This document summarizes research on how the tumor microenvironment influences metastasis at every step of the metastatic cascade. Key points include: (1) the microenvironment can suppress or promote tumorigenesis; (2) the perivascular niche protects disseminated tumor cells and keeps them dormant; (3) targeting the integrin receptors that mediate tumor cell interactions with the vascular niche can sensitize dormant tumor cells to chemotherapy and prevent metastasis without increasing toxicity.

1. Cyrus M. Ghajar, PhD
Associate Member
Public Health Sciences Division/Translational Research Program
Human Biology Division
Fred Hutchinson Cancer Research Center
Metastatic breast cancer
and the
tumor microenvironment

2. Paget’s Theory:
The “seed” and “soil” hypothesis
Paget, The Lancet, 1889

3. RSV in the embryo
Dolberg and Bissell, Nature, 1984
Dolberg et al, Science, 1985
Siewke et al.Science, 1990
Stoker et al, JCB, 1990

4. RSV as a case study:
The need for a permissive microenvironment
RSV in the adult

5. An activated microenvironment can
initiate cancer
Sternlicht et al., Cell 1999

6. Some conclusions…
1. The microenvironment can suppress
tumorigenesis; an oncogene is not enough.
2. The microenvironment can promote
tumorigenesis, too (even in the absence of an
oncogene, initially).
3. Context is everything!
4. This applies to metastasis, also (seed and soil)

7. The tumor microenvironment:
a definition
The other “normal” cells, insoluble factors, soluble
species— including hormones— that surround a
given (tumor) cell.
But also consider the systemic nature of disease…
…and the macro-environment.

8. How does the microenvironment
influence metastasis?
• Greek for “displacement”
METASTASIS
NEXTPLACEMENT
• Refers to the spread (and growth) of a
tumor from one organ site to another.

9. The metastatic cascade:
Guo and Giancotti, Nat Rev MCB 2004
Toolbox
A little help from
some friends
An Exception to the
“rule”
Colonization

10. Invasion
Sabeh et al., JCB 2009

11. Invasion- Toolbox
1. Something to attach you to the environment around you
2. Something to pull yourself through this environment
3. Something to cut through the jungle
Integrins
Contractile Machinery
MMPs

12. Integrins
Giancotti & Ruoslahti, Science 1999
Guo and Giancotti, Nat Rev MCB 2004
Hynes, Cell 2002

13. Focal Adhesions
Focal Adhesions: multifunctional organelles that regulate cell adhesion, force
transmission, cytoskeletal regulation and signaling.
Kanchanawong et al., Nature 2010
Provenzano et al., BMC Med 2006

14. Matrix Metalloproteinases (MMPs):
Egeblad & Werb., Nat Rev Cancer 2002
Sabeh et al., JCB 2009
MT1-MMP is particularly crucial for invasion
through type I Collagen in the stroma

15. Invasion- A little help from some
friends
Gaggioli et al., Nat Cell Biol 2007

16. Invasion- An exception to the rule
Are MMPs ALWAYS necessary?
Wolf et al., JCB 2003
Wolf et al., Nat Cell Biol 2007

17. The metastatic cascade:
Guo and Giancotti, Nat Rev MCB 2004
Toolbox
A little help from
some friends
An Exception to the
“rule”
Colonization

18. Angiogenesis- Toolbox
Hanahan and Folkman, Cell 2002
The formation of new microvasculature from pre-existing
vasculature.
“If a tumor can be held indefinitely in the nonvascularized dormant state, there are a number
of theoretical benefits … it seems appropriate to speculate that “anti-angiogenesis” may
provide a form of cancer therapy worthy of serious exploration.” - J. Folkman, New England
Journal of Medicine 1971

19. Angiogenesis- A little help from
some friends
Hanahan and Coussens, Cancer Cell 2012
Tumors are
“Wounds that
do not heal.”
- H. Dvorak,
NEJM 1986

20. Angiogenesis- An exception
to the rule
Vasculogenic Mimickry:
Maniotis et al., Am J Path 1999

21. The metastatic cascade:
Guo and Giancotti, Nat Rev MCB 2004
Toolbox
A little help from
some friends
An Exception to the
“rule”
Colonization

22. Intra- and Extravasation: Toolbox
Intravasation
Extravasation
• E-selectin-mediated
adhesion
• Integrin-dependent
adhesion to ECs
• Cdc42 and Rac-driven
extrusion through EC
junctions
• Induction of EC
junction opening
• FAK and Integrin-
dependent invasion
• MMP1 required for paracellular
intravasation
• EC ADAM12 can disrupt EC
junctions to foster tumor cell
entry
• Macrophages can facilitate
entry
• Induced remodeling of
endothelium creates pore-like
structures for tumor cells to
enter transcellularly
Reymond et al., Nat Rev Cancer 2013

23. Intravasation and Extravasation- A
little help from some friends
Baluk et al., Curr Opin Genetics & Dev 2005
Irregularities in tumor microvasculature facilitate intravasation:

24. Intravasation and Extravasation- A
little help from some friends
Qian et al., Nature 2011
Inflammatory macrophages
Inflammatory (CCL2+) macro-
phages help tumor cells extra-
vasate from blood vessels.

25. Intra- and Extravasation-
An exception to the “rule”
Lu et al., Cancer Cell 2012
Glioblastoma utilizes perivascular invasion to colonize different
parts of the brain:

26. The metastatic cascade:
Guo and Giancotti, Nat Rev MCB 2004
Toolbox
A little help from
some friends
An Exception to the
“rule”
Colonization

27. Evidence that dissemination is an
early event- mouse models
CK +
HER-2 +
Balb-NeuT mice
CK HER-2 Merge
Husemann et al., Cancer Cell 2008

28. Braun et al., New England Jrnl Med 2005
Sanger et al., International Journal of Cancer 2011
Evidence that dissemination is an
early event- humans

29. Pan et al., NEJM, 2017
Aguire-Ghiso, Nature Reviews Cancer 2007
Current therapies are not effective
against disseminated tumor cells!

30. Tumor cells exist in distant organs in
one of three states:
Goss and Chambers, Nat Rev Cancer 2010
Sanger et al., International Journal of Cancer
2011

31. Survival
Reversibly
Growth-
arrested
Chemo-
resistance
What are the properties of dormant DTCs?

32. Ghajar, Nature Reviews Cancer 2015
Metastasis prevention by targeting
dormant cells

33. Where do chemoresistant DTCs reside?
Orthotopic
injection of
Mammary
Carcinoma Cells
Adjuvant Therapy
1w
X
AC AC AC AC AC
2.5w
Resect Primary
Tumor

34. dbvdMK
dosteoblast
Measuring localization to distinct niches

35. Chemoresistant DTCs preferentially occupy the
perivascular niche in vivo
2.5w
Resect Primary
Tumor
Adjuvant Therapy
Orthotopic
injection of
Mammary
Carcinoma Cells
1w
X
AC AC AC AC AC
GFP
Pan-laminin
DNA
Ki67
Bone Marrow
a b c
a
b
c
0
100
200
300
0
200
400
600
DTC (case-by-base)Distance(um)
103AC
to Blood Vessel
to Megakaryocyte
to Osteoblast
Carlson et al., Nature Cell Biology 2019

36. 7d
10d
YFP Breast Tumor CellsStromal cells
mCherry E4ORF1
ECs
Growth Medium Supplement-Free Medium
or
LrECM Drip
Modeling the microvascular niche in culture
Ghajar et al, Nature Cell Biology, 2013

37. Strom
a
Stroma+ECs
T4-2
TUNEL
Ki67
DNA
T4-2
TUNEL
Ki67
DNA
Ctrl 625nM Dox 2500nM Dox
The perivascular niche protects chemoprotects DTCs
0nM156nM312nM625nM1250nM2500nM
0
20
40
60
80
Doxorubicin
TUNEL-positiveCells(%)
***
0nM156nM312nM625nM1250nM2500nM
0
20
40
60
80
Doxorubicin
TUNEL-positiveCells(%)
N.S.
LrECM Drip
10
days
12
days
YFP Breast Tumour Cells
Bone marrow stromal cells
mCherry endothelial
cells (ECs)
5 daysor
Doxorubicin
or Paclitaxel
Carlson et al., Nature Cell Biology 2019

38. Unbiased profiling of the microvascular niche reveals a
potential role for integrin-ECM signaling
-5-4-3-2-10
Collagen binding
Hormone activity
Molecular transducer activity
Protein binding
Receptor activity
Cytokine activity
Cell adhesion molecule binding
Transmembrane RTK activity
Integrin binding
Receptor binding
Calcium ion binding
RTK activity
false discovery rate (Log10)
Enrichment: Integrin-Cell
Surface Interactions
(REACTOME)
Row MaxRow Min
MVN Enriched
ECM Targeted Proteomics:
Bone marrow stroma Bone marrow MVN
Peter Nelson
Ilsa Coleman
Kirk Hansen
Alexander Barrett
Carlson et al., Nature Cell Biology 2019

39. Does interfering with receptor-mediated interactions between DTCs
and vascular basement membrane sensitize
dormant DTCs to chemotherapy?
Prime with
receptor-i
LrECM Drip
10
days
12
days
YFP Breast Tumour Cells
Bone marrow stromal cells
mCherry endothelial
cells (ECs)
5 daysor
Doxorubicin
or Paclitaxel
Carlson et al., Nature Cell Biology 2019

40. Targeting b1- and avb3- integrins simultaneously optimizes
chemosensitization of quiescent tumor cells
Pan-
CK
TUNE
L
Ki67
DNA
IgG
a-
Itgavb3
a-Itgb1
a-Itgb1+Itgavb3
IgG
Itgb1Itgavb3
Itgb1+Itgavb3
0
20
40
60
80
100
TUNEL-positive
singlecells(%)
+Doxorubicin
IgG
Itgb1Itgavb3
Itgb1+Itgavb3
0
20
40
60
80
100
TUNEL-positive
Clusters(%)
+Doxorubicin
IgG
Itgb1Itgavb3
Itgb1+Itgavb3
0
20
40
60
80
100
Ki67+Cells(%)
N.S.
Carlson et al., Nature Cell Biology 2019

41. Can we target DTCs in vivo, and will this prevent metastasis?
Bone metastasis prevention trial:
Intracardiac Inject
ER+ BCCs in
athymic nude mice
1w
AC AC AC AC
Cohort 2: MFS
Cohort 1:
• BLI of each organ of interest
• Bone Marrow DTC Burden
• Lung DTC Burden
ffluc-eGFP MCF-7
= AIIB2 +/- LM609
X X OVX
Carlson et al., Nature Cell Biology 2019

45. Bone metastasis prevention trial:
Integrin inhibition primes bone marrow DTCs for chemotherapy
ffluc-eGFP MCF7
pan-laminin
IgG+AC
AIIB2+AC
AIIB2+LM609+AC
IgG
+A
CA
IIB
2+A
C
A
IIB
2+LM
609+A
C
0.0
5.0×10-6
1.0×10-5
1.5×10-5
2.0×10-5
DTCs/AreaBoneMarrow
** IgG Control
Intracardiac
Inject ER+
BCCs in
athymic nude
mice
1w
AC AC AC AC
Cohort 2: MFS
Cohort 1:
• BLI of each organ of interest
• Bone Marrow DTC Burden
• Lung DTC Burden
ffluc-eGFP MCF-7
= AIIB2 +/- LM609
X X OVX
Carlson et al., Nature Cell Biology 2019

46. Reduction in DTC burden results in improved
metastasis-free survival
Statistical
Test
p Value
Mantel-Cox test 0.0387
Gehan-
Breslow-
Wilcoxon test
0.0339
IgG
+AC
AIIB2
+AC
AIIB2+
LM609
+AC
Intracardiac Inject
ER+ BCCs in
athymic nude mice
1w
AC AC AC AC
Cohort 2: MFS
Cohort 1:
• BLI of each organ of interest
• Bone Marrow DTC Burden
• Lung DTC Burden
ffluc-eGFP MCF-7
= AIIB2 +/- LM609
X X OVX
0 50 100
0
25
50
75
100
Days
MetastasisFreeSurvival
(Percentage)
IgG+AC (n=11)
AIIB2+AC (n=10)
AIIB2+LM609+AC
(n=9)
Carlson et al., Nature Cell Biology 2019

47. Integrin inhibition does not enhance
chemotherapy-associated toxicities
0 7 14 21 28 35
22
24
26
28
30
Day (post-injection)
BodyWeight(g)
IgG+AC
AIIB2+AC
AIIB2+LM609+AC
NS
Body Weight
IgG
+A
CA
IIB
2+A
C
A
IIB
2+LM
609+A
C
0
5
10
15
20
25
Pan-Laminin
AreaPercentage
IgG Control
Vascular Density
Bone Marrow Cellularity
U
ntreated
IgG
AIIB2
IgG
+
AC
AIIB2
+
AC
1.5×106
2.0×106
2.5×106
3.0×106
3.5×106
4.0×106
Viablecells(count)
U
ntreated
IgG
AIIB2
IgG
+
AC
AIIB2
+
AC
0
1×106
2×106
3×106
4×106
CD45+Cells(count)
U
ntreated
IgG
AIIB2
IgG
+
AC
AIIB2
+
AC
0
1×106
2×106
3×106
4×106
CD11b+Cells(count)
* **
U
ntreated
IgG
AIIB2
IgG
+
AC
AIIB2
+
AC
0
2×104
4×104
6×104
8×104
1×105
CD3+Cells(count)
*
U
ntreated
IgG
AIIB2
IgG
+
AC
AIIB2
+
AC
0
2×105
4×105
6×105
8×105
1×106
B220+cells(count)
** ***
Carlson et al., Nature Cell Biology 2019

48. 1. “Transformation” on its own is not sufficient; progression requires a
permissive microenvironment.
2. The microenvironment is co-opted at every step of the metastatic
cascade…
3. … but we cannot target every step, because dissemination is an early
event.
4. The niche around vasculature (the perivascular niche) is responsible for
keeping disseminated breast tumor cells dormant
5. We can target the niche of disseminated tumor cells in order to make
them sensitive to chemotherapy…
6. Which prevents metastasis without exacerbating chemotherapy-
associated side effects
Summary + Conclusions