1. Erik Rogers – Comprehensive Exam
08.16.2012
• Hypoxia Driven Induction of M2 Tumor
Associated Macrophages and their
Contributions to EMT and Invasiveness in
Carcinoma Cells
2. 2011 Cancer Statistics
• US: 1.6 million reported cancer cases
• Global: WHO estimates 13.1 million cancer
deaths
• NCI: cancer – “diseases in which abnormal
cells divide without control and are able to
invade other tissues”
4. Epithelial to Mesenchymal Transition
Epithelial cells:
•Apical/Basal polarity
•Selectively permeable barriers
•Anchored to BM
•Cell to cell junctions
Mesenchymal cells:
•Amoeboid movement
•Lamellipodia & Filopodia
•Chemotactic
5. Innate immune response to cancer
• Interactions of 3 cell types are focus of proposal:
– Tumor cells
– Tumor associated macrophages (TAMs: M1 & M2
phenotypes)
– CD4+ T helper cells (TH1 & TH2 phenotypes)
• Carcinoma cells produce cytokines and chemokines that
attract myeloid cells and activate resident myeloid cells
• Naïve T cells (TH0) are induced to TH1 and/or TH2
phenotype by interactions with microenvironment
• Naïve macrophages (Mφ) are induced to M1 and/or M2
phenotype by interactions with microenvironment
6. M1 vs. M2 TAMs
• TH1 cytokines – TNF-α, IFN-γ, IL-1β
– M1 TAM phenotype – inflammatory and cytotoxic
– Primarily localized to invasive front
• TH2 cytokines – IL-4, IL-10, IL-13
– M2 TAM phenotype- immunosuppresive and promote
tumor invasiveness (EMT & metastasis)
– Primarily located intratumorally
• Other microenvironmental activators M2 TAM phenotype
• Hypoxia induces many adaptations in expression patterns
7. Hypoxia M2 TAM TGF-β EMT
• Myeloid Derived Suppressor Cell experiments
– Hypoxia results in MDSCs M2 TAMs
• M2 TAMs co-localize with intraepithelial
fibroblastoid cells
– Correlation between M2 TAM and EMT
• M2 TAMs produce TGF-β
– Snail/Slug, Twist, Smad3/7, PI3K, Notch, GS3Kβ,
NF-ΚB, Hey1, Hes1, etc.
8. HIF-1 Regulation
• Constitutively expressed β-subunit
• α-subunit stabilized in hypoxic conditions
– Normoxia = PHDs 1-3 + pVHL degradation
– PHDs O2 and Fe+2 dependent
– ROS & NO inhibit PHDs oxidize Fe+2 to Fe+3
• 60+ genes regulated by HREs (NCGTG)
– Metabolic adaptation, apoptosis resistance,
angiogenesis and metastasis
9. Model for Hypoxia Driven Feedback
Loop = CD4+TH1
= M1 TAM
= CD4+TH2
= M2 TAM
CCL-2
CXCL-12
CCL-5
CXCL-8
CXCL-12
CCL-2
IL-1
HIF-1
VEGF
EGF
TGFβ
MMP-2
MMP-9IL-4
IL-10
IL-13
10. Specific Aim 1: Questions
• Is hypoxia activated HIF-1 the fundamental
regulator of the induction of Mφ to the M2
phenotype?
• Are M2s required contributors to EMT in
tumor cells?
11. Experimental Design
• Culture M2s in hypoxic conditions –
– Assess for stabilized HIF-1α via Western blot
• Culture wt Mφ in hypoxic conditions –
– Assess for M2 markers via qRT-PCR
• Culture Hif-1αfl/flCre+ Mφ in hypoxic conditions –
– Assess for M2 markers via qRT-PCR
• Co-culture M2s/tumor cells –
– Assess for EMT markers in tumor cells via qRT-PCR
and Immunofluorescence staining
12. Does hypoxia stabilize HIF-1α in M2s?
Mφ M2
--------------β-Actin--------------
•Is hypoxia activated HIF-1 the fundamental
regulator of the induction of the M2
phenotype?
•FACS isolated M2s were cultured
in hypoxic incubator chambers for 4 days
•Mφ were cultured in normoxic conditions
for control comparison
•Western blot analysis indicates presence
of stabilized HIF-1α monomer in M2s
13. Does hypoxia activated HIF-1 induce M2?
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
F4/80 IL-1 IL-6 IL-23 IL-4 IL-10 IL-13
FoldDifference
4 Days in Hypoxia •Is hypoxia activated HIF-1 the
fundamental regulator of the
induction of the M2 phenotype?
•wt Mφ were cultured in hypoxic
incubator chambers for 4 days
•F4/80 pan macrophage marker
set as comparative baseline
•qRT-PCR shows significantly
higher transcription of M2
associated cytokines
14. Does hypoxia induce M2 in
Hif-1αfl/flCre+ Mφs?
0
0.2
0.4
0.6
0.8
1
1.2
F4/80 IL-1 IL-6 IL-23 IL-4 IL-10 IL-13
FoldDifference
4 Days in Hypoxia •Is hypoxia activated HIF-1 the
fundamental regulator of the
induction of the M2 phenotype?
•Hif-1αfl/flCre+ Mφ were cultured in
hypoxic incubator chambers for
4 days
•F4/80 pan macrophage marker
set as comparative baseline
•qRT-PCR shows significantly
higher transcription of M2
associated cytokines
15. M2 TAMs induce ‘Cadherin switch’ in
carcinoma cells
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
E-cadherin N-cadherin vimentin
FoldDifference
EMT Marker Expression
Time 0 48 Hours
•Are M2 TAMs required contributors
to EMT in tumor cells?
•FACS isolated M2 TAMs were co-
cultured with NMuMG cells for 48
hours
•qRT-PCR analysis of mRNA from
sorted NMuMG cells shows down-
regulation of E-cadherin and up-
regulation of N-cadherin and
Vimentin
16. Co-culture of carcinoma cells with M2
TAMs suggests EMT
pMEC cells ---------------E-cadherin-------------- NMuMG cells
pMEC cells --------------N-cadherin--------------- NMuMG cells
•Are M2 TAMs required
contributors to EMT in tumor
cells?
•Co-culture of M2 TAMs with
two different carcinoma cell
lines for 48 hours
•Immunofluorescent staining
Shows down-regulation of
E-cadherin and up-regulation
of N-cadherin
17. Alternative Approaches
• As a means of qualifying the influence of M2
TAMs on NMuMG and pMEC cells, we could
evaluate separate hypoxic cultures of both cell
lines for EMT markers via qRT-PCR.
• Evaluate M2 TAM production of ROS and NO
– 27-dichlorofluorescin diacetate (DCFCA)
• non-fluorescent when reduced
• fluorescent when oxidized
18. Model for Hypoxia Driven Feedback
Loop = CD4+TH1
= M1 TAM
= CD4+TH2
= M2 TAM
CCL-2
CXCL-12
CCL-5
CXCL-8
CXCL-12
CCL-2
IL-1
HIF-1
VEGF
EGF
TGFβ
MMP-2
MMP-9IL-4
IL-10
IL-13
19. Matrix Metalloproteinases
• Three distinct functional domains
– Hemopexin-like C terminal domain
– Catalytic domain
– Pro-domain
• Inactive zymogens via Cys-Zn2+ interaction
– Activated by proteases and/or ROS oxidation
• Activate pro-forms of growth factors
• Release ECM bound growth factors
• Tissue remodeling – EMT, angiogenesis,
metastasis
21. Specific Aim 2: Questions
• Are M1 TAMs polarized to an M2 TAM phenotype in
the tumor microenvironment by IL-4 produced by
CD4+ TH2 lymphocytes?
• Are M2 TAMs indispensable sources of MMP-2 and
MMP-9?
• Are MMP-2 and MMP-9 produced by M2 TAMs
required for tumor invasiveness?
22. Experimental Design
• Evaluate expression of M1 vs. M2 markers after co-culture
of Mφ with CD4+ TH2 cells with and without functional IL-4
• Assess M2 TAM contribution to MMP-2/MMP-9 in tumor
microenvironment
• In vitro invasion assay to assess invasiveness of carcinoma
cells in presence of M2 TAM conditioned media with and
without MMP-2 and MMP-9
• In vivo angiogenesis assay to assess neovascularization in
MMTV-PyMT tumors with and without MMP-2 and MMP-9
23. Is IL-4 critical to induce M1 TAMs to
M2 TAM phenotype?
0 2 4 6 8 10
F4/80
IL-1
IL-6
IL-23
IL-4
IL-10
IL-13
Fold Difference
M1 vs. M2 functional IL-4 activity Are M1 TAMs polarized to M2 TAMs in the
tumor microenvironment by IL-4 produced
by CD4+ TH2 lymphocytes?
4 day co-cultures of M1 TAMs with CD4+ TH2
cells (with and without functional IL-4)
Expression levels of TH1 vs. TH2 cytokines
evaluated in sorted TAMs via qRT-PCR
Sorted TAMs from 4-day co-cultures with
functional IL-4 show predominately TH2
expression pattern
Sorted TAMs from 4-day co-cultures without
functional IL-4 show predominately TH1
expression pattern0 1 2 3 4 5 6
F4/80
IL-1
IL-6
IL-23
IL-4
IL-10
IL-13
Fold Difference
M1 vs. M2 with shRNA Inhibition
of IL-4
24. Are the majority of MMP-2 & MMP-9
produced by M2 TAMs
0.85 0.9 0.95 1 1.05
HCC MMP-2
M2 TAM MMP-2
HCC MMP-9
M2 TAM MMP-9
Fold Difference
Relative MMP Expression
Are M2 TAMs indispensable sources of
MMP-2 and MMP-9?
Expression levels of MMP-2 & MMP-9 were
assessed using RNA isolated from M2 TAM
cultures
Expression levels of MMP-2 & MMP-9 were
assessed using RNA isolated from M2 TAMs
co-cultured with cells obtained from
digested mammary epithelial tumors
Heterogeneous cell cultures (HCC)
The ratio of MMP-2 production in M2 TAMs
compared to HCCs ~1:0.95 and the ratio of
MMP-9 production in M2 TAMs compared
to HCCs ~1:0.93
25. M2 TAM conditioned media – MMPs,
chemoattractants and growth factors
TGF-β EGF VEGF
IL-4 IL-8 IL-10 IL-13 MSF
--------------------------------------------β-actin------------------------------------------
------------------β-actin-----------------
MMP-2 MMP-9
-------------Β-actin-------------
M2 TAM conditioned media in
bottom wells of Boyden Chambers
for in vitro invasion assays
Western blot analysis confirms that
M2 TCM contains cytokines, MMPs
and growth factors
Predicts sufficient stimuli to trigger
invasion of carcinoma cells in upper
wells of Boyden Chamber assays
27. M2 produced MMP-2 / MMP-9
increase angiogenesis
M2/CD4+ TH2 functional MMP2/MMP-9
M2/CD4+ TH2 inhibited MMP2/MMP-9
Are MMP-2 and MMP-9 produced
by M2 TAMs required for tumor
invasiveness?
MMTV-PyMT tumors injected with
M2 TAMs and CD4+ TH2 cells (top).
MMTV-PyMT tumors injected with
M2 TAMs and CD4+ TH2 cells with
inclusion of AG3340 to inhibit
MMP-2/MMP-9 activity (bottom).
Neovascularization assessed by
IF staining for CD-31 (left) and
IHC staining for VEGF:VEGFR on
endothelial cells (right).
28. Alternative Approaches
• Replace M2 TAMs with Mφ in the MMTV-PyMT
tumor injections
• Replace CD4+ TH2 cells with CD8+ cytotoxic T cells in
the MMTV-PyMT tumor injections
• M2 TAMs +/- CD4+ TH2 with ascites fluid from
MMTV-PyMT tumors injected intraperitoneally
and/or sub-cutaneously to assess invasiveness
29. Thank You
• Comprehensive Exam Chair: Dr. Douglas Lake
• Committee Chair: Dr. Alan Rawls
• Committee member: Dr. Jeanne Wilson-Rawls
• Committee member: Dr. Kenro Kusumi
• Thanks to everyone for the time, patience and
guidance
