nat. rev. canc, 7, 834. premetastatic micrometastases (pre angiogenic) metastases looking at...

Nat. Rev. Canc, 7, 834

Premetastatic

Micrometastases(pre angiogenic)

Metastases

Looking at secondary tumor sites:

Tumor cells are:IntravasatingCirculatingHoming/ArrestingExtravasatingInitiating & maintaining growth in new environment

<-- From where?

--> Dormancy?

“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 flow14% fewer mets than expected20% more mets than expected

3. Therefore a combined theory…Blood flow then compatibility

-1992, Weiss

• 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?”- 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 flow14% fewer mets than expected20% more mets than expected

3. Therefore a combined theory…Blood flow then compatibility

-1992, Weiss

• Premetastatic Niche conditioning

“Class Action”/ Community effects

Heterogeniety of cells in tumor population

Clustered Migration of Tumor CellsECM degradationParacrine Loops

(signaling /adhesion)Temporal Cooperation

successive waves of cancer cells passing byinduce 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: 310-329

Tumor cells and endothelium: integrins

adhesionat rest

adhesionunder flow

Orr FW et.al., Am J Pathol 190: 310-329

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

Tumor cells and endothelium: direct activation

IL-1 TNFalungliverboth

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

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 tumorF4/80 (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: 347-354


Rinderknecht M, Detmar M J Cell Physiol 216: 347-354

Keratin 14-driven GFP+VEGFA or VEGFC;chemical-induced melanoma model


Hirakawa S et.al., J Exp Med 201: 1089-1099

Lymphatics/Lyve1 HEV/CD31

BrdU Prox1Tumor 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: 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

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



-what does tumor secrete to induce S100A8/9 expression?apparently, TNFa/TGFb/VEGFA:



-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)



-blocking S100A8/9 reduces # myeloid cells and metastasis (never show late time points, though - 24hr maximum)



TNFa, MIP2, TGFb,S100A8/9

QuickTime™ and a decompressor

are needed to see this picture.

Premetastatic Niche:Timing of molecular and cellular changes

HPC express VEGFR1Induction not dependent on presence of tumor cells

Intradermal, SubQ & tail vein injection tumor models

LLC- metastasize to lungB16- metastasize to lung, liver, spleen, kidney

using 2 x 106 cells

Focus on the recruitment of HPCs in the premetastatic nicheHistology/ImmunohistochemistryFACS

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 14BMD clusters

>irradiation<LLC implantn

lung

Day 23µmets

met

BMB16

Day 18V

EG

FR1

Day 14

VEGFR1BM

VEGFR1CD133

VEGFR1CD117

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

VEGFR1Id3

> cluster

Release s Kit-ligand VEGF-A

Xpression enhanced byFN intxn

Id < integrins

Anti alpha 4 integrinMMP-9 KO --> decrease clusters, µmetId3 KO decrease R1+ HPC in circ (654 v 3283)

rescue cluster/µmet w/HPCs from wt

Rescue in Id3 KO

No tumor Day 3 Day 14

FNPDGFR

LLC

qRT-PCRlungs

B16

FN

Conditioned media experiments:Established clusters after MCM treatment/ Tail vein B16

Media alone B16 conditioned media(lone Tumor cell) BMDCs/Tumor

HPC clusters

4 days

1 day

Treat mice with conditioned B16 media before and afterIntradermal LLC- redirect metastases

Spleen Kidney IntestineOviduct

What redirects?



Pre-metastatic niche in lymph node Micrometastatic

/metastatic 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)…

Niche stroma ligand HSC re c e pto r

Endothelia l (E)- select in E- selecti n ligand (ESL) 1 P- selectin glucoprotein ligand - 1 (PSGL)

CD62L

Vascular cell adhesi on molecule - 1 (VCAM- 1), fibronectin

Very la t e antige n- 4 (VLA- 4) ( 4 1)

Urokinase - type plas minogen act ivatio n receptor (uPAR)

Urokinase binding t o uPAR modulate s integrin a ctivat ion

Hyalurona n (HA) CD44, receptor for h yaluronan -mediated motil ity (RHAMM)

N- cadheri n N- cadheri n B- catenin Surface p rotein s including axin Kit lig and c- Kit Osteo pont in (OPN) - and - integrins Stromal d erived fact or - 1 (SDF- 1) Chemokine receptor - 4 (CXCR4)

Molecular machinery

HSCs and/or HPCs Cancer stem cells

Origin Primitive haemangioblast precursor shared with EPC.

Can derive from normal stem cells or from more-differentiated cells re-programmed to express the properties of stem cells.

Microenvironment

Supportive niche within mesenchymal-derived BM stroma (e.g. fibroblasts, osteoblasts and adipocytes). Binding to osteopontin promotes quiescence although also mediates trans-marrow migration. A dysfunctional niche might confer aberrant properties to HSCs

Similar highly specific and specialized microenvironment supported by carcinoma-associated fibroblasts. Unlike normal stroma, components might remain perpetually activated [94]. Production of osteopontin by malignant cells confers a migratory phenotype (m

Adhesion molecules

Necessary for retention in niche and can mediate homing (e.g. VLA-4) [96] (Table 1).

Redistribution and/or downregulation of epithelial adhesion molecules (including E-cadherin and cytokeratins) and expression of mesenchymal molecules (including N-cadherin) mediate epithelial-mesenchymal transition (EMT) and metastatic invasion [97].

Proteolytic degradation enzymes

MMP-9 activates cytokines (e.g. sKitL) and is necessary for release of SDF-1 from platelets and important in retaining HSCs in the BM.

MMP-9 and MMP-3 degrade basement membrane promoting EMT for primary tumour growth and metastatic invasion 97 and 98.

Growth factors

VEGF and VEGF receptor signaling involved in adhesion, survival and migration [99]. Transforming growth factor- (TGF- ), FGF-4, FGF-2 and PDGF also involved.

VEGF and VEGF receptor signaling support tumour expansion [100] and mediate invasion and growth advantage observed with cancer stem cells over non-stem-cell-like cancer cells [101]. TGF-, PDGF, FGF expressed by cancer cells and correlate with progression

Chemokine signaling

CXCR4+-mediated homing. CXCR4 signal transduction might be diminished in pathological conditions.

CXCR4-directed migration in response to peripheral SDF-1 gradients.

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