Extracellular vesicles secreted from bone metastatic renal cell carcinoma promote angiogenesis and endothelial gap formation in bone marrow in a time-dependent manner in a preclinical mouse model

1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer

statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36

cancers in 185 countries. CA Cancer J Clin (2018) 68:394–424. doi: 10.3322/caac.21492

3. Fan Z, Huang Z, Huang X. Bone metastasis in renal cell carcinoma patients: Risk

and prognostic factors and nomograms. J Oncol (2021) 2021:5575295. doi: 10.1155/

2021/5575295

2. Makhov P, Joshi S, Ghatalia P, Kutikov A, Uzzo RG, Kolenko VM. Resistance to

systemic therapies in clear cell renal cell carcinoma: Mechanisms and management

strategies. Mol Cancer Ther (2018) 17:1355–64. doi: 10.1158/1535-7163.MCT-17-1299

4. McKay RR, Kroeger N, Xie W, Lee JL, Knox JJ, Bjarnason GA, et al. Impact of

bone and liver metastases on patients with renal cell carcinoma treated with targeted

therapy. Eur Urol (2014) 65:577–84. doi: 10.1016/j.eururo.2013.08.012

Frontiers in Oncology

13

frontiersin.org

Takeda et al.

10.3389/fonc.2023.1139049

5. Negishi T, Furubayashi N, Takamatsu D, Ieiri K, Nishiyama N, Kitamura H, et al.

Radiographical efficacy of systemic treatment for bone metastasis from renal cell

carcinoma. Oncol Lett (2020) 20:267. doi: 10.3892/ol.2020.12130

25. Nakamura E, Abreu-e-Lima P, Awakura Y, Inoue T, Kamoto T, Ogawa O, et al.

Clusterin is a secreted marker for a hypoxia-inducible factor-independent function of

the von hippel-lindau tumor suppressor protein. Am J Pathol (2006) 168:574–84.

doi: 10.2353/ajpath.2006.050867

6. Zekri J, Ahmed N, Coleman RE, Hancock BW. The skeletal metastatic

complications of renal cell carcinoma. Int J Oncol (2001) 19:379–82. doi: 10.3892/

ijo.19.2.379

26. Ikeda A, Nagayama S, Sumazaki M, Konishi M, Fujii R, Saichi N, et al. Colorectal

cancer-derived CAT1-positive extracellular vesicles alter nitric oxide metabolism in

endothelial cells and promote angiogenesis. Mol Cancer Res (2021) 19:834–46.

doi: 10.1158/1541-7786.MCR-20-0827

7. Chen SC, Kuo PL. Bone metastasis from renal cell carcinoma. Int J Mol Sci (2016)

17. doi: 10.3390/ijms17060987

27. Okuda S, Watanabe Y, Moriya Y, Kawano S, Yamamoto T, Matsumoto M, et al.

jPOSTrepo: an international standard data repository for proteomes. Nucleic Acids Res

(2017) 45:D1107–D11. doi: 10.1093/nar/gkw1080

8. Lipton A, Fizazi K, Stopeck AT, Henry DH, Brown JE, Yardley DA, et al.

Superiority of denosumab to zoledronic acid for prevention of skeletal-related events: a

combined analysis of 3 pivotal, randomised, phase 3 trials. Eur J Cancer (2012)

48:3082–92. doi: 10.1016/j.ejca.2012.08.002

28. Risha Y, Minic Z, Ghobadloo SM, Berezovski MV. The proteomic analysis of

breast cell line exosomes reveals disease patterns and potential biomarkers. Sci Rep

(2020) 10:13572. doi: 10.1038/s41598-020-70393-4

9. Kurata T, Nakagawa K. Efficacy and safety of denosumab for the treatment of

bone metastases in patients with advanced cancer. Jpn J Clin Oncol (2012) 42:663–9.

doi: 10.1093/jjco/hys088

29. Peng W, Zhang Y, Zhu R, Mechref Y. Comparative membrane proteomics

analyses of breast cancer cell lines to understand the molecular mechanism of breast

cancer brain metastasis. Electrophoresis (2017) 38:2124–34. doi: 10.1002/

elps.201700027

10. Bielenberg DR, Zetter BR. The contribution of angiogenesis to the process of

metastasis. Cancer J (2015) 21:267–73. doi: 10.1097/PPO.0000000000000138

11. Zetter BR. Angiogenesis and tumor metastasis. Annu Rev Med (1998) 49:407–

24. doi: 10.1146/annurev.med.49.1.407

30. Liu Y, Cao X. Characteristics and significance of the pre-metastatic niche.

Cancer Cell (2016) 30:668–81. doi: 10.1016/j.ccell.2016.09.011

12. Raymaekers K, Stegen S, van Gastel N, Carmeliet G. The vasculature: a vessel for

bone metastasis. Bonekey Rep (2015) 4:742. doi: 10.1038/bonekey.2015.111

31. Santini D, Procopio G, Porta C, Ibrahim T, Barni S, Mazzara C, et al. Natural

history of malignant bone disease in renal cancer: final results of an Italian bone

metastasis survey. PloS One (2013) 8:e83026. doi: 10.1371/journal.pone.0083026

13. Wortzel I, Dror S, Kenific CM, Lyden D. Exosome-mediated metastasis:

Communication from a distance. Dev Cell (2019) 49:347–60. doi: 10.1016/

j.devcel.2019.04.011

32. György B, Szabó TG, Pá sztó i M, Pá l Z, Misjá k P, Aradi B, et al. Membrane

vesicles, current state-of-the-art: emerging role of extracellular vesicles. Cell Mol Life Sci

(2011) 68:2667–88. doi: 10.1007/s00018-011-0689-3

14. Zomer A, van Rheenen J. Implications of extracellular vesicle transfer on cellular

heterogeneity in cancer: What are the potential clinical ramifications? Cancer Res

(2016) 76:2071–5. doi: 10.1158/0008-5472.CAN-15-2804

33. Gemel J, Kilkus J, Dawson G, Beyer EC. Connecting exosomes and connexins.

Cancers (Basel) (2019) 11. doi: 10.3390/cancers11040476

15. Kosaka N, Iguchi H, Hagiwara K, Yoshioka Y, Takeshita F, Ochiya T. Neutral

sphingomyelinase 2 (nSMase2)-dependent exosomal transfer of angiogenic

microRNAs regulate cancer cell metastasis. J Biol Chem (2013) 288:10849–59.

doi: 10.1074/jbc.M112.446831

34. Ozkocak DC, Phan TK, Poon IKH. Translating extracellular vesicle packaging

into therapeutic applications. Front Immunol (2022) 13:946422. doi: 10.3389/

fimmu.2022.946422

16. Zeng Z, Li Y, Pan Y, Lan X, Song F, Sun J, et al. Cancer-derived exosomal miR25-3p promotes pre-metastatic niche formation by inducing vascular permeability and

angiogenesis. Nat Commun (2018) 9:5395. doi: 10.1038/s41467-018-07810-w

35. Liang W, Gao B, Xu G, Weng D, Xie M, Qian Y. Possible contribution of

aminopeptidase n (APN/CD13) to migration and invasion of human osteosarcoma cell

lines. Int J Oncol (2014) 45:2475–85. doi: 10.3892/ijo.2014.2664

17. 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) 71:5346–56. doi: 10.1158/

0008-5472.CAN-11-0241

36. Lu C, Amin MA, Fox DA. CD13/Aminopeptidase n is a potential therapeutic

target for inflammatory disorders. J Immunol (2020) 204:3–11. doi: 10.4049/

jimmunol.1900868

37. Du Y, Lu C, Morgan RL, Stinson WA, Campbell PL, Cealey E, et al. Angiogenic

and arthritogenic properties of the soluble form of CD13. J Immunol (2019) 203:360–9.

doi: 10.4049/jimmunol.1801276

18. Sato S, Vasaikar S, Eskaros A, Kim Y, Lewis JS, Zhang B, et al. EPHB2 carried on

small extracellular vesicles induces tumor angiogenesis via activation of ephrin reverse

signaling. JCI Insight (2019) 4. doi: 10.1172/jci.insight.132447

38. van Hensbergen Y, Broxterman HJ, Hanemaaijer R, Jorna AS, van Lent NA,

Verheul HM, et al. Soluble aminopeptidase N/CD13 in malignant and nonmalignant

effusions and intratumoral fluid. Clin Cancer Res (2002) 8:3747–54.

19. Dai J, Escara-Wilke J, Keller JM, Jung Y, Taichman RS, Pienta KJ, et al. Primary

prostate cancer educates bone stroma through exosomal pyruvate kinase M2 to

promote bone metastasis. J Exp Med (2019) 216:2883–99. doi: 10.1084/jem.20190158

39. Gong T, Zhang X, Peng Z, Ye Y, Liu R, Yang Y, et al. Macrophage-derived

exosomal aminopeptidase n aggravates sepsis-induced acute lung injury by regulating

necroptosis of lung epithelial cell. Commun Biol (2022) 5:543. doi: 10.1038/s42003-02203481-y

20. Ni J, Zhang X, Li J, Zheng Z, Zhang J, Zhao W, et al. Tumour-derived exosomal

lncRNA-SOX2OT promotes bone metastasis of non-small cell lung cancer by targeting

the miRNA-194-5p/RAC1 signalling axis in osteoclasts. Cell Death Dis (2021) 12:662.

doi: 10.1038/s41419-021-03928-w

40. Claesson-Welsh L, Dejana E, McDonald DM. Permeability of the endothelial

barrier: Identifying and reconciling controversies. Trends Mol Med (2021) 27:314–31.

doi: 10.1016/j.molmed.2020.11.006

21. Wang J, Chen A, Yang C, Zeng H, Qi J, Guo FJ. A bone-seeking clone exhibits

different biological properties from the ACHN parental human renal cell carcinoma in

vivo and in vitro. Oncol Rep (2012) 27:1104–10. doi: 10.3892/or.2011.1572

41. Ma Q, Liang M, Wu Y, Dou C, Xu J, Dong S, et al. Small extracellular vesicles

deliver osteolytic effectors and mediate cancer-induced osteolysis in bone metastatic

niche. J Extracell Vesicles (2021) 10:e12068. doi: 10.1002/jev2.12068

22. Xie C, Schwarz EM, Sampson ER, Dhillon RS, Li D, O'Keefe RJ, et al. Unique

angiogenic and vasculogenic properties of renal cell carcinoma in a xenograft model of

bone metastasis are associated with high levels of vegf-a and decreased ang-1

expression. J Orthop Res (2012) 30:325–33. doi: 10.1002/jor.21500

42. Yu L, Sui B, Fan W, Lei L, Zhou L, Yang L, et al. Exosomes derived from

osteogenic tumor activate osteoclast differentiation and concurrently inhibit

osteogenesis by transferring COL1A1-targeting miRNA-92a-1-5p. J Extracell Vesicles

(2021) 10:e12056. doi: 10.1002/jev2.12056

23. Jingushi K, Uemura M, Ohnishi N, Nakata W, Fujita K, Naito T, et al.

Extracellular vesicles isolated from human renal cell carcinoma tissues disrupt

vascular endothelial cell morphology via azurocidin. Int J Cancer (2018) 142:607–17.

doi: 10.1002/ijc.31080

43. Wu K, Feng J, Lyu F, Xing F, Sharma S, Liu Y, et al. Exosomal miR-19a and IBSP

cooperate to induce osteolytic bone metastasis of estrogen receptor-positive breast

cancer. Nat Commun (2021) 12:5196. doi: 10.1038/s41467-021-25473-y

24. Yamasaki T, Kamba T, Kanno T, Inoue T, Shibasaki N, Arakaki R, et al. Tumor

microvasculature with endothelial fenestrations in VHL null clear cell renal cell

carcinomas as a potent target of anti-angiogenic therapy. Cancer Sci (2012)

103:2027–37. doi: 10.1111/j.1349-7006.2012.02412.x

Frontiers in Oncology

44. Jang SC, Economides KD, Moniz RJ, Sia CL, Lewis N, McCoy C, et al.

ExoSTING, an extracellular vesicle loaded with STING agonists, promotes tumor

immune surveillance. Commun Biol (2021) 4:497. doi: 10.1038/s42003-021-02004-5

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Supplementary Table 1

Category

Patients with bone metastasis

Patients with locally advanced disease

(N=6)

(N=6)

Age(mean, range)

Gender

63.8(55-72)

63.5(52-76)

Male

Female

Male

Female

Laterality

Left

Right

Left

Right

Histology

Clear cell

Clear cell

Biopsy

RN

Biopsy

RN

Clinical T stage

cT1

cT2

cT3

cT1

cT2

cT3

Clinical N stage

cN0

cN2

cN0

cN2

Metastatic site

Bone

Lung

Liver

Pathological T stage

pT1

pT2

pT3

N/A

Types of Procedure

None

pT1

pT2

pT3

Tumor thrombus(n, %)

2(33.3%)

3(50%)

Presurgical Treatment(n, %)

2(33.3%)

2(33.3%)

Supplementary Figure 1

CD63

100nm

786-O luc EV

786-O BM EV

TSG101

786-O BM EV

786-O luc EV

Particle concentration (x108/ml)

Particle concentration (x108/ml)

Size (nm)

NS

Particle size (nm)

150

100

50

786-O luc EV

**

3.0

** P<0.05

2.0

1.0

0.2

* P<0.05

0.15

0.1

0.05

786-O luc EV

786-O BM EV

786-O BM EV

NS

20

Particle protein

ratio (x105/mg)

Protein concentration (x108)

Particle concentration (µg/µl)

Size (nm)

15

10

786-O luc EV

786-O BM EV

786-O luc EV

786-O BM EV

Supplementary Figure 2

786-O luc EV

Liver

Lung

Spleen

Kidney

786-O BM EV

Supplementary Figure 3

shControl EV

B Bone

one m

e t a s ta s isfree

f r e survival

e s u r v iv(%)

a l (% )

metastasis

shANPEP EV

1100

00

NS

50

50

s hshControl

C o n tro l EV

EV

shANPEP

sh

A N P E P EV

EV

(n=9)

00

00

220

40

40

DDays

ays

660

Supplementary Table 2

Description

Abundance Ratio (log2):

Protein Localization

(BM EV) / (luc EV)

Gap junction alpha-1 protein OS=Homo sapiens OX=9606 GN=GJA1 PE=1 SV=2

6.64

Mitofusin-1 OS=Homo sapiens OX=9606 GN=MFN1 PE=1 SV=3

6.64

IC, M

Protein S100-A14 OS=Homo sapiens OX=9606 GN=S100A14 PE=1 SV=1

6.64

IC

SLIT-ROBO Rho GTPase-activating protein 2 OS=Homo sapiens OX=9606 GN=SRGAP2 PE=1 SV=3

6.64

IC

Protein S100-A8 OS=Homo sapiens OX=9606 GN=S100A8 PE=1 SV=1

5.83

IC, S

Alpha-crystallin B chain OS=Homo sapiens OX=9606 GN=CRYAB PE=1 SV=2

5.34

IC, M

Protein-arginine deiminase type-3 OS=Homo sapiens OX=9606 GN=PADI3 PE=1 SV=2

4.77

IC

Serpin B5 OS=Homo sapiens OX=9606 GN=SERPINB5 PE=1 SV=2

3.83

IC

ADP/ATP translocase 2 OS=Homo sapiens OX=9606 GN=SLC25A5 PE=1 SV=7

3.67

10 kDa heat shock protein, mitochondrial OS=Homo sapiens OX=9606 GN=HSPE1 PE=1 SV=2

3.47

IC

Hephaestin-like protein 1 OS=Homo sapiens OX=9606 GN=HEPHL1 PE=2 SV=2

3.24

Beta-hexosaminidase subunit beta OS=Homo sapiens OX=9606 GN=HEXB PE=1 SV=3

3.2

IC

Vitronectin OS=Homo sapiens OX=9606 GN=VTN PE=1 SV=1

3.13

Fatty acid-binding protein, adipocyte OS=Homo sapiens OX=9606 GN=FABP4 PE=1 SV=3

2.81

IC

Vesicle-associated membrane protein 3 OS=Homo sapiens OX=9606 GN=VAMP3 PE=1 SV=3

2.82

Aminopeptidase N OS=Homo sapiens OX=9606 GN=ANPEP PE=1 SV=4

2.54

Immunoglobulin heavy constant alpha 1 OS=Homo sapiens OX=9606 GN=IGHA1 PE=1 SV=2

2.29

IC

Cell surface glycoprotein MUC18 OS=Homo sapiens OX=9606 GN=MCAM PE=1 SV=2

2.24

Serpin B13 OS=Homo sapiens OX=9606 GN=SERPINB13 PE=1 SV=2

2.18

IC,M

Phosphate carrier protein, mitochondrial OS=Homo sapiens OX=9606 GN=SLC25A3 PE=1 SV=2

2.14

IC,M

Supplementary Table 3

Description

# Unique Peptides

Score Mascot

Score Sequest HT

Gap junction alpha-1 protein OS=Homo sapiens OX=9606 GN=GJA1 PE=1 SV=2

42

2.69

ADP/ATP translocase 2 OS=Homo sapiens OX=9606 GN=SLC25A5 PE=1 SV=7

97

8.06

Vesicle-associated membrane protein 3 OS=Homo sapiens OX=9606 GN=VAMP3 PE=1 SV=3

26

3.87

Aminopeptidase N OS=Homo sapiens OX=9606 GN=ANPEP PE=1 SV=4

301

13.97

Cell surface glycoprotein MUC18 OS=Homo sapiens OX=9606 GN=MCAM PE=1 SV=2

256

18.62

...