Targeting tumour associated macrophages in anti-cancer therapies. Annamaria Gal Seminar Series on Drug Discovery Budapest 5 January 2018

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1 Targeting tumour associated macrophages in anti-cancer therapies Annamaria Gal Seminar Series on Drug Discovery Budapest 5 January 2018

2 Macrophages: Professional phagocytes of the myeloid lineage APC, phagocytosis

3 Macrophages activation Classically activated macrophages (M1) Alternatively activated macrophages (M2) T h 1 cell NK cell LPS TLR4 T h 2 cell Granulocyte APC Wound healing Tissue repair Tissue macrophage Tissue macrophage Antibacterial Regulatory T cell Anti-inflammatory Tissue macrophage Nature Reviews Immunology (Modified)

4 Macrophage polarization M1 M2 inos TNFa, IL12, IL-1b, IL-6, CCL2 (MCP-1) CXCL9, CXCL10 Arginase1 (Arg1) Mannose receptor (MR) Resistin-like alpha (Fizz-1) Chitinase 3-like 3 (Ym1) IL-10 Modified after Martinez FO et al Annu Rev Immunol 27:451-83

5 Tumour associated macrophages >80% of cancers poor prognosis Breast, ovarian, gastric, bladder, oral, pancreatic and thyroid cancer are associated with worse overall survival <10% of cancers good prognosis Colorectal cancer is associated with better overall survival Macrophages in pancreatic cancer Macrophages promote pancreatic cancer progression and invasion Promoting angiogenesis, metastasis, Suppressing anti-tumour immune responses Contribute to resistance to chemotherapy and to the emergence of cancer stem-like cells Gardian K. et al. J Cancer 2012; 3:

6 Macrophage polarization Anti-tumour Pro-tumour M1 M2 inos TNFa, IL12, IL-1b, IL-6, CCL2 (MCP-1) CXCL9, CXCL10 Arginase1 (Arg1) Mannose receptor (MR) Resistin-like alpha (Fizz-1) Chitinase 3-like 3 (Ym1) IL-10 Modified after Martinez FO et al Annu Rev Immunol 27:451-83

7 Methods In vitro In vivo Analysis of macrophage plasticity Isolation of bone marrow from C57BL/6 mice Differentiation of myeloid progenitors into macrophages (conditioned media from L292 cells) Polarization of bone marrow derived macrophages Macrophage polarization in experimental metastasis of mouse melanoma Injection of B16F10 mouse melanoma cells into the tail vein of C57BL/6 mice Lung harvest at early (72h) and late (21d) stage of pulmonary metastasis M1 LPS + IFNg M2 IL-4 Fluorescence activated cell sorting (FACS) of alveolar and infiltrating macrophages from the lungs Customized TaqMan PCR array of mouse immune response and macrophage polarity genes FACS analysis of macrophage surface markers Luminex assay to reveal the secretion profile of melanoma cells, macrophages and the metastatic lung

8 Phenotype and gene expression of in vitro polarized bone marrow derived macrophages FITC-labelled mannosylated-bsa TRITC-Phalloidin, DAPI Induced genes Unpolarized M1 LPS+IFNg M2 IL-4

9 Regulated genes of in vitro polarized bone marrow derived macrophages M1 M2

10 Tumour associated macrophages in an experimental metastasis model of melanoma C57BL/6 B16F10 mouse melanoma cells Lung harvest Fluorescence activated cell sort of alveolar and infiltrating macrophages at early (72h) and late stage (21d) of lung metastasis Alveolar (resident) macrophages F4/80 + CD11c + Infiltrating (recruited) macrophages F4/80 + CD11b +

11 Macrophage sort from control and metastases bearing lungs CD11c Ctrl (Unchallenged) Early stage 72h Late stage 21d F4/80 + CD11c + F4/80 + CD11c + F4/80 + CD11c + F4/80 + CD11b + F4/80 + CD11b + F4/80 + CD11b + CD11b

12 Sorted macrophage numbers from metastasis bearing lungs 3d 21d 6.0 F4/80 + gate Cell number x CD11b+ Infiltrating CD11c+ Alveolar 0.0 Ctrl 72h 21d

13 Induced gene expression of sorted macrophages from metastasis bearing lungs Alveolar macrophages Infiltrating macrophages

14 Regulated genes of sorted alveolar macrophages from metastasis bearing lungs Alveolar macrophages

15 Regulated genes of sorted infiltrating macrophages from metastasis bearing lungs Infiltrating macrophages

16 Chemokine ligands for CCR1, CCR2 and CCR5 secreted by tumour cells / present in blood Secretion profile of B16F10 cells Chemokine and colony stimulating factor levels in blood CCL2 receptor: CCR2 CCL4 receptors: CCR1 and CCR5 CCL5 receptors: CCR1, CCR3 and CCR5

17 Chemokines and their cognate receptors

18 Regulation of chemokine receptors in sorted macrophages from metastasis bearing lungs Alveolar macrophages

19 Regulation of chemokine receptors in sorted macrophages from metastasis bearing lungs Infiltrating macrophages

20 Administration of chemokine receptor antagonists C57BL/6 B16F10 mouse melanoma cells CCR1 antagonist: J , 10 mg/kg i.p. per day, n=5 CCR2 antagonist: RS , 4 mg/kg p.o. per day, n=5 CCR5 antagonist: DAPTA, 0.01 mg/kg/day, s.c., n=5 Mice were treated for 3 or 21 days. Lung harvest Colony count CTRL J RS DAPTA FACS analysis of surface markers of alveolar and infiltrating macrophages at early (72h) and late stage (21d). 21 days after B16F10 cells injection

21 Total surface colony counts Lung colony counts after 21-day administration of chemokine receptor antagonists Surface colony counts CCR1 blockage is effective to suppress metastatic growth ** * Ctrl Ctrl J RS DAPTA * J RS DAPTA *p=0.006 **p= CCR1 antag CCR2 antag CCR5 antag 0 n= 4-5 < >1.5 p<0.05 mm

22 Macrophage response to chemokine receptor inhibition in the course of lung colonization/ metastasis progression Infiltrating macrophages Alveolar macrophages

23 Percentage of CD45 + cells CCR1 inhibition is effective to suppress metastatic growth of melanoma CTRL CD3e+ NK1.1+CD3e- CD11c+F480+ CD45 + gate *** * ** CCR1 antag Gr1+ CD11b+F480+ CD11b+CD11c+F480+ *p=0.019 **p=0.013 ***p=0.009 n=4-5

24 Blood secretion profile of mouse melanoma metastasis 4000 Ctrl Secretion of IL-15 and IL-18 by AM Concentration (pg/ml) IL-1b IL-5 72h 24d IL-6 IL-9 IL-10 IL-12(p40) IL-12(p70) IL-13 IL-15 IL-17 IL-18 MCP-1 MIP-1b RANTES KC G-CSF GM-CSF TNF-a IFN-g VEGF

25 Summary Recruited macrophages in the pulmonary metastasis of mouse melanoma show a plasticity phenotype from early to late stage of metastasis progression that doesn t follow closely the in vitro macrophage polarization scheme At early stage a primarily pro-inflammatory phenotype exerts anti-tumour actions At late stage a primarily anti-inflammatory phenotype promotes metastasis growth Resident alveolar macrophages show adaptation to the changing pulmonary environment in response to tumour challenge Chemokine receptors CCR1, CCR2 and CCR5 are involved in the recruitment of circulating monocytes/ macrophages, and upregulated during metastasis progression Chemokine receptors CCR1, CCR2 and CCR5 expressed by alveolar macrophages at very low level, and are not regulated significantly during metastasis progression CCR1, CCR2 and CCR5 inhibition results in reduced infiltration of F4/80+CD11b+ macrophages at early stage of lung colonization CCR1 inhibition also results in reduced infiltration of F4/80+CD11b+ macrophages at late stage of metastasis progression and contributes to reduced colony growth The number of alveolar F4/80+CD11c+ macrophages significantly increase with the reduction of recruited macrophages in response to CCR1 inhibition, and release cytokines important for anti-tumour actions CCR1 inhibition via alveolar macrophage stimulation leads to an increase of NK cell infiltration of the metastasis bearing lung, and contributes to reduced colony growth

26 Acknowledgement Prof Ruth J Muschel Gray Institute for Radiation Oncology & Biology University of Oxford Andrew Worth Jenner Institute University of Oxford Dr Thomas Tapmeier Gray Institute for Radiation Oncology & Biology University of Oxford