Author’s Proof

Carefully read the entire proof and mark all corrections in the appropriate place, using the Adobe Reader commenting tools (Adobe Help). Do not forget to reply to the queries.

We do not accept corrections in the form of edited manuscripts.

In order to ensure the timely publication of your article, please submit the corrections within 48 hours.

If you have any questions, please contact health.production.office@frontiersin.org

Author Queries Form

Query No. Details required Author’s Response

Q1 The citation and surnames of all of the authors have been highlighted. Please check all of the names carefully and indicate if any are incorrect. Please note that this may affect the indexing of your article in repositories such as PubMed.

Q2 Confirm that the email address in your correspondence section is accurate.

Q3 Verify that all the equations and special characters are displayed correctly.

Q4 Ensure, if it applies to your study, the ethics statement is included in the article.

Q5 Ensure that all the figures, tables and captions are correct.

Q6 If you decide to use previously published, copyrighted figures in your article, please keep in mind that it is your responsibility, as the author, to obtain the appropriate permissions and licenses and to follow any citation instructions requested by third-party rights holders. If obtaining the reproduction rights involves the payment of a fee, these charges are to be paid by the authors.

Q7 Please check if the abbreviation “Lamp2b” is appropriate for the expansion “lysosome-associated membrane protein 2.”

Q8 Please confirm if the details in Ref. (2, 14, 26, 72, 81) are fine and also provide the DOI number.

Q9 Please provide the page range for Ref. (2, 19, 38, 43, 81).

Q10 Ensure to add all grant numbers and funding information, as after publication this is no longer possible.

051

052

053

054

055

056

057

058

059

060

061

062

063

064

065

066

067

068

069

070

071

072

073

074

075

076

077

078

079

080

081

082

083

084

085

086

087

088

089

090

091

092

093

094

095

096

097

098

099

100

001

002

003

004

005

006

007

008

009

010

011

012

013

014

015

016

017

018

019

020

021

022

023

024

025

026

027

028

029

030

031

032

033

034

035

036

037

038

039

040

041

042

043

044

045

046

047

048

049

050

July 2018 | Volume 9 | Article 15341

Mini Reviewpublished: xx July 2018

doi: 10.3389/fimmu.2018.01534

Frontiers in Immunology | www.frontiersin.org

Edited by: Martin Herrmann,

Universitätsklinikum Erlangen, Germany

Reviewed by: Silvano Sozzani,

University of Brescia, Italy Dalil Hannani,

UMR5525 Techniques de l’Ingénierie Médicale et de la

Complexité Informatique, Mathématiques et Applications, Grenoble (TIMC-IMAG), France

Giovanna Schiavoni, Istituto Superiore di Sanità, Italy

*Correspondence:Valeriya V. Solovyeva

solovyovavv@gmail.com

Specialty section: This article was submitted to

Molecular Innate Immunity, a section of the journal

Frontiers in Immunology

Received: 23 March 2018Accepted: 21 June 2018Published: xx July 2018

Citation: Chulpanova DS, Kitaeva KV,

James V, Rizvanov AA and Solovyeva VV (2018) Therapeutic

Prospects of Extracellular Vesicles in Cancer Treatment.

Front. Immunol. 9:1534. doi: 10.3389/fimmu.2018.01534

Therapeutic Prospects of extracellular vesicles in Cancer TreatmentDaria S. Chulpanova1, Kristina V. Kitaeva1, Victoria James 2, Albert A. Rizvanov1 and Valeriya V. Solovyeva1*

1 Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan, Russia, 2 School of Veterinary Medicine and Science, University of Nottingham, Nottingham, United Kingdom

Extracellular vesicles (EVs) are released by all cells within the tumor microenvironment, such as endothelial cells, tumor-associated fibroblasts, pericytes, and immune system cells. The EVs carry the cargo of parental cells formed of proteins and nucleic acids, which can convey cell-to-cell communication influencing the maintenance and spread of the malignant neoplasm, for example, promoting angiogenesis, tumor cell invasion, and immune escape. However, EVs can also suppress tumor progression, either by the direct influence of the protein and nucleic acid cargo of the EVs or via antigen presentation to immune cells as tumor-derived EVs carry on their surface some of the same antigens as the donor cells. Moreover, dendritic cell-derived EVs carry major histocompatibility complex class I and class II/peptide complexes and are able to prime other immune system cell types and activate an antitumor immune response. Given the relative longevity of vesicles within the circulation and their ability to cross blood–brain barriers, modification of these unique organelles offers the potential to create new bio-logical-tools for cancer therapy. This review examines how modification of the EV cargo has the potential to target specific tumor mechanisms responsible for tumor formation and progression to develop new therapeutic strategies and to increase the efficacy of antitumor therapies.

Keywords: extracellular vesicles, tumor microenvironment, tumor cells, immune cells, stromal cells, vaccination, cancer therapy

inTRODUCTiOn

Extracellular vesicles (EVs) are of particular interest due to their ability to mediate intercellular communication, influencing multiple cellular processes. EVs can be categorized based upon their biogenesis and divided into exosomes, microvesicles (MVs), and apoptotic bodies (ABs) (1, 2). Exosomes are small vesicles 40–100  nm in diameter, formed as part of the endocytic pathway. Exosomes carry the donor cell cargo, represented by various proteins and nucleic acids [DNA, mRNA, miRNA, and other non-coding RNAs (ncRNAs)] (Figure 1C) (3, 4). Exosomes are stable in biological fluids and small enough to pass through the blood–brain barrier (5). MVs have a diameter of 100–1,000 nm and are released by directly budding from the plasma membrane (6). MVs also carry cargos of proteins and nucleic acids, although their functional roles in cell-to-cell communica-tion remains less well studied than the exosome population (7). In contrast to exosomes and MVs, which are formed continuously by cells, ABs are formed as part of the fragmentation process of cells undergoing apoptosis, the process of programmed cell death (1) (Figure 1A).

Q1

Q2

Q3

Q4

158

159

160

161

162

163

164

165

166

167

168

169

170

171

172

173

174

175

176

177

178

179

180

181

182

183

184

185

186

187

188

189

190

191

192

193

194

195

196

197

198

199

200

201

202

203

204

205

206

207

208

209

210

211

212

213

214

101

102

103

104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

125

126

127

128

129

130

131

132

133

134

135

136

137

138

139

140

141

142

143

144

145

146

147

148

149

150

151

152

153

154

155

156

157

FigURe 1 | Extracellular vesicle (EV) properties and application in antitumor treatment. (A) EVs can be classified based upon their biogenesis and are divided into exosomes, microvesicles (MVs), and apoptotic bodies (ABs). Exosomes are formed as part of the endocytic pathway by inward budding of endosomal membranes, resulting in accumulation of early endosomes and formation of large multivesicular bodies (MVBs) which release their contents (exosomes) into the extracellular space. MVs are released by directly budding from the plasma membrane. ABs are formed as part of the fragmentation process of cells undergoing apoptosis. (B) EVs derived from native or primed/genetically modified cells can be used in antitumor treatment. (C) Different types of EVs contain various proteins, lipids, and nucleic acids and have specific membrane markers. Exosomes have tetraspanin (such as TSPAN29 or TSPAN30), endosomal sorting complex required for transport (ESCRT) components, milk fat globule-EGF factor 8 protein (MFGE8), programmed cell death 6 interacting protein (PDCD6IP), tumor susceptibility gene 101 protein (TSG101), and flotillin molecules on their surface. Exosome content include mRNAs, microRNAs, and other non-coding RNAs (ncRNAs), cytoplasmic and membrane proteins including receptors and major histocompatibility complex (MHC) molecules. MVs carry integrins, selectins, and CD40 ligand on their surface, and also contain mRNAs, microRNAs, ncRNAs, cytoplasmic and membrane proteins. ABs have extensive amounts of phosphatidylserine and contain various parts of the apoptotic cell such as proteins, lipids, nuclear fragments, and cell organelles. Cargo and biogenesis of EVs have been comprehensively discussed elsewhere (8, 9).

2

Chulpanova et al. EVs in Cancer Treatment

Frontiers in Immunology | www.frontiersin.org July 2018 | Volume 9 | Article 1534

The tumor microenvironment is often a very complex and dynamic niche containing not only neoplastic cells but also a multitude of non-malignant stromal cells such as endothelial cells, tumor-associated fibroblasts, pericytes, and immune cells (10). In addition to stromal cells, the extracellular matrix and surrounding tumor adipose tissue also make an important contribution to tumor progression as they contain adipocytes and progenitor cells [preadipocytes and mesenchymal stem cells (MSCs)] (10, 11), as well as a variety of soluble cytokines, growth factors, and metabolites produced the stromal cells within the tumor microenvironment (10, 12). As EVs are believed to medi-ate cell-to-cell communication in the tumor microenvironment

and induce phenotypic modification in recipient cells, there is a growing interest in the potential role of EVs as key mediators of tumor progression and the spread of malignant neoplasm (13–16). Since EV functions are related to the donor cell type and the imparted cargo of proteins and nucleic acids, EVs of different origins exhibit different features. However, as these have been comprehensively discussed elsewhere (17), this review focuses on the use and efficacy of EVs as antitumor therapies. For instance, as a result of the unique properties of MSCs, the EVs produced by stem cells retain the ability to migrate toward tumor niches (18), they also posses the same low immuno genicity of the donor MSCs (19). Therefore, the use of MSC-derived EVs as non-cell

Q5

Q6

272

273

274

275

276

277

278

279

280

281

282

283

284

285

286

287

288

289

290

291

292

293

294

295

296

297

298

299

300

301

302

303

304

305

306

307

308

309

310

311

312

313

314

315

316

317

318

319

320

321

322

323

324

325

326

327

328

215

216

217

218

219

220

221

222

223

224

225

226

227

228

229

230

231

232

233

234

235

236

237

238

239

240

241

242

243

244

245

246

247

248

249

250

251

252

253

254

255

256

257

258

259

260

261

262

263

264

265

266

267

268

269

270

271

3

Chulpanova et al. EVs in Cancer Treatment

Frontiers in Immunology | www.frontiersin.org July 2018 | Volume 9 | Article 1534

structures, in place of MSCs themselves, allows the avoidance of the risk of unlimited cell growth, undesirable transformation, and potential tumor formation (20). The ability to act as multi-signal messengers makes EVs a prospective new class of therapeutic agents to modulate the processes occurring in the tumor micro-environment (21) (Figure 1B).

TUMOR CeLL-DeRiveD evs

Intercellular EV-mediated signaling by tumor cells has been linked with maintain angiogenesis, invasion, immune escape (22) and to develop an aggressive phenotype and chemo- and radio-therapy resistance (16, 23–25). The extent of the contribution of EVs in tumor maintenance has been demonstrated through the study of EV inhibition, following which malignancy is suppressed and cancer cells show enhanced sensitivity to proton-pump inhibitor (omeprazole) and cisplatin (26, 27). As EV traffic is regulated by an acidic microenvironment, a common feature of all solid tumors, altering intracellular pH is an effective means of modulating exosome release. Changes in intracellular pH alters the lipid composition of the cells membrane and subsequently modulates both exosome release and fusion/uptake (28). In addi-tion, the lower extracellular pH can promote tumor resistance to cytotoxic drugs through neutralization of those antitumor drugs that are weak bases or isolating drugs in acidic vesicles and/or eliminating them through an exocytotic pathway (29).

Extracellular vesicles may also promote tumor progression through the transfer of their specific cargos, for example, dur-ing the formation of a pre-metastatic niche (PMN), where the transfer of EV-cargos to stromal cells, induce molecular and cellular changes that promote PMN development (30, 31). For example, the tumor exosomal transport of miR-494 and miR-542p to stromal cells and lung fibroblasts leads to cadherin-17 downregulation and matrix metalloproteinase upregulation (30), while proangiogenic RNAs contained within MVs trigger angiogenesis to promote PMN formation (32).

The ability of tumor cell-derived EVs to fuse with recipient cells through endocytosis and release their cargo into the recipient cell cytoplasm makes EVs a promising biological vector for targeted delivery of various antitumor agents (33). This is exemplified by the use of EVs derived from LNCaP and PC-3 prostate cancer cell lines modified to transport paclitaxel (PTX) into recipient cells through the endocytic pathway, significantly increasing PTX cytotoxicity in  vitro (33). Furthermore, U-87 MG (brain neuronal glioblastoma–astrocytoma) derived EVs primed with doxorubicin (DOX) or PTX significantly decreased the viability of recipient U-87 MG cells by 70 and 50%, respectively, at the highest tested concentration of exosomes (200 µg/mL) in vitro (34).

Tumor-derived EVs can be used for therapeutic drug delivery to reduce systemic toxicity by targeting the tumor microenviron-ment. It was shown that in vitro and in vivo, doxorubicin-loaded exosomes (exoDOX) derived from MDA-MB-231 (breast adenocarcinoma) and HCT-116 (colorectal carcinoma) cell lines did not reduce DOX efficacy. Simultaneously, exoDOX treated nude mice did not show the cardiotoxicity observed in their free-DOX-treated counterparts. Mass spectrometry confirmed that DOX accumulation in the heart was reduced by approximately

40% when DOX was delivered via exosomes (exoDOX) (35). The reduced cardiotoxicity achieved when delivering DOX via modi-fied exosomes would allow for a higher concentration of exoDOX to be used, thus offering the potential to increase DOX efficacy. Similar findings have also been reported for in  vivo models of breast (MDA-MB-231) and ovarian (STOSE) cancer (36).

Tumor cell-derived EVs carry on their surface the same antigens as the cell that produced them (the donor cell), such as HER2/neu, melan-A, Silv, carcinoembryonic antigen (CEA), mesothelin, and others (37). Thus, they can act to prime immune cells by antigen presentation. The delivery of dendritic cells (DCs) in vitro primed with exosomes isolated from the mesothelioma cell line AB1 within a BALB/c mouse mesothelioma model, resulted in increased mean and overall survival times in vivo (38). Similarly, DCs primed with exosomes isolated from rat glioblas-toma cells, induced a strong antitumor response and significantly increased median survival times in glioblastoma-bearing rats when used in combination with α-galactosylceramide (39).

The efficacy of priming immune cells can be improved by com-bining their use with immune cell stimulating drugs. For instance, exosomes derived from the pancreatic cancer cell line UNKC6141 were co-delivered with DCs (DCs/Exo) to UNKC16141 xenograft mice. Tumor onset was delayed in these animals and subsequently a significant increase in survival was observed. When the same assay was repeated, but with the inclusion of all-transretinoic acid (ATRA) alongside the delivery of DCs/Exo, increased lym-phocyte proliferation within lymph nodes was reported which coincided with increased cytotoxic T-cell activity in comparison with untreated or DCs/Exo only treated animals. However, the inclusion of ATRA had no further effect on prolonging survival and only modest changes in metastasis to distant organs were observed. The combination of DCs/Exo with sunitinib in these animal models also led to an increase in cytotoxic activity which in these assays did lead to significantly prolonged survival times in DCs/Exo/sunitinib compared to animals treated only with free sunitinib therapy. Similar increases in survival time and a reduction in metastatic spread was also observed when DCs/Exo use was combined with gemcitabine treatment (40).

To increase the therapeutic potential and immunogenicity of EV-based tumor vaccines, tumor cells producing the EVs can be modified to express specific cytokine/chemokine genes that have an immunomodulating effect. Dai et  al. reported that exosomes derived from LS-174T cells genetically modified to express IL-18 CEA (Exo/IL-18), had a more pronounced effect on specific antitumor immunity when compared with exosomes from native LS-174T  cells. Exo/IL-18 promoted proliferation of peripheral blood mononuclear cells and induced cytokine secretion by T-lymphocytes and DC in vitro, as well as inducing the phenotypic and functional maturation of DCs (41). Similar results were obtained by Yang et al. using in vivo experiments, whereby exosomes were derived from IL-2-modified ovalbumin (OVA)-expressing EL-4 lymphoma cells (Exo/IL-2). Vaccination of C57BL/C mice with Exo/IL-2 more effectively inhibited tumor growth (42).

The modification of tumor cells through the aberrant expres-sion of tumor suppressor genes, apoptosis inductors, and ncRNAs has also been shown to impart a potential therapeutic benefit to the resulting EVs. YUSAC 2 melanoma cells were engineered

386

387

388

389

390

391

392

393

394

395

396

397

398

399

400

401

402

403

404

405

406

407

408

409

410

411

412

413

414

415

416

417

418

419

420

421

422

423

424

425

426

427

428

429

430

431

432

433

434

435

436

437

438

439

440

441

442

329

330

331

332

333

334

335

336

337

338

339

340

341

342

343

344

345

346

347

348

349

350

351

352

353

354

355

356

357

358

359

360

361

362

363

364

365

366

367

368

369

370

371

372

373

374

375

376

377

378

379

380

381

382

383

384

385

4

Chulpanova et al. EVs in Cancer Treatment

Frontiers in Immunology | www.frontiersin.org July 2018 | Volume 9 | Article 1534

to overexpress a dominant-negative mutant form of Survivin (Survivin-T34A). Exosomes derived from Survivin-T34A- modified YUSAC 2 cells, in combination with gemcitabine, sig-nificantly increased apoptosis in pancreatic adenocarcinoma MIA PaCa-2 cells in comparison with gemcitabine alone (43). Rivoltini et  al. showed that exosomes derived from K562 leukemia cells modified with TNF-related apoptosis-inducing ligand (TRAIL) [TRAIL(+) exosomes], induced apoptosis in TRAIL-death recep-tor (DR)5(+) SUDHL4 lymphoma and INT12 melanoma cells in vitro. In in vivo experiments of TRAIL(+) exosomes demon-strated homing of the exosomes to the tumor sites and significant suppression of tumor growth by 58% in SUDHL4-B-cell lym-phoma bearing mice (44). Li et al. investigated exosomes derived from glioblastoma multiforme (GBM) cells with overexpression of the tumor suppressor gene LRRC4 (Exo/LRRC4). Exo/LRRC4 induced significant chemotaxis and expansion of CD4+CCR4+ T cells, inhibited the proportion of Ti-Treg cells, and promoted Ti-Teff cell expansion through cytokines release in vitro (45).

The Rab GTPases control many stages of membrane traffick-ing, including the formation and release of vesicles. Ostrowski et al. identified Rab GTPases Rab2b, Rab9a, Rab5a, Rab27a, and Rab27b that promote exosome secretion in HeLa cells (46), indicating the possibility of manipulating the secretion of Rab proteins to control exosome production. Exosomes, derived from Rab27a-overexpressing A549 cells (exo/Rab27a), exhibited the ability to regulate major histocompatibility complex (MHC) class II molecules and co-stimulatory molecules CD80 and CD86 on DCs. Furthermore, DCs primed with exosomes derived from Rab27-overexpressing A549 cells significantly increased CD4+ T  cell proliferation in  vitro. In vivo immunization with exo/Rab27a inhibited tumor growth in a tumor mouse model (47).

At present ncRNAs are actively being studied as potential anti-tumor agents. However, when developing miRNA-based therapies there are problems with specific targeting of tumor cells and target cells within the tumor microenvironment. Tumor-derived EVs can be used for delivering a variety of potentially therapeutic ncRNAs, for instance miR-134 (48), miR-29a, and miR-29c microRNAs (49), as well as short interfering RNAs (siRNAs) (50) (Table 1).

iMMUne CeLL-DeRiveD evs

Exosomes from immature dendritic cells (imDCs) can be used to deliver chemotherapeutic agents such as DOX. For instance, imDCs were modified to express lysosome-associated membrane protein 2 (Lamp2b) fused to the αv-integrin-specific iRGD pep-tide. It was shown that modified imDC-derived exosomes (Exo/iRGD) loaded with DOX, effectively targeted and delivered DOX to αv-integrin+ MDA-MB-231 breast cancer cells in vitro. Exo/iRGD intravenous injection in BALB/c mice led to inhibition of breast tumor cell growth without any apparent toxic effects (52).

A new approach for cancer immunotherapy is the combina-tion of exosomes and the invariant NKT immune cell ligand α-galactosylceramide (αGC) (53). Loaded with αGC and OVA-model antigen exosomes induced potent NK and γδ T-cell innate immune responses in  vitro and in  vivo. In an OVA-expressing mouse model of melanoma treatment of tumor-bearing mice with αGC/OVA-loaded exosomes decreased tumor growth, increased

antigen-specific CD8+ T-cell tumor infiltration, and increased median survival, relative to control mice immunized with solu-ble αGC  +  OVA alone (53). Similarly, exosomes derived from α-fetoprotein (AFP)-expressing DCs (DEXAFP) intravenously injected into hepatocarcinoma-bearing C57BL6 mice prolonged survival to 57 days in 100% of DEXAFP-treated mice (55).

Without modification, DC-derived exosomes alone carry MHC class I and class II/peptide complexes capable of leading to the priming of CD8+ and CD4+ T cells, respectively, and sub-sequent T cell-dependent tumor rejection (13, 54). DC-derived exosomes have also been reported to trigger NK cell proliferation and activation in vitro and in patients, by trans-presentation of IL-15 by IL-15Rα. This mechanism of action was shown to signifi-cantly reduce the number of lung metastases in vivo. Combination of DC-derived exosomes with IL-15Rα and rhIL-15 molecules led to NK cell proliferation and activation and significantly enhanced IFNγ secretion by NK cells in vitro (54).

Phase I clinical trials have demonstrated the safety of using DC-derived exosomes in patients with metastatic melanoma (69) and lung cancer (70). Phase II trials in non-small cell carcinoma patients using modified IFN-γ expressing DCs to produce exosomes have reported an increase in NKp30-dependent NK cell functions, and 32% of participants experienced stabilization for more than 4 months (56).

In addition to DCs, macrophages have also been studied as a source of EVs of potential therapeutic benefit. Derived from RAW 264.7 macrophages, vesicles loaded with PTX (exoPTX) were reported to significantly increase drug cytotoxicity (more than 50 times) in multidrug resistance (MDR) MDCKMDR1, MDCKwt, and 3LL-M27 cells in vitro. Furthermore, when deliv-ered into the airway of mice modeling Lewis lung carcinoma pulmonary metastases, exoPTX were found to have a potent anticancer effect (57). For PTX targeted delivery macrophages can be modified with aminoethylanisamide-polyethylene glycol (AA-PEG) a vector moiety to target the σ-receptor which is overexpressed by lung cancer cells (58). Jang et al. developed a bioinspired exosome-mimetic nanovesicles that can be modi-fied to deliver DOX, gemcitabine, or carboplatin to the tumor tissue after systemic administration. Chemotherapeutic-loaded nanovesicles, derived from monocytes or macrophages, induced TNF-α-stimulated endothelial cell (HUVECs) death in a dose-dependent manner in vitro. DOX-loaded nanovesicles increased apoptosis and reduced the number of proliferating cells in CT26 colorectal cancer murine models (59) (Table 1).

MSC-DeRiveD evs

Extracellular vesicles released from MSCs have been reported to exhibit variable effects on tumor growth, indicating the influence of EVs is dependent on cargo and the donor cell type (71, 72). Delivered by MSC-derived exosomes molecules of different types of RNA can induce adipogenesis, angiogenesis, apoptosis, and proteolysis in recipient cells (15). Exosomes from gastric cancer-derived MSCs were found to deliver miR-221 to HGC-27 gastric cancer cells, promoting their proliferation and migration in vitro (73). Other biomolecules carried by exosomes such as onco-genic proteins, cytokines, adhesion molecules, and anti-apoptotic

Q7

500

501

502

503

504

505

506

507

508

509

510

511

512

513

514

515

516

517

518

519

520

521

522

523

524

525

526

527

528

529

530

531

532

533

534

535

536

537

538

539

540

541

542

543

544

545

546

547

548

549

550

551

552

553

554

555

556

443

444

445

446

447

448

449

450

451

452

453

454

455

456

457

458

459

460

461

462

463

464

465

466

467

468

469

470

471

472

473

474

475

476

477

478

479

480

481

482

483

484

485

486

487

488

489

490

491

492

493

494

495

496

497

498

499 TAB

Le 1

| Th

e us

e of

ext

race

llula

r ve

sicl

es (E

Vs)

with

or

with

out m

odifi

ed c

argo

for

antit

umor

ther

apy.

vesi

cle

sour

ceve

sicl

e ty

pe

Pur

ifica

tio

n st

rate

gy

Car

go

Mec

hani

sm o

f ac

tio

nM

od

elR

efer

ence

Can

cer

cells

Glio

blas

tom

a–as

troc

ytom

a U

-87

MG

cel

ls

Exo

som

esE

xoso

me

isol

atio

n re

agen

t (In

vitr

ogen

)D

OX

or P

TXC

ell v

iabi

lity

decr

ease

In v

itro

U-8

7 M

G c

ell c

ultu

re(3

4)

LNC

aP a

nd P

C-3

pr

osta

te c

ance

r ce

lls

Exo

som

es

and

mic

rove

sicl

es

Diff

eren

tial c

entr

ifuga

tion

PTX

PTX

cyt

otox

ic e

ffect

incr

ease

In v

itro

PC

-3 a

nd L

NC

aP c

ell c

ultu

re(3

3)

MD

A-M

B-2

31 a

nd

HC

T-11

6 ce

ll lin

esE

xoso

mes

Exo

Qui

ck-T

C™

sol

utio

n (S

yste

m

Bio

Sci

ence

s)D

OX

Car

dio

toxi

city

dec

reas

e, D

OX

effic

acy

incr

ease

MD

A-M

B-2

31 c

ell m

ice

mod

el in

 viv

o(3

5)

MD

A-M

B-2

31 a

nd

STO

SE

cel

l lin

esE

xoso

mes

AB

 cel

l cul

ture

-nan

oves

icle

s so

lutio

n (A

B A

NA

LITI

CA

)D

OX

Bre

ast M

DA

-MB

-231

and

ova

rian

STO

SE

mou

se tu

mor

s in

 viv

o(3

6)

Ora

l can

cer

cells

Exo

som

esU

ltrafi

ltrat

ion

and

affin

ity

chro

mat

ogra

phy

Tum

or-a

ssoc

iate

d an

tigen

sN

K c

ell p

rolif

erat

ion

and

NK

 cel

l cyt

otox

icity

incr

ease

In v

itro

NK

 cel

l cul

ture

(51)

Mou

se m

alig

nant

m

esot

helio

ma

(MM

) AB

1 ce

lls

Exo

som

esS

tepw

ise

ultr

acen

trifu

gatio

nTu

mor

-ass

ocia

ted

antig

ens

Exo

som

e-lo

aded

den

driti

c ce

ll (D

C) i

ncre

ased

med

ian

and

over

all s

urvi

val

AB

1 tu

mor

BA

LB/c

mic

e m

odel

in v

ivo

(38)

Rat

glio

blas

tom

aE

xoso

mes

Exo

RN

easy

Ser

um/P

lasm

a M

axi

Kit

(Qia

gen)

Tum

or-a

ssoc

iate

d an

tigen

s +

 α-g

alac

tosy

lcer

amid

eE

xoso

mes

pul

sed

DC

s in

crea

sed

med

ian

surv

ival

tim

eG

liobl

asto

ma-

bear

ing

rat m

odel

in v

ivo

(39)

UN

KC

6141

(p

ancr

eatic

can

cer)

cells

Exo

som

esS

ucro

se g

radi

ents

ul

trac

entr

ifuga

tion

Tum

or-a

ssoc

iate

d an

tigen

sE

xoso

me-

load

ed D

Cs

dela

yed

tum

or o

nset

and

in

crea

sed

surv

ival

tim

eU

NK

C61

41-b

earin

g m

ice

(40)

DC

s/E

xo +

 all-

tran

sret

inoi

c ac

id in

crea

sed

prol

ifera

tion

of ly

mph

nod

e ce

lls a

nd c

ytot

oxic

T c

ell a

ctiv

ity

DC

s/E

xo a

nd s

uniti

nib

prol

onge

d su

rviv

al ti

me

DC

s/E

xo +

 gem

cita

bine

pro

long

ed s

urvi

val t

ime

Car

cino

embr

yoni

c an

tigen

(CE

A)-

expr

essi

ng

LS-1

74T

tum

or

cells

Exo

som

esS

ucro

se g

radi

ents

ul

trac

entr

ifuga

tion

IL-1

8M

atur

atio

n of

DC

s an

d in

duct

ion

of C

EA

-spe

cific

C

D8+

CTL

DC

s an

d C

TL c

ells

in v

itro

(41)

OVA

-exp

ress

ing

EL-

4 ly

mph

oma

cells

Exo

som

esS

ucro

se g

radi

ents

ul

trac

entr

ifuga

tion

IL-2

Imm

une

resp

onse

indu

ctio

n an

d tu

mor

gro

wth

in

hibi

tion

C57

BL/

C m

ice

mod

el in

 viv

o(4

2)

YU

SA

C 2

m

elan

oma

cells

Exo

som

esS

ucro

se g

radi

ents

ul

trac

entr

ifuga

tion

Sur

vivi

n-T3

4A (S

urvi

vin

bloc

king

pr

otei

n)C

aspa

se a

ctiv

atio

n an

d ap

opto

sis

indu

ctio

nP

ancr

eatic

can

cer

cells

in v

itro

(43)

K56

2 le

ukem

ia

cells

Exo

som

esD

iffer

entia

l cen

trifu

gatio

nTN

F-re

late

d ap

opto

sis-

indu

cing

lig

and

(TR

AIL

)TR

AIL

-rel

ated

apo

ptos

is in

duct

ion

SU

DH

L4 ly

mph

oma

and

INT1

2 m

elan

oma

cells

in v

itro

(44)

Tum

or g

row

th in

hibi

tion

SU

DH

L4-b

earin

g m

ice

A54

9 ce

llsE

xoso

mes

Diff

eren

tial c

entr

ifuga

tion

Rab

27a

Mat

urat

ion

of m

ajor

his

toco

mpa

tibilit

y co

mpl

ex

(MH

C) c

lass

II m

olec

ules

, CD

80 a

nd C

D86

. In

hibi

tion

of tu

mor

gro

wth

DC

s in

 vitr

o, B

ALB

/c m

ice

mod

el in

 viv

o(4

7)

(Con

tinue

d)

5

Chulpanova et al. EVs in Cancer Treatment

Frontiers in Immunology | www.frontiersin.org July 2018 | Volume 9 | Article 1534

614

615

616

617

618

619

620

621

622

623

624

625

626

627

628

629

630

631

632

633

634

635

636

637

638

639

640

641

642

643

644

645

646

647

648

649

650

651

652

653

654

655

656

657

658

659

660

661

662

663

664

665

666

667

668

669

670

557

558

559

560

561

562

563

564

565

566

567

568

569

570

571

572

573

574

575

576

577

578

579

580

581

582

583

584

585

586

587

588

589

590

591

592

593

594

595

596

597

598

599

600

601

602

603

604

605

606

607

608

609

610

611

612

613 vesi

cle

sour

ceve

sicl

e ty

pe

Pur

ifica

tio

n st

rate

gy

Car

go

Mec

hani

sm o

f ac

tio

nM

od

elR

efer

ence

Glio

blas

tom

a m

ultif

orm

e (G

BM

) ce

lls

Exo

som

esD

iffer

entia

l cen

trifu

gatio

nLR

RC

4C

hem

otax

is a

nd e

xpan

sion

of C

D4+

CC

R4+

T c

ells

GB

M c

ells

in v

itro

(45)

Hs5

78T

and

Hs5

78Ts

(i)8

cells

Exo

som

esFi

ltrat

ion

and

ultr

acen

trifu

gatio

nm

iR-1

34C

ellu

lar

mig

ratio

n an

d in

vasi

on re

duct

ion,

dru

gs

sens

itivi

ty e

nhan

cem

ent

Hs5

78Ts

(i)8

cells

in v

itro

(48)

SG

C79

01 c

ells

Mic

rove

sicl

esD

iffer

entia

l cen

trifu

gatio

nm

iR-2

9a a

nd m

iR-2

9cA

ngio

gene

sis

and

tum

or g

row

th s

uppr

essi

onIm

plan

ted

with

SG

C79

01 c

ells

BA

LB/c

m

ice

in v

ivo

(49)

HeL

a an

d H

T108

0 ce

llsE

xoso

mes

Diff

eren

tial c

entr

ifuga

tions

and

m

icro

-filtr

atio

nS

hort

inte

rferin

g R

NA

s (s

iRN

As)

ag

ains

t RA

D51

and

RA

D52

Acc

umul

atio

n of

the

cells

in S

and

G2/

M p

hase

s

of c

ell c

ycle

and

reci

pien

t cel

l dea

th in

duct

ion

HeL

a ce

lls in

 vitr

o(5

0)

imm

une

cells

DC

sE

xoso

mes

Suc

rose

gra

dien

ts

ultr

acen

trifu

gatio

nLa

mp2

b +

 iRG

D +

 DO

XTu

mor

gro

wth

inhi

bitio

nM

DA

-MB

-231

inje

cted

BA

LB/c

nud

e m

ice

mod

el in

 viv

o(5

2)

DC

sE

xoso

mes

Diff

eren

tial c

entr

ifuga

tion

αGC

 + O

VAN

K a

nd γ

δ T-

cell

imm

une

resp

onse

s in

duct

ion

Inva

riant

NK

T ce

lls in

 vitr

o(5

3)

Tum

or g

row

th d

ecre

ase

B16

/OVA

mel

anom

a tu

mor

mod

el

in v

ivo

DC

sE

xoso

mes

Ultr

afiltr

atio

n/di

afiltr

atio

n an

d su

cros

e gr

adie

nts

ultr

acen

trifu

gatio

n

MH

C c

lass

I an

d cl

ass

IIN

K c

ell p

rolif

erat

ion

and

activ

atio

n, IF

Nγ

se

cret

ion

enha

ncem

ent

NK

 cel

ls in

 vitr

o(5

4)

MH

C c

lass

I an

d cl

ass

IIN

K c

ell p

rolif

erat

ion

and

activ

atio

n by

tran

s-pr

esen

tatio

n of

IL-1

5 by

IL-1

5Rα,

num

ber

of

met

asta

ses

redu

ctio

n

Mou

se m

odel

in v

ivo

DC

sE

xoso

mes

Diff

eren

tial c

entr

ifuga

tion

AFP

Sur

viva

l rat

e pr

olon

gatio

nTu

mor

-bea

ring

C57

BL6

mic

e m

odel

in

 viv

o(5

5)

DC

sE

xoso

mes

Ultr

afiltr

atio

n/di

afiltr

atio

n an

d su

cros

e gr

adie

nts

ultr

acen

trifu

gatio

n

IFN

-γN

Kp3

0-de

pend

ent N

K c

ell f

unct

ion

en

hanc

emen

tA

dvan

ced

non-

smal

l cel

l lun

g ca

ncer

pa

tient

s(5

6)

RA

W 2

64.7

m

acro

phag

esE

xoso

mes

Exo

Qui

ck-T

C™

sol

utio

n (S

yste

m

Bio

Sci

ence

s)P

TXD

rug

cyto

toxi

city

incr

ease

, inh

ibiti

on o

f m

etas

tase

s gr

owth

Res

ista

nt m

ultid

rug

resi

stan

ce c

ell

cultu

re in

 vitr

o, L

ewis

lung

car

cino

ma

mou

se m

odel

in v

ivo

(57)

AA

-PE

G +

 PTX

Sup

pres

sion

of m

etas

tase

s gr

owth

and

sur

viva

l tim

e in

crea

seIn

viv

o C

57B

L/6

mic

e lu

ng c

ance

r m

odel

(58)

Mon

ocyt

es o

r m

acro

phag

esE

xoso

me-

mim

etic

na

nove

sicl

es

Iodi

xano

l gra

dien

ts

ultr

acen

trifu

gatio

nD

OX

Apo

ptos

is in

crea

se a

nd n

umbe

r of

pro

lifer

atin

g

cells

redu

ctio

nIn

viv

o m

odel

of m

ouse

CT2

6 co

lore

ctal

ca

ncer

(59)

Mes

ench

ymal

ste

m c

ells

(MS

Cs)

MS

Cs

Exo

som

esD

iffer

entia

l cen

trifu

gatio

nA

nti-m

iR-9

Tem

ozol

omid

e se

nsiti

vity

incr

ease

Tem

ozol

omid

e-re

sist

ant G

BM

 cel

l cu

lture

in v

itro

(60)

TAB

Le 1

| C

ontin

ued

(Con

tinue

d)

6

Chulpanova et al. EVs in Cancer Treatment

Frontiers in Immunology | www.frontiersin.org July 2018 | Volume 9 | Article 1534

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

763

764

765

766

767

768

769

770

771

772

773

774

775

776

777

778

779

780

781

782

783

784

671

672

673

674

675

676

677

678

679

680

681

682

683

684

685

686

687

688

689

690

691

692

693

694

695

696

697

698

699

700

701

702

703

704

705

706

707

708

709

710

711

712

713

714

715

716

717

718

719

720

721

722

723

724

725

726

727 vesi

cle

sour

ceve

sicl

e ty

pe

Pur

ifica

tio

n st

rate

gy

Car

go

Mec

hani

sm o

f ac

tio

nM

od

elR

efer

ence

MS

Cs

Exo

som

esE

xoQ

uick

-TC

™ s

olut

ion

(Sys

tem

B

ioS

cien

ces)

miR

-146

bTu

mor

gro

wth

redu

ctio

nIn

viv

o ra

t mod

el o

f prim

ary

brai

n tu

mor

(61)

MS

Cs

Exo

som

esS

ucro

se g

radi

ents

ul

trac

entr

ifuga

tion

miR

-124

aV

iabi

lity

and

clon

ogen

icity

redu

ctio

nG

liom

a st

em c

ell l

ines

in v

itro

(62)

Pro

long

ed s

urvi

val r

ate

In v

ivo

mod

el o

f mou

se G

SC

267

glio

ma

Bon

e m

arro

w

MS

Cs

(BM

-MS

Cs)

Exo

som

esE

xoQ

uick

-TC

™ s

olut

ion

(Sys

tem

B

ioS

cien

ces)

miR

-340

Tum

or a

ngio

gene

sis

inhi

bitio

n vi

a th

e H

GF/

c-

ME

T si

gnal

ing

path

way

End

othe

lial c

ell c

ultu

re in

 vitr

o(6

3)

MS

Cs

Exo

som

esD

iffer

entia

l cen

trifu

gatio

nP

olo-

like

kina

se 1

(PLK

-1) s

iRN

AC

ance

r ce

ll pr

olife

ratio

n re

duct

ion

by P

LK-1

ge

ne s

ilenc

ing

Bla

dder

can

cer

cells

in v

itro

(64)

MS

Cs

Exo

som

esE

xoQ

uick

-TC

™ s

olut

ion

(Sys

tem

B

ioS

cien

ces)

miR

-122

Ant

itum

or e

ffica

cy o

f sor

afen

ib in

crea

seH

epat

ocel

lula

r ca

rcin

oma

mod

el in

 viv

o(6

5)

BM

-MS

Cs

Mic

rove

sicl

esD

iffer

entia

l cen

trifu

gatio

nP

TXTu

mor

gro

wth

inhi

bitio

nH

uman

pan

crea

tic a

deno

carc

inom

a C

FPA

C-1

cel

ls in

 vitr

o(6

6)

MS

Cs

Mic

rove

sicl

esD

iffer

entia

l cen

trifu

gatio

nP

TX o

r G

CB

Tum

or p

rolif

erat

ion

inhi

bitio

nP

ancr

eatic

can

cer

cells

in v

itro

(67)

MS

Cs

Exo

som

esS

eque

ntia

l ultr

acen

trifu

gatio

n co

mbi

ned

with

0.2

2 µm

ul

trafi

ltrat

ion

TRA

ILA

popt

osis

indu

ctio

nM

231

brea

st c

ance

r ce

lls a

nd o

ther

ca

ncer

cel

l lin

es in

 vitr

o(6

8)

TAB

Le 1

| C

ontin

ued

7

Chulpanova et al. EVs in Cancer Treatment

Frontiers in Immunology | www.frontiersin.org July 2018 | Volume 9 | Article 1534

proteins can also promote tumor progression (74–76), as well as increase tumor resistance to chemotherapy drugs (77).

Exosomes from bone marrow MSCs (BM-MSCs) can transfer miRNAs from the BM, particularly miR-23b, which promote dor-mancy in bone marrow-metastatic human breast cancer through the suppression of a target gene, MARCKS in vivo (78). In support of this, Lee et al. showed that MSC-derived exosomes can sup-press human breast cancer angiogenesis by downregulating the expression of VEGF in tumor cells in vitro and in vivo (79).

In addition to the endogenous effects of MSC-EVs, MSC-derived MVs can be used as delivery vehicles for a variety of potential therapeutic agents, in particular ncRNAs. For example, injection of exosomes derived from miR-146-expressing MSCs into xenograft gliomas in primary brain tumor rat models cause a significant reduction in tumor growth (61). Treatment with MSC-derived exosomes containing miR-124a reduce the viability and clonogenicity of glioma stem cell lines in vitro and increase the survival rate in glioma mouse models up to 50% by silencing FOXA2 (62), while the loading of MSC exosomes with miR-143 acts to significantly reduce the migration of 143B osteosarcoma cells (80). Transfection of bone marrow stromal cells with miR-340 generates exosomes capable of inhibiting tumor angiogenesis via the HGF/c-MET signaling pathway in endothelial cells (63). MSC-derived EVs can also be used to alter the chemosensitivity of tumor cells. Delivery of anti-miR-9 to temozolomide-resistant GBM cells increases cell sensitivity to this drug (60). The sensitiv-ity of hepatocellular carcinoma cells to chemotherapeutic agents (5-fluorouracil and sorafenib) can similarly be altered through the use of miR-122 loaded MSC exosomes in  vivo (65). MSC-derived MVs can also be loaded with various siRNAs that target key genes driving tumorigenesis, for example, MSC exosomes carrying siRNAs against polo-like kinase 1 significantly reduce bladder cancer cell proliferation in vitro (64).

In addition to biomolecules, MSC-derived vesicles can be loaded with chemotherapeutic drugs. BM-MSC-derived MVs primed with high-dose PTX inhibited cell growth by 50% in human CFPAC-1 pancreatic adenocarcinoma cells in vitro (66). This finding was supported by the recent studies of Cocce et al., which showed antitumor activity of MSCs MVs loaded with PTX or gemcitabine (GCB) on pancreatic cancer cells in vitro (67).

Recent studies have also highlighted the potential to deliver TRAIL by MSC-EVs (MSCT). MSCT-EVs induced apoptosis in 11 cancer cell lines in a dose-dependent manner but showed no cytotoxicity in human bronchial epithelial cells in  vitro. Interestingly TRAIL-primed EVs that contain 3.88  ng TRAIL/mL induced significantly more apoptosis in M231 breast cancer cells compared with 100 ng/mL of recombinant TRAIL. TRAIL delivery by MSC-EVs induced significant apoptosis in TRAIL resistant A549 lung adenocarcinoma cells in a dose-dependent manner in vitro (68) (Table 1).

COnCLUSiOn

Extracellular vesicles, which include groups of differing origins such as exosomes and MVs, are released by all cells within the tumor microenvironment during normal cellular activity. EVs carry variable cargos that reflect the composition of the donor

842

843

844

845

846

847

848

849

850

851

852

853

854

855

856

857

858

859

860

861

862

863

864

865

866

867

868

869

870

871

872

873

874

875

876

877

878

879

880

881

882

883

884

885

886

887

888

889

890

891

892

893

894

895

896

897

898

785

786

787

788

789

790

791

792

793

794

795

796

797

798

799

800

801

802

803

804

805

806

807

808

809

810

811

812

813

814

815

816

817

818

819

820

821

822

823

824

825

826

827

828

829

830

831

832

833

834

835

836

837

838

839

840

841

8

Chulpanova et al. EVs in Cancer Treatment

Frontiers in Immunology | www.frontiersin.org July 2018 | Volume 9 | Article 1534

cells, these cargos can be transferred to neighboring cells and thus affect the processes occurring in those recipient cells and subsequently the tumor microenvironment as a whole. In addi-tion to their endogenous ability to influence tumor progression, the ability to modify the EV content makes them a promising tool for cancer therapy. Surface antigens of tumor cell-derived vesicles can be used for immune cell priming. They can also be modified with various agents to directly affect tumor cells or modulate anti-tumor immunity. Genetic modifications can also be performed on MSC-derived vesicles, the main advantage of which is targeted cargo delivery to the tumor microenvironment. From priming the immune response to delivering ncRNAs and antitumor drugs, EVs provide a unique biological means of targeting tumors and their microenvironments, minimizing cytotoxic effects, and increasing the efficacy of treatments at lower drug doses (Table 1). However, despite these many advantages, EVs can have variable effects on tumor progression and the tumor microenvironment dependent upon their protein and nucleic acid cargos. One of the limitations of EV usage is the heterogeneity of the isolated population, since the size of exosomes and MVs overlap, and as yet it is not clear which population carries the greatest potential to elicit functional changes. Furthermore, the inconsistency of the

EV cargo adds an additional caveat to their study and therapeutic use (81). In the case of drug loading, disadvantages include a low transfection efficiency, and, in the case of cell manipulation, there is a high dependence on cell division (82). Therefore, progressing their use as therapeutic tools requires full characterization of such disadvantages and limitations before the promise of MVs in clinical practice is achieved.

AUTHOR COnTRiBUTiOnS

DC wrote the manuscript and made the table. KK created the figure. VJ edited the manuscript. DC, VS, and AR conceived the idea and edited the manuscript and table.

FUnDing

The work is performed according to the Russian Government Program of Competitive Growth of Kazan Federal University. VS was supported by the Russian Foundation for Basic Research (RFBR) grant 16-34-60201. AR was supported by state assign-ment 20.5175.2017/6.7 of the Ministry of Education and Science of Russian Federation and RFBR grant 18-04-01133.

ReFeRenCeS

1. Elmore S. Apoptosis: a review of programmed cell death. Toxicol Pathol (2007) 35(4):495–516. doi:10.1080/01926230701320337

2. Gould SJ, Raposo G. As we wait: coping with an imperfect nomenclature for extracellular vesicles. J Extracell Vesicles (2013) 2.

3. Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ, Lotvall JO. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol (2007) 9(6):654–9. doi:10.1038/ncb1596

4. Wu K, Sharma S, Venkat S, Liu K, Zhou X, Watabe K. Non-coding RNAs in cancer brain metastasis. Front Biosci (Schol Ed) (2016) 8:187–202. doi:10.2741/s457

5. Li X, Tsibouklis J, Weng T, Zhang B, Yin G, Feng G, et al. Nano carriers for drug transport across the blood-brain barrier. J Drug Target (2017) 25(1): 17–28. doi:10.1080/1061186X.2016.1184272

6. Lee Y, El Andaloussi S, Wood MJ. Exosomes and microvesicles: extracellular vesicles for genetic information transfer and gene therapy. Hum Mol Genet (2012) 21(R1):R125–34. doi:10.1093/hmg/dds317

7. Wu K, Xing F, Wu SY, Watabe K. Extracellular vesicles as emerging targets in cancer: Recent development from bench to bedside. Biochim Biophys Acta (2017) 1868(2):538–63. doi:10.1016/j.bbcan.2017.10.001

8. Raposo G, Stoorvogel W. Extracellular vesicles: exosomes, microvesicles, and friends. J Cell Biol (2013) 200(4):373–83. doi:10.1083/jcb.201211138

9. Kalra H, Drummen GP, Mathivanan S. Focus on extracellular vesicles: intro-ducing the next small big thing. Int J Mol Sci (2016) 17(2):170. doi:10.3390/ijms17020170

10. Wang M, Zhao J, Zhang L, Wei F, Lian Y, Wu Y, et al. Role of tumor microenvi-ronment in tumorigenesis. J Cancer (2017) 8(5):761–73. doi:10.7150/jca.17648

11. Jordan BF, Gourgue F, Cani PD. Adipose tissue metabolism and cancer progression: novel insights from gut microbiota? Curr Pathobiol Rep (2017) 5(4):315–22. doi:10.1007/s40139-017-0154-6

12. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell (2011) 144(5):646–74. doi:10.1016/j.cell.2011.02.013

13. Andre F, Chaput N, Schartz NE, Flament C, Aubert N, Bernard J, et  al. Exosomes as potent cell-free peptide-based vaccine. I. Dendritic cell-derived exosomes transfer functional MHC class I/peptide complexes to dendritic cells. J Immunol (2004) 172(4):2126–36. doi:10.4049/jimmunol.172.4.2126

14. Camussi G, Deregibus MC, Bruno S, Grange C, Fonsato V, Tetta C. Exosome/microvesicle-mediated epigenetic reprogramming of cells. Am J Cancer Res (2011) 1(1):98–110.

15. Eirin A, Riester SM, Zhu XY, Tang H, Evans JM, O’Brien D, et al. MicroRNA and mRNA cargo of extracellular vesicles from porcine adipose tissue- derived mesenchymal stem cells. Gene (2014) 551(1):55–64. doi:10.1016/j.gene.2014.08.041

16. Li XJ, Ren ZJ, Tang JH, Yu Q. Exosomal microRNA MiR-1246 promotes cell proliferation, invasion and drug resistance by targeting CCNG2 in breast cancer. Cell Physiol Biochem (2017) 44(5):1741–8. doi:10.1159/000485780

17. Yanez-Mo M, Siljander PR, Andreu Z, Zavec AB, Borras FE, Buzas EI, et al. Biological properties of extracellular vesicles and their physiological functions. J Extracell Vesicles (2015) 4:27066. doi:10.3402/jev.v4.27066

18. Kalimuthu S, Gangadaran P, Li XJ, Oh JM, Lee HW, Jeong SY, et al. In vivo therapeutic potential of mesenchymal stem cell-derived extracellular vesicles with optical imaging reporter in tumor mice model. Sci Rep (2016) 6:30418. doi:10.1038/srep30418

19. Chulpanova DS, Kitaeva KV, Tazetdinova LG, James V, Rizvanov AA, Solovyeva VV. Application of mesenchymal stem cells for therapeutic agent delivery in anti-tumor treatment. Front Pharmacol (2018) 9. doi:10.3389/fphar.2018.00259

20. Gomzikova MO, Rizvanov AA. Current trends in regenerative medicine: from cell to cell-free therapy. BioNanoSci (2017) 7:240–5. doi:10.1007/s12668- 016-0348-0

21. Han L, Xu J, Xu Q, Zhang B, Lam EW, Sun Y. Extracellular vesicles in the tumor microenvironment: therapeutic resistance, clinical biomarkers, and targeting strategies. Med Res Rev (2017) 37(6):1318–49. doi:10.1002/med.21453

22. Aubertin K, Silva AK, Luciani N, Espinosa A, Djemat A, Charue D, et  al. Massive release of extracellular vesicles from cancer cells after photody-namic treatment or chemotherapy. Sci Rep (2016) 6:35376. doi:10.1038/ srep35376

23. Feng Q, Zhang C, Lum D, Druso JE, Blank B, Wilson KF, et al. A class of extra-cellular vesicles from breast cancer cells activates VEGF receptors and tumour angiogenesis. Nat Commun (2017) 8:14450. doi:10.1038/ncomms14450

24. Zhang S, Zhang Y, Qu J, Che X, Fan Y, Hou K, et al. Exosomes promote cetux-imab resistance via the PTEN/Akt pathway in colon cancer cells. Braz J Med Biol Res (2017) 51(1):e6472. doi:10.1590/1414-431X20176472

25. Khan FM, Saleh E, Alawadhi H, Harati R, Zimmermann WH, El-Awady R. Inhibition of exosome release by ketotifen enhances sensitivity of cancer cells to doxorubicin. Cancer Biol Ther (2018) 19(1):25–33. doi:10.1080/15384047.2017.1394544

26. Guan XW, Zhao F, Wang JY, Wang HY, Ge SH, Wang X, et al. Tumor micro-environment interruption: a novel anti-cancer mechanism of proton-pump

Q10

Q8

Q9

956

957

958

959

960

961

962

963

964

965

966

967

968

969

970

971

972

973

974

975

976

977

978

979

980

981

982

983

984

985

986

987

988

989

990

991

992

993

994

995

996

997

998

999

1000

1001

1002

1003

1004

1005

1006

1007

1008

1009

1010

1011

1012

899

900

901

902

903

904

905

906

907

908

909

910

911

912

913

914

915

916

917

918

919

920

921

922

923

924

925

926

927

928

929

930

931

932

933

934

935

936

937

938

939

940

941

942

943

944

945

946

947

948

949

950

951

952

953

954

955

9

Chulpanova et al. EVs in Cancer Treatment

Frontiers in Immunology | www.frontiersin.org July 2018 | Volume 9 | Article 1534

inhibitor in gastric cancer by suppressing the release of microRNA-carrying exosomes. Am J Cancer Res (2017) 7(9):1913–25.

27. Qin X, Yu S, Zhou L, Shi M, Hu Y, Xu X, et al. Cisplatin-resistant lung cancer cell-derived exosomes increase cisplatin resistance of recipient cells in exoso-mal miR-100-5p-dependent manner. Int J Nanomedicine (2017) 12:3721–33. doi:10.2147/IJN.S131516

28. Parolini I, Federici C, Raggi C, Lugini L, Palleschi S, De Milito A, et  al. Microenvironmental pH is a key factor for exosome traffic in tumor cells. J Biol Chem (2009) 284(49):34211–22. doi:10.1074/jbc.M109.041152

29. Luciani F, Spada M, De Milito A, Molinari A, Rivoltini L, Montinaro A, et al. Effect of proton pump inhibitor pretreatment on resistance of solid tumors to cytotoxic drugs. J Natl Cancer Inst (2004) 96(22):1702–13. doi:10.1093/jnci/djh305

30. Rana S, Malinowska K, Zoller M. Exosomal tumor microRNA modulates premetastatic organ cells. Neoplasia (2013) 15(3):281–95. doi:10.1593/neo. 122010

31. Costa-Silva B, Aiello NM, Ocean AJ, Singh S, Zhang H, Thakur BK, et  al. Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver. Nat Cell Biol (2015) 17(6):816–26. doi:10.1038/ncb3169

32. Grange C, Tapparo M, Collino F, Vitillo L, Damasco C, Deregibus MC, et  al. Microvesicles released from human renal cancer stem cells stimulate angiogenesis and formation of lung premetastatic niche. Cancer Res (2011) 71(15):5346–56. doi:10.1158/0008-5472.CAN-11-0241

33. Saari H, Lazaro-Ibanez E, Viitala T, Vuorimaa-Laukkanen E, Siljander P, Yliperttula M. Microvesicle- and exosome-mediated drug delivery enhances the cytotoxicity of Paclitaxel in autologous prostate cancer cells. J Control Release (2015) 220(Pt B):727–37. doi:10.1016/j.jconrel.2015.09.031

34. Yang T, Martin P, Fogarty B, Brown A, Schurman K, Phipps R, et al. Exosome delivered anticancer drugs across the blood-brain barrier for brain cancer therapy in Danio rerio. Pharm Res (2015) 32(6):2003–14. doi:10.1007/s11095- 014-1593-y

35. Toffoli G, Hadla M, Corona G, Caligiuri I, Palazzolo S, Semeraro S, et  al. Exosomal doxorubicin reduces the cardiac toxicity of doxorubicin. Nanomedicine (Lond) (2015) 10(19):2963–71. doi:10.2217/nnm.15.118

36. Hadla M, Palazzolo S, Corona G, Caligiuri I, Canzonieri V, Toffoli G, et al. Exosomes increase the therapeutic index of doxorubicin in breast and ovarian cancer mouse models. Nanomedicine (Lond) (2016) 11(18):2431–41. doi:10.2217/nnm-2016-0154

37. Clayton A, Mason MD. Exosomes in tumour immunity. Curr Oncol (2009) 16(3):46–9. doi:10.3747/co.v16i3.367

38. Mahaweni NM, Kaijen-Lambers ME, Dekkers J, Aerts JG, Hegmans JP. Tumour-derived exosomes as antigen delivery carriers in dendritic cell-based immunotherapy for malignant mesothelioma. J Extracell Vesicles (2013) 2. doi:10.3402/jev.v2i0.22492

39. Liu H, Chen L, Liu J, Meng H, Zhang R, Ma L, et al. Co-delivery of tumor- derived exosomes with alpha-galactosylceramide on dendritic cell-based immunotherapy for glioblastoma. Cancer Lett (2017) 411:182–90. doi:10.1016/ j.canlet.2017.09.022

40. Xiao L, Erb U, Zhao K, Hackert T, Zoller M. Efficacy of vaccination with tumor-exosome loaded dendritic cells combined with cytotoxic drug treatment in pancreatic cancer. Oncoimmunology (2017) 6(6):e1319044. doi:10.1080/2162402X.2017.1319044

41. Dai S, Zhou X, Wang B, Wang Q, Fu Y, Chen T, et al. Enhanced induction of dendritic cell maturation and HLA-A*0201-restricted CEA-specific CD8(+) CTL response by exosomes derived from IL-18 gene-modified CEA-positive tumor cells. J Mol Med (Berl) (2006) 84(12):1067–76. doi:10.1007/s00109-006-0102-0

42. Yang Y, Xiu F, Cai Z, Wang J, Wang Q, Fu Y, et  al. Increased induction of antitumor response by exosomes derived from interleukin-2 gene-modified tumor cells. J Cancer Res Clin Oncol (2007) 133(6):389–99. doi:10.1007/s00432-006-0184-7

43. Aspe JR, Diaz Osterman CJ, Jutzy JM, Deshields S, Whang S, Wall NR. Enhancement of gemcitabine sensitivity in pancreatic adenocarcinoma by novel exosome-mediated delivery of the Survivin-T34A mutant. J Extracell Vesicles (2014) 3. doi:10.3402/jev.v3.23244

44. Rivoltini L, Chiodoni C, Squarcina P, Tortoreto M, Villa A, Vergani B, et al. TNF-related apoptosis-inducing ligand (TRAIL)-armed exosomes deliver proapoptotic signals to tumor site. Clin Cancer Res (2016) 22(14):3499–512. doi:10.1158/1078-0432.CCR-15-2170

45. Li P, Feng J, Liu Y, Liu Q, Fan L, Liu Q, et al. Novel therapy for glioblastoma multiforme by restoring LRRC4 in tumor cells: LRRC4 inhibits tumor- infitrating regulatory T cells by cytokine and programmed cell death 1-containing exosomes. Front Immunol (2017) 8:1748. doi:10.3389/fimmu.2017.01748

46. Ostrowski M, Carmo NB, Krumeich S, Fanget I, Raposo G, Savina A, et al. Rab27a and Rab27b control different steps of the exosome secretion pathway. Nat Cell Biol (2010) 12(1):19–30; sup 11–13. doi:10.1038/ncb2000

47. Li W, Mu D, Tian F, Hu Y, Jiang T, Han Y, et al. Exosomes derived from Rab27a-overexpressing tumor cells elicit efficient induction of antitumor immunity. Mol Med Rep (2013) 8(6):1876–82. doi:10.3892/mmr.2013.1738

48. O’Brien K, Lowry MC, Corcoran C, Martinez VG, Daly M, Rani S, et  al. miR-134 in extracellular vesicles reduces triple-negative breast cancer aggres sion and increases drug sensitivity. Oncotarget (2015) 6(32):32774–89. doi:10.18632/oncotarget.5192

49. Zhang H, Bai M, Deng T, Liu R, Wang X, Qu Y, et al. Cell-derived microve-sicles mediate the delivery of miR-29a/c to suppress angiogenesis in gastric carcinoma. Cancer Lett (2016) 375(2):331–9. doi:10.1016/j.canlet.2016. 03.026

50. Shtam TA, Kovalev RA, Varfolomeeva EY, Makarov EM, Kil YV, Filatov MV. Exosomes are natural carriers of exogenous siRNA to human cells in vitro. Cell Commun Signal (2013) 11:88. doi:10.1186/1478-811X-11-88

51. Wang Y, Qin X, Zhu X, Chen W, Zhang J, Chen W. Oral cancer-derived exosomal NAP1 enhances cytotoxicity of natural killer cells via the IRF-3 pathway. Oral Oncol (2018) 76:34–41. doi:10.1016/j.oraloncology.2017.11.024

52. Tian Y, Li S, Song J, Ji T, Zhu M, Anderson GJ, et al. A doxorubicin deliv-ery platform using engineered natural membrane vesicle exosomes for targeted tumor therapy. Biomaterials (2014) 35(7):2383–90. doi:10.1016/j.biomaterials.2013.11.083

53. Gehrmann U, Hiltbrunner S, Georgoudaki AM, Karlsson MC, Naslund TI, Gabrielsson S. Synergistic induction of adaptive antitumor immunity by codelivery of antigen with alpha-galactosylceramide on exosomes. Cancer Res (2013) 73(13):3865–76. doi:10.1158/0008-5472.CAN-12-3918

54. Viaud S, Terme M, Flament C, Taieb J, Andre F, Novault S, et al. Dendritic cell-derived exosomes promote natural killer cell activation and proliferation: a role for NKG2D ligands and IL-15Ralpha. PLoS One (2009) 4(3):e4942. doi:10.1371/journal.pone.0004942

55. Lu Z, Zuo B, Jing R, Gao X, Rao Q, Liu Z, et al. Dendritic cell-derived exosomes elicit tumor regression in autochthonous hepatocellular carcinoma mouse models. J Hepatol (2017) 67(4):739–48. doi:10.1016/j.jhep.2017.05.019

56. Besse B, Charrier M, Lapierre V, Dansin E, Lantz O, Planchard D, et  al. Dendritic cell-derived exosomes as maintenance immunotherapy after first line chemotherapy in NSCLC. Oncoimmunology (2016) 5(4):e1071008. doi:10.1080/2162402X.2015.1071008

57. Kim MS, Haney MJ, Zhao Y, Mahajan V, Deygen I, Klyachko NL, et  al. Development of exosome-encapsulated paclitaxel to overcome MDR in can-cer cells. Nanomedicine (2016) 12(3):655–64. doi:10.1016/j.nano.2015.10.012

58. Kim MS, Haney MJ, Zhao Y, Yuan D, Deygen I, Klyachko NL, et  al. Engineering macrophage-derived exosomes for targeted paclitaxel delivery to pulmonary metastases: in vitro and in vivo evaluations. Nanomedicine (2018) 14(1):195–204. doi:10.1016/j.nano.2017.09.011

59. Jang SC, Kim OY, Yoon CM, Choi DS, Roh TY, Park J, et  al. Bioinspired exosome-mimetic nanovesicles for targeted delivery of chemotherapeutics to malignant tumors. ACS Nano (2013) 7(9):7698–710. doi:10.1021/ nn402232g

60. Munoz JL, Bliss SA, Greco SJ, Ramkissoon SH, Ligon KL, Rameshwar P. Delivery of functional anti-miR-9 by mesenchymal stem cell-derived exo-somes to glioblastoma multiforme cells conferred chemosensitivity. Mol Ther Nucleic Acids (2013) 2:e126. doi:10.1038/mtna.2013.60

61. Katakowski M, Buller B, Zheng X, Lu Y, Rogers T, Osobamiro O, et  al. Exosomes from marrow stromal cells expressing miR-146b inhibit glioma growth. Cancer Lett (2013) 335(1):201–4. doi:10.1016/j.canlet.2013.02.019

62. Lang FM, Hossain A, Gumin J, Momin EN, Shimizu Y, Ledbetter D, et  al. Mesenchymal stem cells as natural biofactories for exosomes carrying miR-124a in the treatment of gliomas. Neuro Oncol (2018) 20(3):380–90. doi:10.1093/neuonc/nox152

63. Umezu T, Imanishi S, Azuma K, Kobayashi C, Yoshizawa S, Ohyashiki K, et al. Replenishing exosomes from older bone marrow stromal cells with miR-340 inhibits myeloma-related angiogenesis. Blood Adv (2017) 1(13):812–23. doi:10.1182/bloodadvances.2016003251

1070

1071

1072

1073

1074

1075

1076

1077

1078

1079

1080

1081

1082

1083

1084

1085

1086

1087

1088

1089

1090

1091

1092

1093

1094

1095

1096

1097

1098

1099

1100

1101

1102

1103

1104

1105

1106

1107

1108

1109

1110

1111

1112

1113

1114

1115

1116

1117

1118

1119

1120

1121

1122

1123

1124

1125

1126

1013

1014

1015

1016

1017

1018

1019

1020

1021

1022

1023

1024

1025

1026

1027

1028

1029

1030

1031

1032

1033

1034

1035

1036

1037

1038

1039

1040

1041

1042

1043

1044

1045

1046

1047

1048

1049

1050

1051

1052

1053

1054

1055

1056

1057

1058

1059

1060

1061

1062

1063

1064

1065

1066

1067

1068

1069

10

Chulpanova et al. EVs in Cancer Treatment

Frontiers in Immunology | www.frontiersin.org July 2018 | Volume 9 | Article 1534

64. Greco KA, Franzen CA, Foreman KE, Flanigan RC, Kuo PC, Gupta GN. PLK-1 silencing in bladder cancer by siRNA delivered with exosomes. Urology (2016) 91:242.e1–7. doi:10.1016/j.urology.2016.01.028

65. Lou G, Song X, Yang F, Wu S, Wang J, Chen Z, et al. Exosomes derived from miR-122-modified adipose tissue-derived MSCs increase chemosensitivity of hepatocellular carcinoma. J Hematol Oncol (2015) 8:122. doi:10.1186/s13045-015-0220-7

66. Pascucci L, Cocce V, Bonomi A, Ami D, Ceccarelli P, Ciusani E, et al. Paclitaxel is incorporated by mesenchymal stromal cells and released in exosomes that inhibit in  vitro tumor growth: a new approach for drug delivery. J Control Release (2014) 192:262–70. doi:10.1016/j.jconrel.2014.07.042

67. Cocce V, Balducci L, Falchetti ML, Pascucci L, Ciusani E, Brini AT, et  al. Fluorescent immortalized human adipose derived stromal cells (hASCs-TS/GFP+) for studying cell drug delivery mediated by microve-sicles. Anticancer Agents Med Chem (2017) 17(11):1578–85. doi:10.2174/ 1871520617666170327113932

68. Yuan Z, Kolluri KK, Gowers KH, Janes SM. TRAIL delivery by MSC-derived extracellular vesicles is an effective anticancer therapy. J Extracell Vesicles (2017) 6(1):1265291. doi:10.1080/20013078.2017.1265291

69. Escudier B, Dorval T, Chaput N, Andre F, Caby MP, Novault S, et  al. Vaccination of metastatic melanoma patients with autologous dendritic cell (DC) derived-exosomes: results of the first phase I clinical trial. J Transl Med (2005) 3(1):10. doi:10.1186/1479-5876-3-10

70. Morse MA, Garst J, Osada T, Khan S, Hobeika A, Clay TM, et al. A phase I study of dexosome immunotherapy in patients with advanced non-small cell lung cancer. J Transl Med (2005) 3(1):9. doi:10.1186/1479-5876-3-9

71. Huang WH, Chang MC, Tsai KS, Hung MC, Chen HL, Hung SC. Mesenchymal stem cells promote growth and angiogenesis of tumors in mice. Oncogene (2013) 32(37):4343–54. doi:10.1038/onc.2012.458

72. Ramdasi S, Sarang S, Viswanathan C. Potential of mesenchymal stem cell based application in cancer. Int J Hematol Oncol Stem Cell Res (2015) 9(2): 95–103.

73. Wang M, Zhao C, Shi H, Zhang B, Zhang L, Zhang X, et  al. Deregulated microRNAs in gastric cancer tissue-derived mesenchymal stem cells: novel biomarkers and a mechanism for gastric cancer. Br J Cancer (2014) 110(5):1199–210. doi:10.1038/bjc.2014.14

74. Zhu W, Huang L, Li Y, Zhang X, Gu J, Yan Y, et al. Exosomes derived from human bone marrow mesenchymal stem cells promote tumor growth in vivo. Cancer Lett (2012) 315(1):28–37. doi:10.1016/j.canlet.2011.10.002

75. Roccaro AM, Sacco A, Maiso P, Azab AK, Tai YT, Reagan M, et  al. BM mesenchymal stromal cell-derived exosomes facilitate multiple myeloma progression. J Clin Invest (2013) 123(4):1542–55. doi:10.1172/JCI66517

76. Vallabhaneni KC, Penfornis P, Dhule S, Guillonneau F, Adams KV, Mo YY, et  al. Extracellular vesicles from bone marrow mesenchymal stem/stromal cells transport tumor regulatory microRNA, proteins, and metabolites. Onco­target (2015) 6(7):4953–67. doi:10.18632/oncotarget.3211

77. Ji R, Zhang B, Zhang X, Xue J, Yuan X, Yan Y, et al. Exosomes derived from human mesenchymal stem cells confer drug resistance in gastric cancer. Cell Cycle (2015) 14(15):2473–83. doi:10.1080/15384101.2015.1005530

78. Ono M, Kosaka N, Tominaga N, Yoshioka Y, Takeshita F, Takahashi RU, et al. Exosomes from bone marrow mesenchymal stem cells contain a microRNA that promotes dormancy in metastatic breast cancer cells. Sci Signal (2014) 7(332):ra63. doi:10.1126/scisignal.2005231

79. Lee JK, Park SR, Jung BK, Jeon YK, Lee YS, Kim MK, et al. Exosomes derived from mesenchymal stem cells suppress angiogenesis by down-regulating VEGF expression in breast cancer cells. PLoS One (2013) 8(12):e84256. doi:10.1371/journal.pone.0084256

80. Shimbo K, Miyaki S, Ishitobi H, Kato Y, Kubo T, Shimose S, et al. Exosome-formed synthetic microRNA-143 is transferred to osteosarcoma cells and inhibits their migration. Biochem Biophys Res Commun (2014) 445(2):381–7. doi:10.1016/j.bbrc.2014.02.007

81. Gilligan KE, Dwyer RM. Engineering exosomes for cancer therapy. Int J Mol Sci (2017) 18(6).

82. Gresch O, Engel FB, Nesic D, Tran TT, England HM, Hickman ES, et  al. New non-viral method for gene transfer into primary cells. Methods (2004) 33(2):151–63. doi:10.1016/j.ymeth.2003.11.009

Conflict of Interest Statement: The authors declare that the research was con-ducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Chulpanova, Kitaeva, James, Rizvanov and Solovyeva. This is an open­access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.