
the solution was mixed with 50 µmol of L-arginine for 2 h at 37 ◦ C to allow hydrolysis of
L-arginine by Arg1. The byproduct metabolite urea levels were determined by incubation
with α-isonitrosopropiophenone substrate. The absorbance was measured at 540 nm, and
the Arg1 urea production was calculated in samples from the urea standard curve.

2.7. VEGF, IL-10, and TGFβ Secretion

Polarized macrophages were cultured in RPMI 1640 + 20% heat-inactivated FBS for
24 h. The amount of VEGF, IL-10, and TGFβ was quantified using ELISA (R&D Systems)
following the manufacturer’s procedure.

2.8. RNA Isolation and qRT-PCR

The MirVana Isolation Kit (Ambion, Austin, TX, USA, AM1560) was used to obtain
the total RNA, including miRNA. Reverse transcription was performed using the TaqMan
miRNA reverse-transcription kit (Applied Biosystems, Beverly Hills, CA, USA, 4366596) or
SuperScript II reverse transcriptase (Invitrogen, Waltham, MA, USA, 18064014). TaqMan
gene expression assay and TaqMan microRNA assay (Applied Biosciences, Beverly Hills,
CA, USA, A25576 and 4331182, respectively) were used to detect and quantify mir-506 CD80,
CD86, human MHCII, IL1B, TNFA, NOS2, CD163, CD204, ARG1, IL10, CD206, TGFB1, and
STAT3. Relative expression was normalized to the endogenous control GAPDH or RNU6
using the 2−∆∆Ct method. Experiments were carried out in triplicate.

2.9. Western Blotting

Cells were mixed with RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA,
89901). The whole-cell lysate was then separated on a 10% polyacrylamide gel. The mem-
brane was then blocked in 5% nonfat milk in tris-buffered saline (TBS; BIO-RAD, Hercules,
CA, USA, 1706435, pH7.6) with 0.1% Tween-20 (Sigma-Aldrich, St. Louis, MI, USA, P2287).
Biomedicines 2023, 11, 2874 4 of 16

Samples were then incubated with primary antibodies (anti-phospho-STAT3, Tyr705, Cell
Signaling Technology, Danvers, MA, USA, 9145, 1:250; anti-STAT3, Cell Signaling Technol-
ogy, Danvers, MA, USA, 12640, 1:500) at 4 ◦ C overnight and then with secondary antibodies
(Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-2077 or sc-2375 at a concentration of
1:10,000) at room temperature for 2 h. The proteins were visualized using SuperSignal West
Pico chemiluminescent substrate or Femto Maximum Sensitivity Substrate (Thermo Fisher
Scientific, Waltham, MA, USA, 34080 or 34095).

2.10. Reagents and Antibodies

SiRNAs for STAT3 (SASI_Hs01_00121206 and 00121207) were from Sigma. The miRNA
mimics of miR-506 and control miRNA (miR-ctrl) were bought from Dharmacon (c-300846-
05 and CN-001000-01). Briefly, cells were seeded in 6-well plates (2 × 105 /well) overnight,
after which they were transfected with miR-ctrl, miR-506 mimic, or siRNA for STAT3 using
lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA, 13778150). Total RNA and
protein were collected 48 h after transfection.

2.11. Orthotopic PDAC Mouse Model

Female-specific pathogen-free (SPF) C57BL/6 mice (age, 4 weeks; weight, 20–22 g)
were housed in a specific pathogen-free environment with relative humidity of 50 ± 1%,
temperature of 22 ± 1 ◦ C, and light/dark cycle of 12/12 h. All animal studies were done in
compliance with the regulations and guidelines of Tianjin Medical University institutional
animal care and conducted according to the AAALAC and the IACUC guidelines.
The mouse PDAC cell line Panc02 (2 × 106 ), purchased from ATCC (Rockville, MD,
USA), was injected into the pancreas of C57BL/6 mice. Animals were then randomly
divided into two groups (n = 10 per group): miR-ctrl group and miR-506 group. MiRNAs
were injected intraperitoneally for 4 weeks. Next, mice were euthanized, and the xenograft
tumors were harvested and weighed. The tumors were dissociated into single-cell suspen-
sions and analyzed using flow cytometry analysis (antibodies against mouse M2 TAMs
(F4/80+ /CD206+ ), M1 TAMs (F4/80+ /CD206− ), Tregs (CD4+ CD25+ FOXP3+ ), cytotoxic T
cells (CD8+ /CD45+ ), and apoptotic T cells (annexin V+ /CD8+ ) (BD Biosciences, San Jose,
CA, USA) were used. IFN-γ in mouse tumor tissue was measured by ELISA.

2.12. Statistical Analysis

SPSS 17.0 software (SPSS Inc., Chicago, IL, USA) was used for all statistical analyses.
Data are expressed as the mean ± SD of at least 3 separate experiments performed in tripli-
cate. Differences between groups were analyzed using Student’s t-test. A p value < 0.05
was considered to be statistically significant.

3. Results
3.1. Low Endogenous miR-506 Levels Are Associated with Poor Outcomes in PDAC Patients
To identify the role of miR-506 in the progression of PDAC, we evaluated the cor-
relation between clinical stages and miR-506 expression level in a cohort of 126 patients.
Relative miR-506 expression levels were higher in adjacent normal pancreas tissues than in
PDAC tissues (*** p < 0.001; Figure 1A). Clinicopathological analyses further showed an
inverse correlation between the level of miR-506 and T (primary tumor size) and TNM stage.
Additionally, patients with early-stage tumors had higher miR-506 expression compared to
those with advanced tumor (expression was lower in the T3–T4 group and III–IV group
than in the T1–T2 group and I–II group) (*** p < 0.001, Figure 1B,C). Furthermore, the
patients with low miR-506 showed more advanced T and TNM stages than those with
high miR-506 levels (*** p < 0.001, Figure 1D,E). Finally, Kaplan–Meier analysis indicated
significantly shorter OS in patients with a low level of miR-506 within the tumor com-
pared to those with high miR-506 expression (p < 0.001; Figure 1F). All of these results
suggest that low miR-506 expression may be used as a biomarker for poor prognosis in
PDAC patients.
 to those with high miR-506 expression (p < 0.001; Figure 1F). All of these results su
Biomedicines 2023, 11, 2874 that low miR-506 expression may be used as a biomarker for poor prognosis5 of 16 in P
patients.

Figure 1. MiR-506 downregulation is correlated with disease progression in human PDAC.

Figure 1. MiR-506 downregulation is correlated with disease progression in human PDA
(A) Comparison of miR-506 expression in matched pairs of PDAC tissues and corresponding nontu-
Comparison of miR-506 expression in matched pairs of PDAC tissues and corresponding non
mor tissues via qRT-PCR. RNU6 was used as an internal control (n = 126; *** p < 0.001 by Student’s
tissues via qRT-PCR.
t-test). (B) Comparison RNU6 was used
of miR-506 as anininternal
expression patients control
with stage(nT1–T2
= 126;and***stage
p < T3–T4
0.001 PDAC
by Student’s
(B) Comparison
tissues (n = 126;of***
miR-506
p < 0.001expression in patients
by Student’s t-test). with stage
(C) Comparison T1–T2expression
of miR-506 and stage T3–T4 PDAC
in patients
(n = 126; *** p < 0.001 by Student’s t-test). (C) Comparison of miR-506 expression
with stage I–II and stage III–IV PDAC tissues (n = 126; *** p < 0.001 by Student’s t-test). (D) The in patien
stage distribution
I–II and stageof the III–IV PDAC miR-506
T stage between tissueshigh-
(n =and126;low-expression
*** p < 0.001groups
by Student’s t-test).
(n = 126). (E) (D) The di
The distri-
bution of TNM stage between miR-506 high- and low-expression groups (n = 126).
tion of the T stage between miR-506 high- and low-expression groups (n = 126). (E) The distri (F) Kaplan–Meier
of TNMcurves
stagecomparing
between the overall
miR-506 survival
high-rates
and of low-expression
PDAC patients whose tumors(n
groups expressed
= 126). a(F)
lowKaplan–Meier
or high
level of miR-506. The median value was used as the cutoff point for the definition of low and high
comparing the overall survival rates of PDAC patients whose tumors expressed a low or hig
miR-506-expression groups (n = 126; p < 0.001 by log-rank test). Data represent the mean ± SD of at
of miR-506. The median value was used as the cutoff point for the definition of low and hig
least 3 independent experiments.
506-expression groups (n = 126; p < 0.001 by log-rank test). Data represent the mean ± SD of
3.2. Low Endogenous
3 independent miR-506 Levels Are Associated with Immunosuppressive Microenvironment
experiments.
in Human PDAC
Twenty fresh tissue samples of human PDAC (10 in the miR-506 low group and 10 in
3.2. Low Endogenous miR-506 Levels Are Associated with Immunosuppressive Microenvir
the high miR-506 group) were assessed using flow cytometry to investigate the relationship
ment between
in Human PDAC
miR-506 level and infiltration of immune cells in the human PDAC microen-
vironment. Significantly higher percentages of CD4+ CD25+ FOXP3+ Tregs (*** p < 0.001;
Twenty fresh tissue samples of+ human PDAC (10 in the miR-506 low group and
Figure 2B), higher ratios of M2 (CD68 CD163+ )/M1 (CD68+ CD163− ) TAMs (*** p < 0.001;
the high miR-506
Figure 2A), fewergroup)
CD8+ CD45 were assessed
+ cytotoxic using
T cells (*** p flow cytometry
< 0.001; Figure 2C), atogreater
investigate
percent- the rel
ship age
between miR-506
of apoptotic level
cytotoxic and
T cells (***infiltration of immune
p < 0.001; Figure 2D), and lowcells in the ofhuman
production IFN-γ byPDAC m
cytotoxic T cells (*** p
environment. Significantly higher percentages of CD4+CD25+FOXP3+ found
< 0.001; Figure 2E) in the PDAC microenvironment were Tregsin(*** p <
patients with low miR-506 levels. These data indicate that low miR-506 in PDAC correlates
Figure 2B), higher ratios of M2 (CD68+CD163+)/M1 (CD68+CD163−) TAMs (*** p <
with an immunosuppressive TME.
Figure 2A), fewer CD8+CD45+ cytotoxic T cells (*** p < 0.001; Figure 2C), a greater pe
age of apoptotic cytotoxic T cells (*** p < 0.001; Figure 2D), and low production of
by cytotoxic T cells (*** p < 0.001; Figure 2E) in the PDAC microenvironment were
in patients with low miR-506 levels. These data indicate that low miR-506 in PDAC
lates with an immunosuppressive TME.
dicines 2023, 11, x FOR PEER REVIEW 6
Biomedicines 2023, 11, 2874 6 of 16

Figure 2. MiR-506 downregulation is correlated with immunosuppressive microenvironment in

Figurehuman
2. MiR-506
PDAC. downregulation
(A) Flow cytometryisanalysis
correlated withTAMs
of M2/M1 immunosuppressive
ratio in PDAC tissues microenvironment
from fresh
man PDAC.
surgical (A) Flow
samples withcytometry analysis
low and high of M2/M1
expression TAMs
of miR-506 (CD68 ratio
+ CD163in PDAC
− cells astissues
M1 TAMs from
and fresh su
samples + CD163
CD68with + cells
low and
as M2high
TAMs).expression of miR-506
(B–D) Flow cytometry of CD4++CD163
analysis(CD68 CD25+ FOXP3
− cells+ Tregs
as (B),
M1 TAM
+ + +
CD68 CD8
+ CD163 CD45cells
+ as M2
T cells TAMs).
(C), and (B–D)
apoptotic CD8Flow cytometry
T cells (D) in PDAC analysis of CD4
tissues from + CD25
the same + FOXP3+ Tre
samples.
(E) Production
CD8+CD45 + T cellsof(C),
IFN-γandby apoptotic
cytotoxic T cells
CD8in+ the PDAC(D)
T cells microenvironment
in PDAC tissues from from
the same
thepatient
same sampl
samples analyzed by ELISA (n = 10 per group; *** p < 0.001 by Student’s t-test). Data represent the
Production of IFN-γ by cytotoxic T cells in the PDAC microenvironment from the same p
mean ± SD of at least 3 independent experiments.
samples analyzed by ELISA (n = 10 per group; *** p < 0.001 by Student’s t-test). Data represe
mean 3.3.
± SDMiR-506
of at least 3 independent
Inhibits experiments.
the PDAC Progression and Reverses Its Immunosuppressive
Microenvironment in a Macrophage-Dependent Manner
An orthotopic PDAC mouse model was first constructed by injecting murine Panc02
3.3. MiR-506 Inhibits the PDAC Progression and Reverses Its Immunosuppressive Microen
cells into C57BL/6 mice. Animals were consequently treated with miR-ctrl or miR-506 for
ronment in a Macrophage-Dependent
4 weeks, Manner
after which the effect of miR-506 on the immune microenvironment and tumor
progression
An orthotopicwere PDAC
analyzed. Larger model
mouse and heavier
wastumors and a higher percentage
first constructed by injecting of Ki-67-
murine P
positive PDAC cells were seen in the miR-ctrl group vs. the miR-506 group (*** p < 0.001;
cells into C57BL/6 mice. Animals were consequently treated with miR-ctrl or miR-5
NS, not statistically significant; Figure 3A–D). In addition, miR-506 treatment significantly
4 weeks, after the
prolonged which
OS (pthe effect
< 0.001; of miR-506
Figure 3E). on the immune microenvironment and t
progression were analyzed.
Furthermore, Larger
flow cytometry and heavier
analysis tumors
was performed on and a higher percentage
cell suspensions from the of K
fresh mouse tumor tissues to investigate
positive PDAC cells were seen in the miR-ctrl group the relationship between immune cell infiltration
vs. the miR-506 group (*** p <
in the tumor microenvironment and miR-506. M2 (F4/80+ /CD206+ )/M1 (F4/80+ /CD206− )
NS, notThestatistically
TAM ratio was significant;
significantlyFigure
reduced3A–D).
after theIn addition,
addition miR-506
of MiR-506 (*** ptreatment
< 0.001; NS,signific
prolonged the OS (p
not statistically < 0.001;Figure
significant; Figure 3E).
3F). To further determine the role of macrophages in
miR-506-mediated antitumor
Furthermore, flow cytometry analysis immunity and tumorwas suppression,
performedwe ontreated orthotopic
cell suspensions from
tumor-bearing mice with clodronate liposomes (Clo) or PBS and miR-ctrl or miR-506
fresh mouse tumor tissues to investigate the relationship between immune cell infiltr
(n = 10 per group). MiR-506 significantly increased the infiltration of CD8+ CD45+ cytotoxic
in theT cells
tumor(*** pmicroenvironment andsignificant;
< 0.001; NS, not statistically miR-506. M23H),
Figure (F4/80 + /CD206
decreased
+)/M1 (F4/80+/CD
the infiltration of
The TAMCD4+ CD25
ratio+ FOXP3 + Tregs (*** p < 0.001; NS, not statistically significant; Figure 3G), reduced
was significantly reduced after the addition of MiR-506 (*** p < 0.001
the percentage
not statistically of apoptotic cytotoxic
significant; Figure 3F).T cellsTo p < 0.001;
(***further NS, not statistically
determine the role significant;
of macrophag
Figure 3I) and increased the production of IFN-γ by cytotoxic T cells (*** p < 0.001; NS, not
miR-506-mediated antitumor immunity and tumor suppression, we treated orthotop
statistically significant; Figure 3J) in the mouse PDAC microenvironment. However, we
mor-bearing
found thatmice with clodronate
macrophage depletion by liposomes
Clo significantly (Clo) or PBSthe
overturned and miR-ctrl
reversion or miR-506 (
of miR-506
per group). MiR-506 significantly
on the immunosuppression increased
of the TME. the infiltration
Macrophage of CD8 annulled
depletion effectively + CD45+ cytotoxic
the T
differences in the percentage of Tregs (CD4 CD25 FOXP3 ), cytotoxic T cells (CD8+ CD45+ ),
+ + +
(*** p < 0.001; NS, not statistically significant; Figure 3H), decreased the infiltrati
apoptotic cytotoxic T cells, and the production of IFN-γ by cytotoxic T cells between the miR-
CD4+CD25 +FOXP3+ Tregs (*** p < 0.001; NS, not statistically significant; Figure 3G
ctrl group and miR-506 group (*** p < 0.001; NS, not statistically significant; Figure 3G–J).
ducedTothe sumpercentage of demonstrate
up, these results apoptotic cytotoxic
that miR-506Tinhibits
cells (***
PDAC p progression
< 0.001; NS, andnot statisticall
reverses
its immunosuppressive microenvironment in a macrophage-dependent
nificant; Figure 3I) and increased the production of IFN-γ by cytotoxic T cells (*** p < manner.
NS, not statistically significant; Figure 3J) in the mouse PDAC microenvironment.
ever, we found that macrophage depletion by Clo significantly overturned the reve
of miR-506 on the immunosuppression of the TME. Macrophage depletion effective
nulled the differences in the percentage of Tregs (CD4+CD25+FOXP3+), cytotoxic T
(CD8+CD45+), apoptotic cytotoxic T cells, and the production of IFN-γ by cytotoxic T
between the miR-ctrl group and miR-506 group (*** p < 0.001; NS, not statistically si
cant; Figure 3G–J). To sum up, these results demonstrate that miR-506 inhibits PDAC
medicines 2023, 11, x FOR PEER REVIEW 7 of
Biomedicines 2023, 11, 2874 7 of 16

FigureFigure 3. MiR-506
3. MiR-506 inhibits
inhibits thetumor
the tumor progression
progression and reprograms
and reprograms its immunosuppressive microen- microe
its immunosuppressive
vironment in a macrophage-dependent manner. (A) Panc02 cells were injected into C57BL/6 mice
vironment in a macrophage-dependent manner. (A) Panc02 cells were injected into C57BL/6 m
to establish an orthotropic PDAC model and then treated with miR-ctrl or miR-506. Twenty-eight
to establish an orthotropic PDAC model and then treated with miR-ctrl or miR-506. Twenty-eig
days following cell implantation, mice were euthanized and tumors were harvested. (B–D) The
days following cell implantation, mice were euthanized and tumors were harvested. (B–D) The
tumor size (B), tumor weight (C), and percentage of mitotic Ki-67 tumor cells (D) were measured
mor size (B), tumor weight (C), and percentage of mitotic Ki-67 tumor cells (D) were measured (n
(n = 10 per group; *** p < 0.001 by Student’s t-test). (E) Kaplan–Meier curves comparing overall
10 persurvival
group;rates
*** pbetween
< 0.001miR-ctrl
by Student’s
and miR-506t-test). (E)(n
groups Kaplan–Meier
= 20 per group; ***curves comparing
p < 0.001 by log-rankoverall
test). surviv
rates between
(F) M2/M1 TAM ratio in the PDAC microenvironment from both groups of mice (F4/80 /CD206− test).
miR-ctrl and miR-506 groups (n = 20 per group; *** p < 0.001 by+ log-rank
M2/M1 TAM
cells as M1ratio
TAMsin and
theF4/80
PDAC microenvironment
+ /CD206 + cells as M2 TAMs) from both groups
analyzed of mice (F4/80
by flow cytometry
+/CD206− ce
(n = 10 per
as M1group;
TAMs***and p < F4/80
0.001 by+/CD206
Student’scells
+ as (G–I)
t-test). M2 TAMs) +
CD4 CD25 analyzed
+ FOXP3 by + flow
Tregs cytometry
(G), CD8 CD45(n T=cells
+ + 10 per grou
+ T cells (I) analyzed by+flow cytometry.
*** p < 0.001 by Student’s t-test). (G–I) CD4 CD25 FOXP3 Tregs (G), CD8 CD45 T cells (H), a
(H), and apoptotic CD8 + +
(J) Production of +
IFN-γ by +
cytotoxic
apoptotic CD8
T cells + T PDAC
in the cells (I) analyzed by flow
microenvironment fromcytometry.
both groups(J) of Production
C57BL/6 miceofanalyzed
IFN-γ by by cytotoxic
ELISA T ce
in the (n = 10 per
PDAC group; *** p < 0.001; NS,
microenvironment fromnot both
statistically
groups significant
of C57BL/6 micet-test).
by Student’s analyzedData represent
by ELISA the(n = 10 p
*** p±<SD
group;mean of at least
0.001; NS, 3not
independent experiments.
statistically significant by Student’s t-test). Data represent the mean
SD of 3.4.
at least 3 independent experiments.
MiR-506 Reprograms the Polarization of M2 Macrophages
M1 or M2-polarized macrophages were then transfected with miR-506 mimic or
3.4. MiR-506 Reprograms
miR-ctrl to thethe
further assess Polarization of M2 Macrophages
impact of miR-506 on TAM polarization. The expression of
alternative
M1 activated M2macrophages
or M2-polarized surface markers (CD163,
were thenCD206, and CD204)
transfected withwas significantly
miR-506 mimic or mi
decreased in M2-polarized macrophages transfected with miR-506 mimic compared with
ctrl tothose
further
transfected with miR-ctrl, while most of the classically activated M1 surface markers of alte
assess the impact of miR-506 on TAM polarization. The expression
native(MHC-II,
activated M2and
CD86, surface
CD80)markers (CD163, CD206,
were upregulated. and CD204)
The M2-polarized was significantly
macrophages were d
creased in M2-polarized macrophages transfected with miR-506 mimic compared wi
those transfected with miR-ctrl, while most of the classically activated M1 surface marke
(MHC-II, CD86, and CD80) were upregulated. The M2-polarized macrophages were r
programmed towards M1 phenotypes by miR-506. On the other hand, miR-506 could n
 macrophages were transfected with miR-506 mimic, they displayed a shift toward a tu-
mor-inhibition phenotype. MiR-506 transfection promoted nitric oxide (NO) production
Biomedicines 2023, 11, 2874 8 of 16
and phagocytosis of immunosuppressive M2-polarized macrophages while suppressing
the IL-10, VEGF, TGF-β expression, and Arg1 activity. MiR-506 reversed the functions of
M2-polarized macrophages, but not M1-polarized macrophages, toward cancer-suppres-
reprogrammed towards M1 phenotypes by miR-506. On the other hand, miR-506 could not
sive activity (*** p < 0.001; NS, not statistically significant; Figure 4C–H).
affect the polarization of M1 macrophages (Figure 4A).

Figure
Figure4. MiR-506
4. MiR-506reprograms
reprogramsthethe
polarization
polarization of of
M2M2 macrophages.
macrophages.(A)(A) Human
Human macrophage
macrophagediffer-
entiation and polarization. Human monocytes were differentiated into
differentiation and polarization. Human monocytes were differentiated into nonpolarized nonpolarized M0M0 macro-
phages and then polarized into M1 or M2 macrophages using a conditioned
macrophages and then polarized into M1 or M2 macrophages using a conditioned medium. Polarized medium. Polarized
macrophages
macrophages were
weretreated with
treated withmiR-ctrl
miR-ctrlorormiR-506,
miR-506,and andthetheexpression
expression levels
levels of
of M1
M1 and
and M2M2 mac-
rophage surfacesurface
macrophage markers were detected
markers by flow
were detected cytometry.
by flow (B) The
cytometry. expression
(B) The expressionlevels of M1
levels and M2
of M1
macrophage phenotypic
and M2 macrophage genes forgenes
phenotypic polarized macrophages
for polarized were detected
macrophages by qRT-PCR
were detected by qRT-PCR andand
normal-
izednormalized
to the endogenous control GAPDH
to the endogenous (*** p < (***
control GAPDH 0.001;
p <NS, notNS,
0.001; statistically significant
not statistically by Student’s
significant by
t-test). (C–H) Macrophage phagocytosis, nitric oxide (NO) production, IL-10, VEGF,
Student’s t-test). (C–H) Macrophage phagocytosis, nitric oxide (NO) production, IL-10, VEGF, TGF-β TGF-β expres-
sion, and Arg1 activity measurements were established to characterize the functions
expression, and Arg1 activity measurements were established to characterize the functions of the of the miR-506
experimentally polarized macrophages
miR-506 experimentally (*** p < 0.001;
polarized macrophages (*** pNS, not statistically
< 0.001; significant
NS, not statistically by Student’s
significant by t-
test). Data represent
Student’s therepresent
t-test). Data mean ± SDthe of at least
mean ± SD3ofindependent experiments.
at least 3 independent experiments.

In addition,
3.5. STAT3 when
Is a Direct M2-polarized
Target of miR-506macrophages were transfected
Involved in Macrophage with miR-506 mimic
Polarization
and compared with miR-ctrl, the expression of M2 genes (CD163, CD204, CD206, TGFB1,
The expression level of miR-506 between different macrophage subtypes was ana-
ARG1, and IL10) was downregulated, while the expression of M1 genes ((HLA-DRA, NOS2,
lyzed to establish why miR-506 only affects the polarization of M2-polarized macro-
IL1B, CD80, CD86, and TNFA) was increased (*** p < 0.001; NS, not statistically significant;
phages.
FigureqRT-PCR analysesfunctional
4B). Furthermore, revealedstudies
that miR-506
showed levels were
that when significantly
M2-polarized upregulated in
macrophages
M1were
but not M2 macrophages. This differential expression suggested that miR-506 could
transfected with miR-506 mimic, they displayed a shift toward a tumor-inhibition
be phenotype.
a negative MiR-506
regulator of alternative
transfection polarization
promoted of(NO)
nitric oxide macrophages.
production To
andexamine whether
phagocytosis
of immunosuppressive
miR-506 contributes to the M2-polarized
plasticity ofmacrophages
macrophagewhile suppressing
polarization, wethe IL-10,
used VEGF, con-
different
TGF-β media
ditioned expression, andtoArg1
trying activity. the
reprogram MiR-506 reversed theWhen
subpopulation. functions of M2-polarized
culturing macrophages
with a conditioned medium, cells were repolarized from the M1 phenotypeactivity
macrophages, but not M1-polarized macrophages, toward cancer-suppressive to the M2
(*** p < 0.001; NS, not statistically significant; Figure 4C–H).
phenotype. The M1-to-M2 conversion decreased the M1 macrophage markers and in-
creased the expression
3.5. STAT3 of M2ofmacrophage
Is a Direct Target markers,
miR-506 Involved which resulted
in Macrophage in an increased expres-
Polarization
sion of miR-506. Conversely,
The expression M1-conditioned
level of miR-506 medium
between different drove M2subtypes
macrophage macrophage repolariza-
was analyzed
tiontoto M1; the M2-to-M1 conversion increased the M1 markers and decreased
establish why miR-506 only affects the polarization of M2-polarized macrophages. qRT- the expres-
sionPCRof M2 markers,
analyses which
revealed thatalso decreased
miR-506 miR-506
levels were expression
significantly (*** p < 0.001;
upregulated in M1 NS, not sta-
but not
tistically significant;This
M2 macrophages. Figure 5A,B).expression
differential These results indicate
suggested that miR-506
that miR-506 could beisa negative
dynamically
regulator
changing of alternative
according polarization phenotype
to macrophage of macrophages.and To
mayexamine
drive whether miR-506 con-
the repolarization of M2
tributes to the plasticity
macrophages with low miR-506. of macrophage polarization, we used different conditioned media
trying to reprogram the subpopulation. When culturing macrophages with a conditioned
 PCR revealed that the expression of STAT3 was different in a subtype of macropha
inversely correlated with miR-506. Additionally, STAT3 levels were significant
downregulated in M1 than in M2 macrophages (*** p < 0.001; NS, not statistically
cant; Figure 5D), which validated why miR-506 only reverses the polarization of
Biomedicines 2023, 11, 2874 larized macrophages 9 of 16

Transfection with miR-506 mimics significantly reduced STAT3 levels in M2

phages. In contrast, no significant change in the expression levels of STAT3 was o
medium, cells were repolarized from the M1 phenotype to the M2 phenotype. The M1-to-
for M1 macrophage (*** p < 0.001; NS, not statistically significant; Figure 5E). To e
M2 conversion decreased the M1 macrophage markers and increased the expression of M2
the regulation
macrophage of STAT3
markers, by miR-506,
which resulted a luciferase
in an increased reporter
expression assay was
of miR-506. performed. m
Conversely,
significantly suppressed
M1-conditioned medium drove theM2luciferase
macrophage reporter activity
repolarization of the
to M1; theM2-to-M1
wild-type con-but not
tant STAT3 3′-UTR, whereas anti-miR-506 treatment increased the luciferase ac
version increased the M1 markers and decreased the expression of M2 markers, which also
decreased miR-506 expression
STAT3, indicating (*** pis
that STAT3 <a0.001; NS,target
direct not statistically
of miR-506 significant;
(*** p <Figure
0.001;5A,B).
NS, not stat
These results indicate that miR-506 is dynamically changing according to macrophage
significant; Figure 5F).
phenotype and may drive the repolarization of M2 macrophages with low miR-506.

Figure
Figure5. 5.STAT3
STAT3is a direct target of
is a direct miR-506
target of involved
miR-506ininvolved
macrophage inpolarization.
macrophage (A) The expression (A) Th
polarization.
levels of M1 and
sion levels M2 macrophage
of M1 phenotypic genes
and M2 macrophage for repolarized
phenotypic genesmacrophages
for repolarizeddetected by qRT-
macrophages det
PCR and normalized to the endogenous control GAPDH (*** p < 0.001; NS,
qRT-PCR and normalized to the endogenous control GAPDH (*** p < 0.001; NS, not sta not statistically significant
by Student’s t-test). (B) The expression levels of miR-506 for repolarized macrophages detected
significant by Student’s t-test). (B) The expression levels of miR-506 for repolarized macr
by qRT-PCR (*** p < 0.001; NS, not statistically significant by Student’s t-test). (C) An miR-506
detected by qRT-PCR (*** p < 0.001; NS, not statistically significant by Student’s t-test). (C)
binding site predicted in the 30 -UTR of STAT3 by TargetScan. (D) The expression levels of STAT3 for
506 binding site predicted in the 3′-UTR of STAT3 by TargetScan. (D) The expression levels
repolarized macrophages detected by qRT-PCR and normalized to the endogenous control GAPDH
for repolarized macrophages detected by qRT-PCR and normalized to the endogenou
(*** p < 0.001; NS, not statistically significant by Student’s t-test). (E) Polarized macrophages were
GAPDH (***
transfected withpmiR-ctrl
< 0.001;orNS, not statistically
miR-506. The expressionsignificant by Student’s
levels of STAT3 t-test).macrophages
for repolarized (E) Polarized macr
were transfected with miR-ctrl or miR-506. The expression levels
detected by qRT-PCR and normalized to the endogenous control GAPDH (*** p < 0.001; NS, of STAT3 for not
repolarize
phages detected
statistically by by
significant qRT-PCR
Student’sand normalized
t-test). to thereporter
(F) A luciferase endogenous controlthat
assay showed GAPDH
miR-506(*** p < 0
not statistically significant0 by Student’s t-test). (F) A luciferase reporter assay
directly targeted the STAT3 3 -UTR (*** p < 0.001; NS, not statistically significant by Student’s t-test). showed that
directly
Data targeted
represent the meanthe ±STAT3 3′-UTR
SD of at (*** p < 0.001;
least 3 independent NS, not statistically significant by Student
experiments.
Data represent0 the mean ± SD of at least 3 independent experiments.
Next, the 3 -untranslated region (UTR) of STAT3 mRNA was predicted to contain a
potential binding site of miR-506; this site is highly conserved among different species
(Figure 5C). Furthermore, STAT3 is involved in multiple aspects of mechanisms governing
tumor escape from immune surveillance and is a direct target of miR-506 in PDAC cells.
Biomedicines 2023, 11, 2874 10 of 16

qRT-PCR revealed that the expression of STAT3 was different in a subtype of macrophages
and inversely correlated with miR-506. Additionally, STAT3 levels were significantly
more downregulated in M1 than in M2 macrophages (*** p < 0.001; NS, not statistically
significant; Figure 5D), which validated why miR-506 only reverses the polarization of
M2-polarized macrophages
Transfection with miR-506 mimics significantly reduced STAT3 levels in M2 macrophages.
In contrast, no significant change in the expression levels of STAT3 was observed for M1
macrophage (*** p < 0.001; NS, not statistically significant; Figure 5E). To examine the
regulation of STAT3 by miR-506, a luciferase reporter assay was performed. miR-506
significantly suppressed the luciferase reporter activity of the wild-type but not the mutant
STAT3 30 -UTR, whereas anti-miR-506 treatment increased the luciferase activity of STAT3,
indicating that STAT3 is a direct target of miR-506 (*** p < 0.001; NS, not statistically
significant; Figure 5F).

3.6. MiR-506 Reprograms the Polarization of M2 Macrophage through the STAT3

Signaling Pathway
STAT3 is critical in tumor immune evasion and is persistently activated in most
malignancies. STAT3 overactivation in tumor-infiltrating immune cells impairs antitumor
immunity by compromising native immune responses via multiple mechanisms, including
polarized macrophages toward the M2 phenotype. Loss-of-function studies were carried
out to confirm that repolarization of M2 macrophages triggered by miR-506 was mediated
by repression of STAT3.
First, we treated macrophages with a selective STAT3 inhibitor (Stattic). Following
the decline of phosphorylated STAT3 (p-STAT3; Y705) expression, iNOS increased and
Arg1 decreased in M2 macrophages, while the expression of p-STAT3, iNOS, and Arg1
did not significantly change in M1 macrophages. In addition, qRT-PCR analyses revealed
decreased expression of alternative activated M2 phenotype genes and upregulation of
most M1 phenotype genes were upregulated in M2 macrophages after Stattic treatment.
In contrast, there was no significant change in the expression of phenotype genes in M1
macrophages (*** p < 0.001; NS, not statistically significant; Figure 6A top).
Silencing of STAT3 in M2 macrophages by small nterfering RNA (siRNA) (si-STAT3-2
and si-STAT3-1) significantly upregulated the expression of iNOS and downregulated
the expression of Arg1, STAT3, and p-STAT3. Additionally, siRNA knockdown of STAT3
suppressed M2 genes and promoted most of the M1 genes compared to miR-ctrl, which
reprogrammed M2 macrophages polarized toward the tumor-suppressive M1 phenotype
(*** p < 0.001; NS, not statistically significant; Figure 6A bottom).
Next, we randomly treated an orthotopic PDAC mouse with vehicle or Stattic at 3.75
mg/kg/day to investigate whether STAT3 suppression modulates tumor progression and
the immune microenvironment in vivo. After treatment, smaller and lighter tumors and a
lower percentage of Ki-67-positive PDAC cells were seen in the Stattic group vs. the control
group (** p < 0.01, *** p < 0.001; Figure 6B–D). Stattic treatment also prolonged OS in an
independent cohort of mice (n = 20 per group; p < 0.001; Figure 6E).
Next, we performed a flow cytometry analysis. A significantly decreased percentage of
apoptotic cytotoxic T cells, lower M2 (F4/80+ /CD206+ )/M1 (F4/80+ /CD206− ) TAM ratio
decreased infiltration of CD4+ CD25+ FOXP3+ Tregs, increased infiltration of CD8+ CD45+
cytotoxic T cells, and increased production of IFN-γ by cytotoxic T cells in the PDAC
microenvironment were seen after Stattic treatment (** p < 0.01, *** p < 0.001; Figure 6F–J).
Biomedicines 2023, 2023,
Biomedicines 11, x 11,
FOR PEER REVIEW
2874 11 of 16
11 of 16

Figure 6. MiR-506
Figure 6. MiR-506 reprograms
reprograms thethe
polarization
polarizationofofM2M2macrophages
macrophages through STAT3 signaling
through the STAT3 signaling
pathway.
pathway.(A)(A)
TheTheexpression
expression levels
levelsofofmacrophage
macrophagephenotypic
phenotypicgenes
genes for
for macrophages repolarized
macrophages repolarized
by inhibiting
by inhibitingor knocking
or knocking downdownSTAT3
STAT3 detected
detectedby byqRT-PCR
qRT-PCRand and normalized
normalized to to the
the endogenous
endogenous
control GAPDH
control GAPDH (***(***
p <p0.001; NS,
< 0.001; NS,notnotstatistically
statisticallysignificant
significantby
by Student’s t-test). (B)
Student’s t-test). (B) Panc02
Panc02cells
cells
were injected into C57BL/6 mice to establish an orthotropic PDAC model and
were injected into C57BL/6 mice to establish an orthotropic PDAC model and then treated with then treated with
Stattic or vehicle. Twenty-eight days following cell implantation, mice were euthanized and tumors
Stattic or vehicle. Twenty-eight days following cell implantation, mice were euthanized and tumors
were harvested. (B–D) The tumor weight (B), tumor size (C), and percentage of mitotic Ki-67 tumor
were harvested. (B–D) The tumor weight (B), tumor size (C), and percentage of mitotic Ki-67 tumor
cells (D) were measured (n = 10 per group; ** p < 0.01; *** p < 0.001 by Student’s t-test). (E) Kaplan–
cells (D) were measured (n = 10 per group; ** p < 0.01; *** p < 0.001 by Student’s t-test). (E) Kaplan–
Meier curves comparing overall survival rates between Stattic and vehicle groups (n = 20 per group;
Meier curves comparing overall survival rates between Stattic and vehicle groups (n = 20 per group;
p < 0.001 by log-rank test). (F) M2/M1 TAM ratio in the PDAC microenvironment from both groups
p < 0.001 by+ log-rank− test). (F) M2/M1 TAM ratio in+the PDAC microenvironment from both groups
of mice (F4/80 /CD206 cells as M1 TAMs and F4/80 /CD206+ cells as M2 TAMs) analyzed by flow
of mice (F4/80 + /CD206− cells as M1 TAMs and F4/80+ /CD206+ cells as M2 TAMs) analyzed
cytometry. (G–I) CD4 CD25 FOXP3 Tregs (G), CD8 CD45 T cells (H) and apoptotic CD8+ T cells
+ + + + +
by flow cytometry. (G–I) CD4 + + FOXP3+ Tregs (G), CD8+ CD45+ T cells (H) and apoptotic
(I) analyzed by flow cytometry. (J)CD25
Production of IFN-γ by cytotoxic T cells in the PDAC microen-
CD8+ Tfrom
vironment cells both
(I) analyzed
groups by flow cytometry.
of C57BL/6 (J) Production
mice analyzed by ELISAof IFN-γ
(n = 10by cytotoxic
per group; **T cells in the
p < 0.01; ***
PDACby
p < 0.001 microenvironment
Student’s t-test). from
Databoth groupsthe
represent of mean
C57BL/6 mice
± SD analyzed
of at by ELISA (n =experiments.
least 3 independent 10 per group;
** p < 0.01; *** p < 0.001 by Student’s t-test). Data represent the mean ± SD of at least 3 indepen-
3.7.dent experiments. Repolarization of Macrophages Sensitizes Them to Anti-PD-1 Immuno-
MiR-506-Induced
therapy
Biomedicines 2023, 11, x FOR PEER REVIEW
Biomedicines 2023, 11, 2874
12 of 16
12 of 16

Anti-PD-1
3.7. immuneRepolarization
MiR-506-Induced checkpoint of blockade therapy
Macrophages can suppress
Sensitizes Them to PD-L1-induced T cell
Anti-PD-1
inhibition to Immunotherapy
strengthen the antitumor immune response. However, anti-PD-1 immuno-
therapyAnti-PD-1
is unlikelyimmune checkpoint
to be effective blockade
in PDAC therapy
because can suppress
human pancreaticPD-L1-induced
cancer exhibits T cell
very
inhibition to strengthen the antitumor immune response. However, anti-PD-1
low CTL infiltration in the tumor microenvironment. Our results indicated that the tumor immunother-
apy is unlikely toremodeling
microenvironment be effective in PDAC
effects ofbecause
miR-506 human pancreatic
reprogram cancer exhibits
macrophage very
polarization
andlow CTL infiltration
subsequently in the CTL
promote tumor microenvironment.
infiltration. Next, weOur results indicated
evaluated whetherthat the tumor
miR-506 sensi-
microenvironment remodeling effects of miR-506 reprogram macrophage polarization and
tizes pancreatic cancer to anti-PD-1 immunotherapy. Orthotopic PDAC mouse were
subsequently promote CTL infiltration. Next, we evaluated whether miR-506 sensitizes
treated with miR-ctrl iso, miR-ctrl PD1, miR-506 iso, and miR-506 PD1 (n = 10 per group).
pancreatic cancer to anti-PD-1 immunotherapy. Orthotopic PDAC mouse were treated
Thewith
analysis of the
miR-ctrl iso,tumors
miR-ctrlindicated that combined
PD1, miR-506 miR-506PD1
iso, and miR-506 and(nanti-PD-1 immunother-
= 10 per group). The
apyanalysis
have significantly
of the tumors greater efficacy
indicated thatthan eithermiR-506
combined miR-506and or anti-PD-1
anti-PD-1 Ab alone. Further-
immunotherapy
more,
havesmaller and lighter
significantly greatertumors, a lower
efficacy than percentage
either miR-506 orof Ki-67-positive
anti-PD-1 PDAC
Ab alone. cells, and
Furthermore,
moresmaller and lighter tumors, a lower percentage of Ki-67-positive PDAC cells, andthe
production of IFN-γ by CTLs in the tumor microenvironment were seen in miR-
more
506production
PD1 groupofvs. otherbygroups
IFN-γ CTLs in (nthe
= 10 per group;
tumor ** p < 0.01, ***
microenvironment p <seen
were 0.001;
in Figure 7A–D).
the miR-506
As PD1 groupmiR-506
expected, vs. other+ groups
anti-PD-1(n =Ab 10 treatment
per group;also** p significantly
< 0.01, *** p <prolonged
0.001; Figure
OS 7A–D).
in an in-
As expected,
dependent cohort miR-506
of mice+(n anti-PD-1
= 15 per Ab treatment
group; also significantly
** p < 0.01, *** p < 0.001;prolonged
Figure 7E).OS in an
independent cohort of mice (n = 15 per group; ** p < 0.01, *** p < 0.001; Figure 7E).

Figure 7. MiR-506 induced repolarization of M2 macrophage sensitizes pancreatic cancer to anti-

Figure
PD17.therapy.
MiR-506(A)induced
Panc02 repolarization of M2
cells were injected macrophage
in C57BL/6 micesensitizes pancreatic
to establish cancerPDAC
an orthotropic to anti-
PD1model
therapy. (A) Panc02 cells were injected in C57BL/6 mice to establish an orthotropic
and then treated with miR-ctrl or miR-506 with or without anti-PD-1 therapy. Twenty-eight PDAC
model and then treated with miR-ctrl or miR-506 with or without anti-PD-1 therapy.
days following cell implantation, mice were euthanized, and tumors were harvested. (A–C) The Twenty-eight
daystumor
following
weightcell
(A),implantation,
tumor size (B),mice
and were euthanized,
percentage and
of mitotic tumors
Ki-67 tumorwere
cellsharvested. (A–C) The
(C) were measured.
tumor
(D) Production of IFN-γ by cytotoxic T cells in the PDAC microenvironment from both mousemeasured.
weight (A), tumor size (B), and percentage of mitotic Ki-67 tumor cells (C) were groups
(D) analyzed
Production of IFN-γ by cytotoxic T cells in the PDAC microenvironment from both mouse
by ELISA (n = 10 per group; ** p < 0.01; *** p < 0.001 by Student’s t-test). (E) Kaplan–Meier
groups analyzed by ELISA (n = 10 per group; ** p < 0.01; *** p < 0.001 by Student’s t-test). (E) Kaplan–
curves comparing overall survival rates between groups (n = 15 per group; ** p < 0.01; *** p < 0.001 by
Meier curves comparing overall survival rates between groups (n = 15 per group; ** p < 0.01; *** p <
log-rank test). Data represent the mean ± SD of at least 3 independent experiments.
0.001 by log-rank test). Data represent the mean ± SD of at least 3 independent experiments.

4. Discussion
Biomedicines 2023, 11, 2874 13 of 16

4. Discussion
In the present study, we combined clinical data and basic experiments to explore
the relationship between miR-506 and macrophages of TME in PDAC patients. First, we
discovered that lower expression of miR-506 was associated with poor prognosis and
immunosuppressive TME in patients with PDAC. Then, to further explore the detailed
mechanism, we established the PDAC mouse model and found that miR-506 could repro-
gram the polarization of M2-like macrophages by targeting the STAT3 signaling pathway.
MiR-506 could also sensitize PDAC to anti-PD-1 immunotherapy. Unlike previous studies,
this study first investigated the miR-506 and TME, especially M2-like macrophages, thus
providing novel insights into mechanisms of tumor progression and potential immunother-
apeutic targets for further clinical treatment.
PDAC is a highly aggressive lethal malignancy due to limited treatment response
and a lack of early diagnosis. Although adjuvant chemotherapy or neoadjuvant therapy
has a significant survival benefit in patients with resected PDAC, most patients fail to
receive therapy due to poor patient performance status, postoperative complications, or
early disease progression [30]. Therefore, researchers are gradually turning to immune-
targeted therapy to treat PDAC. Studies have found that multiagent cytotoxic regimens
significantly affect advanced PDAC [31,32]. This approach requires activating the host
immune system through cytotoxic T cells, which recognize tumor antigens [33]. However,
in most PDAC patients, the TME is characterized by the absence of CD8+ T cells and abun-
dant macrophages (especially TAMs) and regulatory T cells. These unique characteristics
enhance the immunosuppression and drug resistance of PDAC, pointing to the need for
effective and novel therapeutic targets for PDAC patients.
Macrophages originate from bone marrow hematopoietic stem cells (HSCs), which
then differentiate into monocytes in the blood. During injury, monocytes are recruited and
differentiated into macrophages with great plasticity [34]. Macrophages can differentiate
into proinflammatory M1-like phenotypes with antitumor immune responses through
classically activated pathways and anti-inflammatory M2-like phenotypes with immuno-
suppressive responses through alternatively activated pathways [35–37]. Meanwhile, fully
polarized subgroups can repolarize and transform due to different TMEs [38]. Studies have
shown that the TME of PDAC is abundant in M2 macrophages in patients with advanced
PDAC, promoting tumor progression and metastasis [39,40]. Related clinical trials also
proved that higher M macrophage infiltration is strongly correlated with a poorer progno-
sis and shorter OS. The density of M2 macrophage infiltration could be an independent
prognostic factor for PDAC patients [41].
MiR-506 has an important role in tumorigenesis [42,43]. Yang et al. found that the
lower expression of miR-506 in ovarian cancer patients is associated with poor progno-
sis [44]. Subsequent studies confirmed the tumor-suppressive function of miR-506 in
other types of cancer [44–48]. As a multiagent regulatory gene, miR-506 can modulate cell
differentiation through STAT3, flotillin 1, cell adhesion and migration through integrin
beta 1 and 3, cell proliferation through cyclin-dependent kinase 4 and 6, and so on [49].
Among these targeted factors, STAT3 is critical for cell differentiation, proliferation, and
angiogenesis [17].
STAT3 has been demonstrated to be a key mediator in contributing to the local im-
munosuppressive TME because of its activation within various immunosuppressive cells,
including TAMs. Our previous study established that activated STAT3 can augment the
expression and secretion of its downstream mediator IL-10, which promoted TAM po-
larization toward an M2 phenotype. Inhibiting STAT3 activation in tumor cells triggers
macrophage production of NO and leads to macrophage-mediated, nitrite-dependent cyto-
static activity against tumor cells. Our previous research confirmed that miR-506 can induce
cell death through the STAT3 signaling pathway in PDAC patients. In this study, we further
found that miR-506 can also repolarize M2-like macrophages, acting as a tumor-suppressive
regulatory molecule by targeting the STAT3 pathway.
Biomedicines 2023, 11, 2874 14 of 16

5. Conclusions
To sum up, our data suggest that miR-506 can reprogram the polarization of M2-like
macrophages toward an M1-like phenotype through targeting STAT3. MiR-506 can also
sensitize PDAC to anti-PD-1 immunotherapy and overcome immunotherapy resistance
in PDAC because of the tumor microenvironment remodeling effects of miR-506 repro-
gramming macrophage polarization and subsequently promoting CTL infiltration. These
findings suggest a strong association between miR-506 and tumor stage, prognosis, and
drug sensitivity of PDAC, thus providing novel insights into mechanisms of tumor pro-
gression and potential immunotherapeutic targets for further clinical treatment. These
data provide a rationale for clinical trials to evaluate the combination therapy of miR-506
and immune checkpoint therapy, particularly in immunotherapy-resistant tumors, such
as PDAC, which lack infiltration of CTLs. However, this study on the method of adminis-
tration, treatment process, dosage, toxicity and side effects of combination therapy needs
further verification.

Author Contributions: T.Y., Y.H. and J.C. designed the study and drafted the manuscript; Y.H. and
J.C. performed the experiments and analyzed the data; X.L. and L.S. supervised the study and edited
the manuscript. All authors have read and agreed to the published version of the manuscript.
Funding: L.S. was supported by the Tianjin Health Science and Technology Project (TJWJ2022MS002),
the National Natural Science Foundation of China (81702410) and the Natural Science Foundation of
Tianjin (17JCQNJC11100).
Institutional Review Board Statement: The use of health donor- or patient-derived material was
approved by the institutional research ethics committee of TMUGH. In addition, all patients provided
signed informed consent. All animal studies were done in compliance with the regulations and
guidelines of Tianjin Medical University institutional animal care and conducted according to the
IACUC and AAALAC guidelines.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Chandana, S.; Babiker, H.M.; Mahadevan, D. Therapeutic trends in pancreatic ductal adenocarcinoma (PDAC). Expert Opin.
Investig. Drugs 2019, 28, 161–177. [CrossRef]
2. Cai, J.; Chen, H.; Lu, M.; Zhang, Y.; Lu, B.; You, L.; Zhang, T.; Dai, M.; Zhao, Y. Advances in the epidemiology of pancreatic cancer:
Trends, risk factors, screening, and prognosis. Cancer Lett. 2021, 520, 1–11. [CrossRef]
3. Schnelldorfer, T.; Adams, D.B.; Warshaw, A.L.; Lillemoe, K.D.; Sarr, M.G. Forgotten pioneers of pancreatic surgery: Beyond the
favorite few. Ann. Surg. 2008, 247, 191–202. [CrossRef] [PubMed]
4. Barugola, G.; Partelli, S.; Marcucci, S.; Sartori, N.; Capelli, P.; Bassi, C.; Pederzoli, P.; Falconi, M. Resectable pancreatic cancer:
Who really benefits from resection? Ann. Surg. Oncol. 2009, 16, 3316–3322. [CrossRef] [PubMed]
5. Semaan, A.; Bernard, V.; Lee, J.J.; Wong, J.W.; Huang, J.; Swartzlander, D.B.; Stephens, B.M.; Monberg, M.E.; Weston, B.R.;
Bhutani, M.S.; et al. Defining the Comprehensive Genomic Landscapes of Pancreatic Ductal Adenocarcinoma Using Real-World
Endoscopic Aspiration Samples. Clin. Cancer Res. 2021, 27, 1082–1093. [CrossRef]
6. Apte, M.V.; Park, S.; Phillips, P.A.; Santucci, N.; Goldstein, D.; Kumar, R.K.; Ramm, G.A.; Buchler, M.; Friess, H.; McCarroll, J.A.;
et al. Desmoplastic reaction in pancreatic cancer: Role of pancreatic stellate cells. Pancreas 2004, 29, 179–187. [CrossRef]
7. Neesse, A.; Michl, P.; Frese, K.K.; Feig, C.; Cook, N.; Jacobetz, M.A.; Lolkema, M.P.; Buchholz, M.; Olive, K.P.; Gress, T.M.; et al.
Stromal biology and therapy in pancreatic cancer. Gut 2011, 60, 861–868. [CrossRef]
8. Hosein, A.N.; Brekken, R.A.; Maitra, A. Pancreatic cancer stroma: An update on therapeutic targeting strategies. Nat. Rev.
Gastroenterol. Hepatol. 2020, 17, 487–505. [CrossRef]
9. Feig, C.; Gopinathan, A.; Neesse, A.; Chan, D.S.; Cook, N.; Tuveson, D.A. The pancreas cancer microenvironment. Clin. Cancer
Res. 2012, 18, 4266–4276. [CrossRef]
10. Hinshaw, D.C.; Shevde, L.A. The Tumor Microenvironment Innately Modulates Cancer Progression. Cancer Res. 2019, 79,
4557–4566. [CrossRef] [PubMed]
11. Murray, P.J.; Allen, J.E.; Biswas, S.K.; Fisher, E.A.; Gilroy, D.W.; Goerdt, S.; Gordon, S.; Hamilton, J.A.; Ivashkiv, L.B.; Lawrence, T.;
et al. Macrophage activation and polarization: Nomenclature and experimental guidelines. Immunity 2014, 41, 14–20. [CrossRef]
[PubMed]
Biomedicines 2023, 11, 2874 15 of 16

12. Liao, X.; Sluimer, J.C.; Wang, Y.; Subramanian, M.; Brown, K.; Pattison, J.S.; Robbins, J.; Martinez, J.; Tabas, I. Macrophage
autophagy plays a protective role in advanced atherosclerosis. Cell Metab. 2012, 15, 545–553. [CrossRef] [PubMed]
13. Wu, K.; Lin, K.; Li, X.; Yuan, X.; Xu, P.; Ni, P.; Xu, D. Redefining Tumor-Associated Macrophage Subpopulations and Functions in
the Tumor Microenvironment. Front. Immunol. 2020, 11, 1731. [CrossRef] [PubMed]
14. Mantovani, A.; Biswas, S.K.; Galdiero, M.R.; Sica, A.; Locati, M. Macrophage plasticity and polarization in tissue repair and
remodelling. J. Pathol. 2013, 229, 176–185. [CrossRef] [PubMed]
15. Yang, Y.; Guo, Z.; Chen, W.; Wang, X.; Cao, M.; Han, X.; Zhang, K.; Teng, B.; Cao, J.; Wu, W.; et al. M2 Macrophage-Derived
Exosomes Promote Angiogenesis and Growth of Pancreatic Ductal Adenocarcinoma by Targeting E2F2. Mol. Ther. 2021, 29,
1226–1238. [CrossRef]
16. Lin, E.Y.; Li, J.F.; Gnatovskiy, L.; Deng, Y.; Zhu, L.; Grzesik, D.A.; Qian, H.; Xue, X.N.; Pollard, J.W. Macrophages regulate the
angiogenic switch in a mouse model of breast cancer. Cancer Res. 2006, 66, 11238–11246. [CrossRef]
17. Hanlon, M.M.; Rakovich, T.; Cunningham, C.C.; Ansboro, S.; Veale, D.J.; Fearon, U.; McGarry, T. STAT3 Mediates the Differential
Effects of Oncostatin M and TNFα on RA Synovial Fibroblast and Endothelial Cell Function. Front. Immunol. 2019, 10, 2056.
[CrossRef] [PubMed]
18. Rawlings, J.S.; Rosler, K.M.; Harrison, D.A. The JAK/STAT signaling pathway. J. Cell Sci. 2004, 117 Pt 8, 1281–1283. [CrossRef]
19. Johnson, D.E.; O’Keefe, R.A.; Grandis, J.R. Targeting the IL-6/JAK/STAT3 signalling axis in cancer. Nat. Rev. Clin. Oncol. 2018, 15,
234–248. [CrossRef]
20. Wang, T.; Niu, G.; Kortylewski, M.; Burdelya, L.; Shain, K.; Zhang, S.; Bhattacharya, R.; Gabrilovich, D.; Heller, R.; Coppola, D.;
et al. Regulation of the innate and adaptive immune responses by Stat-3 signaling in tumor cells. Nat. Med. 2004, 10, 48–54.
[CrossRef]
21. Zou, S.; Tong, Q.; Liu, B.; Huang, W.; Tian, Y.; Fu, X. Targeting STAT3 in Cancer Immunotherapy. Mol. Cancer 2020, 19, 145.
[CrossRef] [PubMed]
22. Nefedova, Y.; Huang, M.; Kusmartsev, S.; Bhattacharya, R.; Cheng, P.; Salup, R.; Jove, R.; Gabrilovich, D. Hyperactivation of
STAT3 is involved in abnormal differentiation of dendritic cells in cancer. J. Immunol. 2004, 172, 464–474. [CrossRef] [PubMed]
23. Zorn, E.; Nelson, E.A.; Mohseni, M.; Porcheray, F.; Kim, H.; Litsa, D.; Bellucci, R.; Raderschall, E.; Canning, C.; Soiffer, R.J.; et al.
IL-2 regulates FOXP3 expression in human CD4+CD25+ regulatory T cells through a STAT-dependent mechanism and induces
the expansion of these cells in vivo. Blood 2006, 108, 1571–1579. [CrossRef]
24. Aveta, A.; Cilio, S.; Contieri, R.; Spena, G.; Napolitano, L.; Manfredi, C.; Franco, A.; Crocerossa, F.; Cerrato, C.; Ferro, M.; et al.
Urinary MicroRNAs as Biomarkers of Urological Cancers: A Systematic Review. Int. J. Mol. Sci. 2023, 24, 10846. [CrossRef]
[PubMed]
25. Fathi, M.; Ghafouri-Fard, S.; Abak, A.; Taheri, M. Emerging roles of miRNAs in the development of pancreatic cancer. Biomed.
Pharmacother. 2021, 141, 111914. [CrossRef]
26. Cheng, R.F.; Wang, J.; Zhang, J.Y.; Sun, L.; Zhao, Y.R.; Qiu, Z.Q.; Sun, B.C.; Sun, Y. MicroRNA-506 is up-regulated in the
development of pancreatic ductal adenocarcinoma and is associated with attenuated disease progression. Chin. J. Cancer 2016,
35, 64. [CrossRef]
27. Li, J.; Wu, H.; Li, W.; Yin, L.; Guo, S.; Xu, X.; Ouyang, Y.; Zhao, Z.; Liu, S.; Tian, Y.; et al. Downregulated miR-506 expression
facilitates pancreatic cancer progression and chemoresistance via SPHK1/Akt/NF-κB signaling. Oncogene 2016, 35, 5501–5514.
[CrossRef]
28. Borchardt, H.; Kogel, A.; Kalwa, H.; Weirauch, U.; Aigner, A. Therapeutic miR-506-3p Replacement in Pancreatic Carcinoma
Leads to Multiple Effects including Autophagy, Apoptosis, Senescence, and Mitochondrial Alterations In Vitro and In Vivo.
Biomedicines 2022, 10, 1692. [CrossRef] [PubMed]
29. Sun, L.; Zhang, X.; Song, Q.; Liu, L.; Forbes, E.; Tian, W.; Zhang, Z.; Kang, Y.; Wang, H.; Fleming, J.B.; et al. IGFBP2 promotes
tumor progression by inducing alternative polarization of macrophages in pancreatic ductal adenocarcinoma through the STAT3
pathway. Cancer Lett. 2021, 500, 132–146. [CrossRef]
30. Park, W.; Chawla, A.; O’Reilly, E.M. Pancreatic Cancer: A Review. JAMA 2021, 326, 851–862. [CrossRef]
31. Wang-Gillam, A.; Li, C.P.; Bodoky, G.; Dean, A.; Shan, Y.S.; Jameson, G.; Macarulla, T.; Lee, K.H.; Cunningham, D.; Blanc, J.F.; et al.
Nanoliposomal irinotecan with fluorouracil and folinic acid in metastatic pancreatic cancer after previous gemcitabine-based
therapy (NAPOLI-1): A global, randomised, open-label, phase 3 trial. Lancet 2016, 387, 545–557. [CrossRef]
32. Conroy, T.; Desseigne, F.; Ychou, M.; Bouché, O.; Guimbaud, R.; Bécouarn, Y.; Adenis, A.; Raoul, J.L.; Gourgou-Bourgade, S.; de la
Fouchardière, C.; et al. FOLFIRINOX versus gemcitabine for metastatic pancreatic cancer. N. Engl. J. Med. 2011, 364, 1817–1825.
[CrossRef]
33. Grant, T.J.; Hua, K.; Singh, A. Molecular Pathogenesis of Pancreatic Cancer. Prog. Mol. Biol. Transl. Sci. 2016, 144, 241–275.
34. Li, P.; Hao, Z.; Wu, J.; Ma, C.; Xu, Y.; Li, J.; Lan, R.; Zhu, B.; Ren, P.; Fan, D.; et al. Comparative Proteomic Analysis of Polarized
Human THP-1 and Mouse RAW264.7 Macrophages. Front. Immunol. 2021, 12, 700009. [CrossRef]
35. Martinez, F.O.; Gordon, S. The M1 and M2 paradigm of macrophage activation: Time for reassessment. F1000Prime Rep. 2014, 6,
13. [CrossRef]
36. Fong, C.H.; Bebien, M.; Didierlaurent, A.; Nebauer, R.; Hussell, T.; Broide, D.; Karin, M.; Lawrence, T. An antiinflammatory role
for IKKbeta through the inhibition of “classical” macrophage activation. J. Exp. Med. 2008, 205, 1269–1276. [CrossRef]
Biomedicines 2023, 11, 2874 16 of 16

37. Gordon, S.; Martinez, F.O. Alternative activation of macrophages: Mechanism and functions. Immunity 2010, 32, 593–604.
[CrossRef]
38. Shapouri-Moghaddam, A.; Mohammadian, S.; Vazini, H.; Taghadosi, M.; Esmaeili, S.A.; Mardani, F.; Seifi, B.; Mohammadi, A.;
Afshari, J.T.; Sahebkar, A. Macrophage plasticity, polarization, and function in health and disease. J. Cell. Physiol. 2018, 233,
6425–6440. [CrossRef]
39. Xia, Y.; Rao, L.; Yao, H.; Wang, Z.; Ning, P.; Chen, X. Engineering Macrophages for Cancer Immunotherapy and Drug Delivery.
Adv. Mater. 2020, 32, e2002054. [CrossRef]
40. Sica, A.; Mantovani, A. Macrophage plasticity and polarization: In vivo veritas. J. Clin. Investig. 2012, 122, 787–795. [CrossRef]
41. Hu, H.; Hang, J.J.; Han, T.; Zhuo, M.; Jiao, F.; Wang, L.W. The M2 phenotype of tumor-associated macrophages in the stroma
confers a poor prognosis in pancreatic cancer. Tumour Biol. 2016, 37, 8657–8664. [CrossRef]
42. Bentwich, I.; Avniel, A.; Karov, Y.; Aharonov, R.; Gilad, S.; Barad, O.; Barzilai, A.; Einat, P.; Einav, U.; Meiri, E.; et al. Identification
of hundreds of conserved and nonconserved human microRNAs. Nat. Genet. 2005, 37, 766–770. [CrossRef]
43. Zhao, Y.; Liu, H.; Li, Y.; Wu, J.; Greenlee, A.R.; Yang, C.; Jiang, Y. The role of miR-506 in transformed 16HBE cells induced by
anti-benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide. Toxicol. Lett. 2011, 205, 320–326. [CrossRef]
44. Yang, D.; Sun, Y.; Hu, L.; Zheng, H.; Ji, P.; Pecot, C.V.; Zhao, Y.; Reynolds, S.; Cheng, H.; Rupaimoole, R.; et al. Integrated analyses
identify a master microRNA regulatory network for the mesenchymal subtype in serous ovarian cancer. Cancer Cell 2013, 23,
186–199. [CrossRef]
45. Zhang, Z.; Ma, J.; Luan, G.; Kang, L.; Su, Y.; He, Y.; Luan, F. MiR-506 suppresses tumor proliferation and invasion by targeting
FOXQ1 in nasopharyngeal carcinoma. PLoS ONE 2015, 10, e0122851. [CrossRef]
46. Du, J.; Zheng, X.; Cai, S.; Zhu, Z.; Tan, J.; Hu, B.; Huang, Z.; Jiao, H. MicroRNA-506 participates in pancreatic cancer pathogenesis
by targeting PIM3. Mol. Med. Rep. 2015, 12, 5121–5126. [CrossRef]
47. Yang, F.Q.; Zhang, H.M.; Chen, S.J.; Yan, Y.; Zheng, J.H. MiR-506 is down-regulated in clear cell renal cell carcinoma and inhibits
cell growth and metastasis via targeting FLOT1. PLoS ONE 2015, 10, e0120258. [CrossRef]
48. Li, Z.; Liu, Z.; Dong, S.; Zhang, J.; Tan, J.; Wang, Y.; Ge, C.; Li, R.; Xue, Y.; Li, M.; et al. miR-506 Inhibits Epithelial-to-Mesenchymal
Transition and Angiogenesis in Gastric Cancer. Am. J. Pathol. 2015, 185, 2412–2420. [CrossRef]
49. Li, J.; Ju, J.; Ni, B.; Wang, H. The emerging role of miR-506 in cancer. Oncotarget 2016, 7, 62778–62788. [CrossRef]

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content.