A. Pessina,2017

AUGUSTO PESSINA

Department of Biomedical, Surgical and Dental Sciences

By Swiss Stem Cell Foundation

***************************

June the 13th 2017,

Techno Park Zurich, Technoparkstrasse 1, Zurich

ASCs cell therapy for antitumor drug

delivery

A. Pessina,2017

A summary of main characteristics of MSCs

• Isolated by Friedenstein et al. (1974) , named CFU-F.

• Adherent cells with fibroblast-like morphology (PDT=

33-48 hours).

• Positive for CD44, CD73, CD90, CD105, STRO-1, CD166, HLA-I and many other aspecific markers.

• Negative for hematopoietic markers as CD14, CD34 and CD45.

• Present in all organs and tissues.

• Showing high plasticity (multipotency).

• Having significant immunosuppressive /anti-

inflammatory activity.

2

• Easy to isolate.

• Easy to expand and manipulate in vitro.

• Have migration capacity (homing tissues across

endothelium).

A. Pessina,2017 3

The characteristics of MSCs strongly increased the interest

to use them to deliver antitumor agents by creating

engineered MSCs able to secrete therapeutic cytokines or

inhibitory factors

About MSCs and drugs delivery

MSCs have been

genetically modified

to become vehicles

of anti-tumoral

molecules:

interleukins,

interferons,

prodrugs, oncolitic

viruses,

anti-angiogenic agents,

pro-apoptotic proteins

and growth factors

antagonists 3 (From Shah K, 2012).

A. Pessina,2017

Our past observations from studies on drug dependent myelotoxicity in cancer treatment (Pessina et al., Life Sci, 1999)

1. Murine bone marrow stromal cells (SR4987) showed high resistance to

Doxorubicin

2. They were able to internalize DRX without showing any sign of toxicity.

3. BM-HSCs (very sensitive to DXR) co-coltured with stromal cells ( treated

with DXR) were dramatically inhibited in their proliferation

These data suggested that

4

a. MSCs were able to uptake and then release anti-tumoral drugs.

b. That MSCs ( without any genetic manipulation) could be a vehicle for drug delivery.

A. Pessina,2017

Why Paclitaxel as first line drug for our studies

• Paclitaxel (PTX) is one of the most effective chemotherapeutic drug used to treat

a broad range of cancers (lung, breast, ovarian) and also having an important

anti-angiogenic activity

• PTX blocks the cell division BUT NOT INTERFERE WITH the mechanism of cell

uptake/release.

• PTX formulated in organic solvents are known to cause serious side effects

(hypersensitivity reactions, anemia and cardiovascular events)

5

The development of a PTX cell delivery system could

- increase in situ drug concentration and bioavailability

- reduce systemic toxicity.

A. Pessina,2017

The uptake-release capacity of MSCs has been confirmed in different experimental conditions

6

In DIFFERENT TUMOR MODELS

• in vitro : on mieloma, glioma, pancreatic ca, sarcoma, mesothelioma….

• in vivo : on leukemia, prostatic ca, melanoma and lung metastasis….

THIS PRESENTATION WILL REPORT ONLY THE MAIN RESULTS ON

PACLITAXEL THAT WAS THE MORE EXTENSIVELY INVESTIGATED

DRUG

By using MSCs FROM DIFFERENT TISSUES

•bone marrow,

•adipose tissues,

•gingival papilla,

•cord blood ,

•skin fibroblasts

•placenta

•circulating CD14 + dendritic cells

By using DIFFERENT DRUGS

•paclitaxel,

•vincristine

•doxorubicin,

•gemcitabine

A. Pessina,2017

IN VITRO STUDIES

A. Pessina,2017

First Step: THE SENSITIVITY OF hMSCs TO PACLITAXEL

This suggested to prime hMSCs with 2,000 ng/ml in order to

- block the proliferation

- maintain 80% cell viability allowing cells to uptake/release the drug.

Proliferation Cytotoxicity

ng/ml IC50 IC90

BM-MSCs 4.07±1.75 > 1000

AT-MSCs 2.55±1.02 > 1000

0

20

40

60

80

100

120

0 0,1 1 10 100 1000

Ce

ll P

rolif

era

tio

n (

% o

f co

ntr

ol)

Paclitaxel (ng/ml)

BM-MSCs

AT-MSCs

0

20

40

60

80

100

120

0 100 1000 10000

Ce

ll vi

abili

ty (

% o

f co

ntr

ol)

Paclitaxel (ng/ml)

BM-MSCs

AT-MSCs

7

hMScs are SENSITIVE TO THE ANTI-

PROLIFERATIVE ACTIVITY But RESISTANT

TO PTX DIRECT CYTOTOXICITY

(> 10,000 ng/ml)

A. Pessina,2017

The procedure for loading MSCs with PTX and testing their anticancer efficacy

Paclitaxel

2,000 ng/ml

Sub confuent

MSCs cultures

+

PTX loaded MSCs (MSCsPTX)

24 h incubation 37°C, 5% CO2

Trypsinized and Washed

PTX release into culture medium ( PTX-CM)

8

Subcultured

In vitro studies

- anti-cancer

- anti-angiogenic potential of MSCsPTX

In vivo studies

(murine models)

Biological dosage of PTX-CM activity on

A. Pessina,2017

MASS SPECTROPHOTOMETRY excluded the

presence of the two main hydroxylated

metabolites but IDENTIFIED THE PRESENCE OF

7-EPITAXOL (A PTX ISOMER HAVING

PHARMACOLOGICAL ACTIVITY IDENTICAL TO PTX).

THE RELEASE OF PACLITAXEL

IN THE CONDITIONED MEDIA WAS

CONFIRMED BY HPLC ANALYSIS .

I.S.

PTX

↓

hMSCsPTX-CM

I.S.

hMSCs-CM CTR

I.S.

PTX

↓

Standard PTX in PBS

I.S.=internal standard (cephalomannine)

XIC of +MRM (4 pairs): 854.0/509.0 amu from Sample 48 (Campione 3 ) of Datataxolo terreni 27... Max. 5.0 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5Time, min

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

2400

2600

2800

3000

3200

3400

3600

3800

3955

In

te

ns

ity

, c

ps

3-OH/6-OH-PTX exclusion

XIC of +MRM (4 pairs): 854.0/509.0 amu from Sample 27 (terreno 20ptx d5 10ptx) of Datatax... Max. 1340.0 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5Time, min

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5405

In

te

ns

ity

, c

ps

1.94

PTX

d5

PTX d5 NH4

PTX

PTX NH4

Adduct NH4 exclusion

XIC of +MRM (2 pairs): 854.0/509.0 amu from Sample 18 (Campione 6 ) of Datataxolo terren... Max. 1012.0 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5Time, min

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

6000

In

te

ns

ity

, c

ps

1.74

1.99

PTX d5

PTX PTX2

PTX 2 peak?

PTX

7-epi-PTX

A. Pessina,2017

The activity of PTX released in the conditioned medium was

evaluated by a “biological dosage assay” on leukemia cells

as target

The CM from PTX primed hMSCs produced a dose-dependent inhibition of MOLT-4 cells.

A PTX equivalent concentration (PEC) of

the drug released in the conditioned

medium was estimated by

an algorithm

that compare the IC50 values of pure PTX

and that of the conditioned medium .

PEC (ng/mL) = (IC50PTX/V50) x 100

PEC (pg/cell) = PEC (pg/ml) x CM vol(ml)/nr

cells seeded

The release of PTX is time-dependent

Anti-proliferative effect on MOLT-4

0 1/128 1/64 1/32 1/16 1/8 1/4 1/2 1

0

20

40

60

80

100

120

0 0,098 0,195 0,391 0,781 1,563 3,125 6,25 12,5

Dilution Factor of CM

Ce

ll P

rolif

era

tio

n (

% o

f co

ntr

ol)

Paclitaxel (ng/ml)

PTX

AT-MSCs

BM-MSCs

9

PEC range: from 0.2 to 0.8 pg/cell

Therefore :

10<6 cells can deliver from 200 to 800 ng of PTX.

A. Pessina,2017

In vitro anti tumor activity of conditioned medium (---------) from PTX primed hMSCs was confirmed on four human cancer cell lines

1/128 1/64 1/32 1/16 1/8 1/4 1/2 1

0

20

40

60

80

100

0,39 0,78 1,56 3,13 6,25 12,5 25 50

Dilution Factor of CM

Cell

prol

ifera

tion

(% o

f con

trol

)

PTX (ng/ml)

CFPAC-1

PTX (standard curve)

BM-MSCsPTX-CM

IC50= 3.97±4.48 ng/ml

IC90= 39.34±34.61 ng/ml

1/128 1/64 1/32 1/16 1/8 1/4 1/2 1

0

20

40

60

80

100

0,39 0,78 1,56 3,13 6,25 12,5 25 50

Dilution Factor of CM

Cell

prol

ifera

tion

(% o

f con

trol

)

PTX (ng/ml)

DU-145

PTX (standard curve)

BM-MSCsPTX-CM

IC50= 9.13±4.54 ng/ml

IC90= 22.05±14.24 ng/ml

1/128 1/64 1/32 1/16 1/8 1/4 1/2 1

0

20

40

60

80

100

0,39 0,78 1,56 3,13 6,25 12,5 25 50

Dilution Factor of CM

Cell

prol

ifera

tion

(% o

f con

trol

)

PTX (ng/ml)

SK-ES-1

PTX (standard curvel)

BM-MSCsPTX-CM

IC50= 1.70±1.11 ng/ml

IC90= 2.81±2.55 ng/ml

1/128 1/64 1/32 1/16 1/8 1/4 1/2 1

0

20

40

60

80

100

0,39 0,78 1,56 3,13 6,25 12,5 25 50

Dilution Factor of CM

Cell

prol

ifera

tion

(% o

f con

trol

)

PTX (ng/ml)

T98G

PTX (standard curve)

BM-MSCsPTX-CM

IC50= 5.43±3.3 ng/ml

IC90= 20.11±18.43 ng/ml

13

A. Pessina,2017 13

0

20

40

60

80

100

120

CTRL 1:100 1:10 1:1

Ce

ll P

rolif

era

tio

n (

% o

f co

ntr

ol)

hMSCs/MOLT-4 ratio

MOLT-4

hMSCs normal hMSCs/PTX CTRL

*

** **

*

0

20

40

60

80

100

120

CTRL 1:100 1:10 1:1

Ce

ll P

rolif

era

tio

n (

% o

f co

ntr

ol)

hMSCs/MOLT-4 ratio

MOLT-4

BM-MSC normal

BM-MSC/PTX

CTRL

*

** **

*

MOLT-4

0

20

40

60

80

100

120

CTRL 1:100 1:10 1:1

Ce

ll P

rolif

era

tio

n (

% o

f co

ntr

ol)

hMSCs/DU145 ratio

DU-145

BM-MSC normal

BM-MSC/PTX

CTRL**

***

*

DU145

0

20

40

60

80

100

120

CTRL 1:100 1:10 1:1C

ell

Pro

life

rati

on

(%

of

con

tro

l)

hMSCs/T98G ratio

T-98G

BM-MSC normal

BM-MSC/PTX

CTRL

*

**

**

**

T98G

0

20

40

60

80

100

120

140

CTRL 1:100 1:10 1:1

Ce

ll P

rolif

era

tio

n (

% o

f co

ntr

ol)

SR4987/B16 ratio

B16

SR4987 normal

SR4987PTX

CTRL

**

**

B16

0

20

40

60

80

100

120

140

CTRL 1:100 1:10 1:1

Ce

ll P

rolif

era

tio

n (

% o

f co

ntr

ol)

SR4987/B16 ratio

B16

SR4987 normal SR4987PTX CTRL

**

**

hMSCsPTX inhibited proliferation of MOLT-4, T98G and DU145 in a dose-

dependent way.

murine cells “per se” are able to inhibit B16 proliferation.

THE DIRECT IN VITRO ANTICANCER ACTIVITY OF PTX LOADED MSCs

WAS TESTED IN A CO-COLTURE ASSAYS ( MSCs + Tumor cells)

A. Pessina,2017

B A C

Co-colture of hMSCs and MOLT-4

hMSCs and MOLT-4. hMSCsPTX and MOLT-4.

15

In vitro killing of leukemia cells by hMSCsPTX studied by

rosetting phenomenon (Pessina et al., JBH, 2013)

When rosettes were subcultured on

slides. ONLY rosettes obtained with

PTX primed MSCs indicated clear

damages of leukemia cells .

MSCs mixed with leukemia

and maintained in

suspension formed a

central nucleus of hMSCs

surrounded by a “crown” of

LCs

A. Pessina,2017

Several areas of contact between hMSCs and LCs, but no apparent specialization of the plasma membrane such as junctional structures at cell-cell interface. LCs bound to untreated hMSCs maintained a round-shaped phenotype.

hMSCs + LCs

hMSCsPTX + LCs

TEM

LCs adhering to hMSCsPTX, showed marked changes of degeneration in nuclear and cytoplasmic ultrastructure (vacuoles, condensed chromatin, cell emptying and lysis). 16

Trasmission Electron microscopic analysis of Rosette formed by hMSCs and LCs

A. Pessina,2017

SEM

hM

SCs

+ LC

s h

MSC

sPTX

+ L

Cs

LCs displayed a villous appearance and were randomly attached, individually or in groups to hMSCs.

LCs adhering to hMSCsPTX undergone great degeneration or death.

17

Scanning Electron microscopic analysis of Rosette formed by hMSCs and LCs

A. Pessina,2017

DIRECT EFFECT OF PTX PRIMED MSCs WAS STUDIED ON

MYELOMA IN A 3D CULTURE SYSTEM Hematology Oncology Bonomi et al. 2016, DOI 10.1002/hon 2360

INCREASE OF

CELL

MORTALITY

AND SIGNS OF

CELL DEATH

A. Pessina,2017 18

CTRL

+ hMSCs-CM

+ hMSCsPTX-CM

+ PTX 5 ng/ml

PTX ng/ml

IC50=4.69 ng/ml

20

25

30

35

40

100 10 1

nr

of

ECs

x10

4

CTRL HUVECs HMECs

** **

** **

45

ECs seeded (day 0)

HUVECs HMECs

20

25

30

35

40

1:2 1:4 1:8

nr

of

HM

ECs

x10

4

CM dilutions

** **

*

**

HMECs seeded (day 0)

45

20

25

30

35

40

1:2 1:4 1:8

nr

of

HU

VEC

s x1

04

CM dilutions

45

**

**

*

**

HUVECs seeded (day 0)

20

40

60

80

100

1:2 1:4 1:8

CM dilutions

nr

of

cap

illar

ies/

rin

g

**

**

160 **

CTRL medium

+hMSCs-CM

+hMSCsPTX-CM

VEGFa 20ng/ml

CTRL medium +hMSCs-CM 1:2 +hMSCsPTX-CM 1:2

ECs sensitivity to PTX ECs proliferation with

hMSCsPTX-CM

ANTI-ANGIOGENIC ACTIVITY OF CM FROM PTX PRIMED MSCS WAS

CONFIRMED ON UVEC AND DERMA MICROVASCULAR ECS

Aorta ring assay : after 7 days of culture CM of

MSCsPTX induced capillary regression and

zones of vessel necrosis

A. Pessina,2017

THE DIRECT ANTI-ANGIOGENIC ACTIVITY OF hMSCS/PTX

WAS CONFIRMED IN CO-COLTURE WITH ENDOTHELIAL

CELLS

20

25

30

35

40

1:1 1:5 1:10 hMSCs/HUVECs ratio

nr

of

HU

VEC

s x1

04

**

**

*

45

HUVECs seeded (day 0)

HUVECs

20

25

30

35

40

1:1 1:5 1:10 hMSCs/HMECs ratio

nr

of

HM

ECs

x10

4

**

**

*

45

HMECs seeded (day 0)

HMECs CTRL

+hMSCs

+hMSCsPTX

72h

20x

48h

20x

24h

20x

Time 0

20x

hMSCsPTX/HMECs 1:5

19

At ratio 1:5 primed hMSCs start to kill endothelial

cells during the first 24h of culture .

72h

20x

48h

20x

24h

20x

Time 0

20x

72h

20x

48h

20x

24h

20x

Time 0

20x

After 72 hs very few

cells survived

A. Pessina,2017

IN VIVO STUDIES

A. Pessina,2017

** p< 0.01 versus saline treated mice (controls)

All mice treated with LCs either mixed with control hMSCs or with PTX (2µg/ml), developed a solid tumor.

20

Experimental group Nr of LC

injected

Tumor

model

Ratio

BM-MSCs/TCs

Tumor

weight

(grams±sd)

Tumor/tot

al#

CO-INJECTION BM-MSCs and TCs

Control

2x106 MOLT-4

- 2.55 ±

0.430 5/5

+ PTX 2000 ng/ml - 2.10 ±

0.212 3/3

+ BM-MSCs 1:5 2.32 ±

0.456 5/5

+BM-MSCsPTX 1:5 -** 0/5

The firtst preliminary study Subcutaneous co-inoculum with leukemia cells

Mice injected with LCs mixed with

hMSCsPTX LCs were 100% tumor free

A. Pessina,2017

Second Study : Subcutaneous co-inoculum with human prostate Ca (DU145) and mouse melanoma (B16)

20

40

60

80

100

12 15 17

DU145

DU145+hMSCs

DU145+hMSCsPTX

% o

f m

ice

wit

h t

um

or

Days after cells transplant

**

19 21

0

** *

8 6 4 0 0

B16

B16+hMSCs

B16+hMSCsPTX

*

*

Days after cells transplant 6 8 10 13

Tum

or

volu

me

/ m

m3

50

100

200

400

600

800

1,000

1,200 B16 B16+hMSCs

B16+hMSCsPTX

**

*

B16

+hMSCs

+hMSCsPTX 1 cm

Days after cells transplant

Tum

or

volu

me

/ m

m 3

19 39

50

100

200

400

600 800

1,000

1,200 1,400

1,600

21 23 25 27 29 31 33 35 37

DU145 DU145+hMSCs DU145+hMSCsPTX

**

* **

1 cm

° °

°

DU145

+hMSCsPTX

+hMSCs

Tumor growth of DU145 in nod scid mice

Tumor growth of B16 in

nude mice

A. Delay in tumor takes of DU145 and B16 in mice

21

0.468 ± 0.102*

0.657 ± 0.245*

C. 25% OF MICE DID

NOT DEVELOP

TUMORS

B. TUMOR GROWTH

WAS DRAMATICALLY

REDUCED.

A. Pessina,2017

Third Study : Subcutaneous co-inoculum of GFP+MSCs and RFP+ human glioblastoma

EFFECT OF GFP+SR4987 ON RFP+U87MG

XENOGRAFTS

RFP+ U87

RFP+ U87/

GFP+

SR4987

RFP+ U87/

GFP+SR4987- PTX

RFP+U87/

GFP+SR4987

RFP+ U87/

GFP+SR4987-

PTX

vimenti

n H&E

0

2

4

6

8

10

12

14

16

18

20

2 4 6T

um

or

dia

me

ter

(mm

)

weeks after grafting

RFP+ U87

RFP+ U87/GFP+ SR4987

RFP+ U87/GFP+ SR4987-PTX

0

2

4

6

8

10

12

14

16

18

20

2 4 6

Tu

mo

r d

iam

ete

r (m

m)

weeks after grafting

RFP+ U87

RFP+ U87/GFP+ SR4987

RFP+ U87/GFP+ SR4987-PTX

*

*

*

**

*

Tumor histology at 2 weeks

Tumor histology at 4 weeks

23

The not primed stromal cells appear to be

mixed with the glioblastoma cells without

any propensity to organize into secondary

structures

The primed stromal cells (green)

arranged themselves in strands and

columns to form nets entrapping red

glioblastoma cells.

The xenograph

glioblastoma

+primed

stromal cells

were

significantly

smaller

A. Pessina,2017

1. GREEN MSCs GRAFTED IN BRAIN ADJACENT TO GLIOMA RED CELLS SHOWED A REMARKABLE TROPISM TOWARDS THE TUMOR

2. PTXMSCs RETAIN SOME TROPISM AND PRODUCED MULTI SPINDLE

MITOSIS , CENTROSOME NUMBER ALTERATIONS AND NUCLEAR

FRAGMENTATION (Pacioni et al. Stem Cell Res Ther, 6,194,2015)

A. Pessina,2017 Coccè V., 2013

After two months when the tumor mass was detectable : Intratumor (i.t.) treatment with hMSCsPTX inhibit the growth of an established tumour nodule, and reduce tumor vascularization.

*Four injections at day 0,7,14,21. ** p< 0.01 versus saline treated mice (controls)

24

Experimental group

Nr of MOLT-

4 s.c.

injected

Nr of hMSCs i.t.

injected

Tumor weight

(grams±sd) Tumor/total#

INTRA-TUMOR INJECTION of BM-MSCs

Control

6x105

- 4.78±1.67 6/6

+ PTX 2000 ng/ml - 3.12± 1.98 5/5

+ BM-MSCs 3x105 cellsx4

times

3.01±1.79 6/6

+BM-MSCsPTX 3x105 cellsx4

times

0.78±0.72**

5/6

FIRST THERAPEUTIC EXPERIMENT Injection of hMSCs or hMSCsPTX into established tumor

masses of leukemia

A. Pessina,2017

LUNG MELANOMA METASTASIS SUPPRESSION BY

INTRAVENOUSLY INJECTED PTX LOADED MSCS

(Pessina et al.J.Exp.Clin Cancer Res.34,82,2015)

I.V. SR4987-PTX localize in lung

parenchyma of mice in vessels nearby

metastatic nodule.

A. Pessina,2017

MSCs loaded with PTX

24 h incubation 37°C, 5% CO2

Cell subculture

Primed MSCs release PTX in the culture medium

30

Paclitaxel 2,000 ng/ml

Sub confuent MSCs cultures

+

Collection of CM

Centrifuge at 2,500 x g, 15 min

(to eliminate residual cells and

debris)

Discard pellet and keep SN

(SN1)

SN2

Centrifuge 16,500 x g, 20min

Discard pellet and keep SN

(SN2)

Ultracentrifuge 110,000 x g,

70 min

Pellet with Exosomes/MVs

SN1

Two questions: How is Paclitaxel internalized and released from MSCs ?

Have microvesicles/ exosomes a role ?

Pascucci L. et al …J. Controlled Release 192,262–270, 2014

A. Pessina,2017

1 h

ou

r 2

4 h

ou

rs

24

+24

ho

urs

MASK PTX-F BODIPY

20 µm

PTX-F: fluorescent PTX (Oregon Green 488 Taxol).BODIPY® TR ceramide: Golgi specific marker (red).

Coccè V., 2013 27

PTX internalization in hMSCs: STUDY OF UPTAKING PHASE by Fluorescent PTX.

1. Internalization was appreciable

after 1 h co-localized with Golgi

apparatus or Golgi-derived vesicles. Staining was intense and enriched inside vesicles

at the end of the priming (24h).

2.After 24h, the distribution of F-

PTX was confined to the vesicles. Many of vesicles close to the cell membrane,

suggesting a possible secretion.

A. Pessina,2017

b

n nu

ck

c m

m

a d

G

ck

Molt-4

e

Molt-4

h

v v Molt-4

f

G

G

m

Molt-4 g

cv

v

cv

hMSCs

hMSCsPTX

PTX primed hMSCs don’t show significant morphological alterations but only an increment of vacuoles .

n= nucleus, nu= nucleoli, ck=cytoskeleton filaments, m= mitochondria, G= golgi complexes, v=membrane-bounded vacuoles, cv= confluent vacuoles

Coccè V., 2013 28

TEM analysis of hMSCs after PTX priming

A. Pessina,2017

A C B

D E F

31

Biogenesis and release of exosomes was observed

in PTX loaded MSCs ( SR4987)

Multivesicular body

Fusion of a multivesicular body with plasma membrane

Exosomes release

“Vacuole-like” structures inside treated SR4987

Nascent and neo-formed shedding vesicles

A. Pessina,2017

MVs were isolated from CM of SR4987PTX and untreated cells and analysed by TEM and SEM

32

TEM SEM

Morphology of MVs from SR4987PTX

Morphology of MVs from SR4987

TEM SEM

1. Round-shaped membrane-bounded vesicles isolated or aggregated in small groups. 2. MVs from control stromal cells and PTX primed MSCs had a similar size (from 10 to 150 nm ) and the same shape and electron-density.

A. Pessina,2017 1800 800 4000 2800

MVs CTR

MVs PTX

PTX C=O

//

//

/

/

//

Wavenumber (cm-1)

Ab

so

rba

nce

A

Wavenumber (cm-1) 3000 2800

Absorb

ance

2924

2850

B

Wavenumber (cm-1) 1770 1720

Absorb

ance

C

CH2/CH3

33

ATR-Fourier Infrared micro- spectroscopy analysis of MVs

confirmed that MVs released by drug primed cells contained

molecules of PTX

NOTE

Attenuated Total Reflection (ATR)-

Fourier transformed infrared (FTIR)

micro-spectroscopy is a technique

that provides the highest possible

level of accuracy.

However it can not exactly define if

PTX is within the MVs or bound on

their membrane.

For that, prudentially, we

prefer to speak about

MVs associated PTX.

A. Pessina,2017

B

THE PHARMACOLOGICAL ACTIVITY OF PTX

WAS NOT AFFECTED DURING THE

PHYSIOLOGICAL BIOGENESIS OF MVS.

IC50 PTX (ng/ml) = 5.89± 0.79; PEC (ng/ml)= 19.02; Single cell release: 0.0012 pg/cell

0

10

20

30

40

50

60

70

80

90

100

110

PTX (ng/ml)

CF-

PA

C p

rolif

era

tio

n (

% v

s C

TRL)

MV/media dilutions

SR4987-PTX MV (% vs CTRL MV) PTX (ng/ml)

SR4987-MV SR4987PTX-MVs

PTX (ng/ml)

0

10

20

30

40

50

60

70

80

90

100

110

PTX (ng/ml)

MV/media dilutions

CF

PA

C-1

pro

lifera

tio

n

(% v

s C

TR

L)

34

PTX PTX

SR4987-MV SR4987PTX-MVs

MVs from control cells have “per se” some anticancer

activity , confirming finding of other authors NORMALIZED vs SR4987-MVs

MVs SECRETED BY SR4987PTX CELLS EXERTED A SIGNIFICANT ANTI-

PROLIFERATIVE ACTIVITY AGAINST A PANCREATIC CANCER CELL

LINE.

SR4987-MV SR4987PTX-MVs

PTX (ng/ml)

A. Pessina,2017

THE PTX DRUG DELIVERY MEDIATED BY MVs HAS BEEN

CONFIRMED ALSO BY USING A FLUORESCENT

IMMORTALIZED HUMAN ADIPOSE DERIVED STROMAL

CELLS (HASCS-TS/GFP+) (ACAMC,2017).

A. Pessina,2017

25

Main conclusions

1. MSCs from different tissues are able to uptake drugs and release them

in active form

2. PTX can be released both as free molecule and associated to

microvesicles and exosomes

3. Drug loaded MSCs, their conditioned medium and microvesicles

produced a significant inhibition of tumor proliferation in vitro.

4. The anticancer activity is exerted also in vivo. ( Intravenously injection

of PTX loaded MSCs reduce of 90% the number of lung metastasis

from melanoma in mice)

5. Mesenchymal stromal cells :

• can be considered an interesting tool for “cell drug

delivery” without the need of genetic manipulations.

• they can be used to produce PTX-associated MVs working

as a new” biotech drugs” ( eg. with increased homing

capacity and efficacy ).

A. Pessina,2017

Sic ergo quaeramus tamquam inventuri,

et sic inveniamus tamquam quaesituri. ( Augustine,De Trinitate IX .1.1)

Let us seek

with the desire to find,

and find

with the desire to seek still more. ( Augustine, De Trinitate IX,1.1)

I wish to thank you with the auspice of S. Augustin

that is interesting for science and for our personal life .

A. Pessina,2017

Arianna Bonomi,Valentina Cocce’ Loredana Cavicchini, Francesca Sisto

Gianpietro Bondiolotti Dept of Biomedical Surgical Dental Sciences

Graziella Cappelletti Dept of Biosciences University of Milan

Giulio Alessandri,Emilio Ciusani Eugenio Parati

Department of Cerebrovascular Diseases, Neurological Institute C. Besta, Milan

Luisa Pascucci Dep. of Biopathological Sciences

and Hygiene of Animal and Food Productions, University of Perugia

I thank you for your attention.

My gratitude to the people that contributed to obtain the

results here presented.

Roberto Pallini Institutes of Neurosurgery, Università Cattolica del Sacro Cuore

,Rome

Maria Laura Falchetti , Simone Pacioni CNR-Institute of Cell Biology and Neurobiology (IBCN),Rome

Nathalie Steimberg, Anna Benetti, Angiola Berenzi , Giovanna Mazzoleni

Department of Clinical and Experimental Sciences, University of Brescia, Italy

Carlo Leonetti Experimental Chemotherapy Laboratory, Regina

Elena National Cancer Institute, Rome, Italy

Diletta Ami , Silvia Maria Doglia Department of Biotechnology and Biosciences, University of Milano Bicocca.