Chemotherapeutic tumor microparticles elicit a neutrophil ... · 7/11/2020 · 1 Chemotherapeutic...

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Chemotherapeutic tumor microparticles elicit a neutrophil response targeting

malignant pleural effusions

Pingwei Xu1,6

, Ke Tang1,6

, Jingwei Ma1, Huafeng Zhang

1, Dianheng Wang

1, Liyan

Zhu1, Jie Chen

1, Keke Wei

1, Jincheng Liu

1, Haiqing Fang

1, Liang Tang

1, Yi Zhang

2,

Jing Xie2, Yuying Liu

2, Rui Meng

3, Li Liu

3, Xiaorong Dong

3, Kunyu Yang

3, Gang

Wu3, Fei Ma

4, Bo Huang

1,2,5*

1Department of Biochemistry and Molecular Biology, Tongji Medical College,

Huazhong University of Science and Technology, Wuhan 430030, China.

2Department of Immunology, Institute of Basic Medical Sciences & State Key

Laboratory of Medical Molecular Biology, Chinese Academy of Medical Sciences

and Peking Union Medical College, Beijing 100005, China.

3Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of

Science and Technology, Wuhan 430022, China.

4National Cancer Center, State Key Laboratory of Molecular Oncology, Cancer

Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College,

Beijing 100005, China.

5Clinical Immunology Center, Chinese Academy of Medical Sciences, Beijing 100005,

China

6 These authors contributed equally

Running title: Microparticles elicit a neutrophil response to kill tumors

Keywords: microparticles, neutrophils, malignant pleural effusions

*Correspondence: Bo Huang, Chinese Academy of Medical Sciences, 5 Dong Dan

San Tiao, Beijing 100005, China. Phone: 86-10-69156447; Fax: 86-10-65229258;

E-mail: [email protected]

Research. on September 22, 2020. © 2020 American Association for Cancercancerimmunolres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 13, 2020; DOI: 10.1158/2326-6066.CIR-19-0789

http://cancerimmunolres.aacrjournals.org/

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Abstract

Malignant pleural effusion (MPE) is a frequent complication of various cancers and

often leads to a poor quality of life, prognosis, and life expectancy, and its

management remains palliative. New approaches that can effectively treat MPE are

highly desirable. Here, we show that methotrexate (MTX)-packaging, tumor

cell–derived microparticles (MTX-MP) act as an effective immunotherapeutic agent

to treat patients with MPE by mobilizing and activating neutrophils. We find that

MTX-MPs perfusion via a pleural catheter elicits the recruitment of neutrophils in

patients through macrophage-released CXCL1 and CXCL2. By performing ex vivo

experiments, we find that the recruited neutrophils are activated and release reactive

oxygen species (ROS) and neutrophil extracellular trap (NET) to kill tumor cells.

Neutrophil-released NETs were also able to seal-off the damaged endothelium,

facilitating MPE resolution in vitro and in tumor-bearing mice. These findings reveal

the potential for use of cell-derived materials to package drugs as an

immunotherapeutic agent against MPE.




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Introduction

Despite adaptive antitumor immunity, the mobilization of the innate immune system

in cancer immunotherapies is lagging. Neutrophils, the most abundant innate immune

cells, represent 50–70% of all leukocytes, and more than 1011

neutrophils undergo

death daily in our body (1). Maintaining this innate cell death without activation is

presumed to prevent ubiquitous infections. However, this physiological process

presents an opportunity to modulate neutrophils to exert an antitumor effect before

their death. Indeed, antitumor responses from neutrophils can be elicited by Coley

toxin and Bacillus Calmette-Guérin in certain cancer patients (2,3). However,

neutrophils can also be polarized to become tumor-promoting in particular tumor

microenvironments, where factors such as inhibitory cytokines, hypoxia, low pH and

potassium overabundance can redirect neutrophils, thus limiting the treatment

efficiency (4). Studies have revealed the double-edged sword role of neutrophils in

tumor microenvironments (5,6) but mobilizing neutrophils in cancer immunotherapy

remains a challenge.

Solid tumor cells are likely to metastasize to the pleura through the bloodstream and

invade the visceral pleura, resulting in an effusion in the pleural cavity, referred to as

malignant pleural effusion (MPE)(7,8). About 40% of lung cancer patients develop

MPE (9). Patients with refractory MPE commonly suffer dyspnea, followed by chest

discomfort, coughing, and poor quality of life and may have a reduced life span. Lung

cancer patients with MPE display a decreased rate of survival compared with patients

without pleural effusions (9,10). Despite the progress in cancer treatment, the current

management of MPE remains palliative. Thus, approaches that can efficiently target

MPE with minimal side effects are highly desirable. MPE is stemmed from the




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damaged blood vessels, where a relatively physiological state with average oxygen

pressure, pH value, and electrolyte balance may be present. The increased capillary

permeability may facilitate the entry of leukocytes into the effusions. Thus, we asked

whether we could use this to mobilize neutrophils into malignant effusions where the

neutrophils could avoid malignant re-education and exert an antitumor effect.

Tumor cells are able to release extracellular vesicles, which are labeled as tumor

microparticles (T-MPs)(11). We previously reported that T-MPs can stimulate DCs

using the cGAS/STING pathway and function as a vaccination platform (12,13), but

such T-MPs also stimulate M2 macrophage polarization to exert a tumor-promoting

effect (14). We also found that T-MPs can function as natural carriers to deliver

chemotherapeutic drugs or oncolytic viruses to tumor cells, effectively killing

malignant cells (15,16). T-MPs, packaged with chemotherapeutic drugs, can

reprogram M2 macrophages toward M1 phenotype (17). Thus, the incorporation of

drugs into T-MPs may yield chemo-immunotherapeutic, dual-functional MPs. In this

study, we provide evidence that methotrexate (MTX)-packaging T-MPs recruited

neutrophils to the pleural cavity where they exerted a therapeutic effect against MPE

in cancer patients.

Materials and Methods

Patient recruitment and study design

The clinical trial was registered in the Chinese Clinical Trial Register (No.

ChiCTR-ICR-15006304), as a multicenter, randomized, double-blind,

parallel-treatment study. The trial was conducted in accordance with the Declaration

of Helsinki, the relevant International Conference on Harmonization Good Clinical

Practice guidelines, and all applicable regulatory and ethical requirements. The




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Clinical Trials Ethics Committee of the Huazhong University of Science and

Technology approved the clinical trial (NO: [2015] 0702-2). Sixty-two patients, who

completed the whole treatment schedule (Supplementary Table S1) were used for

sample collection. The main inclusion criteria were age 18-70 years and diagnosed

with non-squamous non-small cell lung cancer with primary malignant pleural

effusion and pathologically confirmed non-squamous advanced non-small cell lung

cancer with pleural effusion cytology showing tumor cells. Main exclusion criteria

included pregnancy, lactation, allergy to multiple drugs, severe comorbidities,

psychological diseases, severe infection including pleural infection, previously

received chemotherapy or local intrathoracic administration, and participation in other

clinical trials within the past three months. All patients were recruited from the Hubei

Cancer Hospital or Union Hospital affiliated to Tongji Medical College of Huazhong

University of Science and Technology. Patients were received pemetrexed (PEM, 500

mg/m2，Nanjing Pharmaceutical, China) and cisplatin (DDP, 75 mg/m

2，Qilu

Pharmaceutical, China) i.v. administration at day 1 in 21-day cycles according to

NCCN guidelines, and treated with 50 mL methotrexate tumor cell–derived

microparticles (MTX-MPs; 5×107 MPs containing 25 μg MTX preparation discussed

below) or 50 mL saline at day 5, 7, 9, 11, 13, and 15 by intrathoracic (i.t.) injection

every other day (QOD), see schedule in Fig. 1A. MPE was collected at day 5 (before

MTX-MP treatment) and day 7 (after first time of MTX-MP treatment) for cell

population analysis. 10 mL MPE were collected at 2, 4, 8, 12, 24, and 48 hours after

MTX-MP or saline treatment (n=6) for chemokine detection. All MPE samples were

transferred to the lab for analysis no more than 1 hour after drainage. All patients

signed informed consent forms to participate in the study.




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Healthy donor

Following informed consent, peripheral blood samples (10 mL) were collected from

healthy volunteers (n=9). Blood samples were transferred to the lab for analysis no

more than 15 minutes after blood draw.

Animals and cell lines

Female wild-type C57BL/6 and BALB/c mice (6-8 weeks) were purchased from the

Center of Medical Experimental Animals of Hubei Province (Wuhan, China). Female

nude mice (6-8 weeks) were purchased from Beijing Vital River Laboratory Animal

Technology Co. Ltd. C57BL/6 CD45.1 mice (B6.SJL-PtprcaPepcb/BoyJ) were

purchased from Peking University Health Science Center. All animal experiments

were conducted in accordance with a protocol approved by the Animal Care and Use

Committee of Tongji Medical College.

The human tumor cell line A549 was purchased from ATCC. Murine

hepatocarcinoma cell line H22, Lewis murine lung carcinoma (LLC) cell line, and

human umbilical vein endothelial cell (HUVEC) line CRL-1730 were purchased from

the China Center for Type Culture Collection (Beijing, China). A549, H22, and

CRL-1730 cells were cultured in RPIM-1640 medium (Thermo Fisher Scientific) with

10% fetal bovine serum (Gibco), and Lewis cells were cultured in MEM medium

(Thermo Fisher Scientific) with 10% fetal bovine serum (Gibco). Cells were tested for

mycoplasma detection, interspecies cross contamination, and authenticated by

isoenzyme and short tandem repeat (STR) analyses in the Cell Resource Centre of

Peking Union Medical College before the beginning of the study and spontaneously

during the research, and the cells were kept in culture for no more than 10 passages.

All cells were cultured in a humidified atmosphere containing 5% CO2 at 37 °C.




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Preparation of MTX packaging MPs

MTX-MPs were prepared according to our previously published paper (15). In brief,

2×108 A549 cells containing 2 mg/mL MTX (Heng Rui Pharmaceutical, China) were

exposed to ultraviolet irradiation (UVB, 300 J/m2) for one hour in biosafety cabinet

(Thermo Fisher Scientific), and then cultured in RPIM-1640 medium for 18 hours.

The supernatants were collected for centrifugation: first 5 minutes at 500 × g, and

then 2 minutes at 14,000 × g to clear cell debris. The supernatants were then

centrifuged for 60 minutes at 14,000 × g to pellet MTX-MPs. The pellets were

washed and resuspended in saline (0.8~1.5 × 106/mL) for injection. The size, drug

concentration, and sterility of MTX-MPs were analyzed as described previously (18).

For clinical trials, the process was conducted according to the good manufacturing

practice (GMP) quality standard. For each preparation, equal amounts of MTX treated

the same number of tumor cells under the same conditions. This was done to maintain

the stability and reproducibility of MTX packaging MPs.

CT imaging of patient

Computed Tomography (CT) was carried out at day -1 (before PEM and DDP

treatment) and day 43 by DiscoveryTM

CT750 HD (General Electric Company, GE).

MPE volumes were analyzed through three-dimensional imaging by GE Advance

Workstation 4.5.

Tumor models

In the ascites model, H22 cells (3×105 cells were suspended in 200 μL PBS) were

injected into the peritoneal cavity. Three days later, the mice were i.p injected with




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MTX-MPs (1×107) or neutrophils (1×10

6, bone marrow derived), and the control

group were i.p injected with PBS once per day for 5 days. Part of mice (n=5) were

sacrificed for analysis of ascites volume. The remaining mice (n=10) were used for

long-term survival analysis.

For the establishment of the MPE model, mice were anesthetized using isoflurane

before all procedures. LLC (1×105 cells suspended in 50 μL PBS) were injected into

the pleural cavity through the left tenth or eleventh intercrostal space at the

midaxillary line. The animals were observed until complete recovery. The procedure

was not associated with any mortality or morbidity.Three days later, the mice were

intrapleurally injected with MTX-MPs (5×106) or PBS (50 μL) once per day for 5

days. A portion of mice (n=5) were sacrificed for analysis of MPE volume. The

reamining mice (n=10) were used for long-term survival analysis.

For neutrophil depletion, mice were i.p injected with H22 cells, and three days later,

the mice were i.p injected with Ly-6G antibody (100 μg per mouse, twice a week,

#100763, Biolegend). IgG (#400565, Biolegend) was used as control.

BrdU (Bromodeoxyuridine) incorporation

BrdU (1mg/20g) was i.v injected to BALB/c mice, and 24 hours later, MTX-MPs

were i.p injected. 24 hours later, neutrophils were isolated (as described below) from

peritoneal cavity, peripheral blood (retro-orbital collection), and bone marrow

(harvested from tibias and femurs) and fixed with 4% polyformaldehyde (PFA). Cells

were then washed with HBSS, resuspended to 1×106/100 μL, and incubated with

0.5% Triton-X 100 (X100PC, Sigma-Aldrich) for 15 minutes at room temperature.

After washing with HBSS, cells were labeled with APC-conjugated BrdU (#364114,




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Biolegend), then analyzed by flow cytometry (Canto II, BD), and the data were

analyzed by FlowJo (version 7.6.1).

Neutrophil isolation

For isolating human neutrophils from MPE and peripheral blood, the MPE or

peripheral blood was centrifuged, and the sediments were washed in Hank’s balanced

Salt Solution (HBSS) three times. Neutrophils were isolated by Ficoll Hypaque

solution (#LTS1092P, TBD) according to the manufacturers’ instructions. Mouse

neutrophils were isolated from mouse bone marrow by using Percoll (#17-0891-01,

GE) gradient density centrifugation.

Neutrophil negative magnetic sorting

Female wild-type BALB/c mice (6-8 weeks) were sacrificed, and tibias and femurs

were harvested. After lysis of RBCs, bone marrow neutrophils were sorted by

MojoSort™ mouse neutrophil isolation kit (#480058, Biolegend) according to the

manufacturer’s guidelines.

Neutrophil adoptive transfer

2×106 neutrophils were isolated from bone marrow of CD45.1 mice and injected to

CD45.2 mice via tail vein, followed by intrapleural injection of 1×107 MTX-MPs or

PBS. 18 hours later, the CD45.1 neutrophils in pleural cavity were analyzed by flow

cytometry.

Isolation of human monocytes




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Human peripheral blood mononuclear cells (PBMCs) were isolated from human

peripheral blood using Ficoll density gradient separation. Monocytes were purified by

human CD14 Micro-Beads (#130-50-201, MACS), according to the manufacturer’s

guidelines (~98% were monocytes), and then cultured in complete RPMI 1640

medium containing recombinant human M-CSF (20 ng/mL; #300-25, PeproTech) for

the induction of macrophages. Seven days later, human macrophages were harvested

and stimulated with A549 cells derived MTX-MPs at a ratio 1:20 (cell:MP) for 2

hours, the using of cells and supernatant described below.

CD45 – cell isolation

Total cells were collected from MPE, washed with PBS three times, and CD45- cells

were purified using human CD45 Micro-Beads (#130-045-801, MACS) according to

the manufacturer’s guidelines (~98% were CD45-negative). Cytology examination by

H&E staining was performed as described below.

Flow cytometric analysis

For a phenotype analysis of cells, human cells were stained with surface antibodies:

CD3 (#300318), CD4 (#357404), CD8 (#980904), CD11b (#301308), CD15

(#125606), CD66b (#305116), CD68 (#333806), CD56 (#318304), CD45 (#368508),

CD44 (#338806), CD62p (#304904), CD62e (#330012), CD162 (#328806), CD62L

(#304810), EpCAM (#324204), CD54 (#353120), CD31 (#303106). Mouse cells were

stained with antibodies: Ly6G (#127606), NK1.1 (#108707), CD19 (#152403), F4/80

(#123127), CD3 (#100236), CD138 (#142505), PD-1 (#135214), CD45.1 (#110713),

CD45.2 (#109806), Ki67 (#151212), CD11b (#101255), IL12 (#505203) or CD31

(#102510).




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For intracellular cytokine staining, lymphocytes prepared from mouse peritoneal

cavity using CD4/CD8 (TIL) MicroBeads (#130-117-114, MACS) according to

manufacturer’s guidelines. Cells were restimulated with phorbol 12-myristate

13-acetate (PMA; 20 ng/mL, #P1585, Sigma-Aldrich) and ionomycin (1μg/mL,

#I3909, Sigma-Aldrich) in vitro for 4 hours with monensin (1 μg/mL, #1445481,

Sigma-Aldrich), and then stained for surface CD3 and CD8. After surface staining,

cells were treated with Fix/Perm solution (#426803, Biolegend) and stained with

anti-IFNγ (#505808) and IL10 (#112706). All antibodies were purchased from

Biolegend, and flow cytometric was performed with BD Canto II.

Confocal microscopy

Neutrophils (2×106) isolated from MPE were cocultured with 1×10

5 CD44-labeled

A549 (described above) tumor cells on glass-bottom cell culture dishes (#801001,

NEST Biotechnology) for 2 hours, washed with HBSS. 100μL HBSS was added, and

cells were then incubated with Sytox green (5 nM; #425303, Biolegend)) for 30

minutes at temperature. Cells were visualized by two-photon fluorescent microscopy

(ZEISS 780). All groups were assessed with same threshold and parameters.

Neutrophil extracellular trap staining

1×106 neutrophils from healthy mouse bone marrow were cocultured with 1×10

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Lewis derived MTX-MPs for 2 hours in the presence or absence of DPI (10μM,

#D2926, Sigma-Aldrich), and NETs were analyzed using Sytox green (#425303,

Biolegend) staining. For endothelial cell assays, 1×105 HUVEC were labeled with

CD31, and seeded on glass bottom cell culture dishes for 24 hours in RPIM-1640

medium with 10% FBS, and neutrophils from patient MPE after MTX-MP or saline




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treatment were seeded with the HUVEC layer for 2 hours. The cells were then stained

with Sytox green and visualized by two-photon fluorescent microscopy as described

above.

Neutrophil chemotaxis

Neutrophils isolated from patient peripheral blood (5×105) were seeded in the upper

chamber of 24-well plate with a Millicell Hanging Cell Culture Insert. The lower

chamber was MPE double diluted by RPMI 1640 with or without CXCL1

(#MAB275-SP, R&D), CXCL2 (#ab89324, Abcam), GM-CSF (#502319, Biolegend)

or IL1β (#MAB601-SP, R&D) neutralization antibody. After one hour, cells from the

lower chamber were collected and counted by flow cytometry (Accuri C6, BD).

Real-time PCR

Total RNA extraction was prepared with TRIzol reagent (#15596026, Invitrogen), and

cDNA were generated by ReverTra Ace qPCR RT Kit (FSQ-101, Toyobo). Real-time

PCR analysis was performed with 2μg of cDNA as a template. A SYBR Green mix

(Applied Bioscience) and an Agilent Technologies Stratagene M-x3500 Real-time

PCR system were used. Relative quantification was performed using 2-ΔCt

. The

procedure was repeated in at last three biologically independent samples. The primer

sequences are shown as follows: Arginase-1, 5’-TCATCTGGG

TGGATGCTCACAC-3’ (sense) and 5’-GAGAATCCTGGCACATCGGGAA-3’

(antisense); MPO, 5’-GAGCAGGACAAATACCGCACCA-3’ (sense) and 5’-AGAG

AAGCCGTCCTCATACTCC-3’ (antisense); NOS2, 5’-GCTCTACACCTCCAATG

TGACC-3’ (sense) and 5’-CTGCCGAGATTTGAGCCTCATG-3’ (antisense); NOX2,

5’-CTCTGAACTTGGAGACAGGCAAA-3’ (sense) and




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5’-CACAGCGTGATGACAACTCCAG-3’ (antisense); GAPDH,

5’-GTCTCCTCTGACTTCAACAGCG-3’ (sense) and 5’-ACCA

CCCTGTTGCTGTAGCCAA-3’ (antisense). Reduced glyceraldehyde-phosphate

dehydrogenase (GAPDH) was used for normalization.

Cytotoxicity assay

A549 cells were stained with carbox fluorescenceindiacetate succinimidyl ester

(CFSE; Sigma-Aldrich, #21888) and cocultured with neutrophils isolated from patient

peripheral blood or MPE (48 hours after MTX-MPs treatment) at a ratio of 1:20 for

16 hours, with or without N-acetylcysteine (NAC; 20 mM, #A0737, Sigma-Aldrich),

diphenylene iodonium (DPI; 10 μM, #D2926, Sigma-Aldrich), or Cl-amine (10μM,

#S8141, Selleck). CFSE-labeled cells were collected and stained with propidium

iodide (PI) and APC-Annexin V (#640932, Biolegend) to assess apoptosis by flow

cytometry (Canto II, BD) and analyzed by FlowJo 7.6.1.

Detection of hydrogen peroxide and ROS

Hydrogen peroxide (H2O2) in MPE patient was detected by a hydrogen peroxide

detection kit (#s0038, Beyotime biotechnology), and ROS in neutrophils was

measured using the CellROX probe (#C10422, Thermo Fisher Scientific).

Mitochondria-originated ROS was detected with the MitoSOX probe (#M36008,

Thermo Fisher Scientific) according to the manufacturers’ instructions. The cells were

then stained with Hochest (#94403, Sigma-Aldrich) for one hour at room temperature

and visualized by two-photon fluorescent microscopy, as described above.

Evans blue leakage




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1×105 H22 tumor cells were i.p. injected into C57BL/6 mice, and 3 days later, the

mice were i.p injected with 5×106 MTX-MPs, Cl-amidine (1 μg/g), or

MTX-MPs/Cl-amidine (1 μg/g) daily for three days. Normal saline was used as a

control. Twenty-four hours after the last treatment, mice were injected with 1% Evans

blue (5 mg/kg, #E2129, Sigma-Aldrich) via tail vein injection. One hour after the

Evans blue injection, the mice were sacrificed, and 2 mL PBS was used to flush the

peritoneal cavity. The flushed fluid was collected and the supernatant was quantified

spectrophotometrically by microplate reader (Biotek) according to standard curve.

Western blot assay

Protein from MPE patient neutrophils was extracted, the protein concentrations were

determined by BCA kit (#23235, Thermo Fisher Scientific). Then the protein (20 μg)

was run on an SDS-PAGE and transferred to nitrocellulose membranes.

Nitrocellulose membranes were blocked in 5% bovine serum albumin (BSA) and probed

with anti-human arginase 1 (1:1,000 dilution, Cell Signaling Technology, #79404),

anti-human MPO (1:1,000 dilution, Cell Signaling Technology, #79623), anti-human

NOX2 (1:1000 dilution, Cell Signaling Technology, #4312S), iNOS (1:1,000 dilution,

Cell Signaling Technology, #39898) or anti-human β-actin antibodies (1:1,000

dilution, Cell Signaling Technology, #4967) overnight at 4℃, followed by a second

horseradish peroxidase-coupled antibody incubation at room temperature for 1 hour.

Blots were visualized by enhanced chemiluminescence according to the

manufacturer’s instructions (ECL kit, Pierce).

PKH26 labeling



https://www.cst-c.com.cn/products/primary-antibodies/inos-antibody/39898?site-search-type=Products


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MPs were suspended in diluent C solution, and incubated with PKH26 (MINI26,

Sigma-Aldrich) for 30 minutes at room temperature followed by quenching with an

equal volume of FBS (Gibco) and washing twice with PBS.

Cellular uptake assay

To investigate the cellular internalization of MPs, we carried out uptake assays using

cells from MPE patients as recipient cells. Briefly, T cells, B cells, macrophages,

neutrophils, and tumor cells in MPE were cocultured with PKH26-labeled MPs for 30

minutes or 2 hours. Cells were then washed with PBS, and analyzed by flow

cytometry. For in vivo uptake assay, PKH26-labeled MPs were intrapleural or i.p

injected, and 2 hours later, the cells were collected, and uptake of MP was analyzed

by flow cytometry.

Cytokine detection assay

Human macrophages were harvested and stimulated with A549 cell-derived

MTX-MPs at a ratio of 1:20 (cell:MP) for 2 hours. The supernatant wascentrifuged at

14, 000 × g for 30 minutes to remove MTX-MPs, and then 50 μL supernatant was

used for ELISAs.

MPE was collected before and 2, 4, 8, 24, and 48 hours after MTX-MPs or saline

treatment. Supernatants were centrifuged at 3000 × g for 8 minutes, then 50 μL was

used for ELISA analysis. CXCL1 and CXCL2 concentrations were assessed using a

human GRO-alpha/MGSA (CXCL1) Mini ELISA kit (#900-M38, PeproTech) and

human GRO-beta (CXCL2) ELISA kit (#900-M120, PeproTech) according to

manufacturer’s instructions.




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H&E staining

Neutrophils were collected and fixed in 4% paraformaldehyde (#158127, Sigma-

Aldrich) at room temperature for 15 minutes. 1×105 cells were smeared on glass slide

and dried at 50℃. Hematoxylin staining for one minute and eosin staining for 10

seconds was performed, and samples were analyzed by microscope (Leica, DMi8) at

200× and 400× magnification.

Electron microscopy

Neutrophils were collected and fixed in 4% glutaric dialdehyde solution (#G7651,

Sigma-Aldrich) at room temperature for 2 hours. The pellets were then dehydrated in

a graded ethanol series, treated with propylene oxide, and embedded with Spurr’s

epoxy resin. cut sections were stained with uranyl acetate and lead citrate, and then

imaged using JEM1010 electron microscope (JEOL).

For scanning electron microscopy assay, 1×106 neutrophils were collected from MPE,

and fixed with 4% glutaric dialdehyde solution at room temperature for 2 hours. MPE

was then incubated with 1% osmium tetroxide and dehydrated with an ascending

ethanol series. After critical-point drying, the samples were coated with gold and

analyzed by S4800 scanning electron microscopy (Hitachi).

Endothelial permeability detection

1×105 HUVECs were seeded in the upper chamber of 24-well plate with a Millicell

Hanging Cell Culture Insert (pore size 0.4 μm). 5×105 neutrophils isolated from

patient MPE in saline- or MTX-MP-treated groups after were seeded on the HUVEC

layer for 2 hours in the presence or absence of 10 μM Cl-amidine (CLAM). 100 μL

FITC-dextran (1mg/ml，#FD40, Sigma-Aldrich) was then added into the upper




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chamber. One hour later, FITC-dextran in lower chamber was detected by Microplate

System (Biotek).

Immunofluorescence imaging

1×107 MTX-MPs or PBS were i.p injected to MPE mice for 18 hours, Sytox green

(1:1000 diluted by PBS, 200μL) was injected through tail vein for 2 hours. Mice were,

sacrificed, and frozen sections of pleural vessel were stained with PE-anti CD31

(#102407, Biolegend) and analyzed by fluorescence microscope (Leica).

Endothelium-neutrophil contact detection

2×105 HUVEC were seeded in 24-well culture plate (Corning) for 48 hours, then

1×106 neutrophils were collected from MPE and seeded on HUVEC layer for 24

hours in the presence or absence of 1×107 MTX-MPs. Endothelium-neutrophil contact

was observed by phase contrast microscope after washing.

Quantification and statistical analysis

All experiments were performed at least three times. Results are expressed as mean ±

SEM and were analyzed by Student’s t-test. The survival rates were determined by

Kaplan-Meier survival analysis. The p value < 0.05 was considered statistically

significant. The analysis was conducted using the Graph pad 8.0 software. Sample

exclusion was never carried out.

Results

MTX-MPs efficiently treat MPE, which correlates with neutrophil recruitment




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To assess the treatment efficacy of a pleural injection of MTX-MPs, newly diagnosed

NSCLC patients with MPE were recruited and received routine systemic

chemotherapy in advance. A schematic of the clinical treatment procedure was briefly

depicted in Fig.1A. Because drug-free T-MPs can promote M2 tumor-associated

macrophage development (14,19), we used saline instead of drug-free T-MPs as the

control. MTX-MPs were characterized by multiple aspects, including size, plasma

membrane origin, mitochondrial and genomic DNA fragments, drug concentration,

stability and sterility (Supplementary Fig. S1-2), which was consistent with previous

reports (15). The amelioration of the color and turbidity of malignant fluids was

observed following the initial treatment (Supplementary Fig. S3). In line with this

observation, CD45– tumor cells, which were not mesothelial cells (Supplementary Fig.

S4), were efficiently removed from the MPE in the MTX-MPs treated patients,

whereas abundant CD45+ immune cells were present in the fluids (Fig. 1B and C).

However, in the saline-treated patients, both CD45– and CD45

+ cells were not altered,

but CD45+ cell numbers slightly decreased after saline treatment (Fig. 1D and E).

Four weeks after treatment, analysis of pleural effusions showed that MTX-MP

treatment resulted in a significant decrease of MPE compared to the saline treatment

(Fig. 1F). Given the presence of overabundant immune cells in the MPE after

MTX-MP treatment, we asked which immune cell type(s) were mainly present. The

flow cytometric analysis showed that the number of CD4+ and CD8

+ T cells was

increased, but the number of CD56+ NK cells, CD68

+ macrophages, and

CD11b+CD33

+CD14

– myeloid-derived suppressor cells (MDSCs) was not altered (Fig.

1G). Robust CD11b+CD15b

+ neutrophils were collected in the effusions following the

MTX-MP treatment (Fig. 1H and I). Functionally, CD11b+CD15b

+ neutrophils did

not affect T-cell proliferation, whereas CD11b+CD33

+CD14

- MDSCs inhibited T-cell




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proliferation (Supplementary Fig. S5). 48 hours after the initial MTX-MP treatment,

the neutrophil number showed a correlation with the reduction of MPE in the lung

cancer patients (Fig. 1J), Together, these results suggested that MTX-MPs efficiently

treat MPE, which correlated with CD11b+CD15

+ neutrophil recruitment.

Neutrophil recruitment enhances MTX-MP treatment efficiency

The above treatment efficacy was likely due to direct killing of tumor cells, consistent

with our previous report (18). However, recruiting numerous neutrophils to the MPE

raised a possible role of neutrophils against MPE. We thus tested this using H22

hepatocellular carcinoma-induced ascites in BALB/c mice to mimic a malignant

effusion. Mice were i.p. injected with tumor cells, followed by the intraperitoneal

injection of MTX-MPs. We found that a large number of neutrophils were present in

the ascites (Fig. 2A). However, we did not find alteration of T cells, B cells, NK cells,

and macrophages (Supplementary Fig. S6). 20% of macrophages but not T cells, B

cells, NK cells, or neutrophils underwent apoptosis following MTX-MP treatment

(Supplementary Fig. S6), which might be due to a high uptake of MTX-MPs by

macrophages. BrdU was incorporated into proliferating myeloid progenitors in the

bone marrow that subsequently differentiated into neutrophils (Fig. 2B). These

recruited neutrophils showed low Ki67 staining (Fig. 2C), suggesting that

MTX-MP-recruited neutrophils lost their proliferating capability. We also found that

the recruited neutrophils were not MDSCs through H&E staining (Fig. 2D). Also, the

recruited neutrophils did not suppress CD8+

T-cell proliferation and did not affect the

expression of IFNγ，IL10, and PD-1 in the T cells (Supplementary Fig. S7). Using a

Ly6G-depleting antibody to deplete neutrophils, we found that the therapeutic effect

of MTX-MPs was abrogated, as demonstrated by that the increased volume of ascites




20

and decreased survival (Fig. 2E). We isolated neutrophils from healthy mouse bone

marrow and i.p. injected them to the H22 ascites in mice. The ascites volume was

50% reduced and long-term survival was prolonged (Fig. 2F). In line with the Ficoll

isolation method, neutrophils isolated with negative magnetic sorting also showed

antitumor effects (Supplementary Fig. S8). The i.p. injection of MTX-MPs did not

affect the frequency of neutrophils in peripheral blood and bone marrow

(Supplementary Fig. S9). This anti-ascites effect of neutrophils was T

cell-independent, because similar results were also obtained in nude mice (Fig. 2G).

Consistently, MTX-MP treatment also showed antitumor effects in nude mice

(Supplementary Fig. S10). Using a LLC induced MPE, we further demonstrated that

neutrophil depletion impaired the effect of MTX-MPs on the long-term survival of the

mice (Fig. 2H). Intravenous injection of CD45.1+

neutrophils to CD45.2 mice showed

that MTX-MP treatment attracted neutrophils to the pleural effusion (Supplementary

Fig. S11). Together, these results suggested that neutrophil recruitment enhanced

MTX-MP treatment efficacy.

MTX-MPs induce chemokines for neutrophil recruitment

Next, we investigated how neutrophils were attracted to the MPE. We isolated the

cells from the MPE of patients and incubated them with MTX-MPs. We found that

MTX-MPs could be taken up by CD68+ macrophages, CD11b

+CD15

+ neutrophils and

CD45– tumor cells (Fig. 3A). Macrophages showed the strongest fluorescent intensity

(Fig. 3A). These macrophages rather than the neutrophils or tumor cells could take up

the MPs within 30 minutes, as demonstrated by the increased MFI of PKH26 (Fig.

3B). Using a LLC-induced MPE or H22-induced ascites model, a consistent uptake by

macrophages was observed (Supplementary Fig. S12). We then asked if the cells




21

following MTX-MP uptake produced neutrophil chemoattractant(s). A trans-well

assay showed that only the supernatants from a two-hour co-incubation of MTX-MPs

with patient MPE macrophages could recruit (Fig. 3C). A 2-hour incubation was

based on the observation that the number and proportion of neutrophils in MPEs

increased 2 hours after MTX-MP injection (Fig. 3D). GM-CSF and IL-1β are known

chemoattractants of neutrophils (20,21). However, the addition of GM-CSF- or

IL-1β-neutralizing antibody did not affect the above supernatant-mediated attraction

of neutrophils (Fig. 3E). Other CXCL chemokines including CXCLs1-3 and

CXCLs5-8 also attract neutrophils (22, 23). CXCL1 and CXCL2 were upregulated in

MTX-MP-treated macrophages (Fig. 3F) and were detected in the supernatants of the

two-hour co-incubation (Fig. 3G). In line with these results, clinical MPE samples

with a high neutrophil infiltration (n=6) also showed increased CXCL1 and CXCL2

two hours after MTX-MP injection (Fig. 3H and I). CXCL1 and CXCL2 were not

detected in the macrophages in the MPE before MTX-MP injection but were induced

2 hours after MTX-MP treatment (Fig. 3J). CXCL1 or CXCL2 neutralization

abrogated the attraction of neutrophils to the lower chamber in the above trans-well

assay (Fig. 3K). Despite the chemokine-mediated effect, recruiting neutrophils also

need endothelial activation, which provides binding molecules for the rolling,

tethering and arrest of neutrophils to the endothelium (23,24). We found that

MTX-MPs not only upregulated CD62e and CD62p in the human vein endothelial

cells (Fig. 3L), but also upregulated P-selectin glycoprotein ligand 1 (PSGL1) in

neutrophils (Fig. 3M). In line with this result, the promotion of neutrophil-endothelial

cell contact by MTX-MPs was observed in H22 ascites (Supplementary Fig. S13).

Also, MTX-MPs promoted the tight contact of patient MPE-derived neutrophils with

endothelial cells in vitro (Fig. 3N). These results suggest that MTX-MPs treatment not




22

only induced macrophages in the MPE to release CXCL1 and CXCL2 but also

activated endothelial cells, allowing for neutrophil recruitment.

Attracted neutrophils possess antitumor properties in the MPE of patients

Neutrophils can have a N1 or N2 phenotype to exert an antitumor or protumor effect

(25-27). To investigate this, we analyzed the neutrophils in patients’ MPE after

MTX-MP treatment. We found that the forward scatter (FSC) of neutrophils was

reduced after MTX-MP treatment (Fig. 4A) but was not altered in the saline-treated

patients (Fig. 4B). Lower FSC phenotype may reflect an antitumor property of

neutrophils (28). In addition to the low FSC, the activated phenotype

(CD66b+CD62L

low or CD54

+CD62L

low) might also relate to the antitumor property of

neutrophils (29). We found that neutrophils in the MTX-MP-treated MPE upregulated

the expression of CD11b, CD66b and CD54 but downregulated the expression of

CD15 compared to the cells before MTX-MP treatment (Fig. 4C). However,

neutrophils in the saline group did not show the phenotype alteration (Fig. 4D). In line

with the surface markers, oxidative enzymes NADPH oxidase 2 (NOX2),

myeloperoxidase (MPO), and inducible nitric oxide synthase (NOS2) were

upregulated but arginase 1 was downregulated after MTX-MP treatment (Fig. 4E).

Morphologically, a more vacuolated structure and degranulation in MTX-MP-treated

neutrophils were observed under transmission electron microscope (TEM) (Fig. 4F),

and more cytoplasmic azurophilic granules and hypersegmented nuclei were observed

(Fig. 4G and H), which are a signature for lysosome activation (28), further

suggesting a bias toward an antitumor phenotype (24, 29). Using MTX-MPs to treat

neutrophils isolated from the MPE, we found that the surface markers CD11b, CD66b,

and CD54 and the enzymes iNOS, NOX2, and MPO were upregulated but CD15 and




23

arginase 1 were downregulated (Fig. 4I and J). Supernatants from the

MTX-MP-treated A549 cells could stimulate neutrophils to upregulate CD11b,

CD66b, CD54, iNOS, NOX2, and MPO but to downregulate CD15 and arginase 1

(Fig. 4K and L). Together, the injection of MTX-MP not only recruited neutrophils to

the MPE of patients but also conferred antitumor properties.

Attracted neutrophils are cytotoxic to tumor cells by utilizing ROS and NETs

Next, we investigated the antitumor role of attracted neutrophils. We isolated CD45–

tumor cells and neutrophils from the MPE before MTX-MP treatment. We also

isolated neutrophils from the MPE after the treatment in the same patient. Then, we

co-incubated the isolated neutrophils and tumor cells, respectively, and found that

only neutrophils from the treatment setting were able to kill tumor cells (Fig. 5A). We

further found that such neutrophils also eliminated A549 cancer cells in vitro (Fig.

5B). We observed that the tumor cells were surrounded by neutrophils (Fig. 5C),

suggesting that the neutrophil-mediated killing of tumor cells may be in a cell-cell

contact manner (28). Such cell-cell contact was also observed in the MTX-MP-treated

MPE in patients, but not from the saline-treated MPE (Fig. 5D). ROS is a mechanism

neutrophils use to kill tumor cells. In line with the upregulation of iNOS, NOX2, and

MPO, ROS expression in neutrophils was elevated in the MPE of patients treated with

MTX-MPs, but not in the MPE of the saline group (Fig. 5E). We also found that the

H2O2 concentration in the MTX-MP-treated MPE of patients was elevated

(Supplementary Fig. S14). Both the mitochondria and lysosomes are the main

organelles that generate ROS in a cell. However, we only detected minor ROS in

mitochondria of the MTX-MP-treated neutrophils by fluorescent microscopy and flow

cytometry (Fig. 5F), suggesting that the ROS was mainly produced from the




24

lysosomes. Consistently, the use of ROS scavenger N-acetylcysteine (NAC) or NOX2

inhibitor diphenylene iodonium (DPI) could lower the ROS levels (Supplementary

Fig. S15) and also inhibited the killing of tumor cells by neutrophils isolated from

clinical MPE samples (Fig. 5G). We found that lysosomal biogenesis was enhanced in

MTX-MP-treated neutrophils (Supplementary Fig. S16).

Besides ROS, neutrophils also use neutrophil extracellular traps (NETs) to induce

tumor cell NETosis. This NET formation is regulated by ROS, especially from

MPO-processed ROS (30), which, however, relies on the NOX2 system-derived H2O2

(31, 32). Using an H22 ascites model, we stained the MTX-MP-treated neutrophils

with Sytox green, a DNA binding dye, which showed presence of extracellular traps,

and 35.8% of neutrophils released NETs (Supplementary Fig. S17). As expected, the

blockade of ROS generation resulted in the inhibition of the NET formation in

MTX-MP-treated murine neutrophils (Supplementary Fig. S18). We also found NETs

surrounding tumor cells in the MTX-MP-treated MPE of patients but not in saline

group (Fig. 5H), which was also confirmed by scanning electron microscopy (SEM)

(Fig. 5I), suggesting that NETs might be involved in the killing of tumor cells. Indeed,

the inhibition of NETs by Cl-amidine inhibited the killing of A549 tumor cells by the

MTX-MP-recruited neutrophils in vitro (Fig. 5J). Together, these results suggest that

neutrophils may use ROS and NETs to kill tumor cells in the MPE of patients

following MTX-MP treatment.

NETs prevent vascular leakage by sealing off the damaged endothelium

The formation of MPE is due to the damage of pleural capillaries caused by tumor

invasion and soluble factors, which leads to excessive plasma leakage through

hyper-permeable pleural vasculature networks (33). Histological analysis of the




25

mouse MPE model showed that some vascular endothelial cells from the pleural

vasculature underwent apoptosis, and the structure of endothelium was not integrative

(Fig. 6A). Regarding filamentous net-like structure and the stickiness of NETs, we

further hypothesized that besides the killing of tumor cells, NETs had a repairing

effect on the vascular leakage in pleural cavity. To test this hypothesis, we seeded

human endothelial cells in the upper chamber of transwells, and MTX-MP- or

saline-treated neutrophils were placed on an endothelial cell layer. We found that

saline-treated neutrophils could not prevent FITC-Dextran (molecular 40, 000) from

entering the bottom chamber by crossing the endothelial layer (Fig. 6B). However,

MTX-MP-treated neutrophils effectively prevented this process (Fig. 6C), which was

abrogated by Cl-amidine (Fig. 6D). Using fluorescence staining, we found that NETs

intertwined with the membrane of endothelial cells in the MTX-MP-treated neutrophil

group but not in the saline control group (Fig. 6E). In the MPE mouse model, we also

found that Evans blue i.v. injected could be detected in the MPE of the saline-treated

mice, but very little detected in the MTX-MP-treated mice (Fig. 6F). Similarly, the

administration of NET inhibitors increased the vascular permeability and allowed

Evans blue to enter the pleural cavity (Fig. 6G). In line with these results, the frozen

section analysis showed that neutrophils anchored to blood vessels and abundant NET

were located between the endothelial gap (Fig. 6H). We further evaluated the

therapeutic effect of the NETs on MPE by treating the MPE mice with NET inhibitors

instead of neutrophil depletion. Under this condition, we found that the therapeutic

effect of MTX-MPs on pleural effusions was also inhibited (Fig. 6I) and the mice

survival was reduced (fig. 6J). Together, these results suggest that MTX-MP-recruited

neutrophils may release NETs to seal off the damaged endothelium, thus inhibiting

plasma leakage.




26

Discussion

A way to effectively mobilize endogenous neutrophils and promote their intrinsic

antitumor activities as an immunotherapeutic approach is not available. In this study,

we showed that tumor cell-derived MPs packaging MTX mobilize neutrophils to the

MPE of NSCLC patients, where the neutrophils display an antitumor phenotype, not

only exerting a tumor cell-killing effect but also releasing NETs to seal off the

damaged endothelium, leading to the effective treatment of MPE with low adverse

effects. During the treatment process, only some patients developed a fever and did

not exhibit other symptoms, consistent with our previous reports (18).

Many factors contribute to the chemoattraction of neutrophils to inflammatory sites

(20-22,34). In this study, we found that CXCL1 and CXCL2, released by

MTX-MP-stimulated macrophages, played a role in the attraction of neutrophils to the

MPE. Macrophages are commonly polarized to tumor-associated M2 macrophages

with anti-inflammatory properties in MPEs. It is unclear how MTX-MP treatment

switches the M2 phenotype to a pro-inflammatory M1 with the release of CXCL1 and

CXCL2 for neutrophil recruitment. However, our previous studies have shown that

drug-packaging tumor cell-derived MPs are able to reset tumor-associated

macrophages towards the M1 phenotype (17). Notably, drug-free tumor cell-derived

MPs promote M2 tumor-associated macrophage development (14). The key point lies

in the very low amount of chemotherapeutic drug in the MPs. Such little drug may not

exert a cytotoxic effect but act as a lysosomal regulator because MPs enter lysosomes

following their uptake. How drug-packaging MPs regulate macrophage M1 phenotype

is under investigation.




27

In addition to recruitment, the effect of MTX-MPs also confers neutrophils antitumor

properties, which seems not to be mediated by MTX-MP-triggered CXCL1 and

CXCL2 (35). MTX-MPs can exert an indirect effect on neutrophil activation through

the macrophage pathway. Macrophages upon taking up MTX-MPs may release

soluble factors, thus inducing neutrophil polarization towards a N1 phenotype. These

MTX-MP-triggered neutrophils are not terminally differentiated but are immature

cells, allowing BrdU incorporation. Although we propose that these immature

neutrophils are mobilized from the bone marrow by MTX-MPs, an alternative

possibility is that MTX-MPs induce the differentiation of MDSCs to become

neutrophils with the ability to proliferate. In this regard, whether MTX-MPs induce

MDSC differentiation to become neutrophils is worthy of investigation.

We found in this study that MTX-MP-recruited neutrophils released NETs in the

MPE. NETs comprise a web of fibers composed of chromatin and serine proteases

that trap and kill microbes extracellularly (36) and also serve as a physical barrier,

preventing the further spread of pathogens. Besides the antimicrobial properties,

NETs may have pathological effects on organ injury such as acute lung injury (37,38).

Studies have also shown a proposed role for NETs in cancer (39). In preclinical

models, NETs show a tumor-promoting effect, including facilitating metastasis.

However, in this study we found that NETs have a tumor-inhibiting effect. NETs may

mobilize their DNA fiber, histone structure proteins and the released proteases to

exert this antitumor effect, similar to their bactericidal process. In the MPE of NSCLC

patients, NETs were found to surround tumor cells, which may prevent tumor cells

from migrating and benefit the killing of tumor cells by nearby neutrophils.

Besides the inhibitory effect on tumor cells, NETs are also able to interfere with the

vascular leakage, leading to the effective treatment of malignant fluids. The primary




28

cause of MPE lies in the loss of endothelial integrity such as tight junction alteration

and endothelial cell apoptosis. However, MTX-MP treatment may enhance the

interaction of endothelial cells and neutrophils by upregulating the expression of

endothelial E-selectin and P-selectin. Thus, the anchored neutrophils may release and

allow NETs to seal off the damaged endothelium. An important issue about NETs is

the manner of how MTX-MPs trigger neutrophils to release NETs in the MPE of

NSCLC patients. MTX-MPs may stimulate macrophages to produce CXCL1 and

CXCL2. These two chemokines not only attract neutrophils but also facilitate

neutrophils to release NETs.

Malignant pleural effusion poses a significant clinical problem with a poor treatment

efficacy. However, based on the present findings and our previous studies (15,17,18),

we propose that drug-packaging MPs may represent a unique approach, which can

efficiently treat MPEs in NSCLC patients through the processes, as shown in Fig. 7.

All in all, our data showed that drug-packaging MPs, by virtue of their ability to

recruit neutrophils, lead to a robust antitumor innate immunity. These findings

provide insights into the versatile antitumor function of neutrophils, especially in the

control of malignant fluids.

Acknowledgments

This work was supported by National Natural Science Foundation of China

(81788101 ， 81530080, 91742112), CAMS Initiative for Innovative Medicine

(2016-I2M-1-007), Young Elite Scientists Sponsorship Program by CAST (YESS20

160166), Program for HUST Academic Frontier Youth Team (2018QYTD01),

National Major Science and Technology Projects of China (2019ZX09301001).




29

Author Contributions

B.H. conceived the project. K.T., P.X., J.W., H.Z., D.W., L.Z., J.C., K.W., J. L., H.F.,

Y.Z., J.X. and Y.L. performed the experiments. B.H., P.X., K.T., H.Z. and J.M.

developed methodology. B.H., K.T. and P.X. performed data analysis, provided

administrative, technical, or material support. B.H. and K.T. wrote the manuscript with

input from all authors.

Declaration of Interests

U.S. Patent No. 9,351,931 and China Patent No. ZL201110241369.8 held by Hubei

Soundny Bio-Tech Co. Ltd. cover the pharmaceutical preparation for “Tumor

cell-derived microparticles packaging of chemotherapeutic drugs”. All authors declare

that they have no competing interests.

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Figure legends

Figure 1. MTX-MPs treat MPE and is coordinated with neutrophil recruitment.

(A) Graphical outline of the treatment protocol. MPE patients received intravenous

injection (i.v.) of PEM (500 mg/m2) and DDP (75 mg/m

2) on day 1 (blue

arrow) ,followed by intrapleural treatment with MTX-MPs or saline on days 5, 7, 9,

11, 13 and 15 (red arrow). ; CT images were performed at day –1 and 43 (purple

arrow). (B-E) Cells were collected from the MPE of (B,C) MTX-MP-treated or (D,E)

saline-treated patients on day 5 and 7 and analyzed by flow cytometry (n=16/group).

(F) Left top: CT image thorax of control and treated patients with MPE on day -1 and

day 43 (see purple arrows in A). The white arrows indicate pleural effusion. Left

bottom: Pleural effusion assessment of the same patients by CT analysis. Right: MPE

volume ratio (volume on day 43/volume on day -1) was analyzed (n=32/group). (G-I)

MPE samples (n=16) were analyzed by flow cytometry on day 5 and 7 to assess (G,H)

numbers of CD3+CD4

+ T cells, CD3

+CD8

+ T cells, CD16

+CD56

+ NK cells,

CD11b+CD68

+ macrophages, CD11b

+CD14

–CD33

+ MDSCs, CD11b

+CD15

+CD49d

–

neutrophils and (I) the population of neutrophils. (J) Correlation analysis between

neutrophil number on day 7 and decreased MPE volume (volume on day 43 minus

volume on day -1). For all graphs, data present mean±SEM for at least three

independent experiment. *P<0.05, **P<0.01, ***P<0.001, ns: not significant by

paired Student’s t-test (C, E, G, H, and I) or unpaired Student’s t-test (F).

Figure 2. Neutrophil recruitment is required for MTX-MP treatment efficiency.

(A) H22 tumor cells were i.p. injected into BALB/c mice (n=6), followed by the

intraperitoneal injection of MTX-MPs (MP) or saline (Control: Ctrl) one day later.




36

After 18 hours, peritoneal CD11b+Ly6G

+ cells were analyzed. (B) BrdU (1 mg/20g)

was i.v. injected into mice (n=6) for 24 hours, followed by saline or MTX-MPs i.p.

injection. Peritoneal neutrophils were analyzed by flow cytometry. Bone marrow

(BM)-derived neutrophils were used as a positive control. (C,D) H22 cells were i.p

injected into mice (n=6), followed by MTX-MPs or saline. 18 hours later, the

neutrophils were analyzed by (C) flow cytometry for Ki67 expression and (D) H&E

stain to assess neutrophils (Neu) and MDSCs. Scale bar 10 μm. (E) H22 cells were i.p

injected into mice, followed by i.p. injected MTX-MPs once every day for 6 times.

Ly6G-depleting antibody was used to deplete neutrophils. IgG acted as a control.

Ascites volume (left, collected on day 15 after tumor inoculation, n=5) and long-time

survival (right, n=10/group) are shown. **P<0.01 versus IgG group, #P<0.05 versus

MP/Ly6G antibody group. (F, G) H22 cells were i.p. injected into (F) wild-type or (G)

nude mice for 3 days. The mice (n=15) were treated by i.p. infusion of BM-derived

neutrophils daily. Some mice (n=5) were used to analyze CD45– tumor cell number

and the ascites volume. The remaining mice (n=10) were used for long-time survival.

(H) LLC cells were i.p. injected into C57BL/6 mice, followed by i.p. injection

MTX-MPs daily. Ly6G-depleting antibody were used, and long-time survival was

analyzed (n=10/group). **P<0.01 versus IgG group, ##P<0.01 versus MP/Ly6G

antibody group. **P<0.01, ***P<0.001, ****P<0.0001 by unpaired Student’s t-test

(C, F, G), one-way ANOVA (E). The data represent mean±SEM for three

independent experiments. The survival rates were determined by Kaplan-Meier

survival analysis.

Figure 3. MTX-MPs induce chemokines for neutrophil recruitment




37

(A,B) Cells from MPE of patients were incubated with PKH26-labeled MTX-MPs

(cell:MP=1:10) for (A) 2 hours or (B) 30 minutes. MFIs for tumor cells (Tumor),

macrophages (Mac), neutrophils (Neu), T and B cells were analyzed by flow

cytometry. (C) Macrophages, neutrophils and tumor cells from (A,B) were incubated

with MTX-MPs for 2 hours, and cultured supernatants were used for a trans-well

assay. Neutrophil migration to the lower chamber was counted. (D) MPE samples of

patients (n=6) were collected at different time points following the first MTX-MP

injection. CD11b+CD15

+ neutrophils in MPE were analyzed. (E) MPE supernatant

from MTX-MP-treated patients were loaded to the lower chamber with or without

GM-CSF- or IL1β-neutralizing antibody. Neutrophils were in the upper chamber. IgG

was used as control. (F, G) PBMC-derived macrophages were incubated with

MTX-MPs for 2 hours. CXCL1 and CXCL2 was analyzed by (F) real-time PCR

(n=9), and (G) culture supernatants were analyzed by ELISA (n=3). (H,I) MPE was

collected from patients at different times after the first MTX-MP injection. (H)

CXCL1 and (I) CXCL2 was measured by ELISA (n=6). (J) Macrophages isolated

from untreated MPE of patients were incubated with MTX-MPs. Four hours later,

CXCL1 and CXCL2 in the supernatant were analyzed (n=6). (K) MPE supernatants

from patients were collected 48 hours after MTX-MP treatment for the trans-well

experiments (Lower chamber: supernatant with or without anti-CXCL1 or CXCL2.

Upper chamber: peripheral blood derived neutrophils). (L) HUVECs (CRL-1730)

were cocultured with MTX-MPs for 4 hours. CD62p and CD62e were analyzed. (M)

Neutrophils were isolated from the MPE of patients untreated or 24 hours after

treatment. CD162 was analyzed (n=3). . (N) Neutrophils from MPE of patients

without MTX-MPs treatment were added onto the HUVEC layer for 24 hours with or

without MTX-MPs. Endothelium-neutrophil contact was assessed. Scale bar: 20 μm.




38

**P<0.01, ***P<0.001, ****P<0.0001 by unpaired Student’s t-test (A, B, F, G, J, L,

M and N), or one-way ANOVA (E and K), the data present mean±SEM for three

independent experiments.

Figure 4. Attracted neutrophils possess antitumor properties in the MPE of

patients. (A-D) Neutrophils from MPE of patients (n=16) before and 48 hours after

treatment with (A,C) MTX-MPs or (B,D) saline were analyzed by flow cytometry. (E)

MPO, iNOS (NOS2), NOX2 (CYBB), and ARG1 in neutrophils (n=6) was analyzed by

real-time PCR. (F-H) The above neutrophils were analyzed by (F) TEM and (G,H)

H&E staining. Black arrows: vacuolated structure; red arrows: lobulated nucleus.

Scale bar: 10 μm. (I, J) Neutrophils from the untreated MPE patients (n=3) were

incubated with MTX-MPs for 12 hours. CD11b, CD66b, CD54, and CD15 were

analyzed by (I) flow cytometry and (J) real-time PCR. (K, L) Neutrophils from

untreated MPE patients (n=4) were cocultured with the supernatants of

MTX-MP-treated A549 cells for 12 hours. CD11b, CD66b, CD54, and CD15 were

analyzed by (K) flow cytometry and (L) western blot. *P<0.05, **P<0.01,

***P<0.001, ****P<0.0001 by unpaired Student’s t-test (A-D and H-K), or paired

student’s t-test (E). The data present mean±SEM for three independent experiments.

Figure 5. Recruited neutrophils are cytotoxic to tumor cells by utilizing ROS and

NETs. (A,B) CFSE-labeled (A) tumor cells from untreated MPE patients (n=3) or (B)

A549 cells were incubated with neutrophils from MPE patients for 16 hours. CFSE+

cells were analyzed by flow cytometry. Blank: no neutrophils; Ctrl: neutrophils from

saline-treated MPE; MP: neutrophils from MTX-MP-treated MPE; PBN: neutrophils

from peripheral blood of untreated patients. (C) A549 cells were incubated with




39

neutrophils from the treated MPE. Cells were observed under microscope. Blank:

without neutrophils. Scale bar: 20 μm. (D) Fresh MPE from MTX-MP-treated

patients was stained with anti-EpCAM and anti-CD15 for fluorescent microscopic

analysis. Scale bar: 20 μm. (E,F) Neutrophils from MPE patients (control: no

treatment) were collected. MFIs for (E) total ROS and (F) mitochondrial ROS were

analyzed by microscopy and flow cytometry. Scale bars: 10 μm. (G) Neutrophils from

MPE patients (n=3) were incubated with A549 cells with or without of NAC (20 mM)

or DPI (10 μM). Apoptosis of tumor cells was detected by flow cytometry. (H)

Neutrophils from treated MPE patients were incubated with CD44-stained A549 cells

in serum-free medium. NETs stained by Sytox green were analyzed using confocal

microscopy. Scale bar: 30 μm. (I) Neutrophils in MPE were isolated from patients

with or without MTX-MP treatment and observed by SEM. Scale bar: 10 μm. (J)

Neutrophils from treated patients’ MPE (n=3) were incubated with CFSE-labeled

A549 cells with or without Cl-amidine (CLAM). A549 cell apoptosis was analyzed by

flow cytometry. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by unpaired

Student’s t-test (E and F) or one-way ANOVA (A, B, G and J). The data present

mean±SEM for three independent experiments.

Figure 6. NETs prevent vascular leakage by sealing off damaged endothelium. (A)

LLCs were i.p. injected into mice (n=6) for 10 days. The pleural vasculature was

analyzed by H&E staining (normal: healthy mice). Scale bar: 300 μm. (B-D)

HUVECs were seeded on the upper chamber. Neutrophils isolated from MPE (n=5)

from (B) saline- or (C) MTX-MP-treated patients were seeded on the HUVEC layer

for 2 hours (D) with or without Cl-amidine. Dextran was then added into the upper

chamber for colorimetric analysis. (E) Neutrophils from MTX-MP- or saline-treated




40

MPE patients (n=6) were added onto the HUVEC layer (anti-CD31) for 2 hours.

NETs were assessed with Sytox green by confocal microscopy. 3D and 2D images are

shown on the left and right, respectively. Scale bar: 20 μm. (F,G) LLCs were

intrapleurally injected into mice (n=4), followed by intrapleural injection of

MTX-MPs (F) without or with Cl-amidine (G). Evans blue dye was i.v. injected 1

hour before mice were scarified. The extravascular Evans blue in pleural cavity was

quantified spectrophotometrically. (H) LLCs were inoculated in the pleural cavity for

10 days, followed by intrapleural injection of MTX-MPs. 18 hours later, Sytox green

was i.v. injected and the mice were scarified 2 hours later. Vessel sections were

stained with PE-labeled CD31 and assessed for NETs (n=5). Scale bar: 100 μm. (I, J)

LLCs were intrapleurally injected into C57BL/6 mice for 3 days, followed by the

treatment of saline or MTX-MPs, Cl-amidine, or MTX-MPs plus Cl-amidine. On day

12, (I) MPE volume was measured (n=5) and (J) the long-term survival was analyzed

(n=10/group), ***P<0.001 versus control group, ###P<0.001 versus MP/Cl-amidine

group, Kaplan-Meier survival analysis was used (J). *P<0.05, **P<0.01,

***P<0.001 by unpaired Student’s t-test (B-D and F) or one-way ANOVA (G and I),

the data present mean±SEM for three independent experiments (B-I).

Figure 7. Schematic diagram for MTX-MP treatment of MPE.

(1) Following the injection into the MPE via pleural catheter, drug-MPs are taken up

by and kill tumor cells. (2) Drug-MPs are taken up by and induce macrophages to

release CXCL1 and CXCL2, thus recruiting neutrophils to the MPE. (3) Neutrophils

are activated in drug-MP-remodeled malignant environments. (4) Activated

neutrophils launch the second wave of killing tumor cells. (5) Neutrophils release




41

NETs to seal off the damaged endothelium, facilitating the regression of the MPE.

Red arrows: increased expression. A: steps 1-3; B: steps 4 and 5.




Published OnlineFirst July 13, 2020.Cancer Immunol Res Pingwei Xu, Ke Tang, Jingwei Ma, et al. response targeting malignant pleural effusionsChemotherapeutic tumor microparticles elicit a neutrophil

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