Erik Rogers – Comprehensive Exam08.16.2012

• Hypoxia Driven Induction of M2 Tumor Associated Macrophages and their Contributions to EMT and Invasiveness in Carcinoma Cells

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”

Cancer Progression Simplified

EMT metastases MET Very complex, multi-step process

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

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

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

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.

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

Model for Hypoxia Driven Feedback Loop= CD4+TH1

= M1 TAM

= CD4+TH2

= M2 TAM

CCL-

2

CXCL

-12

CCL-

5

CXCL

-8

CXCL-12CCL-2IL-1

IL-1IL-2IFNγ

HIF-1

IL-4IL-10IL-13

MMP-2

MMP-9

VEGF

EGFTGFβ

MMP-2MMP-9

Snail

IL-4

IL-1

0IL

-13

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?

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

Does hypoxia stabilize HIF-1α in M2s?

Mφ M2

--------------β-Actin--------------

•Is hypoxia activated HIF-1 the fundamental regulator of the induction of the M2phenotype?

•FACS isolated M2s were culturedin hypoxic incubator chambers for 4 days

•Mφ were cultured in normoxic conditionsfor control comparison

•Western blot analysis indicates presenceof stabilized HIF-1α monomer in M2s

Does hypoxia activated HIF-1 induce M2?

F4/80 IL-1 IL-6 IL-23 IL-4 IL-10 IL-130

0.5

1

1.5

2

2.5

3

3.5

4

4.5

4 Days in Hypoxia

Fold

Diff

eren

ce

•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 significantlyhigher transcription of M2associated cytokines

Does hypoxia induce M2 in Hif-1αfl/flCre+ Mφs?

F4/80 IL-1 IL-6 IL-23 IL-4 IL-10 IL-130

0.2

0.4

0.6

0.8

1

1.2

4 Days in Hypoxia

Fold

Diff

eren

ce

•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 significantlyhigher transcription of M2associated cytokines

M2 TAMs induce ‘Cadherin switch’ in carcinoma cells

E-cad

herin

N-cadherin

vimen

tin

E-cadherin

N-cadherin

vimen

tin0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

EMT Marker Expression

Time 0 48 Hours

Fold

Diff

eren

ce

•Are M2 TAMs required contributors to EMT in tumor cells?

•FACS isolated M2 TAMs were co-cultured with NMuMG cells for 48hours

•qRT-PCR analysis of mRNA from sorted NMuMG cells shows down-regulation of E-cadherin and up-regulation of N-cadherin and Vimentin

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 withtwo different carcinoma cell lines for 48 hours

•Immunofluorescent staining Shows down-regulation of E-cadherin and up-regulation of N-cadherin

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

Model for Hypoxia Driven Feedback Loop= CD4+TH1

= M1 TAM

= CD4+TH2

= M2 TAM

CCL-

2

CXCL

-12

CCL-

5

CXCL

-8

CXCL-12CCL-2IL-1

IL-1IL-2IFNγ

HIF-1

IL-4IL-10IL-13

MMP-2

MMP-9

VEGF

EGFTGFβ

MMP-2MMP-9

Snail

IL-4

IL-1

0IL

-13

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

MMP-2 & MMP-9• MMP2 / MMP-9 proteolytically activate TGF-β• MMP2 / MMP-9 release ECM bound EGFR

ligands– EGF:EGFR – proliferation, survival, angiogenesis,

tumor metastasis, MMP-9 expression• MMP-9 degradation of E-cadherin• MMP2 / MMP-9 increase available VEGF– Angiogenesis and Lymphangiogenesis

• MMP-2 / MMP-9 increase vessel permeability

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?

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

Is IL-4 critical to induce M1 TAMs to M2 TAM phenotype?

F4/80IL-1IL-6

IL-23IL-4

IL-10IL-13

0 1 2 3 4 5 6 7 8 9 10

M1 vs. M2 functional IL-4 activity

Fold Difference

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 cytokinesevaluated in sorted TAMs via qRT-PCR

Sorted TAMs from 4-day co-cultures withfunctional IL-4 show predominately TH2expression pattern

Sorted TAMs from 4-day co-cultures withoutfunctional IL-4 show predominately TH1expression pattern

F4/80IL-1IL-6

IL-23IL-4

IL-10IL-13

0 1 2 3 4 5 6

M1 vs. M2 with shRNA Inhibition of IL-4

Fold Difference

Are the majority of MMP-2 & MMP-9 produced by M2 TAMs

HCC MMP-2

M2 TAM MMP-2

HCC MMP-9

M2 TAM MMP-9

0.88 0.9 0.92 0.94 0.96 0.98 1 1.02

Relative MMP Expression

Fold Difference

Are M2 TAMs indispensable sources of MMP-2 and MMP-9?

Expression levels of MMP-2 & MMP-9 wereassessed using RNA isolated from M2 TAMcultures

Expression levels of MMP-2 & MMP-9 wereassessed using RNA isolated from M2 TAMsco-cultured with cells obtained from digested mammary epithelial tumors Heterogeneous cell cultures (HCC)

The ratio of MMP-2 production in M2 TAMscompared to HCCs ~1:0.95 and the ratio ofMMP-9 production in M2 TAMs comparedto HCCs ~1:0.93

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 Chambersfor in vitro invasion assays

Western blot analysis confirms thatM2 TCM contains cytokines, MMPsand growth factors

Predicts sufficient stimuli to triggerinvasion of carcinoma cells in upperwells of Boyden Chamber assays

Does M2 conditioned media induce invasion of carcinoma cells?

wtM

EC n

o BM

wtM

EC w

ith B

M

wtM

EC n

o BM

wtM

EC w

ith B

M

wtM

EC n

o BM

wtM

EC w

ith B

M

NMuM

G no

BM

NMuM

G w

ith B

M

NMuM

G no

BM

NMuM

G w

ith B

M

NMuM

G no

BM

NMuM

G w

ith B

M

pMEC

no

BM

pMEC

with

BM

pMEC

no

BM

pMEC

with

BM

pMEC

no

BM

pMEC

with

BM

0

20

40

60

80

100

120In Vitro Invasion Assay

% In

vasio

n

-M2CM +M2CM +AG3340 -M2CM +M2CM +AG3340 -M2CM +M2CM +AG3340

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 withM2 TAMs and CD4+ TH2 cells (top).MMTV-PyMT tumors injected withM2 TAMs and CD4+ TH2 cells withinclusion of AG3340 to inhibitMMP-2/MMP-9 activity (bottom).

Neovascularization assessed byIF staining for CD-31 (left) and IHC staining for VEGF:VEGFR onendothelial cells (right).

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

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