The FEBS Journal - 2022 - Liu

IL-35 enhances angiogenic effects of small extracellular
vesicles in breast cancer

Jia Liu, Nana Dong, Ning Li, Hui Zhao, Yali Li, Huihui Mao, Hanxiao Ren, Yimin Feng, Jie Liu,
Lutao Du and Haiting Mao
Department of Clinical Laboratory, The Second Hospital, Cheeloo College of Medicine, Shandong University, Jinan, China
Keywords As an indispensable process for breast cancer metastasis, tumour angiogene-

angiogenesis; breast cancer; HUVECs; sis requires a tight interaction between cancer cells and endothelial cells in
IL-35; sEVs
tumour microenvironment. Here, we explored the participation of small
extracellular vesicles (sEVs) derived from breast cancer cells in modulating
Correspondence
H. Mao, Department of Clinical Laboratory, angiogenesis and investigated the effect of IL-35 in facilitating this process.
The Second Hospital, Cheeloo College of Firstly, we characterized breast cancer cells-derived sEVs untreated or treated
Medicine, Shandong University, Jinan, with IL-35 and visualized the internalization of these sEVs by human umbili-
Shandong Province, 250033, China cal vein endothelial cells (HUVECs). Breast cancer cells-derived sEVs pro-
Tel: +86-531-85875068 moted endothelial cell proliferation through facilitating cell cycle progression
E-mail: maohaiting@sdu.edu.cn
and enhanced capillary-like structures formation and microvessel formation.
Subsequent results proved that IL-35 further reinforced the angiogenic effect
Jia Liu and Nana Dong contributed equally.
induced by breast cancer cells-derived sEVs. Moreover, sEVs from breast
(Received 6 October 2021, revised 15 cancer cells significantly enhanced tumour growth and microvessel density in
December 2021, accepted 14 January 2022) breast tumour-bearing mice model. Microarray analysis showed that IL-35
might alter the mRNA profiles of sEVs and activate the Ras/Raf/MEK/
doi:10.1111/febs.16359 ERK signalling pathway. These findings demonstrated that IL-35 indirectly
promoted angiogenesis in breast cancer through regulating the content of
breast cancer cells-derived sEVs, which could be internalized by HUVECs.
Introduction
As the leading cause of malignant tumour-related several sequential steps, including endothelial cell acti-
death among females, breast cancer accounts for 15% vation, proliferation, migration and tube formation [6].
of the total cancer deaths in 2018 [1]. The poor prog- This complex process requires a tight interaction
nosis of breast cancer is mainly due to its propensity between endothelial cells and their surrounding envi-
to metastasize to distant organs, such as lung, bone ronment [7,8]. In tumour angiogenesis, extracellular
and brain [2–4]. In cancer metastasis, angiogenesis is vesicles which contain the angiogenic factors act as
indispensable for the escape of tumour cells into critical medium between tumour cells and endothelial
bloodstream and for distant metastasis establishment cells [9–13].
[5]. As a procedure of neo-vascular formation from Extracellular vesicles are now extensively studied as
pre-existing blood vessels, angiogenesis comprises vital mediators for intercellular communication which
Abbreviations
BDMCs, blood-derived monocytes; Breg, regulatory B cell; CALM1, calmodulin 1; CAM, chick embryo chorioallantoic membrane; CCK8, cell
counting kit 8; CDK2, cyclin-dependent kinase 2; DAPI, 4’,6-diamidino-2-phenylindole; DEGs, differentially expressed genes; ERK,
extracellular signal-regulated kinase; GO, gene ontology; HUVECs, human umbilical vein endothelial cells; IL-35, interleukin-35; INSR, insulin
receptor precursor; KDR, kinase insert domain receptor; KEGG, kyoto encyclopedia of genes and genomes; MEK, mitogen-activated protein
kinase kinase; PTEN, phosphatase and tensin homolog; qRT-PCR, quantitative real-time PCR; RA, rheumatoid arthritis; Raf, rapidly
accelerated fibrosarcoma; Ras, rat sarcoma; sEVs, small extracellular vesicles; THBS1, thrombospondins 1; VEGFA, vascular endothelial
growth factor A.
The FEBS Journal 289 (2022) 3489–3504 ª 2022 Federation of European Biochemical Societies 3489
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IL-35 enhances the role of extracellular vesicles in breast cancer J. Liu et al.
facilitates the exchange of proteins and nucleic acids In this study, we not only found that sEVs secreted
between cells [14,15]. Here, we focused on the function by breast cancer cells promoted angiogenesis in vitro
of small extracellular vesicles (sEVs) with diameter from and in vivo but also proved the participation of IL-35
50 nm to 200 nm which are secreted by almost all cells in modulating breast cancer angiogenesis through
and frequently classified generally as “exosomes” in a altering the mRNA profile of breast cancer cells-
number of previous studies [9,11,15,16]. Increasing evi- derived sEVs. In terms of mechanism, we demon-
dence has demonstrated that sEVs are concerned with strated that IL-35 could enhance the effect of breast
the regulation of tumour microenvironment via the pro- cancer cells-derived sEVs in promoting HUVECs pro-
motion of tumorigenesis, metastasis, angiogenesis and liferation and angiogenesis through improving the
signalling pathway activation [10,12,17]. And studies on expression of CALM1 and activating the Ras/Raf/
breast cancer have demonstrated that sEVs transfer spe- MEK/ERK signalling pathway. Our study explores
cific messages to the recipient cells and regulate their the specific molecular mechanism of metastasis under-
biological behaviour [18,19]. However, the angiogenic lying breast cancer cells and identifies novel therapeu-
effect of sEVs in tumour microenvironment of breast tic target for tumour angiogenesis.
cancer remains poorly understood.
On the other hand, our previous studies demon-
Results
strated that both breast tumour cells and tumour-
infiltrating lymphocytes expressed and secreted inhibi-
Isolation and characterization of sEVs derived
tory cytokine IL-35 [20,21]. Moreover, IL-35 further
from breast cancer cells
promoted tumour progression through mediating the
conversion of tumour infiltrating Tconv cells into iTr35 To view the sEVs and IL-35-sEVs derived from breast
cells [21] and increased IL-35 level in breast cancer cancer cells, electron microscopy analysis was applied
microenvironment was considered as a predictor of to identify the morphology of purified sEVs (Fig. 1A).
poor prognosis [20,22]. Nanoparticle tracking analysis observed that the
Fig. 1. Characterization of the purified sEVs. Transmission electron micrographs of sEVs derived from breast cancer cell lines stimulated by
IL-35 or not. Scale bar: 200 nm (A). The nanoparticle concentration and size distribution of the sEVs were analysed by NTA (B). The protein
levels of CD9 and TSG101 in sEVs were determined by western blot (C).
3490 The FEBS Journal 289 (2022) 3489–3504 ª 2022 Federation of European Biochemical Societies
J. Liu et al. IL-35 enhances the role of extracellular vesicles in breast cancer
diameter of purified extracellular vesicles was around SEVs from breast cancer cells promoted HUVECs
100 nm, typical of exosomes (Fig. 1B). Meanwhile, proliferation and cell cycle progression and IL-35
exosomal markers (CD9 and TSG101) were expressed facilitated this effect
in all purified sEVs (Fig. 1C). Results showed that
Endothelial cell proliferation is an important step of
there was no significant difference in the characteriza-
angiogenesis [5]. In the proliferation analysis, we firstly
tion between sEVs and IL-35-sEVs.
stimulated the HUVECs with sEVs from breast cancer
cells. Results of CCK8 showed that breast cancer cells-
Verification of sEVs internalization derived sEVs significantly promoted HUVECs prolifera-
tion in comparison with control group (Fig. 3A). The
To verify the interaction between the purified sEVs
flow cytometry analysis indicated that breast cancer
and HUVECs, sEVs labelled with PKH26 were co-
cells-derived sEVs accelerated the transition from G0/
incubated with HUVECs for 4 h. Images captured
G1 phase of the cell cycle to S phases in HUVECs and
by confocal microscope showed that the red-labelled
led to an increased S and G2/M population (Fig. 3B).
sEVs, isolated from breast cancer cells treated with IL-
Subsequently, we stimulated the breast cancer cells in
35 or not, were all internalized by HUVECs and pre-
advance with IL-35 (25 ngmL 1) or not for 3 days.
dominantly enriched in the cytoplasm of endothelial
Then, sEVs from IL-35-treated breast cancer cells (IL-
cells (Fig. 2).
35-sEVs) or controls (con-sEVs) were collected and
Fig. 2. Uptake of sEVs derived from MDA-

MB-231 (231) and MCF-7 cells by HUVECs.
Fluorescence microscopy images showed
the internalization of sEVs by HUVECs.
Blue: Nucleus stained with DAPI. Red:
PKH26-labelled sEVs. Each experiment was
repeated three times (n = 3). Scale bar:
10 µm.
Fig. 3. SEVs and IL-35 induced the proliferation and cell cycle progression in HUVECs. HUVECs were incubated with IL-35-sEVs or con-
sEVs. Then, the proliferation of HUVECs was detected by CCK8 assay (A). DNA content analysis by flow cytometry showed the
percentages of cells in the G0/G1, S and G2/M phases of the cell cycle (B). Cell-cycle-related proteins were detected by western blot in
different treating HUVECs (C). Experiments were repeated three times in triplicate. The Student’s t-test was used to analyse the difference.
Data were presented as mean standard deviation (SD), n = 3. *: P < 0.05.
incubated with HUVECs. The results from proliferation IL-35 accelerated the angiogenic effects of breast
and cell cycle analysis showed that IL-35-sEVs played a cancer cells-derived sEVs
better role in promoting cell proliferation and cell cycle
progression than that of con-sEVs (Fig. 3A,B). To confirm the angiogenic effects of sEVs from breast
To better explore the effect of breast cancer cells- cancer cells on HUVECs, tube formation assays were
derived sEVs in cell cycle, we also examined the performed to evaluate their vascular formation abili-
expression of cell cycle-related proteins, including ties. As shown in Fig. 4A, sEVs from breast cancer
cyclinD1, cyclinE1 and CDK2. As depicted in Fig. 3C, cells obviously promoted the angiogenesis of HUVECs
the levels of these G1/S phase-related proteins were by enhancing total branch length in network struc-
significantly increased in HUVECs after treatment tures. Moreover, HUVECs incubated with IL-35-sEVs
with sEVs. And compared with con-sEVs, the expres- formed more capillary-like structures than those incu-
sion of these G1/S phase-related proteins in HUVECs bated with unstimulated sEVs (con-sEVs) (Fig. 4A,B).
stimulated by IL-35-sEVs was dramatically elevated. Next, we used the in vivo CAM model to further
These results suggested that IL-35-sEVs promoted the verify the in vitro findings. Similarly, exposure of the
G1/S transition in HUVECs. CAM to con-sEVs or IL-35-sEVs both enhanced the
Fig. 4. SEVs and IL-35 facilitated tube formation of HUVECs in vitro and increased the vessel density in CAM models. HUVECs were plated
on Matrigel and co-cultured with IL-35-sEVs or con-sEVs. Representative pictures were taken after stained with Calcein-AM. Scale bar:
200 µm (A). Tube formation ability was quantified by measuring the total branch length (B). Chick chorioallantoic membranes were treated
with sEVs. Representative pictures of the vascularized areas in CAM were depicted. Scale bar: 100 µm (C). The blood vessel density was
quantified by the number of branch points (D). The Student’s t-test was used to analyse the difference. Data were presented as
mean standard deviation (SD) from three independent experiments, n = 3. *: P < 0.05.
formation of branched blood vessels. Furthermore, IL- IL-35-sEVs further increased the growth rate of subcu-
35-sEVs-stimulated CAM formed more microvessels taneous tumours in mice model compared with those
than those exposed to con-sEVs (Fig. 4C,D). injected with sEVs from unstimulated breast cancer
cells (con-sEVs) (Fig. 5A).
In support of these data, immunohistochemical
In vivo validation of IL-35 promoting breast
results from mouse breast cancer tissue proved that
cancer cells-derived sEVs effects
sEVs administration obviously increased the positive
In this study, we constructed the breast tumour- rate of nuclear proliferation associated antigen Ki67
bearing mice model to further observe the angiogenic and CD31 protein that was generally regarded as the
action of sEVs and regulatory effect of IL-35. As specific endothelial marker and represented as micro-
shown in Fig. 5A, the treatment of sEVs from 231 vessel density. Moreover, IL-35-sEVs-treated group
cells significantly enhanced the tumour growth than showed higher vascularization in tumour microenvi-
PBS control group (sEVs-). It is worth noting that ronment and tumour cell proliferation (Fig. 5B,C).
Fig. 5. SEVs and IL-35 promoted the tumour growth and angiogenesis in mice model of breast cancer. Xenograft transplanted breast
tumour models were established and treated with PBS (sEVs-), IL-35-sEVs or con-sEVs. The tumour size was measured every 5 days (A).
After mice were sacrificed, tumours were taken out for immunohistochemistry analysis. Representative pictures of Ki67 were shown and
quantified for cell proliferation. Scale bar: 100 µm (B). Representative pictures of CD31 were shown and quantified for vascular density.
Scale bar: 100 µm (C). The Student’s t-test was used to analyse the difference. Data were presented as mean standard deviation (SD),
n = 3. *: P < 0.05.
Differential analysis of mRNA expression profiles on the angiogenesis, HUVECs were stably transfected
between con-sEVs and IL-35-sEVs with the siCALM1 plasmid to achieve silent expression
and siNC was transfected as control. The effectiveness
Previous researches confirmed that mRNAs delivered
of CALM1 interference was determined by qRT-PCR
by sEVs were actually functional and could be trans-
(Fig. 7B). The results of tube formation assays showed
lated into protein in the recipient cells [23,24]. In the
that the down-regulation of CALM1 significantly
present study, by comparing global mRNA-expression
inhibited the vascular formation abilities of HUVECs.
profiles between IL-35-sEVs and con-sEVs from
Additionally, the negative effect of siCALM1 on
MDA-MB-231 cells, we identified a series of differen-
angiogenesis of HUVECs could be offset by adding
tially expressed genes (DEGs). According to the data,
IL-35-sEVs distinctly (Fig. 7C).
a total of 1841 genes were up-regulated and 1667 genes
Moreover, a large number of studies indicate that
were down-regulated in IL-35-sEVs compared to
Ras/Raf/MEK/ERK signalling pathway not only pro-
con-sEVs (Fig. 6A). The heat maps were depicted to
motes angiogenesis by up-regulating the expression
present the top 50 up- or down-regulated DEGs
of angiogenic factors, but also plays an important
(Fig. 6B,C).
role in cell proliferation [27–29]. Therefore, we further
In order to elucidate the potential functions of
assessed the effect of different sEVs on Ras/Raf/
DEGs, GO biological process enrichment and KEGG
MEK/ERK signalling pathway in HUVECs. Results
signalling pathway analysis were applied for data min-
showed that, compared to the con-sEVs, the protein
ing. Results suggested that DEGs mainly involved in
levels of Ras, Raf, p-MEK and p-ERK in HUVECs
GO terms, such as angiogenesis and cell proliferation.
were notably increased after exposing to IL-35-sEVs
Then, we plotted the PPI network of DEGs enriched
(Fig. 7D).
in angiogenesis and identified the hub genes (THBS1,
Taken together, these results revealed essential func-
KDR, PTEN and VEGFA) (Fig. 6D). As a result of
tions of CALM1 derived from IL-35-sEVs in promot-
their location in the network, these genes were consid-
ing the angiogenic ability of HUVECs. All these data
ered topologically important to the structure of the
supported that IL-35 might modulate the angiogenic
network and played critical roles in modulating angio-
gene CALM1 in the sEVs from breast cancer cells via
genesis (Fig. 6E). Similarly, DEGs also played a role
Ras/Raf/MEK/ERK signalling pathway, and hence
in KEGG terms, such as RAS signalling pathway
altered the properties of recipient HUVECs which con-
which was demonstrated to be related with angiogene-
tributed to angiogenesis.
sis (Fig. 6F) [25]. PPI network of DEGs showed that
RRAS, INSR, CALM1 and KDR were hub genes
which played dominant roles in regulating RAS signal- Discussion
ling pathway (Fig. 6G). Angiogenesis is a multistep physiological process by
which the new vasculature is formed and it is closely
related to the distant metastasis and poor prognosis
IL-35-sEVs promoted tumour angiogenesis by
of tumour [30,31]. At present, angiogenesis inhibitors
transferring CALM1 to HUVECs and activating
are considered ideal antitumour treatments and target-
the Ras/Raf/MEK/ERK signalling pathway
ing tumour angiogenesis becomes a widely accepted
Based on the above analysis, the mRNA level of treatment in clinic. However, existing antitumour ther-
RRAS, INSR, CALM1, THBS1, KDR, PTEN and apies are not particularly effective because of the
VEGFA was detected in HUVECs treated with differ- incomplete suppression of tumour angiogenesis [32,33].
ent sEVs to verify the results of microarray. Results Therefore, it is necessary to explore the mechanism
showed that RRAS, INSR, CALM1, KDR and of affecting tumour angiogenesis and seek new targets
VEGFA were significantly up-regulated and THBS1 for anti-angiogenesis treatment. In tumour microenvi-
and PTEN were obviously down-regulated in ronment, angiogenesis is achieved by intercellular com-
HUVECs co-cultured with IL-35-sEVs (Fig. 7A). And munication between endothelial cells and tumour cells.
the results in Fig. 7A could validate part of the tran- Studies on breast cancer indicated that this complex
scriptomic microarray analysis. According to the sequential process was regulated by a series of chemo-
changes of the expression of differential molecules in kines and cytokines in tumour microenvironment. Che-
Fig. 7A, CALM1 was not only obviously and stably mokines, such as C-X-C motif chemokine ligand-12
differential expressed, but also it was closely related to (CXCL12) and Vasohibin-2 (VASH2), facilitated the
angiogenesis and mediated various signal transduction proliferation, migration or tube formation of endothe-
processes [26]. To further explore the effect of CALM1 lial cells in tumour microenvironment [34–36].
Fig. 6. Microarray analysis revealed DEGs between IL-35-sEVs and con-sEVs and Function annotation. Scatter plot of DEGs variations in IL-
35-sEVs and con-sEVs secreted by MDA-MB-231 (231) cells. Dots above the top line and below the bottom line indicated the fold change
of the DEGs were more than 2.0 between the two groups (A). Heat map of the top 50 up-regulated (B) and down-regulated (C) DEGs in IL-
35-sEVs compared with con-sEVs. The results of GO biological process enrichment (D) and KEGG signalling pathway analysis (F) were
presented as bubble chart. The size and colour of nodes varied from fold change of DEGs. And the different colours of edges were
displayed upon degree of connectivity of the node. The PPI network for DEGs that enriched in angiogenesis (E) and RAS signalling pathway
(G) were plotted.
As critical extracellular messengers, extracellular ves- mRNAs and miRNAs [37,38]. Extracellular vesicles
icles also mediate tumour microenvironment cross- are a collective term covering various subtypes of cell-
talking through the horizontal transfer of proteins, released membranous structures, such as exosomes,
microparticles, microvesicles, ectosomes and so on proliferation, autophagy and reduced the apoptosis of
[14]. The term “exosomes” commonly refers to a spe- breast cancer cells under hypoxia and serum depriva-
cific class of sEVs which are released by the exocytosis tion environment, and hence facilitated breast cancer
of multivesicular bodies and is different from “ecto- survival (data not published). Here, the regulatory role
somes” which are released from the plasma membrane of IL-35 on breast cancer cells was further explored.
or other similarly sized extracellular vesicles with We found that sEVs secreted by IL-35-stimulated
unknown biogenesis [39–41]. As the overlap between breast cancer cells had a stronger effect on promoting
the ranges of size of exosomes and ectosomes, these angiogenesis and indirectly led to tumour development
two extracellular vesicle types cannot be differentiated and progression than the unstimulated group.
by size alone [14,16]. Hence, according to the Minimal Recently, some studies have also paid increasing
information for studies of extracellular vesicles 2018 attention to the modulatory role of IL-35 in angiogen-
(MISEV2018), the term “sEVs” is the best descriptor esis. In pancreatic ductal adenocarcinoma, tumour-
for the target population of extracellular vesicles that derived IL-35 recruited the blood-derived monocytes
we discuss here (< 200 nm) [14]. In this study, the (BDMCs) into the tumour environment. These
diameter of these breast cancer cells-derived sEVs was BDMCs further contributed to angiogenesis through
around 100 nm and owned exosomal markers CD9 secreting angiogenesis factors, including vascular endo-
and TSG101. Therefore, we think that the main ingre- thelial growth factor A (VEGFA), tumour necrosis
dients of these sEVs are exosomes. Data from Zhang factor alpha (TNF-a) and basic fibroblast growth fac-
et al. [42] demonstrated that exosomal HMGB3 pro- tor [46–48]. In plasmacytoma, tumour-derived IL-35
tein secreted by nasopharyngeal carcinoma cells pro- increased myeloid-derived suppressor cells accumula-
moted tumour metastasis by inducing angiogenesis. tion which was a known cell type that promoted
Deng et al. [43] clearly showed that exosomal miR-155 angiogenesis via production of VEGFA [48–50].
derived from gastric carcinoma promoted angiogenesis According to the studies aforementioned, IL-35 was
by targeting the c-MYB/VEGF axis of endothelial involved in angiogenesis indirectly through targeting
cells. However, there were few studies illustrating the the immune cells in tumour microenvironment. In this
mechanism involving sEVs in breast cancer study, we confirmed that IL-35 accelerated the tumour
angiogenesis. angiogenesis via altering the mRNA profile of breast
In this study, we first isolated breast cancer cells- cancer cells-derived sEVs. However, studies in non-
derived sEVs and demonstrated that these sEVs could tumour disease displayed diverse effects of IL-35 in
be internalized by HUVECs and promoted HUVECs angiogenesis process. In rheumatoid arthritis (RA), IL-
proliferation, cell cycle progression and branched 35 inhibited angiogenesis by decreasing VEGF expres-
blood vessels formation ability. Similarly, in vivo sion along with its downstream Ang2 secretion in RA
experiments on mice also certified that local injection synovial tissue explants [51]. In the synovial tissues
of breast cancer cells-derived sEVs accelerated tumour from collagen-induced arthritis mice, IL-35 treatment
growth and angiogenesis in tumour tissue. depressed VEGF expression and two of its receptors,
Our previous researches showed that both tumour- Flt-1 and Flk-1 [52]. Based on these results, we
infiltrating lymphocytes (TILs) in breast cancer tissue hypothesized that IL-35 might act as a dual-directional
and breast cancer cells expressed and secreted inhibi- regulation factor depending on its target cells and the
tory cytokine IL-35. On the one hand, IL-35+ suppres- microenvironment, which deserves further exploration.
sive TILs (mainly IL-35+ Tregs) induced Bregs, while Actually, tumour angiogenesis induced by tumours
Breg-derived IL-35 also sustained Treg cells or CD8+ has been certified to suppress immune responses. One
Tregs [44,45]. On the other hand, breast cancer cells- of the mechanisms is to suppress leucocyte–endothelial
derived IL-35 mediated the Treg cells conversion to cell interactions, which causes tumour cells to escape
iTr35 in tumour microenvironment which led to cancer from immune surveillance. And anti-angiogenesis may
progression [20,21]. Thus, these different IL-35- recover leucocyte–endothelial cell interactions and
producing populations might promote reciprocal activ- increase the infiltration of immune cells [53]. The
ity through a positive feedback mechanism in the high effects of antitumour can be achieved by either inhibit-
IL-35 milieu. Meanwhile, IL-35 could create favour- ing vessels or increasing tumour lymphocyte infiltra-
able conditions for tumour cell growth and metastasis tion. The enhanced interaction between lymphocytes
by indirectly increasing the microvascular density [46]. and endothelial cell can let more immune cells enter
In addition to the above effects, we also demonstrated [54,55]. According to our study, immunohistochemistry
that IL-35 receptor was expressed on the surface of analysis can be used to detect the expression of vascu-
human breast cancer cells and IL-35 promoted lar endothelial cell marker CD31 in con-sEVs and
The FEBS Journal 289 (2022) 3489–3504 ª 2022 Federation of European Biochemical Societies
J. Liu et al.
IL-35 enhances the role of extracellular vesicles in breast cancer
3498
Fig. 7. Hub genes of the DEGs and signalling pathway identification. The mRNA levels of hub genes (RRAS, INSR, CALM1, THBS1, KDR,
PTEN and VEGFA) in HUVECs co-cultured with different sEVs were verified by qRT-PCR (A). qRT-PCR analysis of CALM1 expression in
HUVECs treated with siNC and siCALM1 (B). Representative images were taken and tube formation ability was quantified by HUVECs
treated with siNC, siCALM1, IL-35-sEVs (MCF-7) + siNC, IL-35-sEVs (MCF-7) + siCALM1, IL-35-sEVs (231) + siNC, IL-35-sEVs
(231) + siCALM1. Scale bar: 200 µm (C). The Ras/Raf/MEK/ERK signalling associated proteins in HUVECs treated by different sEVs
were analysed by western blot. And densitometry analysis was provided (D). Experiments were performed three times independently.
The Student’s t-test was used to analyse the difference. Data were presented as mean standard deviation (SD), (n = 3). *: P < 0.05, **:
P < 0.01, ***: P < 0.001.
IL-35-sEVs treated tumour tissues from nude mouse contributed to the stabilization of microtubule cyto-
model, which is used to assess whether the tumour skeleton in endothelial cells and led to blood vessels
shows more leaky and abnormal vessels after treated regeneration [63]. PTEN was defined as an orchestra-
by IL-35-sEVs. tor of vessel density by regulating Notch-PTEN signal-
It has been demonstrated that IL-35 has an effect ling axis [64]. The functions of INSR, CALM1, KDR
on site-specific H3K14 (histone 3 lysine 14) acetylation and VEGFA on angiogenesis and oncogenicity were
[56]. The addition of acetyl groups on histone tails also illustrated in many studies [26,65–67]. In the pre-
neutralizes the histone charges which weaken histone– sent study, the expression of RRAS, INSR, CALM1,
DNA interaction and relax the chromatin structure THBS1, KDR, PTEN and VEGFA was detected in
[57,58]. Through influencing site-specific H3K14 acety- HUVEC cells that co-cultured with sEVs. According
lation, IL-35 increases the binding of transcription fac- to the results, we found that IL-35-sEVs elevated the
tor AP-1 in the promoter of certain genes which lead proliferation and angiogenic ability of HUVECs by
to gene transcription activation [56]. Hence, IL-35 may the down-regulation of THBS1 and PTEN and up-
change the mRNA profile of both cells and sEVs regulation of RRAS, INSR, CALM1, KDR and
through histone acetylation and transcription factors VEGFA. Moreover, we silenced the expression of
activation. To elucidate the mechanism underlying the CALM1 in HUVECs before treated with IL-35-sEVs
promoting angiogenic effect of IL-35, we further ana- and the negative effect of siCALM1 on angiogenesis
lysed the mRNA profile of sEVs from breast cancer of HUVECs could be offset by adding IL-35-sEVs dis-
cells stimulated by IL-35 or not. Through comparing tinctly. In brief, our data certified that breast cancer
global mRNA expression profiles between IL-35-sEVs cells-derived sEVs facilitated angiogenesis and IL-35
and con-sEVs, we identified a series of DEGs. GO bio- strengthened the effect by modulating the expression
logical process enrichment and KEGG signalling path- of angiogenesis-related genes and activating Ras/Raf/
way analysis were conducted in this study to clarify MEK/ERK signalling pathway.
potential functions of these DEGs. According to the
results, DEGs mainly participated in biological pro- Materials and methods
cesses, such as cell proliferation and angiogenesis via
signalling pathways, including RAS and others. Ras, Ethics statement
Raf, MEK and ERK are the main cascading signals in
the RAS signalling pathway, and many researches The study involving animal was approved by Institutional
have been done to show that Ras/Raf/MEK/ERK sig- Animal Care and Use Committee of The Second Hospital,
nalling pathway promotes angiogenesis by contributing Cheeloo College of Medicine, Shandong University.
to the proliferation and invasion of endothelial cells
[28]. Our experiments confirmed that IL-35-sEVs acti- Cell lines and cell culture
vated the pathway via promoting the expression of
Human umbilical vein endothelial cells (HUVECs), breast
Ras, Raf, p-MEK and p-ERK. Moreover, we also cancer cell lines MDA-MB-231 and MCF-7 were purchased
identified the hub genes which were considered topo- from American Type Culture Collection. HUVECs were
logically important to the structure of the PPI network grown in Endothelial Cell Medium (ECM) (Science cell,
[59]. Among these hub genes, THBS1 has been widely USA). Two breast cancer cell lines, including MDA-MB-231
characterized in the tumour microenvironment. and MCF-7, were cultured in Roswell Park Memorial Insti-
THBS1 interacted with VEGF and MMPs and modu- tute (RPMI) 1640 Medium (Gibco, USA) supplemented with
lated the balance between pro- and anti-angiogenic sig- 10% fetal bovine serum (FBS) and 1% penicillin/streptomy-
nal transduction pathways, such as PI3K/AKT cin (Gibco, USA). All cells were cultured at 37 °C in a
pathway [60–62]. Moreover, PI3K/AKT signalling humidified incubator with 5% CO2. Recombinant human
pathway could be activated by RRAS which IL-35 was purchased from Peprotech (Rocky Hill, USA).
To avoid the influence of other extracellular vesicles, (1 : 1000, #2922), CyclinE1 (1 : 1000, #20808), p-ERK
FBS was centrifuged using an ultracentrifuge (Beckman (1 : 1000, #4370) and ERK (1 : 1000, #4695) were pur-
Coulter, USA). After centrifuged at 100 000 9 g for 12 h, chased from Cell Signaling Technology (Beverly, MA).
the pellet which contains extracellular vesicles was dis- CDK2 (1 : 1000, CY5020), Ras (1 : 1000, CY6672) and
carded and the supernatant was sEVs-depleted FBS. MEK (1 : 1000, CY5168) were obtained from Abways
(Shanghai, China). Raf1 (1 : 1000, E-AB-60020) was pur-
chased from Elabscience (Wuhan, China). P-MEK
Isolation and characterization of sEVs (1 : 1000, bs-3270R) was acquired from Bioss (Beijing,
Breast cancer cells stimulated by IL-35 (25 ngmL 1) or China).
PBS were cultured into RPMI 1640 Medium containing
10% FBS without EVs. The culture supernatants were col-
Quantitative real-time PCR
lected after 3 days. The achieved supernatants were differ-
entially centrifuged at 300 9 g for 10 min and 16 500 9 g Total RNA was extracted using TRIzol reagent (Invitro-
for 0.5 h, all at 4 °C. After filtration through a 0.2 lm fil- gen, USA) and reverse transcribed using ReverTra Ace Kit
ter, supernatants were ultracentrifuged at 100 000 9 g for (TOYOBO, Shanghai, China). Quantitative real-time PCR
2 h at 4 °C and sEVs collected from the pellets were resus- was conducted using SYBR Green Realtime PCR Master
pended with 10-mL PBS followed by another ultracentrifu- Mix (TOYOBO, Shanghai, China). Primer synthesis was
gation at 100 000 9 g at 4 °C for 2 h. Finally, the sEVs completed according to the sequence from Primer Bank
were resuspended in 200-lL PBS following quantization (https://pga.mgh.harvard.edu/primerbank/). GAPDH was
using RIPA Lysis Buffer (Beyotime, Haimen, China) and applied as the internal control standard for data
BCA Protein Assay Kit (Beyotime, Haimen, China). normalization.
Nanoparticle tracking analysis (NTA) Cell proliferation assay and cell cycle analysis
Nanosight tracking system (Zeta view, Germany) was Firstly, HUVECs (3000 cellswell 1) were seeded in a 96-
applied to assess the size distribution of sEVs. Isolated well flat-bottom plate and cultured with sEVs-depleted
sEVs were firstly resuspended with sterile phosphate saline FBS. Then, HUVECs were co-cultured with sEVs
buffer and then were analysed according to the manufac- (2.5 lgwell 1) from breast cancer cells stimulated by IL-35
turer’s recommendations. (IL-35-sEVs) or not (con-sEVs) for 24 h. For cell prolifera-
tion assay, each well was supplemented with 20 lL of
CCK8 solution (Beyotime, Haimen, China) and cultured
Transmission electron microscopy
for another 2 h. The proliferation of HUVECs was
Transmission electron microscopy was applied to observe assessed according to the absorbance values obtained from
the morphology of purified sEVs. Firstly, sEVs suspension the Microplate Reader (Bio-Rad, USA). To evaluate the
was loaded into a carbon-coated electron microscopy grid cell cycle distribution, HUVECs were firstly fixed and dyed
and fixed with glutaraldehyde and paraformaldehyde. After with propidium iodide/RNase buffer (Beyotime, Haimen,
stained with 2% methylamine tungstate, samples were China). Samples were analysed with a BD FACS Calibur
photographed under a Zeiss transmission electron micro- flow cytometer while data were processed with MODFIT
scope (Zeiss, Germany). software.
Western blot assay Labelling and internalization of sEVs

As we have previously depicted, samples were lysed using Initially, breast cancer cells were stimulated with IL-35
RIPA buffer. The isolated protein was quantified with (25 ngmL 1) or PBS in advance for 3 days and different
BCA Protein Assay Kit. Protein was separated with SDS- sEVs were collected from supernatants. SEVs (20 lg) iso-
polyacrylamide gel electrophoresis and transferred onto a lated from breast cancer cell culture supernatants were
polyvinylidene fluoride membrane (Merk-Millipore, Ger- firstly dyed with PKH26 Red Fluorescent (Sigma-Aldrich,
many). Subsequently, the polyvinylidene fluoride membrane USA) and then purified using an ultracentrifuge [68]. Sub-
was blocked with 5% non-fat milk. After blockage, the sequently, PKH26-labelled sEVs were resuspended in PBS
membrane was incubated with primary and secondary anti- and added into sEVs-free ECM medium that contained
bodies respectively. At last, bands were probed with chemi- HUVECs and incubated together at 37 °C for 4 h. To
luminescence [44]. Primary antibodies against CD9 examine the subcellular localizations of the sEVs, HUVECs
(1 : 1000, ab92726) and TSG101 (1 : 1000, ab125011) were were fixed with 4% paraformaldehyde and then were
obtained from Abcam (Cambridge, UK). CyclinD1 stained with DAPI (Solarbio, USA) for nucleus. Samples
were photographed under a Zeiss Laser Scanning Confocal peroxidase-conjugated secondary antibodies. Whereafter,
Microscope (Zeiss, Germany). the expression of Ki67 and CD31 was visualized using
DAB substrate kits.
In vitro tube formation assay
In vitro vascular formation analysis was evaluated with a Microarray analysis and data mining
Matrigel (BD, USA)-coated, flat bottom, 96-well plate. The sEVs were isolated from breast cancer cells and RNA
HUVECs (2 9 104 cellswell 1) were plated on the top of was extracted using trizol (Invitrogen, USA). RNA micro-
Matrigel and treated with sEVs or not. After incubating array analysis was conducted using Agilent Human
for 4 h, HUVECs were stained with Calcein-AM (Sigma- lncRNA+mRNA Array V4.0 by Shanghai Biotechnology
Aldrich, USA). Subsequently, cells were visualized under a Corporation. GENE SPRING Software was applied for subse-
Zeiss fluorescence inverted microscope (Zeiss, Germany). quent data pre-processing (Agilent Technologies, USA).
Tube formation ability was quantified based on the total Differentially expressed mRNAs (DEG) between the two
branch length of microscopic field which was calculated groups were identified with the following criteria: Fold
with IMAGE J software. changes ≥ 2 and Probe raw intensity ≥ 200. DEGs were
annotated using the Kyoto Encyclopedia of Genes and
In vivo Chick Embryo Chorioallantoic Membrane Genomes (KEGG) signalling pathway analysis and gene
(CAM) angiogenesis assay ontology (GO) biological process enrichment. The numbers
of DEGs included in each GO or KEGG term were
Fertilized chicken eggs were incubated at 38 °C with 70% counted, and the EASE score was calculated. Based on
humidity for 7 days. After disinfection of the shell centre EASE score, False Discovery Rate (FDR) and Enrichment
outside the air sac with benzalkonium bromide, a hole was Score were identified to assess the biological significance of
drilled above the vascularized areas. Sterilized filter-paper DEGs [69]. To further evaluate the protein–protein interac-
disks absorbed with suspension of sEVs (20 lg) were tion (PPI) information, DEGs were analysed using Search
implanted on the CAMs. Eggs were incubated for addi- Tool for the Retrieval of Interacting Genes [70]. Hub genes
tional 72 h at 38 °C incubator. After incubation, the vascu- were identified based on the node connectivity (degree)
larized areas of CAM were photographed using a using Cytoscape (University of California, USA) [59].
stereomicroscope (JNOEC, Nanjing, China). The numbers
of branch points in the vessels were counted to quantify
the blood vessel density using IMAGE J software. All animal Statistical analysis
studies were performed in accordance with protocols
GRAPHPAD PRISM 6 software was applied to perform statistical
approved by the Animal Investigation Committee of The
analysis. The Student’s t-test was used to assess the difference
Second Hospital, Cheeloo College of Medicine, Shandong
between groups and a P value < 0.05 was considered signifi-
University.
cant. Data were presented as mean standard deviation
(SD).
Mice model construction and in vivo experiment
Female Balb/c nude mice (4 weeks old) were purchased Acknowledgments
from Beijing Vital River Laboratory Animal Technology
This project was supported by the Natural Science
Co., Ltd. MDA-MB-231 cells (3 9 106) were injected into
Foundation of China (Grant No: 82172342, 31570919
the breast fat pad. After the subcutaneous tumour volume
growth exceed 100 mm3, PBS (sEVs-), sEVs from 231 cells
and 31270970), Natural Science Foundation of Shan-
and sEVs from 231 cells cultured with IL-35 (10 lg) were dong Province of China (ZR2019MC064) and Science
then injected into the subcutaneous tumour every 2 days. and Technology Innovation Project of Clinical Medi-
The tumour sizes were measured every 5 days. Tumour vol- cine of Jinan, P.R. China (202019146).
umes were calculated with the formula: length 9 width2 9
0.5. Forty-five days later, mice were sacrificed and tumours Conflict of interest
were fixed with formalin for further study.
The authors declare no conflict of interest.
Immunohistochemistry
Author contributions
Formalin fixed tissues were embedded in paraffin. Sections
were blocked by 3% hydrogen peroxide and incubated JL and ND. performed the experiments, researched
with primary antibodies, including Ki67 (Abcam, USA) the data and drafted the paper. NL, HZ, YL, HM and
and CD31 (Abcam, USA) followed by the horseradish HR contributed to data analysis and interpretation.
YF, JL, LD and HM contributed to the manuscript carcinoma-derived exosomes mediates angiogenesis by
critical revision, HM conceived and supervised the repressing a novel target gene TSGA10. Oncogene.
project, secured funds and edited the paper. All 2018;37:2873–89.
authors approved the final submitted manuscript. 10 Kong X, Li J, Li Y, Duan W, Qi Q, Wang T, et al. A
novel long non-coding RNA AC073352.1 promotes
metastasis and angiogenesis via interacting with YBX1
Peer review in breast cancer. Cell Death Dis. 2021;12:670.
The peer review history for this article is available at 11 Liu Y, Luo F, Wang B, Li H, Xu Y, Liu X, et al.
STAT3-regulated exosomal miR-21 promotes
https://publons.com/publon/10.1111/febs.16359.
angiogenesis and is involved in neoplastic processes of
transformed human bronchial epithelial cells. Cancer
Data availability statement Lett. 2016;370:125–35.
12 Lu C, Zhao Y, Wang J, Shi W, Dong F, Xin Y, et
All the DEGs analysed by RNA microarray and other
al. Breast cancer cell-derived extracellular vesicles
data that support the findings of this study are openly transfer miR-182-5p and promote breast
available from the corresponding author (maohai- carcinogenesis via the CMTM7/EGFR/AKT axis. Mol
ting@sdu.edu.cn) upon reasonable request. Med. 2021;27:78.
13 Wang J, He Z, Xu J, Chen P, Jiang J. Long noncoding
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IL-35 enhances angiogenic effects of small extracellular

vesicles in breast cancer

Keywords As an indispensable process for breast cancer metastasis, tumour angiogene-

Fig. 2. Uptake of sEVs derived from MDA-

Western blot assay Labelling and internalization of sEVs

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