Organ specific metastasis

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Organ specific metastasis Breast cancer lymph
nodes, bone, liver, brain and lung Prostate
cancer lymph nodes and bone Colorectal cancer
liver Melanoma same as breast cancer
skin Stephen Paget (1889) seed and soil James
Ewing (1920) hemodynamics - plumbing
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Possible Determinants of Site-Specific
Metastasis Hemodynamics (Plumbing) 1. Blood
flow patterns (How many cells are delivered to an
organ?) 2. Mechanics of cell arrest, growth and
extravasation Intravascular metastatic
cancer cell homotypic aggregation at the sites
of primary attachment to the endothelium.
(Glinsky and Quinn. Cancer Research. 63.
July 2003). Homing 1. Chemokines
Involvement of chemokine receptors in breast
cancer metastasis. (Mueller and Zlotnick.
Nature. 410. March 2001). Expression
of CXC chemokine receptor-4 enhances the
pulmonary metastatic potential of
murine B16 melanoma cells. (Murakami and
Hwang. Cancer Research. 62. December 2002).
Multiple actions of the chemokine CXCL12 on
epithelial tumor cells in human ovarian
cancer. (Scotton and Balkwill. Cancer
Research 62. October 2002). 2. Selectins 3.
Vasculature specificity

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Possible Determinants of Site-Specific Metastasis
Seed and Soil 1. Bone growth factors
Metastasis to bone causes, consequences and
therapeutic opportunities. (Mundy. Nature
Reviews Cancer. 2. August 2002).
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From MacDonald and Chambers. BioEssays. 24.10.
2002.
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Hemodynamics vs. Seed Soil Support for Both
Hypotheses Leonard Weiss (1986) autopsy
studies Breast and prostate cancer a greater
of bone mets. than would be expected based
solely on blood flow patterns. Osteosarcoma,
stomach and testicular cancer a smaller of
skin mets. than would be expected based on blood
flow. - 16 primary tumor types and 8 target
organs were analyzed 66 of tumor-type-organ
met. pairs explained on basis of blood flow 14
of pairs were less than expected 20 of pairs
were more than expected
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Quantitation of Tumor Cell Shedding Into
Blood (Butler and Gullino, 1975) MTW9 mammary
carcinoma grown as s.c. tumor Tumor enveloped
in a paraffin sac Tumor was drained by a single
vein Tumor cells were recovered by cannulating
tumor vein and counted by immunofluorescence
staining for anti-MTW9 12X more tumor cells in
venous vs. arterial blood Total cells released
was 3-4 x 106 cells/g tumor/24 hrs when tumor
was size 2-4 grams.
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Arrest of Circulating Tumor Cells Conventional
Wisdom
Circulating tumors arrest in the first capillary
bed they encounter (mechanical arrest/hemodynamics
). Both lung and liver are very efficient at
arresting cells. Most circulating cells arrested
by size restriction Capillaries 3-8 um in
diameter Cancer cells 20 um in diameter or
more Other factors blood pressure,
deformability of the cell and of the
capillaries. However, leukocytes much smaller
than tumor cells can arrest in blood vessels much
larger in size. Mediated by selectins and
integrins.
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Metastatic Inefficiency (Cameron and MacDonald,
2000) - Injected B16F0 along with fluorescent
microspheres in accounting technique - High
efficiency of cell arrest in the lung and
extravasation. - 80 melanoma survive initial
arrest. - 1/40 inititated growth in the lung. (or
liver) - 1/100 micromets. progress to
macromets. - Inefficiency 1) failure to
initiate growth 2) failure to continue
growth. Overexpression of Ras aided 2 but not
1 in NIH3T3 (Varghese and Chambers, 2002) -
Metastatic and nonmetastatic cells do not
differ in their ability to arrest and
extravasate. (Also, Luzzi and Groom, 1998)
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Intravascular growth of tumors cells (Al-Mehdi
and Muschel, 2000) - Observation of 2.10.10
fibroblast cells 4-6 hours after injection in
lung - Attached to pre-capillary arterioles -
larger in diameter than tumor cell -
Extravasation very rare - before 24hrs, 2
extravasated after 24 hrs., 0 - Found
intravascular colonies - strings not present at
earlier times
Extension - strings
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Intravascular metastatic cancer cell homotypic
aggregation at the sites of primary
attachment to the endothelium.
Aggregate formation on HBMEC monolayer in flow
chamber (A) MDA-MB-435 (breast) (B) DU-145
(prostate) (C) DU-145 intravascular homotypic
adhesion ex vivo - perfused dura mater
microvessels (D) Same w/ MDA-MB-435 (Glinsky
and Quinn, 2003)
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(E) DU-145 forming aggregate in a big
arteriole (F) Multiple DU-145 aggregates (G)
Multiple MDA-MB aggregates in mouse
subpleural precapillary aterioles
(Glinsky and Quinn, 2003)
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12 DU145 aggregates joining together in narrow
capillary Arrow stably adhered tumor
cells (Glinsky and Quinn, 2003)
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Red arrow site of microvascular spasm
(constriction) - Through contortion, cell avoids
mechanical entrapment - Later becomes adhered to
wider part of the microvessels (Glinsky and
Quinn, 2003)
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Mechanism of Aggregation - Carbohydrate
T-antigen mucin-type core 1 disaccharide
GalNAc - Involved w/ hetero- and homo-typic
adhesion of breast and prostate canc. - Binds
galectin-3 on endothelial cells - T-antigen
upreg. on tumor surface upon arrest -
Lactosyl-L-leucine T-antigen mimic -
Lactitol-L-leucine nonfunctional isomer - P30
T-antigen masking agent - Neither affects
confluent cell growth
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Hemodynamics/Mechanics of Metastasis Formation
Summary Many, but not all, cases of primary
tumor/metastasis pairs can be explained by blood
flow. But seed and soil still applies after
arrest - not mutually exclusive. Arrest likely
occurs by trapping or aggregation. Glinsky and
Quinn propose a 2-step mechanism for arrest 1)
Heterotypic adhesion b/t tumor cell and EC 2)
Homotypic adhesion b/t tumor cells to form
aggregates These interactions involve
carbohydrate T-antigen on the tumor and
galectin-3 on blood vessels. Extravasation is
not necessary for tumor growth. Metastasis is
inefficient estimated to be 0.01 of cells form
mets.
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Chemo-attractants and Metastasis
Tumor cells migrate in vitro towards
organ-specific extracts Extracts of bone, brain,
liver and lung - chemoattraction and -invasion
assays - B16 melanoma .......................
migrates to brain extract - T241-PM2
fibrosarcoma ........ migrates to lung
extract - M50 sarcoma ..........................
migrates to liver extract - Preferential
migration ............ 3X over other extracts -
Preferential invasion .............. 4X over
other extracts - Dose-dependent migration -
Reflective of sites that get invaded in vivo
(Hujanen and Terranova, 1985) 12A-8
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ECM Matrix and Metastasis
Integrin ?-v/?-3 increases CHO cell metastasis to
bone - Increased area of bone lesions 5X -
Increased adhesion to collagen and bone
sialoprotein - ?-v/?-3 lig. - MDA-MB231 cells
from bone marrow - increased ?-v/?-3 (Pecheur
and Clezardin, 2002) 12A-17 CT26 murine colon
carcinoma selected for in vitro migration to
fibronectin. - No difference in integrin
levels. - Differences in activation status. -
Positive selection - reduced s.c. growth and lung
metastasis. - Negative selection - increased
s.c. growth and lung metastasis. (Geng and
Rees, 1998) 12A-5
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Homing by Chemokines Role in homing lymphocytes
and hematopoietic cells to specific organs and
sites of inflammation. - Small cytokine-like
proteins - Bind G-protein coupled receptors -
Cause cytoskeletal rearrangement, adhesion to
endothelial cells, directional migration
From Chambers and MacDonald, 2002
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Normal Physiological Functions of Chemokines -
HSCs Express chemokine receptor CXCR4.
Ligand CXCL12 found in bone marrow - osteoblasts,
stroma... KO chemokine/receptor impaired stem
cell migration f/ fetal liver to the bone
marrow - Dendritic Cells Respond to and use
ligands for CCR1/2/5/6/9 and CXCR4 to
extravasate into stroma. Upon activation, they
upregulate CCR7 - helps enter afferent
lymphatics - migrate to draining lymph nodes -
entrance into T-zones of lymphoid tissues -
Other immune effects - T-cell
extravasation - CCL27/CCR10 and CCL17/CCR4 -
skin homing - CCL25/CCR9 - transport of T cells
into the gut - CXCR12 (SDF-1) induces upreg. of
cell adhesion receptors - integrins VLA-4,
VLA-5 LFA-1 - mediate HSC adhesion to BM-ECs
and egress also upreg. MMP-9. - ELR chemokines
are angiogenic ELR- are anti-angiogenic.
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Chemokine-Mediated Arrest of Tumor Cells
(CXCL12)
From Moore, 2001
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Expression of chemokine receptors in (a) 7 human
breast cancer cell lines compared w/ normal
mammary epithelia (b) 12 malignant human melanoma
cell lines compared w/ normal melanocytes (Mueller
and Zlotnick, 2001)
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Common sites of breast cancer metastasis
Expression of chemokine ligands in normal tissue
sites (o) CXCL12 (p) CCL21 (q)
CCL27/CTACK (Mueller and Zlotnick, 2001)
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Migration of MDA-MB-231 Breast Carcinoma to
Organ-Derived Extracts (Mueller and
Zlotnick, 2001)
/- CXCR4 Antibody
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Chemokines As Homing/Adhesion Inducers
Stimulation of IGROV invasion by CXCL12 is
inhibited by Ab anti-TNF-alpha. (Scotton and
Balkwill, 2002)
CXCL12 stimulates adhesion of B16-CXCR4 to DMECs
(neonatal foreskin) (Murakami and Hwang, 2002)
Adhesion to LMECs (lung) -No ligand required!
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Chemokines As Inducers of Tumor
Proliferation (Scotton and Balkwill, 2002)
Growth of IGROV cells after CXCL12
stimulation /- anti-CXCR4 Ab (low serum)
CXCR4 Ab
TNF alpha production by IGROV after CXCL12
stimulation - TNF may be growth factor for
tumor cells (biphasic signaling) - but
neutralizing TNF had no effect on growth after
CXCL12 stimulation
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Lung Metastasis of MBA-MB-231 /- hCXCR4
mAb (Mueller and Zlotnick, 2001)
B16 OE CXCR4 cells have increased lung
mets. (Murakami and Hwang, 2002)
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Chemokines As Inducers of Tumor Proliferation
- B16 overexpressing CXCR4 - Growth under low
serum - T22 CXCR4 inhibitor - Ala Control
peptide - No effects under normal serum
(Murakami and Hwang, 2002)
- p42/p44 MAPK phos. after CXCL12 stimulation
(ERK1/2 - biphasic signaling)
- Akt phosphorylation in ovarian IGROV cells w/
CXCR4 after CXCL12 stimulation.
(Scotton and Balkwill, 2002)
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Chemokines and Metastasis Summary - MDA-MB-231
cells chemotactic to CXCL12/SDF-1 and
CCL21/6Ckine. - Neutralizing CXCL12/CXCR4
interaction inhibits lung metastasis, 73-82. -
CCR10/CCL27 are overexpressed in melanoma, which
metastasize to skin May represent a
skin-specific homing interaction Associated with
homing of memory T cells to skin - Chemotaxis
assays to organ-specific extracts could be
inhibited by antibodies neutralizing chemokine
interactions. - The exact effect of chemokines
is unclear and may involve many mechs. Growth
signaling of CXCR4 to Akt/MAPK indirectly via
TNF-alpha enhanced growth under low serum
conditions Adhesion binding to endothelial
cells (D/LMECs) in flow chamber - Overall,
overexpression of CXCR4 sufficient to increase
lung mets.
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Homing by Selectins Family of adhesion receptors
that bind mucin-type glycoproteins and recruit
leukocytes from circulation to sites of tissue
inflammation or injury.
May be used by tumor cells to form microemboli in
vasculature w/ platelets and/or leukocytes.
Molecular Cell Biology
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Homing by Selectins (Qian and Weissman, 2001),
8-2 L-selectin directs extravasation of
bloodborne lymphocytes into peripheral lymph
nodes and mesenteric lymph nodes via
addressins. RIP-Tag model overexpression of
L-selectin SV40 Beta-Gal ---gtblue cells were
detected in peripheral and mesenteric lymph
nodes Cells overexpressing L-selectin were
isolated and injected i.v. Result increased
homing to lymph nodes. Effect inhibited with a
neutralizing antibody against L-selectin. Subcutan
eous metastasis results also coincide. (Borsig
and Varki, 2002), 8-3 L- and P-selectins enhance
metastasis of mu. colon carcinoma
cells. L/P-selectin KO reduces mets. by LS180
(Rag2 null background) MC-38 expresses
L/P-selectin-ligands Detect by
chimeric-selectins/FACS L-ligands sialylated
mucins P-ligands sulfitides? MC-38 mets.
inhibited by P/L-KO synergistically
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Are Selectins Involved In Metastasis? (Krause and
Turner, 1999)
E-selectins most convincing metastasis data w/
colon carcinoma cells - Expression of selectin
ligands (sLe-x/a) correlated w/ metastasis -
Increased binding of cells to IL-1/TNF activated
Ecs Antibody inhibition studies - Melanoma
cells inconsistent data 12 cell lines have
sLe-x/a ligands without showing
E-selectin-dependent adhesion Ligand groups
might be on wrong glycoprotein - In vivo,
inhibiting E-selectin/ligand generally reduces
lung mets. (some melanoma, HT-29 colon
carcinoma) - B16 cells in mice overexpressing
E-selectin - redirected f/ lung to
liver P/L-selectins less clear - KO mice show
reduced metastasis - L-selectin may mediate
spread to peripheral lymph nodes - P-selectin on
platelets for activation - Chemotherapy using
cytokines like IL-1 or TNF-alpha may actually
aid metastasis by stimulating endothelial
cells to upreg. e.g. E-selectin!
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Organ-Specific Vasculature
Ruoslahti in vivo phage display/selection
identifies peptides that home to specific organ
vasculature. Normal/Malignant Breast
Vasculature CPGPEGAGC (cyclic)
.................. targets Aminopeptidase P, 100X
efficiency. Normal/Malignant Prostate
Vasculature SMSIARL .................... 10-15X
efficiency to prostate coupled to proapoptotic
peptide destroyed vessels in
prostate and delayed TRAMP Normal Lung
Vasculature CGFECVRQCPERC .....................
membrane dipeptidase Lymphatics (LyP-1)
Pasqualini and Ruoslahti. Nature. 1996. pp.
364-6.
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Osteolytic Bone Metastasis Osteolysis net bone
destruction. Majority of breast cancer. Not
caused by direct effects of tumor. Net
Osteolysis Activation of osteoclasts and/or
inhibition of osteoblasts OPG (osteoprotegerin)
a decoy receptor for RANKL. Inhibition Neutraliz
ing Ab anti-PTHrP inhibits osteolysis.
From Mundy, 2002
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Osteoblastic bone metastasis Bone
formation. Majority of prostate cancer. Caused by
direct effects of tumor. Net bone formation
Activation of osteoblasts and/or inhibition
of osteoclasts. Endothelin-1 osteoblast
activation factor uPA (S) protease that
liberates TGF-beta from TGF-beta
binding protein PSA (S) protease that
liberates TGF-beta, IGF TGF-beta
activates osteoblasts, tumor
proliferation
From Mundy, 2002
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Vicious Cycle of Osteolytic Metastases
Breast cancer cells that met. to bone with high
efficiency secrete more PTHrP (Powell, 1991)
From Mundy, 2002
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Breast Cancer In Vivo Bone Selection Experiments
(MDA-MB-231) Yoneda and Nishimura, 2001 -
MDA-231BO larger osteolytic bone
metastases Increased PTHrP secretion Proliferati
on not affected by TGF-beta (inhibited
BR/Par) Proliferation increased by IGF-1 (no
effect on BR/Par) - MDA-231BR mets. only to
brain - Growth did not differ f/ each other or
parental line Kang and Massague, 2003 - Isolated
cell lines w/ increased metastasis to bone or
adrenals - Highly metastatic to bone Cells
overexpress CXCR4, CTGF, IL-11, MMP1 -
Osteopontin nonspecifically overexpressed,
stimulates osteoclast adhesion to
bone matrix (OPN) - Increasing bone
metastasis Overexpress OPN IL11 - increased
bone mets. Overexpress OPN IL11 CXCR4 OR
CTGF - even more bone mets. - Pre-existing
metastatic variants High 5 - highly bone
metastatic High 3-4 - intermedate bone
metastasis