1. Nat. Rev. Canc, 7, 834

2. Premetastatic Micrometastases (pre angiogenic) Metastases Looking at secondary tumor sites: Tumor cells are: Intravasating Circulating Homing/Arresting Extravasating Initiating & maintaining growth in new environment <-- From where? --> Dormancy?

3. “What is it that decides which organ shall suffer in a case of disseminated cancer?” - 1889, Paget 1.“Seed and Soil” organ specificity depends on tumor cell and 2° site 2.Blood Flow Patterns determine sites ( 1920s ) 66% consistent with blood flow 14% fewer mets than expected 20% more mets than expected 3.Therefore a combined theory… Blood flow then compatibility - 1992, Weiss 4.Premetastatic Niche conditioning

4. Chambers et al, Nature Reviews Cancer (2002), 2, 563

5. “What is it that decides which organ shall suffer in a case of disseminated cancer?” - 1889, Paget 1.“Seed and Soil” organ specificity depends on tumor cell and 2° site 2.Blood Flow Patterns determine sites ( 1920s ) 66% consistent with blood flow 14% fewer mets than expected 20% more mets than expected 3.Therefore a combined theory… Blood flow then compatibility - 1992, Weiss 4.Premetastatic Niche conditioning

6. “Class Action”/ Community effects Heterogeniety of cells in tumor population Clustered Migration of Tumor Cells ECM degradation Paracrine Loops (signaling /adhesion) Temporal Cooperation successive waves of cancer cells passing by induce progressive changes

7. Aguirre-Ghiso, J., Nat. Rev. Canc, 7, 834 Head-Neck Tumor Reacquisition/ Presence of uPAR on cells- growth of disseminated cells

8. “pre”metastatic niche - do metastatic tumors affect host tissues, either locally around the tumor, or systemically? tumor cells and:- endothelium (vascular, lymphatic) - immune cells (local, from BM) - fibroblasts/stroma

9. Tumor cells and endothelium: -lots of experiments from the 1990’s show that cytokine injection (IL-1, IL-6, TNFa) activate endothelim and promote subsequent tumor (tail vein met assays) formation Orr FW et.al., Am J Pathol 190: 310-329

10. Tumor cells and endothelium: integrins adhesion at rest adhesion under flow Orr FW et.al., Am J Pathol 190: 310-329

11. Tumor cells and endothelium: selectins Biancone L et.al., J Exp Med 183: 581-587 Lots of data on sel. upregulation by cytokines and tumor cells, but this is nice: a mouse model with transgenic E- selectin + B16 cells engineered to express selectin ligands changes the pattern of tumor metastasis

12. Tumor cells and endothelium: direct activation IL-1TNFa lung liver both E-selectin Injected tumor cells can induce cytokine production and endothelial changes in the liver (met site) Khatib A et.al., Cancer Res 59: 1356-61

13. Tumor cells and endothelium: direct activation Khatib A et.al., Am J Pathol 167: 749-759 Cytokines actually made by immune cells (Kupffer?) in the liver; By confocal microscopy, adhesion molecule induction leads to tumor cell extravasation GFP tumor F4/80 (Kupffer?) TNFa Met. tumor cells Non-met. tumor cells Point though: Known that most of these extravasating cells will not survive

14. Tumor cells and lymphatic metastasis/niche Rinderknecht M, Detmar M J Cell Physiol 216: 347-354

15. Tumor cells and lymphatic metastasis/niche Rinderknecht M, Detmar M J Cell Physiol 216: 347-354 Keratin 14-driven GFP+VEGFA or VEGFC; chemical-induced melanoma model

16. Tumor cells and lymphatic metastasis/niche Hirakawa S et.al., J Exp Med 201: 1089-1099 Lymphatics/Lyve1HEV/CD31 BrdU Prox1 Tumor can influence downstream tissue and make it more hospitable for metastasis. Argue secreted factors, but do they really exclude circulating cells?

17. Tumor cells and bone marrow-derived cells Whetton AD, Graham GJ Trends Cell Biol 9: 233-238 tumor Definite clinical/mouse evidence to indicate that some bone marrow- derived cells (BMDCs) are mobilized into blood in cancer. Not clear: what induces mobilization (secreted factors like VEGF? tumor cells in BM?); what exactly these cells are; what they do for tumor

18. Systemic tumor effect: metastatic niche in the lung Hiratsuka S et.al., Nature Cell Biol 8 (12): 1369-1375 -benign F2; B16; LLC; 3LL highly metastatic cells inj. s.c. into nude mice, extract lungs on d.14, microarray - what is upregulated in “pre-metastatic” (section staining; PCR) lungs? -top two proteins are secreted chemokine-like molecules, S100A8 and A9 -expressed by Mac1+ myeloidy cells and by lung endothelial cell, not by tumor cells -more Mac1+ cells in pre-metastatic lungs

19. Systemic tumor effect: metastatic niche in the lung Hiratsuka S et.al., Nature Cell Biol 8 (12): 1369-1375 -what does tumor secrete to induce S100A8/9 expression? apparently, TNFa/TGFb/VEGFA:

20. Systemic tumor effect: metastatic niche in the lung Hiratsuka S et.al., Nature Cell Biol 8 (12): 1369-1375 -S100A8/9 promote mac. and tumor cell migration in vitro -Mac and endothelial cells stimulated with S100A8 promote tumor cell migration (TNFa, MIP2, TGFb - p38 for migration)

21. Systemic tumor effect: metastatic niche in the lung Hiratsuka S et.al., Nature Cell Biol 8 (12): 1369-1375 -blocking S100A8/9 reduces # myeloid cells and metastasis (never show late time points, though - 24hr maximum)

22. Systemic tumor effect: metastatic niche in the lung Hiratsuka S et.al., Nature Cell Biol 8 (12): 1369-1375 TNFa, MIP2, TGFb, S100A8/9

23. Premetastatic Niche: Timing of molecular and cellular changes HPC express VEGFR1 Induction not dependent on presence of tumor cells

24. Intradermal, SubQ & tail vein injection tumor models LLC- metastasize to lung B16- metastasize to lung, liver, spleen, kidney using 2 x 10 6 cells Focus on the recruitment of HPCs in the premetastatic niche Histology/Immunohistochemistry FACS

25. Day 3 Day 8 Day 14 Day 18 Day 23 FN expression in lungs BMDC/HPCs Solitary Tumor Cells & EPCs Micrometastases FACS -2 days -1-2 days

26.  -gal BM Day 14 BMD clusters >irradiation <LLC implant n lung Day 23 µmets met BM B16 Day 18 VEGFR1

28. Day 14 VEGFR1 BM VEGFR1 CD133 VEGFR1 CD117 BMDC are HPCs with stem/progenitor markers similar VEGFR1 expression in cellular clusters in pre-metastatic human tissue (lymph nodes) breast, lung, gastrointestinal Blockade of VEGFR1+ cells (Ab): decrease clusters, µmet

29. Day 14 VEGFR1  VEGFR1 Id3 > cluster   Release s Kit-ligand VEGF-A Xpression enhanced by  FN intxn Id < integrins Anti alpha 4 integrin MMP-9 KO--> decrease clusters, µmet Id3 KO decrease R1 + HPC in circ (654 v 3283) rescue cluster/µmet w/HPCs from wt

30. Rescue in Id3 KO

31. No tumorDay 3Day 14 FN PDGFR  LLC qRT-PCR lungs B16 FN

32. Conditioned media experiments: Established clusters after MCM treatment/ Tail vein B16 Media aloneB16 conditioned media (lone Tumor cell)BMDCs/Tumor HPC clusters 4 days 1 day

33. Treat mice with conditioned B16 media before and after Intradermal LLC- redirect metastases Spleen Kidney IntestineOviduct What redirects?

34. Pre-metastatic niche in lymph node Micrometastatic/m etastatic niche Primary Breast Tumor Kaplan and Lyden reviews

35. Open questions… Is the premetastatic niche real? What tumor derived signals target BMDCs to tissue-specific sites? Genetic/Chemokine Expression Profile Is BMDC localization random or to specific sites within target organs? ie Selective upregulation of S100A8/A9 or throughout lungs What exactly is the function of VEGFR1+ HPCs in “niche”? Do they remain undifferentiated? Do they return to BM? Do they incorporate into tumor vasculature? What exactly are the Mac1+ cells, are they functioning like HPCs? How exactly are both HPCs and Mac1+ cells enhancing disseminated tumor cell recruitment? changes endothelium…MMP activation (is there)…