Treatment of PDAC mouse models with immunotherapy in combination with cancer exosomes inhibition

Figure 4 – PDAC exosomes impairment increases tumor immune infiltration. (A) Schematic representation of PDAC mouse

model PKT (Ptf1αcre/+; LSL-KRasG12D/+; Tgfβr2flox/flox) Rab27A inducible and conditional knock-out (R26LSL-FLPOERT2/+; Rab27aFrt/Frt). (B)

Immune profile of CD4 positive T cells. (C) Representative images of tumor sections with CD4 staining. (D) Flow cytometry analysis

of NK cells from PKT-iRab27aKO tumors. (E) Flow cytometry analysis of NK cells from PKT-iRab27aKO tumors – gating strategy.

Immunomodulation in Pancreatic Cancer:

Exosomes Take Center Stage

Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer with less than 9% of patients surviving past 5 years (1) due to lack of effective treatment options, being chemotherapy and radiotherapy the only options for

patients (2, 3). Despite low mutation burden, PDAC presents tumor-specific neoepitopes. Immunotherapy has yielded remarkable clinical results, but it is still not applicable to PDAC as the immunosuppressive tumor

microenvironment (TME) suppresses T cell activation (2). This admix of conditions is part of the reason immunotherapy is not beneficial for PDAC patients (2, 3). To determine the impact of the immune system in PDAC

progression, PDAC mouse models were crossed with the Rag2-/- allele to deplete T and B cells, and with the IL2rg-/- allele to deplete NK cells. Interestingly, depletion of NK cells was shown to have an impact in disease

progression, more specifically in enhancing metastasis establishment. Hence, our data suggests that the immune system is not blind to PDAC.

Cancer exosomes are known for their role in reprograming target cells in the tumor microenvironment for tumor benefit (4-6). We hypothesize that PDAC exosomes are instrumental in immunomodulation, establishing and

maintaining an immunosuppressive tumor microenvironment in PDAC. We demonstrate that cancer cells communicate with distinct subtypes of immune cells through exosomes. Considering the potential of

immunotherapy, the quest for strategies that make PDAC immune responsive is imperative. To study whether PDAC exosomes have the ability to mediate changes in the T cell population, we treated spleen-derived T cells

with PDAC exosomes. So far, we have been able to observe a clear increase in T cell viability when PDAC exosomes were added to their culture medium. In order to assess the impact of PDAC exosomes in the

establishment of an immunosuppressive microenvironment, we have developed a new PDAC mouse model that allows the conditional and inducible inhibition of exosomes secretion by tumor cells through the knockout of

Rab27a gene. In this regard, we observe changes in the tumor immune landscape, particularly in the numbers of CD4+ T cells and NKs, when cancer exosomes secretion is impaired. Our data suggests that PDAC

exosomes are instrumental in immunomodulation, and the crosstalk between cancer cells and NKs is involved in disease progression. Our in vivo models will shed light on cancer exosomes’ role in the anti-tumor immune

response, opening new avenues for implementation of immunotherapy in PDAC.

Sofia Quintas1,2,3, Inês A. Batista1,2, Nuno Bastos1,2, Bárbara Adem1,2, José Carlos Machado1,2,3 and Sónia A. Melo1,2,3

1 i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal.2 Instituto de Patologia e Imunologia Molecular da Universidade do Porto, Portugal.3 Faculty of Medicine, University of Porto, Portugal.

Figure 1 – Immune system restrains PDAC tumor progression. (A-B) Schematic representation of PDAC mouse models (A)

KPC Rag2-/- (depleted of B and T cells) and (B) KPC Rag2-/-IL2rg-/- (depleted of B, T and Natural Killer cells). (C) Overall survival

from time of diagnosis of KPC Rag2-/- (n=8) and KPC Rag2-/-IL2rg-/- mice (n=5). (D, F) Prevalence of (D) liver and (F) lung

metastasis observed in KPC Rag2-/- and KPC Rag2-/-IL2rg-/- mice at euthanasia. (E, G) Representative images of H&E stained (E)

liver and (G) lung metastasis of KPC Rag2-/- and KPC Rag2-/-IL2rg-/- mice.

6 Take-home message

A

3 Treatments(1x1011 exos/treatment)

Treatment with

KPC exos Mix1

TCRβ

CD4

CD8

Mix2

CD3

CD4

CD25

Foxp3T cells

LaserDetector

Flow Cytometry

Ex-vivo culture

Pan- T CellIsolation Kit

Wild-type

Spleen

T cells isolationEvaluation of T-cells

subpopulationsA B

Figure 3 – Evaluation of changes in the T cell population upon treatment with PDAC exosomes. (A) Schematic representation

of murine T cells and PDAC derived exosomes isolation followed by T cells activation and exosomes treatment ex vivo. (B)

Percentage of viable cells post-exosomes treatment at day 8 (control n=2, PDAC exosomes n=2).

AMicroenvironmentCancer cell

Cancer exosomes

Immune cells

Frt Frt

R26LSLFLP0ERT2/+

Exon 4Rab27a

*Tamoxifen administration: 6mg, mother oral

gavage (days post birth of pups: 0, 1, 2 and 4).

Rab27a KO

Impairment of cancer

exosomes secretion

Rab27a Rab27a KO

+ TAMOXIFEN*

PKT-iRab27aKO

(KrasG12D/+;

TGFβR2flox/flox;

Ptf1αCre/+;

R26LSLFLP0ERT2/+;

Rab27aFrt/Frt)

+ TAMOXIFEN

Absent cells:

• T cells

• B cells

KO of Rag2

KPC-Rag2-/-

(KrasG12D/+;

p53R172H/+;

Pdx1Cre/+;

Rag2-/-)

Via

ble

ce

lls

(%

)

C o n tr o l K P C E x o s

1 0

1 2

1 4

1 6

U n p a ire d t- te s t

p = 0 .0 1 2 5

M a n n -W h itn e y t- te s t

p = 0 .3 3 3 3

*

p = 0 .0 1 2 5

U n p a ire d t- te s t

0

2 5

5 0

7 5

1 0 0

Pre

va

len

ce

of

Liv

er m

eta

sta

sis

(%

)

K P C R a g 2- / -

K P C R a g 2- / -

IL 2 rg- / -

F is h e r 's e x a c t te s t

p = 0 .0 8 0 8

C h i-s q u a re te s t

p = 0 .0 3 8 4

F is h e r 's e x a c t te s t

p = 0 .0 8 0 8

C h i-s q u a r e te s t w ith Y a te s ' c o r re c tio n

p = 0 .1 4 7 3

3 o f 7

a n im a ls

5 o f 5

a n im a ls

p = 0 .0 8 0 8

F is h e r 's e x a c t te s t

0

2 5

5 0

7 5

1 0 0

Pre

va

len

ce

of

Lu

ng

me

tas

tas

is (

%)

K P C R a g 2- / -

K P C R a g 2- / -

IL 2 rg- / -

F is h e r 's e x a c t te s t

p = 0 .1 5 1 5

C h i-s q u a r e te s t w ith Y a te s ' c o r re c tio n

p = 0 .2 9 4 9

0 o f 7

a n im a ls

2 o f 5

a n im a ls

p = 0 .1 5 1 5

F is h e r 's e x a c t te s t

CB

KPC-Rag2-/-IL2rg-/-

(KrasG12D/+;

p53R172H/+;

Pdx1Cre/+;

Rag2-/- ; IL2rg-/-)

Absent cells:

• T cells

• B cells

KO of Rag2 & IL2rg

• NK cells

D

F

E

G

KPC Rag2-/-IL2rg-/-KPC Rag2-/-

4x 4x

4x 4x

KPC Rag2-/-IL2rg-/-KPC Rag2-/-

LIV

ERLU

NG

Cross-sectional cohorts of KPC, KPC Rag2-/- and Rag2-/- IL2rg-/- (euthanasia at 25 weeks) to:

Assess differences in tumor volume and metastasis formation

Inoculation of tumor cells into wild type C57BL/6, Rag2-/- and Rag2-/-IL2rg-/- mice

Cancer cell

Microenvironment

Cancer exosomes

Immune cells

Impairment of cancer

exosomes secretion

Rab27a Rab27a KOPKT-iRab27aKO

(KrasG12D/+;

TGFβR2flox/flox;

Ptf1αCre/+;

R26LSLFLP0ERT2/+;

Rab27aFrt/Frt)

+ TAMOXIFEN*

*Tamoxifen administration: 6mg, mother oral gavage

(days post birth of pups: 0, 1, 2 and 4).

PKT-Rab27aKO

(KrasG12D/+;

TGFβR2flox/flox;

Ptf1αCre/+;

Rab27aFrt/Frt)

Rab27a

+ TAMOXIFEN*

Rab27a

Control group:

• Lacks R26LSLFLP0ERT2/+

• No impairment of cancer exosomes

secretion upon Tamoxifen treatment

anti-PD-L1**I.p. injections of

10mg/Kg (2x/week, for 2

weeks) of anti–mouse

PDL1 antibody (clone

10F.9G2, BioXcell)

+

Inhibiting exosomes

secretion could potentiate

immunotherapy efficacy

Cancer exosomes are critical modulators

of the immune response against PDAC

HYPOTHESISThe immune system is not blind to PDAC

Depletion of

NK cells

Faster disease progression and

enhanced metastasis establishment

To validate the improved efficiency of immunotherapy to treat PDAC preclinical

models when used in combination with cancer exosomes inhibition

REFERENCES

1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2019. CA Cancer J Clin. 2019;69(1):7-34.

2. Chiaravalli M, Reni M, O'Reilly EM. Pancreatic ductal adenocarcinoma: State-of-the-art 2017 and new therapeutic strategies. Cancer Treat Rev. 2017;60:32-43.

3. 3.Werner J, Combs SE, Springfeld C, Hartwig W, Hackert T, Büchler MW. Advanced-stage pancreatic cancer: therapy options. Nat Rev Clin Oncol. 2013;10(6):323-

33.

4. Zhang X, Yuan X, Shi H, Wu L, Qian H, Xu W. Exosomes in cancer: small particle, big player. J Hematol Oncol. 2015;8:83.

5. Guo W, Gao Y, Li N, Shao F, Wang C, Wang P, et al. Exosomes: New players in cancer (Review). Oncol Rep. 2017;38(2):665-75.

6. Ruivo CF, Adem B, Silva M, Melo SA. The Biology of Cancer Exosomes: Insights and New Perspectives. Cancer Res. 2017;77(23):6480-8.

1 The immune system is not blind to PDAC

5 Future Plans

3 PDAC exosomes may play a role in immunomodulation

4 PDAC exosomes impairment modifies the tumor immune

landscape

PS063

1

Treatment of T cells

with PDAC exosomesIncrease in T cells viability

PDAC exosomes may be able to modulate the immune system

3

4Figure 2 – Immune system restrains PDAC tumor progression. (A) Schematic representation of PDAC mouse model KPC ExoBow (Pdx1cre/+;

Pdx1flp/+; LSL-KRasG12D/+; LSL-Trp53R172H/+; R26CD63-XFP/+). (B) Representative confocal microscopy images of immunofluorescence in the

pancreas tumor of a KPC-ExoBow mouse. (C) Communication of PDAC exosomes with tumor-infiltrating immune cells, CD4+ T cells, CD8+ T

cells, Foxp3+ regulatory T cells (T-regs) and CD68+ cells (monocyte lineage).

CD63-phiYFP

CD63-mCherry

CD63-mTFP

CD63-eGFP

DAPI

200 µm

B

Pancreatic

tumor

tile scan

5 µm

5µm

CD

4C

D8

CD

68

Merge CD63-mTFP

5 µm 5 µm

Foxp

3

5 µm

Merge CD63-mTFPC

Imm

un

e c

ells o

f th

e

tum

or

mic

roen

vir

on

men

t

Microenvironment

Cancer cell

Cancer exosomes

Secretion of color coded

cancer exosomes

KPC-ExoBow

(KrasG12D/+;

p53R172H/+;

Pdx1Cre/+;

Pdx1Flp/+;

R26CD63-XFP/+)

A

2 Cancer cells communicate with immune cells through

cancer exosomes

Rag2-/-C57/BL6 wt

Orthotopic inoculation in

the pancreas

KPC

KPC-Rag2-/-

KPC-Rag2-/-IL2rg-/-

Single cell

suspension

Tumor

collection

Tissue digestion:

• Mechanic

• Enzymatic

Rag2-/- IL2rg-/-

PKT (rapid PDAC progression) mouse model in an immunodeficient background

PKT

(KrasG12D/+;

TGFβR2flox/flox;

Ptf1aCre/+)

MicroenvironmentCancer cell

Cancer exosomesImmune cells

Absent cells:

• T cells

• B cells

KO of Rag2

PKT-Rag2-/-

(KrasG12D/+;

TGFβR2flox/flox;

Ptf1aCre/+;

Rag2-/-)

PKT-Rag2-/-IL2rg-/-

(KrasG12D/+;

TGFβR2flox/flox;

Ptf1aCre/+;

Rag2-/- ; IL2rg-/-)

Absent cells:

• T cells

• B cells

KO of Rag2 & IL2rg

• NK cells

We observe an earlier disease onset and decreased

survival in comparison to PKT mice (n=1).

0 5 1 0 1 5 2 0 2 5

0

2 5

5 0

7 5

1 0 0

T im e fr o m d ia g n o s is (w e e k s )

Su

rv

iva

l (%

)

K P C R a g 2- / -

K P C R a g 2- / -

IL 2 rg- / - *

p = 0 .0 3 9 7

L o g -ra n k te s t

(M a n te l-C o x )

We observe a tendency for higher tumor volume when T and B cells are depleted (KPC-Rag2-/-; n=1) in

comparison to immunocompetent KPC mice (n=2), as well as a higher number of liver metastasis.

Increase in CD4+ T cells

Decrease in NK cells

PDAC exosomes impairment (Rab27a KO) induces changes in

the tumor immune landscape

Cancer exosomes mediate the communication between

cancer cells and immune cells2

Ongoing work (early results):

Note: The orthotopic inoculation of tumor cells from a KPC mouse into wild type C57BL/6,

Rag2-/- and Rag2-/-IL2rg-/- mice was already performed and we are waiting for the results.

ACKNOWLEDGEMENTS

0

5

1 0

1 5

2 0

2 5

CD

4+ c

ell

s /

fie

ld

P K T R a b 2 7 a- / -

P K T iR a b 2 7 a- / -

R 2 6L S L -F L P O E R T 2 /+

p = 0 .0 1 4 1

U n p a ire d t te s t*

B

Pancreas Tumor 10x

PKT Rab27a-/-

CD4

10x

PKT Rab27a-/- R26LSL-FLP0ERT2/+

CD4

Pancreas Tumor

C

0

5

1 0

1 5

2 0

2 5

CD

4+ c

ell

s /

fie

ld

P K T R a b 2 7 a- / -

P K T iR a b 2 7 a- / -

R 2 6L S L -F L P O E R T 2 /+

p = 0 .0 5 1 9

M a n n -W h itn e y te s t

0

1

2

3

4

5

NK

ce

lls

(%

)

P K T R A B 2 7 A- / -

P K T F L P O E R T 2/+

R A B 2 7 A- / -p = 0 .1 2 1 7

U n p a ire d t- te s t

0

5

1 0

1 5

2 0

2 5

CD

4+ c

ell

s /

fie

ld

P K T R a b 2 7 a- / -

P K T iR a b 2 7 a- / -

R 2 6L S L -F L P O E R T 2 /+

p = 0 .0 5 1 9

M a n n -W h itn e y te s t

D E

SS

C-A

FSC-A

FS

C-W

FSC-A

FS

C-H

Comp-PE-A: NK cells