Tumor associated macrophages in endocrine-related cancers Sun Wook Cho, M.D, Ph.D Seoul National University Hospital
Tumor Microenvironment Fibroblasts Immune cells Angiogenesis Tumor microenvironment=cancer cells + stroma Heterogeneous and complex
Cancer Immunology Immunosurveillance Necrotic Cell debris Dendritic cell T cell NK T cell Immune evasion Macrophage Immunoediting
Cancer Immunology Classical immune actions Mature dendritic cell Immature dendritic cell Macropahge MDSC Immune Therapy CD8 T cell Finn, NEJM. 28 Th1 CD4 T cell Treg cell Th2 CD4 T cell Alternative immune modulations Host defense mechanism
Tumor-associated macrophage, TAM Classically activated TNF, IFNγ Microbial LPS Monocytes Alternative activated IL-4, IL-1 M-CSF inos Arginase I M1-macrophage M2-macrophage IL-12, IL-23, TNF, IL-1, CXCL1 Microbicidal Tumoricidal IL-1, IL-13, IL-4, CCL22 scavenger, mannose receptor Cell clearance/wound healing Tumor promotion
Interactions between macrophage and tumor cells M2-macrophage (TAM) B cell TReg cell T2h cell Promote tumor survival
Tumor cell Macrophage Cell growth Cell cycle Other mechanisms Protease, Adhesion molecule ( MMP-1, TIMP-1, ICAM-1) Signal transduction (IL-6) Inflammation (CSF-1, IL-6, IL-8, PDGF) Angiogenesis (CSF-1, IL-6, IL-8)
The relationship between macrophage densities and 5-yr survival rates of human cancers 5-year overall survival (%) 27 24 21 18 15 12 9 6 3 1 8 6 4 2 Average CD163 positive area (%) Thyroid (PTC) Endometrium Kidney Prostate Stomach Breast Ovary Cervix Liver Esophagus Larynx GB Lung Pancreas Thyroid (ATC) CD163 1.8 2.8 2.8 3.6 4.4 4.7 5. 5.6 5.9 6.1 6.3 6.9 6.9 7. 22.9 5yr OS 1 86.5 78.8 92 69.4 91.3 61.6 8.1 28.6 3.2 71.7 27.5 2.7 8.7 NA
TAM densities in thyroid cancers P/D, CD68 P/D, CD163 M/D, CD68 W/D, CD68 High density of TAMs in PDTC correlates with invasion and decreased cancer-related survival. Ryder et al. Endocr Relat Cancer. 28
TAM densities in thyroid tissues Normal Adenoma PTC ATC Macrophage Normal (N=7) Adenoma (n=57) PTC (n=148) ATC (n=17) P-value Negative 6 (86 %) <.1 Low density 1 (14 %) 41 (72 %) 78 (57 %) 3 (18 %) High density 16 (28 %) 58 (43 %) 14 (82 %)
TAM densities in Papillary thyroid cancers TAMs were scored by the number of CD68+ cells/total tumor cells Low (<25%) High (25% to 7%) Kim, Cho et al. EnM 213
Clinical characteristics according to the TAM densities TAM density Low (n=79) CD163 High (n=64) P-value Sex (M/F) 13/66 12/52.72 Age of diagnosis 45.3 ± 15.8 43.5 ± 16.6.5 Tumor size (cm) 2.3 ± 1.1 2.8 ± 1.7.42 Extrathyroidal invasion 15 (22%) 13 (22%).997 LN metastasis 38 (62%) 48 (8%).31 Recurrence or metastasis 5 (16%) 9 (23%).429 CD31 + cell (%) 3 (5%) 11 (19%).16 CD68 + cell (%) 3 (5%) 25 (4%) <.1
Macrophages support tumor growth Tumor cell ±THP-1 injection 7 35 (days) Sac BHP1-3 only BHP1-3/THP-1 Tumor volume (mm 2 ) 7 6 5 4 3 2 1 THP-1(+)/PBS THP-1(-)/PBS 1. 7 2. 14 3. 28 4. 35 Days after implantation
Macrophages support tumor growth Tumor cell ±THP-1 injection 7 35 (days) Sac H&E BHP1-3 only BHP1-3 / THP-1 Area of necrosis (µm 2 ) 4 3 2 1 1 2 BHP1-3 only BHP1-3 / THP-1 Area of necrosis
Clodronate liposome inhibited macrophage-related tumor growth Tumor cell ±THP-1 injection Clodronate liposome Peritumoral injection (3 times/ week) 7 35 (days) Sac Clodronate liposome 7 Tumor volume (mm 2 ) 6 5 4 3 2 1 # # THP-1(+)/PBS THP-1(+)/Clodronate THP-1(-)/PBS THP-1(-)/Clodronate 1.7 2. 14 3. 28 4. 35 Days after implantation
Clodronate liposome inhibited macrophage-related tumor growth Tumor cell ±THP-1 injection Clodronate liposome Peritumoral injection (3 times/ week) 7 35 (days) Sac + THP-1(+)/PBS + THP-1(+)/Clodronate THP-1(-)/PBS THP-1(+)/PBS THP-1(+)/Clodronate Frequency Intensity of CD68 CD68+ (%) 3 2 1, # Day 1 15 Day 2 25, #
TAMs support tumor cell migrations Wound healing assay Transwell assay 3D invasion assay + macrophage
TAM increased angiogenesis in tumor microenvironment Control CM BHP1-3 CM Control CM BHP1-3 CM + macrophage Total tube length (µm/field) 4 3 2 1 1 2 THP-1 (-) THP-1 (+)
Tumor cell Macrophage Tumor growth How it works? Tumor cell migration Angiogenesis
CXCL16 expressions in tumor/macrophage co-cultures H.Tori (normal thyroid cells) BHP1-3, KTC-1 (PTC cells) + macrophage Conditioned medium (CM) + macrophage Normal thyrocyte KTC-1 BHP1-3 CXCL16 (pg/ml) 3 2 1 H tori 1 2 3 4 5 6 7 BHP1-3 KTC-1 macrophage H tori BHP1-3 KTC-1 +macrophage
CXCL16/CXCR6: a regulator of cancer metastasis Transmembrane CXCL16: growth Soluble CXCL16: metastasis
CXCL16 expressions in PTCs Control CM M/BHP1-3 CM Control CM Monocyte CM BHP1-3 CM M/BHP1-3 CM BHP1-3SC Macrophage CXCR6/GAPDH 4 3 2 1 Monocyte 1 BHP1-3SC 2 # CXCL16/GAPDH 7 6 5 4 3 2 1 # Monocyte 1 BHP1-3SC 2 CD163 + cell (No./1 3 µm 2 ) γ 2 =.28, P<.1 CXCR6 expressions in both tumor cell and stroma CXCR6 + cell (No./1 3 µm 2 )
CXCL16 mediated macrophage effects on tumor cell migration Vehicle CXCL16 THP-1 (-) THP-1 (+) Vehicle anti-cxcl16 TAPI-2 Migration length 8 6 4 2 δ # 1 2 THP-1 (-) THP-1 (+) # mtor pakt AKT VEH CXCL16 VEH THP-1 (+) Anti- CXCL16 Densitometry 1.5 1.2.9.6.3 THP-1(-) / VEH THP-1(-) / CXCL16 THP-1(+) / VEH THP-1(+) / anti-cxcl16 # # GAPDH (.) mtor/gapdh 1 pakt/akt 2
CXCL16 mediated macrophage effects on angiogenesis CM Vehicle CXCL16 Control 8 Control CM M/BHP1-3 CM M/BHP1-3 Vehicle anti-cxcl16 Total tube length (µm/field) 6 4 2 1 2 #
? Tumor cell Macrophage CXCL16/CXCR6 signaling Tumor growth Tumor cell migration Angiogenesis
Tumor recruit circulating monocytes and switch into M2- macrophages NOG mouse Bone marrow Tumor Tumor cell injection PKH26-labelled monocyte injection Frequency Intensity of red fluorescence (PKH26) 28 31 (days) Sac PKH+ cell (%) in 1 6 cells Peripheral blood Bone marrow 6.3 ±.7 Tumor 13.3 ± 3.7
Tumor recruit circulating monocytes and switch into M2- macrophages NOG mouse mrna expressions in PKH26+ cell 2.5 2. Bone marrow Tumor Tumor cell injection PKH26-labelled monocyte injection 1.5 1. 28 31 (days) Sac.5. CD163 IL-4 IL-12 TNFα
CD163 IL-1 CD26 Mock Relative gene expression Relative gene expression 2.5 2. 1.5 1. CM 2.5 25 2. 1.5 2 15 1 5 1. CM CM Relative gene expression
CD163 IL-1 CD26 2.5 2.5 25 Mock Anti-CXCL16 Relative gene expression Relative gene expression 2. 1.5 1. CM # 2. 1.5 1. # CM CM 2 15 1 5 # Relative gene expression 2.5 2. 1.5 1. CD163 IL-1 CD26 IL-β inos 1.6.5 Relative mrna expressions (rhcxcl16/vehicle) 1.2.8.4 IL-1β 1.5 1.2.9.6.3 Relative gene expression Relative gene expression. CM 1 2 3 4 5 CM inos M-CSF (-) M-CSF (+).
TAM, as a therapeutic target in cancer therapy Blocking differentiation CSF-1, cfms inhibitors CCL2, monocloncal Ab (MLN122) Repolarizations Pantoprazole IL-12 Killing TAM in the tumor microenvironments Clodronate-loaded liposomes (CLIP) Zoledronic acid
Conclusions TAMs support tumor growth, invasion, and metastasis. CXCL16 signaling mediated TAM actions in human papillary thyroid cancers. TAM might be a potential therapeutic targets of human cancers.
Acknowledgements Seoul National University Pf. Young Joo Park Pf. Kyong Soo Park Pf. Do Joon Park Pf. Young A Kim Gachon University Pf. Byung-Chul Oh Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI) Young Investigater s award from Korean Endocrine society ( 향설연구비 )