Nat. Rev. Canc, 7, 834

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?

“What is it that decides which organ shall suffer in a case of disseminated cancer?” , 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 , Weiss 4.Premetastatic Niche conditioning

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

“What is it that decides which organ shall suffer in a case of disseminated cancer?” , 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 , Weiss 4.Premetastatic Niche conditioning

“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

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

“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

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:

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

Tumor cells and endothelium: selectins Biancone L et.al., J Exp Med 183: 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

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:

Tumor cells and endothelium: direct activation Khatib A et.al., Am J Pathol 167: 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

Tumor cells and lymphatic metastasis/niche Rinderknecht M, Detmar M J Cell Physiol 216:

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

Tumor cells and lymphatic metastasis/niche Hirakawa S et.al., J Exp Med 201: 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?

Tumor cells and bone marrow-derived cells Whetton AD, Graham GJ Trends Cell Biol 9: 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

Systemic tumor effect: metastatic niche in the lung Hiratsuka S et.al., Nature Cell Biol 8 (12): 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

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

Systemic tumor effect: metastatic niche in the lung Hiratsuka S et.al., Nature Cell Biol 8 (12): 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)

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

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

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

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

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

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

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

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

Rescue in Id3 KO

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

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

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

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

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)…