TAM and cancer: from the early days on

Transcription

1 TAM and cancer: from the early days on RUDOLF LUDWIG KARL VIRCHOW ( ) omnis cellula e cellula (Balkwill and Mantovani. Inflammation and Cancer: back to Virchow. Lancet 2001) Cytotoxicity Growth factors, vascular reactions angiogenesis (direct or via IL1/TNF) (direct, via PCA fibrin) (Mantovani, Curr Op Immunol 1990; Mantovani et al, Immunol Today 1992)

2 Polarization of macrophage fuctions: a simplified view Tumor resistance Killing of intracellular parasites Tissue destruction Immunoregulation Angiogenesis Tissue remodeling Th1 Parasite encapsulation Tumor promotion LPS M1 IFN IL10 Immune complexes IL1/LPS M2s CSF1 IL21 IL4, IL13 IL12 high ; IL23 high ; IL10 low ; ROI; RNI; TNF high ; IL1 high ; M1 chemokines (e.g. CXCL10). IL12 low ; IL23 low ; IL10 high ; arginase1; TNF low ; IL1ra high ; decoy IL1 RII high ; MMP9high; scavenger, mannose, galactose receptor high ; M2 chemokines (e.g. CCL22). (modified from Mantovani et al., Blood 2006; Mantovani et al., Trends Immunol and 2004)

3 The interplay between TAM and breast carcinoma cells: lessons from patients and mice MCSF Chemokines (CCL2; CCL5) EGF TAM Recruitment M2 skewing CARCINOMA Progression (adenoma carcinoma), Growth, Invasion, Metastasis 1. High TAM: bad prognosis (6 studies) 2. High MCSF, CCL2, CCL5: bad prognosis (1 study each) 3. TAM are the sole source of EGF in human breast carcinoma (Harris) 4. TAM accumulate in areas of hypoxia/angiogenesis (Harris; Lewis) 5. TAM have a M2like activation phenotype (Sica) 6. MCSF polarizes macrophages towards M2 (Martinez) 7. TAM recruitment (via MCSF) is required for progression in mice (adenoma carcinoma) (Pollard) 8. TAM promote angiogenesis, growth, invasion and metastasis in mice (Pollard)

4 Selected evidence for a protumor function of inflammatory reactions Epidemiological association between inflammation and cancer: thyroid, bladder, cervical, ovarian, prostate, esophagus, stomach, colon Protection against cancer (colon, breast) by NSAIDs and antibiotics Inflammatory cells, cytokines and chemokines are present in the darwinian microenvironment of neoplastic tissues Frequent (but not universal) correlation between high macrphage infiltration and poor prognosis Tumor promotion by adoptive transfer of inflammatory cells Functional polymorphisms of inflammatory cytokines (IL1, TNF) are associate with cancer susceptability and severity Cytokine or NFkB (including myeloidspecific) genetargeted mice are protected against carcinogenes, matastases and lymphoproliferative symdrome (Balkwill and Mantovani, Lancet 2001; Balkwill and Mantovani, Cancer Cell 2005; Mantovani, Nature 2005)

5 The IL1R/TLR superfamily

6 Tuning IL1R and TLR: the decoy receptor TIR8/SIGIRR IL1ra IL1ra LPS LPS IL1 IL1 IL1 IL1 IL 1 TI AcP R Decoy IL1RII AcP TI R MyD88 TI TI IL1RI R R D D AcP TI R MyD88 TI TI IL1RI R R D D TI R TIR8/ SIGIRR TI R MyD88 TI TI R R D D TLR TI R MyD88 TI TI R R D D TI R TIR8/ SIGIRR Human: Mouse: chromosome 11, 10 exs chromosome 7, 9 exs mrna: 1695 bp mrna: 1450 bp protein: 410 aa protein: 409 aa The decoy paradigm: Colotta et al, Science 1993 Mantovani et al, Trends Immunol 2002 TIR8/SIGIRR: Wald et al, Nature Immunol 2003 Polentarutti et al, Eur J Immunol 2003 Garlanda et al, PNAS 2004

7 Increased acute and chronic intestinal inflammation and colon carcinogenesis in the absence of Tir8

8 Histological analysis of colon tumors in Tir8/ mice A) B) C) Sub gross examination reveals the presence of a high grade (asterisk) and a low grade (circle) adenomas A low grade adenoma in Tir8/ and of a high grade adenoma in Tir8/ mice. An healing ulcer characterized by mild inflammatory changes in Tir8/ and an active ulcer characterized by a severe suppurative process in Tir8 /.

9 Cytokines and chemokines in colon lysates from DSSfed mice Lysate Tir8 / Tir8 / P value IL ± ± IL6 30 ± ± CCL ± ± CXCL2 390 ± ± CCL ± ± PTX ± ± TGF 80 ± ± Supernatants IL10 58 ± ± IFN 25 ± ± Values presented are mean ± SE, in pg/mg of proteins (n=8). Unpaired onetailed Student s ttest.

10 Leukocyte infiltration in Tir8/ and Tir8/ tumors Number of cells per field infiltrating Tir8/ and Tir8/ tumors of similar dimension and severity

11 Regulation of colitisassociated cancer by TIR8/SIGIRR IL1ra LPS IL1 Epithelial cells, idc, endothelial cells AcP T I R T I T I IL1RI R R T I R TIR8/ SIGIRR TLR4 T I R T T I I R R T TIR8/ I SIGIRR R TIR8 / MyD88 D D MyD88 D D TIR8/ Proinflammatory cytokines Inflammatory chemokines IL10 TGF IFN Macrophage infiltration

12 Chemokines and cancer Transformation and diversion of adaptive immunity HHV8 KSV Leukocyte recruitment Inflammation Angiogenesis Inflammatory CC or CXC Survival, proliferation, invasion, metastasis CXCR4 CXCL12 vgpcr Mø T cell vmipi, II, III Kaposi sarcoma Mc TAM Eo CCR7 Th2, Treg Tumor promotion Quantity Quality Tumor suppression (Balkwill, Nature Rev Cancer 2004; Mantovani, Seminars in Cancer Biol 2004)

13 The chemokine receptor repertoire of human pancreatic adenocarcinoma 100 Primary Ascites Metastasis Cell line PT45 PaCa44 MiaPaCa2 Panc1 HPAF A8184 AsPC1 CFPAC T3M4 Capan1 HS766T Hs766T CCR2 CCR6 CCR7 CXCR2 CXCR4 CX3CR1 CX3CR1 mrna (fold over HPDE6) CX3CR1 mrna (fold over HPDE6) 10 1, HPDE6 HPDE6 MiaPaCa 2 CFPAC PaCa 44 PT45 HS766T T3M4 HPAF Panc 1 AsPC1 A8184 Ducts PK93 PK96 PK97 PK126 PK132 PK135 PK136 pancreatic tumor surgical samples (Marchesi et al, Cancer Res 2004; and unpublished)

14 CX3CR1 mediates migration and protects tumor cells from apoptosis Migrated cells/10 HPF Migrated cells/10 HPF 50 A * * * AsPC1 ** * CX3CL1 (ng/ml) * * * * * * CX3CL1 (ng/ml) MiaPaCa2 CX3CR1GFP MiaPaCa2 GFP % of Sub G1 Migrated cells/10hpf A8184 * Ctrl CX3CR1 irrelevant antibody CX3CL1 MiaPaCa2 CX3CR1GFP 30% MiaPaCa2 GFP 0 CX3CL1 IL1 /TNF

15 Positive nerves (n) Pancreatic carcinoma cells use CX3CR1 to interactwith CX3CL1 on neuronal cells in vitro Adhesion to neural cells (% of input cells) A SKNBE TNF /IFN CX3CR1 * CX3CR1GFP MiaPaCa2 GFP * * Adhesion to neural cells (% of input cells) GFP/MiaPaCa2 20x CX3CR1GFP/MiaPaCa2 20x SKNBE Ctrl SKNBE TNF /IFN * * CX3CR1GFP 40x 40x MiaPaCa2 and to infiltrate and destroy peripheral nerves in vivo (Marchesi et al, unpublished) Neural invasion percentage GFP R=0.29; p= CX3CR1 expression score

16 Chemokine receptors in the progression of human pancreatic adenocarcinoma CX3CR1 is expressed in tumors but not in normal epithelial cells CX3CL1 (fractalkine) induces migration and survival of tumor cells CX3CL1 promotes migration towards, and interaction with neuronal cells in vitro CX3CL1 mediates interaction with and damage of nerves in vivo in mice In human tumor specimens CX3CR1 expression correlates with nerve invasion CCR2 Leukocyte recruitment (Monti et al, Cancer Res 2003) CCL2 Increased proliferation Resistance to apoptosis CXCL12 (Marchesi et al, Cancer Res 2004) CXCR4 CX3CR1 Perineural invasion and destruction CXCL12 (Marchesi et al, unpublished) Invasion CX3CL1 Migration Distant organs expressing CXCL12

17 Papillary thyroid carcinoma (PTC) and RET PTC is associated to thyroiditis, with a prominent inflammatory infiltrate (Scapino et al., Am J Pathol 2000) Fully fledged malignancy apparently in one step Somatic rearrangements or RET (RET/PTCs) are frequent (30%) RET Y1062 multidocking site (RET/PTC1 Y451) is essential for oncogenic activity

18 An inflammatory programme activated by RET/PTC1 oncogene in thyrocytes 10 Uninfected Ret/Ptc Uninfected RET/PTC1 Y451F RET/PTC CXCL1 CXCL5 MMP10 UPA MCSF CXCL8 NCAM1 CXCL2 GMCSF MMP1 CXCL6 CXCR4 ICAM1 CCL20 GCSF SELP NRCAM CD44 MMP7 UPAR CXCL3 SELL CXCL12 CD26 CCL2 CCR1 IL1A MMP3 IL1B MMP9 SPP1 MMP14 TIMP2 TIMP3 SERPINE1 SERPINE2 MCAM Relative expression Ret/Ptc1Y451F CXCL12 TIMP3 SELP MMP9 PLAU/UPA GMCSF CXCR4 SELL IL1B Expression (Borrello et al., PNAS 2005)

19 Expression of inflammatory molecules in papillary (PTC) versus follicular (FTC) thyroid carcinoma SELL GMCSF MMP9 Relative Expression 10.0 ** ** N PTC FTC ** ** N PTC FTC 15 *** N PTC FTC OSTP PLAU/UPA CXCR4 Relative Expression *** N PTC FTC 300 # N PTC FTC N PTC FTC Multiple massive lymph nodes metastasis (Borrello et al., PNAS 2005)

20 The RET/PTC1 oncogene activates an inflammatory profile in thyrocytes Y451 RET/PTC1 H4 TK RET Uninfected RET/PTC1 RET/PTC1Y451F MCSF GMCSF GCSF Leukocyte recruitment and survival IL1 Inflammation CXCR4 CXCL12 Migration Proliferation Survival CCL2 CCL20 Macrophage and immature Dendritic cell recruitment CXCL8 and related CXC Leukocyte recruitment Angiogenesis L selectin (CXCR4) homing to lymph nodes MMP14,7,9,10 upa, upar TIMP 2, 3 PAI 1, 2 Invasion and metastasis

21 Inflammation and cancer: two pathways EXTRINSIC Inflammatory/infectious conditions (e.g. IBD, H. pylori, HCV, HBV, HPV) INTRINSIC Oncogenetic events (RET/PTC; EGFR; ras; BRAF; Wnt/ catenin; RalB; Myc) Inflammatory cells Cytokines, chemokines, MMPs, PG Proliferation and survival, angiogenesis and lymphangiogenesis, migration, invasion, metastasis INFLAMMATIONRELATED CANCER Inflammation as a risk factor for cancer CANCERRELATED INFLAMMATION Inflammatory microenvironment in cancer unrelated to inflammation (modified from Mantovani and Balkwill, Cell 2006)

22 Inflammation and cancer Istituto Clinico Humanitas & University of Milan Federica Marchesi Paola Allavena Tiziana Schioppa Subhra Biswas Saki Paul Antonio Sica Nadia Polentarutti Cecilia Garlanda Istituto Nazionale Tumori Maria Grazia Borrello Marco Pierotti PathologyUniv. of Milan Manuela Nebuloni Luca Vago HSR Paolo Monti Lorenzo Piemonti Annarita Destro Massimo Roncalli Raffaella Bonecchi Fernando Martinez Massimo Locati Alberto Mantovani

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24 Chemokine Receptor Main targets IL8 GCP2 NAP2 ENA78 GRO GRO GRO IP10 Mig ITAC SDF1 / BCA1 BRAK MCP1 MCP4 MCP3 MCP2 MIP1 MIP1 S MIP1 P RANTES MPIF1 HCC1 HCC2 HCC4 Eotaxin Eotaxin2 Eotaxin3 TARC MDC MIP3 ELC SLC I309 TECK CTACK PARC Lymphotactin SCM1 Fractalkine CXCL8 CXCL6 CXCL7 CXCL5 CXCL1 CXCL2 CXCL3 CXCL10 CXCL9 CXCL11 CXCL12 CXCL13 CXCL16 CXCL14 CCL2 CCL13 CCL7 CCL8 CCL4 CCL3 CCL3LI CCL5 CCL23 CCL14 CCL15 CCL16 CCL11 CCL24 CCL26 CCL17 CCL22 CCL20 CCL19 CCL21 CCL1 CCL25 CCL27 CCL18 XCL1 XCL2 CX3CL1 CXCR1 CXCR2 CXCR3 CXCR4 CXCR5 CXCR6 Unknown CCR2 CCR5 CCR1 CCR3 CCR4 CCR6 CCR7 CCR8 CCR9 CCR10 Unknown XCR1 CX3CR1 Inflammatory (RED), homeostatic (GREEN), mixed (YELLOW) Neutrophil B cell Immature Mature Monocyte Eosinophil Basophil Naive Memory Th1 Th2 Treg pdc NK cell mdc T cell (Mantovani, Bonecchi, Locati. Nature Rev Immunol 2006)

25 Pathways leading to CCL2 production in tumors FIBROBLAST TUMORS JEgene 1 TDCF 2 MCP1 / CCL2 SMCDF 3 SMOOTH MUSCLE CELLS Molecular or biological description: 1) Rollins et al PNAS 85, 3738, ) Bottazzi et al Science 220, 210, ) Valente et al Am. J. Path 117, 409, 1984 Molecular identification: 1) Rollins et al PNAS 85, 3738, ) Yoshimura et al J. Exp. Med. 169, 1449, ) Matsushima et al J. Exp. Med. 169, 1485, 1989

26 Effect oh hypoxia on CXCR4 expression (Schioppa et al, J Exp Med 2003)

27 Multistep navigation of TAM

28 (Gordon S, Taylor PR. Nat Rev Immunol. 2005)

29 M2a polarization: study design Mo 100 ng/ml MCSF 72 hours 100 ng/ml MCSF 72 hours Md3 M0 M M1 100 ng/ml LPS 20 ng/ml IFN 18 hours M2a 20 ng/ml IL4 (Martinez et al J Immunol 2006)

30 GLOBAL DISTANCE MAPPING: Full Transcriptome Principal Component Analysis (Martinez et al J Immunol 2006)

31 Gene expression signatures during macrophage polarization : Interactome of M1 (red) vs M2 (green) Macrophages M1 chemokines Metabolism Scavenger Receptors M2 soluble trophic factors (Martinez et al J Immunol 2006)

32 MONOCYTETO MACROPHAGE DIFFERENTIATION AND POLARIZATION: LESSONS FROM PROFILING MCSF driven monocytetom differentiation is associated with activation of cell cycle genes, possibly a reflection of an underestimated proliferation potential of monocytes MCSF leads to expression of a substantial part of the M2 transcriptome, suggesting that under homeostatic conditions a default shift towards M2 occurs Lipid metabolism is a main category of modulated transcripts, with expected upregulation of Cox2 and SPHK1 in M1 cells and unexpected Cox1 and CERK1 upregulation in M2 cells Each step is characterized by a different repertoire of G proteincoupled receptors, with 5 nucleotide receptors as novel M2 associated genes The chemokinome of polarized M is profoundly different and new differentially expressed chemokines were identified The solute carrier family is widely influenced by M polarization, which suggests a pivotal role of these poorly studied molecules in macrophage pathophysiology

33 Resting TAM express a distinct transcriptional profile compared to peritoneal macrophages (Biswas et al Blood, 2006)

34 Smouldering and polarized macrophagedriven inflammation in extablished neoplasia Proliferation survival VEGF,PlGf FGF, TGF Chemokines MMPs Angiogenesis, lymphangiogenesis, Matrix and remodeling MCSF, VEGF, PlGF, CCL2, CCL3 Recruitment/survival Invasion metastasis CXCL12 TNF low IL6, IL1 CXCR4 TAM / M2 Mø (IL12 low ; IL10 high ; ManR, GalR, SRhigh) IL10, TGF Tumor proliferation, survival, progression Growth factors (EGF) Adaptive immunity (Anergy, suppression, Th2 skewing) TGF, IL10 Tumor cell Response to hormones Resistance to chemotherapy (Balkwill & Mantovani, Lancet 2001; Mantovani et al., Trends Immunol 2002; Balkwill, Charles & Mantovani, Cancer Cell 2005)