Figure 1: High Galectin-1(Gal-1) expression is positively correlated with advanced and highly metastatic phenotype in OSCC, which is further enhanced by radiation and anoxia.

Xerostomia or severe dry mouth, is the most common long-term side effect ofradiotherapy (RT) in patients with head and neck cancer (HNC). RT-damaged salivaryglands fail to produce saliva, resulting in recurrent oral infections, dental decay, speechand swallowing dysfunction, leading to an overall deterioration in quality of lives. Currenttreatment strategies for xerostomia are inefficient in delivering long-lasting and effectiverelief to patients. We and others have demonstrated that salivary glands harbor adultstem/progenitor cells (SSPCs), which are important for regeneration and maintaininghomeostasis. In pursuit of new promising therapeutic agents, we focus on the possiblerole of exosomes in rescuing saliva function post RT. Exosomes regulates unidirectionalflow of information and have been shown to regulate epithelial progenitor expansionduring normal development of salivary glands. Previous studies have revealed thattreatment with extracellular vesicles (EVs), including exosomes and microvesicles,isolated from human cardiosphere-derived cells (CDC) led to reduced inflammation, lessfibrosis and enhanced regeneration in murine cardiac tissues after ischemic damage(Ibrahim et al, 2014). In this study, we evaluate whether CDC-EVs can stimulate SSPCself-renewal and improve saliva function post-RT. CDC-EVs treatment of murine andhuman SSPCs significantly increased salisphere formation, which reflect in vitro self-renewal ability of SSPC. More importantly, the CDC-EVs treatment enhanced salisphereformation in culture post RT when compared to either vehicle control or treatment withfibroblast derived EVs. This effect appeared to be specific to SSPCs, as treatment withCDC-EVs did not enhance proliferation, migration and colony formation of head and neckcancer cells. Work are ongoing to evaluate CDC-EVs’ ability to rescue saliva function invivo when administered locally to the murine submandibular gland either before or afterradiation treatment. Since EVs from other sources are being tested in clinical trials, it hasthe potential to rapidly translate to human study as a novel therapy for the management ofradiation-induced xerostomia in HNC patients.

ABSTRACT

• CDC derived EVs improves the sphere formation efficiency of mouse as well as human SSPCs• CDC derived EVs protect the murine primary as well as cultured SSPCs from the harmful effects of irradiation• CDC derived EVs treatment results in downregulation of pro-apoptotic genes in vitro • CDC derived EVs does not exert any influence on proliferation, migration and colony formation of HNC cells in vitro

SUMMARY OF KEY FINDINGS

Figure 1:A) Graph representing the cost pertreatment versus effectiveness of current treatmentmodalities for Xerostomia (Sasportas et al, 2013). B)Improvement in saliva function and acinarregeneration upon GDNF treatment in vivo (Xiao et al,2014). C) Effect of CDC-EVs on myocardial infractionmodel (Capricor therapeutics, LA, Ibrahim et al, 2014)

Vignesh Viswanathan1, Ahu Turkoz1, Hongbin Cao1, Joshua Bloomstein1, Dhanya Nambiar1, Kiel Peck2, Luis Rodriguez-Borlado2 and Quynh Thu Le1

1Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA.2 Capricor Therapeutics, Beverly Hills, CA.

Evaluation of the role of extracellular vesicles for treatment of Xerostomia in patients with Head and Neck Cancer

BACKGROUND

Murine and human SSPCs can be isolated and maintained in culture in vitro

Figure 3: (A) Image of 2D murine salivaryspheres treated with CDC EVs, HDF EVs andVehicle at day 10.(B) Graph representingquantification of average number of spheresnormalized to the vehicle control (N=4) (C)Human salivary spheres treated with CDC Evs,HDF EVs and vehicle control at day 7.(D)Quantification of average number of humansalivary spheres normalized to vehicle control(N=5). (E) Immunofluorescence imagerepresenting the uptake of FITC labelled EVsby Tomato Red positive murine SSPCs after 3hours of incubation. Error bars represent SEM.

Xerostomia/Dry mouth in Head and Neck cancer patients

• HNC is the 6th most common type of Cancer• Treatment primarily includes either surgery + radiotherapy (RT) or RT +

chemotherapy • Serious side effects such as Xerostomia is associated with RT treatment• Loss of salivary function -> difficulty in chewing, eating, food digestion, dental

decay, bone infection & deterioration of quality of life• Our group has demonstrated the effect of specific growth factors and activator

molecules in protecting salivary gland stem/progenitor cells (SSPCs) from the radiation damage

Figure 2: (A) Representative scheme of isolation of murine SSPCs. (B) Representative images of murine salivary spheres in 2D and3D culture at day 10. (C) FACS analysis of murine SSPCs grown in 2D culture showing maintenance of stem cell phenotyperepresented by EpCAMhigh/CD24high population.

CDC EVs significantly improve the sphere formation efficiency of murine and human SSPCs in vitro

CDC EVs exert a radioprotection role on murine SSPCs

Figure 4: (A) Schematic showing in vitro irradiation of murine SSPCs with 4 Gy followed by EV treatment. Thegraph represents the average number of murine SSPCs normalized to the vehicle control at day 10 (N=3) (B)Schematic showing in vivo irradiation of mouse salivary glands with 15 Gy IR. The glands were isolated 7 dayspost IR and salivary EpCAMhigh cells were treated with CDC EVs and controls. The graph represents the averagenormalized sphere count at day post treatment (N=2) (C) Relative mRNA expression of pro-apoptotic genes bax,puma and noxa in murine SSPCs analyzed 6 hours post exosome treatment in vitro (N=2). Error bars representSEM.

CDC EVs does not effect characteristics of Head and Neck cancer cells

Figure 5: (A) Proliferation assay results of SCC90 and SCC25 cells post treatment with EVs and vehicle (N=3) (B) colonyformation assay showing SCC90 colonies at day 10 post EV treatment fixed and stained with crystal violet.(C) Averagenumber of colonies of SCC90 and FADU at day 10.(D) Graph displaying average of SCC90 cells migrated 24 hours postEV and vehicle treatment. Error bars represent SEM.

A) B)

B)

A)

C)

C) D)

E)

A) B)

C)

RESULTS

A)

B)

C) D)

REFERENCES:• Ibrahim AG, Cheng K, Marban E (2014) Exosomes as critical agents of cardiac regeneration triggered by cell therapy. Stem Cell Reports 2(5):606–619 79.• Sasportas LS, Hosford DN, Sodini MA, Waters DJ, Zambricki EA, Barral JK, et al. Cost-effectiveness landscape analysis of treatments addressing xerostomia in patients receiving head and neck

radiation therapy. Oral Surg Oral Med Oral Pathol Oral Radiol. 2013;116(1):e37–51.• Xiao, N., Lin, Y., Cao, H. et al, Neurotrophic factor GDNF promotes survival of salivary stem cells. J Clin Invest. 2014;124:3364–3377.

**

C)

Ann-Sophie Walravens1, Hocine Rachid2, Kiel Peck1, Linda Marban1, Reem Al-Daccak2, Luis Rodriguez-Borlado1

1Capricor, Inc. 8840 Wilshire Blvd. 2nd Floor. Beverly Hills. CA 90211;2HLA et Medicine Jean Dausset Laboratory, Hopital Saint Louis, Paris, France.

CONCLUSIONS

REFERENCES

CDCs-EVs show immunomodulatory capabilities in vitro and improve

survival and clinical score in a GVHD mouse model

Cardiosphere derived cells (CDCs) have immunomodulatory capabilities, limit fibrosis and promote tissue

regeneration. Previous studies showed that extracellular vesicles from CDCs (CDC-EVs) recapitulate most of

the activities mediated by CDCs both in vitro and in vivo. Here we show that:

• CDC-EVs modulate the immune response of in vitro activated T cells. The results suggest the implication of

PD-L1/PD1 pathway in the immunomodulation observed.

• CDC-EVs upregulate the expression of anti-inflammatory genes in activated mouse macrophages in a dose

dependent manner.

• Repeated systemic dosing of CDCs-EV in a GVHD model reverted the weight loss observed in vehicle

treated mice, increased survival and reduced GVHD score.

Figure 6. CDC-EVs improve GVHD.

A) Experimental design: Mice with

GVHD were treated with vehicle or

two different doses of CDC-EVs. B) GVHD score. Data presented as mean ± SEM. C) Percent body weight relative to Day 0. Data presented as mean ± SEM. D) Survival was

tracked for the duration of the

study and percent survival for all

groups was plotted. * p<0.05

ABSTRACT

1. Aminzadeh et al. (2018). Stem Cell Reports. Vol 10 (3): 942-955

INTRODUCTION

MATERIALS AND METHODS

RESULTSCDC-EVs immuno-modulatory activity on T-

Cells

CDC-EVs immunomodulate Macrophage activity

CDC-EVs improve survival in a GVHD model• CDCs are derived from heart tissue.

0

Days

g radiationFollow up

Sacrifice

Histology

35 43 50 54

Transplant of:5x106 CD3- BM cells2x106 splenocytes

282147

High dose1x1010 EVs

Low dose1x109 EVs

3x109 EVs

n=15 (3x)

A

2 1 2 8 3 5 4 2 4 9 5 6

2

4

6

8

D a y

GV

HD

Sc

ore

V e h ic le

L o w d o s e

H igh D o se

21 28 35 42 49 56Days

8

6

4

2

GV

HD

Sco

re

GVHD Score

Vehicle

Low dose

High dose

B Percentage Weight Change

2 1 2 8 3 5 4 2 4 9 5 6

-3 0

-2 5

-2 0

-1 5

-1 0

-5

D a y

Pe

rc

en

t w

eig

ht c

ha

ng

e

V e h ic le

L o w d o s e

H igh D o se

*

**

**

*

**

*

21 28 35 42 49 56Days

-5

-10

-15

-20

-25

-30

% W

eig

ht

chan

ge

Vehicle

Low dose

High dose

C

2 1 2 8 3 5 4 2 4 9 5 6

0

2 5

5 0

7 5

1 0 0

D a y

Pe

rc

en

t s

urv

iva

l

V e h ic le

L o w d o s e

H ig h D o s e

21 28 35 42 49 56Days

100

75

25

0

% S

urv

ival

50

Vehicle

Low dose

High dose

SurvivalD

3% Brewer’s Thioglycollate (i.p.)

72h Isolation peritoneal macrophages

Plating 2x106

cells / well on a 6-well plate

Gene expression of Arg1 and Nos2

6h1h Dosing CDC/MSC-EVs per cell

K-means (K=3)

MSC1 15dMSC2 15dMSC3 15dMSC4 15dMSC3 48hMSC4 48h

CDC1CDC2CDC8CDC3CDC7CDC9

CDC4.1CDC11

CDC4.2CDC5

CDC10.1CDC10.2

CDC12CDC6

MSC

1 1

5d

MSC

2 1

5d

MSC

3 1

5d

MSC

4 1

5d

MSC

3 4

8h

MSC

4 4

8h

CD

C1

CD

C2

CD

C8

CD

C3

CD

C7

CD

C9

CD

C4

.1C

DC

11

CD

C4

.2C

DC

5C

DC

10

.1C

DC

10

.2C

DC

12

CD

C6

Figure 4. CDC-EVs upregulate anti-

inflammatory genes in activated

mouse macrophages. A)

Experimental design: B) Arg1:Nos2

mRNA expression ratio in activated

macrophages treated with EVs from

MSC or from potent and non-potent CDCs. Data presented as mean ± SEM. * p<0.05

Figure 5. Unsupervised clustering analysis using repeated downsampling datasets. For each sample pair, the color code indicates the percentage of downsamplingtrials where the two samples are grouped into the same cluster. Results for K-means clustering are shown. The number of clusters was set to 3. MSC-EVs clustered separately from CDC-EVs

Vehicle

CDC exosome-treated

Co

un

ts

CFSE

100 101 102 103 104 1050

100

200

300

400 1h

100 101 102 103 104 105

4h

100 101 102 103 104 105

16h

T T

T

T

MoMo

NK

NKB

B

BT

TT

T

Human PBMC T cells

Negative

Selection

PHA

CDC-EVs

T

T T

T

T T

T

T T

Proliferation quantification

Mediu

mPHA

CDC

5x109 E

V/ml

10x109 E

V/ml

20x109 E

V/ml

0

20

40

60

80

100

% p

rolif

erat

ing

cells

CD4+CD8+

*

****

***

****

*

Mediu

mPHA

CDC

5x109 E

V/ml

10x109 E

V/ml

20x109 E

V/ml

0

20

40

60

80

100

% p

rolif

erat

ing

cells

CD4+CD8+

*

****

***

****

*

PHA

0

100

200

300

400

0

100

200

300

400

• Human T lymphocytes activated by PHA were treated

with CDC-EVs. T-cells were labeled with CFSE and

proliferation analyzed by flow cytometry.

• Macrophages were isolated from peritoneal exudates

from mice primed with 3% Brewer’s thioglycolate

and phenotype tested by PCR.

• EVs small RNA sequencing:

• RNA was isolated using Serum/Plasma kit.

• Next Gen 500 Sequencing (Illumina)

• 30 ng of RNA was used per sample

• GVHD was created after delivery of allogeneic bone

marrow cells and splenocytes in mice. Mice were

randomized and systemically injected with CDC-EVs

or vehicle. Mice were sacrificed after 56 days of

follow up.

Donor heart (OPO) Explant formation Cardiosphere formation CDCs

• CDCs improve cardiac function and exercise

capabilities in a Duchenne muscular dystrophy

(DMD) mouse model1.

• Early immune infiltration can contribute

substantially to the initiation and progression of

muscle pathology. Activated T cells and

macrophages play a critical role in muscle wasting.

• CDCs are cleared shortly after administration and

paracrine signals play an essential role in the

activation of the regenerative pathways.

• EVs from CDCs are able to recapitulate most of the

effects mediated by the cells.

• EVs isolation from CDCs and MSCs.

10kDa MWCO0.45 µm PESP5 CDCs Serum-free medium Concentrated CM with EVs

Storage

at -80°C15 d

• CDC-EVs have strong immunomodulatory capabilities on human activated T-lymphocytes

• EVs from CDCs showed a stronger upregulation of anti-inflammatory genes in activated mouse macrophages when compared to MSC.

• Repeat dosing of CDC-EVs is effective increasing survival in a GVHD mouse model and reducing GVHD score.

• These data support the use of CDC-EVs for the treatment of inflammatory indications.

Figure 3. CDC-EVs immunomodulate T cells response. A) CDC-EVs were

labeled with CFSE and incubated with Jurkat T cells (25x103 particles per cell).

EVs uptake was evaluated after O/N culture by flow cytometry. B) Experiment

design to evaluate CDC-EVs immunomodulatory activity on primary human T-cells. C) Immuno-modulation of PHA-induced T cell proliferation by CDC-EVs.

Results are mean percentage ± SEM from 3 different donors each done in triplicate. *

p<0.05, *** p<0.005, **** p<0.001.

A B

C

Arg

1 :

No

s2 r

atio

A

B

*

Exosomal miRNA of CDC-EVs and MSC-EVs

% p

rolif

era

tin

g ce

lls