RUDOLF LUDWIG KARLVIRCHOW (1821-1902)

omnis cellula e cellula

B

(Balkwill and Mantovani. Inflammation and Cancer:back to Virchow. Lancet 2001)

TAM, INFLAMMATION AND CANCER

(Biswas and Mantovani, Nature Immunol 2010)

(Mantovani A., 1978;Acero et al 1984)

(Bottazzi et al Science 1983)

Giavazzi R.et al.Cancer Research 50:4771 (1990)

(Scarpino et al Am J Path 2000)

30

20

10

0

0 2 4 48 72hours

IN VITRO

Control

mF S6 + TAM

Control

mF S6 + TAM

[3H

] Td

Rup

take

(cpm

x 10

-3)

In Vitro and in vivo

(Mantovani et al Immunol Today 1982 )

The evidence that links cancer and inflammation

• Inflammatory disease increases the risk of cancer (e.g. thyroid, bladder, cervical, ovarian, prostate, oesophageal, gastric, intestinal)

• Non-steroidal inflammatory drugs protect against so me cancers (e.g. colon and breast)

• Inflammatory leucocytes, cytokines and chemokines a re present in all (?) experimental and human cancers from the earlies t stages

• Inflammatory pathways are downstream of oncogenic m utations (e.g. ras, myc, RET/PTC)

• Targeting cytokines, chemokines, key transcription factors of inflammation and inflammatory cells, decreases inci dence and spread of cancer (e.g. TNF-αααα, IL-1ββββ, NF-κκκκB, Stats)

• Adoptive transfer of inflammatory cells, or over-ex pression of inflammatory cytokines, promotes tumour development

(Balkwill and Mantovani, Lancet 2001; Balkwill, C harles and Mantovani Cancer Cell 2005;Mantovani, Allavena, Sica and Balkwill, Nature, 20 08 )

(Mantovani Nature 2009; an integration toDouglas Hanahan and Robert A. Weinberg Cell, 200 0)

(Mantovani, Sica, Allavena, Balkwill Nature 2008)

An inflammatorymicroenvironment

eg RET/PTC

PATHWAYS LEADING TO CCL2 (MCP -1)

MCP-1 / CCL2

SMOOTH MUSCLE CELLS

FIBROBLAST TUMORS

JEgene 1 TDCF2

SMCDF3

Molecular or biological description:

1) Rollins et al PNAS 85, 3738, 19882) Bottazzi et al Science 220, 210, 19833) Valente et al Am. J. Path 117, 409, 1984

Molecular identification:1) Rollins et al PNAS 85, 3738, 19882) Yoshimura et al J. Exp. Med. 169, 1449, 19893) Matsushima et al J. Exp. Med. 169, 1485, 19894) Van Damme...Bertini et al EJI 19, 2367, 19895) Bottazzi et al Int J Cancer 45,795, 19906) Zachariae et al J. Exp. Med. 171, 2177, 1990

IL-8GCP-2NAP-2

ENA-78GROαGROβGROγIP-10

MigI-TAC

SDF-1α/βBCA-1

BRAKMCP-1MCP-4MCP-3MCP-2MIP-1β

MIP-1αSMIP-1αPRANTES

MPIF-1HCC-1HCC-2HCC-4

EotaxinEotaxin-2Eotaxin-3

TARCMDC

MIP-3αELCSLC

I-309TECK

CTACKPARC

LymphotactinSCM-1β

Fractalkine

CXCL8CXCL6CXCL7CXCL5CXCL1CXCL2CXCL3CXCL10CXCL9CXCL11CXCL12CXCL13CXCL16CXCL14CCL2CCL13CCL7CCL8CCL4CCL3CCL3LICCL5CCL23CCL14CCL15CCL16CCL11CCL24CCL26CCL17CCL22CCL20CCL19CCL21CCL1CCL25CCL27CCL18XCL1XCL2CX3CL1

CXCR1

CXCR2

CXCR3

CCR4

CCR5

Main targets

CXCR4CXCR5CXCR6

Unknown

CCR1

CCR3

CCR6

CCR7

CCR8CCR9CCR10

Unknown

XCR1

CX3CR1

CCR2

Chemokine Receptor

Neutrophil

Monocyte

Eosinophil

Basophil

NK cell

B cell

Immature

MaturemDC

T cell

pDC

NaiveMemoryTh1Th2Treg

Inflammatory (RED), homeostatic (GREEN), mixed (YELLOW) (Mantovani, Bonecchi ,Locati Nature Rev Immunol 200 6)

(Mantovani , Allavena, Locati and Bonecchi Cytokine Growth Factor Rev 2010)

Skin(cutaneouslymphoma ) Intestine

(melanoma) Liver(colon,

pancreas)

Lymph nodes(breast, stomach,

melanoma)

HPC niches(CLL,AML leukemia)

Lungbreast)

Nerves, ganglia(pancreas)

Brain(ALL leukemia)

Bone(breast, prostate)

(Mantovani , Allavena, Locati and Bonecchi Cytok Gr owth Factor Rev 2009)

Atypical chemokine

receptors

No conventional signaling

(CTX, Ca, MAPK, …)

Signalingchemokine

receptors

Signaling(CTX, Ca, MAPK, …)

The “darc side” of the chemokine system

CXCR7

(Mantovani, Bonecchi and Locati,Nature Rev Immunol 2006; CGFR 2010)

Chemokine decoy receptor D6

CCL11CCL13CCL14CCL17CCL22

CCL2CCL3L1CCL4CCL5CCL7CCL8

degradation

Binding Trafficking

Placenta Sk in lymphatic vessels

FunctionExpression

(Mantovani, Bonecchi and Locati,Nature Rev Immunol 2006; CGFR 2010; Graham et al Trends Immunol 2006; EJI 2009)

D6 is expressed in human Kaposi’s sarcoma lesions

D6LANA 1 (HHV8)

A

(Nebuloni, Vago et al unpublished)

Reduced in vivo growth of D6/KS IMM transfectants

D6/KS-IMM

mock/KS-IMM

(Savino et al Unpublished)

M2-like

Integrated from Biswas and Mantovani Nature Immuno l 2010, Sica and Mantovani J Clin Invest 2012

The yin-yang of macrophage polarization

M1M2-like

- intracellular pathogens- tissue damage- tumor resistance - parasite encapsulation

- tissue remodeling- tumor promotion- immunoregulation

M2

PTX3

Macrophage plasticity and polarization in pathology: in vivo veritas

Sica A and Mantovani A. J.Clin.Invest. 2012

Annual Review Issue - January 2013

INFLAMMATION, WOUND REPAIR & FIBROSIS

Guest Editors: Eric S. White & Alberto R. Mantovani

Articles already available online at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1096-9896/accepted

(Cairo et al Trends Immunol 2011 ; Biswas and Mantovani, Cell Metabolism 2012, in pres s)

MACROPHAGE ORCHESTRATION OF METABOLISM

LPS

NF-κB

IRF-3TNFα, IL-12,CXCL1,-2,-3,-4,-5,-8,-9,-10CCL2,-3,-4,-5,-11,-17, SOCS3

IL-10

CXCL9,-10,-11NOS2CCL5, SOCS-3

STAT1

M2

p65p50

TLR4 IFN-α/βR IFN-γR

IFN-β

IL-1Ra,, SOCS1, ARG1, KLF4PPARγ,

IL-1Ra, IL-4RαARG1, ARG2, NOS2

STAT3p50 p50

NF-κB

STAT6

M1

IFN-β

IL-4 IL-4/IL-13

IL-4Rα γC IL-4Rα IL-13Rα1

IFN-γ

Il-10c-Maf

PPARγFABP4

KLF4ARG1, Mrc1, Fizz1, PPARγ

ARG1HIF-2α

NOS2HIF-1α

Hypoxia

IRF-5IL-12, IL-23

JMJD3

IRF4

c-Myc

(Sica and Mantovani, J Clin Inv,2012) M2-like

Mantovani A et al, Lancet 2008

Postulated interactions between immune and cancer cells at various stages of carcinogenesis and progression

Interplay between tumour-associated macrophages and cancer cells in established tumours

Angiogenesis, lymphangiogenesis,

Matrix and remodeling

TNFlow

IL-6, IL-1

M-CSF, VEGF, PlGF, CCL2, CCL3

Adaptive immunity(Anergy, suppression,

Th2 skewing)

Growth factors (EGF)

Tumorcell

CXCL12

CXCR4

TAM / M2 Mø/MDSC

(IL-12 low ; IL-10 high ;ManR, GalR,

SRhigh )

Response to hormones

Resistance to chemotherapy

Proliferation survival

Invasion metastasis

SMOULDERING AND POLARIZED MACROPHAGE-DRIVENINFLAMMATION IN ESTABLISHED NEOPLASIA

(rev. in Balkwill and Mantovani Lancet 2001; Mantovani et al Trends Immunol 2002; Balkwill, Charles and Mantovani Cancer Cell 2005)

Recruitment/survival

Tumor proliferation,survival, progression

Lancet, 2012

Aspirin and cancer protection – Recent literature

Lancet, 2012

Lancet, 2012

Targeting cancer-related inflammation

Anti-inflammatory properties of theanti-tumor agent Trabectedin

TRABECTEDIN

TRABECTEDIN (ET-743, Yondelis) PharmaMar, Spain

1- Natural product derived from a marine tunicate

2 - Binds the minor groove of DNA, interacts with NFY

3- Effective anti-tumor agent in vivo (phase II and III)

in soft tissue sarcomas, ovarian cancer, and breast

4- In 2008 approved by EMEA for 2nd line therapy of Soft Tissue Sarcoma,

in 2009 for 2nd line therapy of ovarian cancer in combination with

Pegylated Liposomal Doxorubicin

5- Clinical activity characterized by delayed, prolonged responses

(Germano et al Cancer Res 2005; 2010; Cancer Cell, 2013, in press; D’Incalci Mol Cancer Ther 2010)

Trabectedin is selectively cytotoxic for Monocytes and reduces some inflammatory cytokines

Allavena et al Cancer Res, 2005, 2010; Germano et al Cancer Cell, 2013, in press

*

* IC 50 on tumor cells

IL-6

0

250

Ctrl 2.5 5

500

*

**

CCL2

0

10

20

30*

*

**

Ctrl 2.5 51.2

VEGF

0

200

Ctrl 2.5 5

400

Ang2

*

**0

100

200

300

Ctrl 2.5 5

400

*

**0

20

Ctrl 2.5 5

40

TNF

Cleaved-Caspase 8

Actin

Monocytes

2.5 5-

PMNs

2.5 5-

T cells

2.5 5-

Cyt C

Cleaved-Caspase 9

Trabectedin nM

Trabectedin nM

CD68

Untreated Treated

Untreated Treated0

50

100

150

200

250 ***

num

of

CD

68+

cel

ls f

or fi

eld

Untreated Treated0

20

40

60

80

100

***

num

of

CD

3+ c

ells

for

fiel

d

CD3

Tumor-associated macrophages but not T cells are decreased after

Trabectedin

LLCMN/MCAI

Macrophages PMNs T cells0

25

50

75 **

*

% o

f ce

lls /

CD

45+

Macrophages PMNs T cells0

10

20

30

40

50

60

70 **

% o

f C

D45

+ ce

lls

ID8

Macrophages PMNs T cells0

10

20

30

40

50

60

70 *

*

% o

f C

D45

+ ce

lls

Untreated

Treated

MCA-induced fibrosarcoma

Macrophages PMNs T cells0

10

20

30

40

50

60

70 **

% o

f C

D45

+ ce

lls

Germano et al, Cancer Cell 2013 in press)

10 15 20 25 30 350

1000

2000

3000

4000

5000

6000

UntreatedTreated

Time post inocolum (days)

MN/MCAI Res

**

Tum

or

Wei

ght

(mg)

A

Role of macrophage depletion in the anti-tumor activity of Trabectedin

0

10

20

30

40

50

60

MN/MCA1 WT MN/MCA1 Res

Trabectedin - + - + - + - +

pre vivo post vivo pre vivo post vivo

% o

f P

I po

siti

ve c

ells

BMN/MCA1 WT MN/MCA1 RES

UntreatedMacrophagesTrabectedinMacrophages &Trabectedin

10 15 20 25 30 350

1000

2000

3000

4000

5000

6000

UntreatedTreated

Time post inocolum (days)

MN/MCA1WT

**

Tum

or W

eigh

t (m

g)

15 20 250

1000

2000

3000UntreatedTrabectedinMDSCMDSC & Trabectedin

Days

Tu

mo

r We

igh

t (m

g)

Time post inocolum (days)

Resistance to Trabectedin

Germano et al, Cancer Cell 2013 in press)

Tumor macrophages and vessels are reduced in treat ed STS patients

PRE POST

CD31 vessels CD163 macrophages

PRE POST

(PRE: biopsy before surgery; POST: tumor sample at surgery, after therapy)

Correlation with clinical response

p = 0,07

Bloodmonocytes

TARGETING TAM IS A KEY COMPONENT OF THE ANTITUMOR ACTIVITY OF TRABECTEDIN

• Trabectedin is preferentially toxic for cells of th e monocyte-macrophage lineage. In these cells it activates a TRAIL-R dependent extrinsic pathway of apoptosis,

• TAM depletion is sufficient for the anti-tumor acti vity of Trabectedin (resistant lines; macrophage rescue)

• First evidence that targeting tumor-promoting TAM is involved in the anti-tumor activity of a clinica lly approved agent (sarcomas; ovarian carcinoma)

• This finding provides proof of principle for TAM targeting in human cancer treatment and has implications for combination therapy and design

(Germano et al, Cancer Cell 2013 in press)

STRATEGIES TO COUNTERACT THE “MALA EDUCACION” OF TAM

RE-EDUCATION

-IFNg (eg Allavenaet al Int J Cancer 1994), TLR

-CD40 agonist mab(Beatty et al Science 2011)

ELIMINATION

-Blocking recruitmentand survival(eg anti-CCL2, Qian et Nature 2011; CSF-1R)

- Elimination (eg Trabectedin,Cancer Cell 2013, in press)

EMEA- approved for clinical use

Clinical evidence of activity

(Mantovani Nature 2009; an integration toDouglas Hanahan and Robert A. Weinberg Cell, 200 0)

(Mantovani, Sica, Allavena, Balkwill Nature 2008)

An inflammatorymicroenvironment

eg RET/PTC

Hanahan and Weinberg Cell 2011

Emerging Hallmarks of CancerThe six Hallmarks of Cancer

Hanahan and Weinberg Cell 2000

Mantovani A. Nature 2009

The six Hallmarks of Cancer (plus one)

An inflammatorymicroenvironment

Acknowledgments

Istituto Clinico HUMANITAS

Giovanni GermanoCristina BelgiovineSamantha PesceManuela LiguoriAchille Anselmo Nadia PolentaruttiFabio Pasqualini

Alberto Mantovani

Istituto Mario Negri

Roberta FrapolliEugenio ErbaMassimo ZucchettiMaurizio D’Incalci

Istituto Tumori MilanoPaolo CasaliRoberta SanfilippoSilvana PilottiAndrea Anichini

PharmaMarCarlos GalmariniNadia Badri

www.ici2013.org