1. Tumor Microenvironment Network (TMEN) Division of Cancer Biology
Dinah Singer, Ph.D.
Director
Suresh Mohla, Ph.D.
TMEN Program Director
Division of Cancer Biology
1. Although this is a reissuance, its goal is not simply to continue the existing Network, but to build on and extend the success of the original TMEN.
2. present our vision for the new TMEN, which will highlight its differences from the existing one.

2. TMEN : Goals
Generate a comprehensive understanding of the composition of the normal stroma and the role of the stroma in tumor initiation, progression and metastasis
Develop resources and infrastructure critical to the broader research community to advance understanding of the tumor microenvironment (TME)
When TMEN was first established in 2006, it was already understood that there is a complex interplay between the tumor and its surrounding stroma.
However, the composition of the stroma and its role in cancer remained unclear. Although a number of investigators – including some at this table – were making significant progress, it was clear that the advances in the field could be dramatically expedited through a programmatic effort that:
importantly, working together to:
Generate the approaches and critical reagents and resources, such as stromal cell markers, that would

3. TMEN 2006-2011: Current Program
Nine interdisciplinary groups characterizing the TME in major cancer sites, emphasizing human samples
Identifying and translating promising leads
Collaboratively addressing the complexity of TME by assessing:
The co-evolution of tumor-associated stromal cell types and the tumor
Stromal cell interactions with each other and with tumor cells
Generating novel reagents, models and technologies for the research community
The success of the TMEN, as a network has derived from the collaborative efforts of the Network to address the complexity of the TME and to generate novel resources for the community.
Neither of these could have been accomplished through traditional funding mechanisms and depended on the Network infrastructure.

4. TMEN 2006-2011: Areas of Emphasis
Microenvironment
Initiation Progression Metastasis
Tumor initiating cells
Tumor heterogeneity
Stromal cell types and cellular processes
Complex signaling pathways
Genes and genetics
The first TMEN RFA identified 5 broad scientific areas of emphasis for which a collaborative, network approach would give us insights into the role of the microenvironment in initiation, progression and metastasis.
In the 4 years since the TMEN was established, significant progress has been made in each of these areas, much of it deriving from TMEN-supported research.
In the next few slides, I’ll highlight just a few of the accomplishments of the TMEN. However, a more detailed list can be found in the concept and other documentation that you’ve been provided. 4

5. TMEN 2006-2011: Scientific Accomplishments
Tumor Initiating Cells:
Combined invasive gene signatures in tumor initiating cells and stromal wound repair response genes robustly predict metastasis-free and overall survival in breast, medulloblastoma, lung and prostate cancer patients
New therapeutic approaches to circumvent the chemo- and radio-resistance of tumor initiating cells
Stromal Cells:
Delineation of mechanisms to describe the roles of the various bone marrow-derived cell lineages in tumor growth, invasion, inflammation, angiogenesis and metastasis
Stromal genes and factors:
Cancer-associated stromal gene signatures predict progression to biochemical recurrence in human prostate cancer
Cancer-associated neurogenesis in human prostate predicts aggressive disease
Understanding the signaling and metabolic pathways that distinguish TIC’s has allowed new therapeutic approaches.
Targeting the Notch ligand Delta can reverse chemoresistant of CSCs. Phase I trial initiated.
Low ROS levels account for redioresisitance of CSC and inhibitor studies showed that reversal may make CVSC radiation sensitive. 5

6. TMEN 2006-2011: Scientific Accomplishments
Tumor Initiating Cells:
Combined signatures of invasive and wound repair response genes in tumor initiating cells robustly predicts relapse and overall survival in breast, medulloblastoma, lung and prostate cancer patients
New therapeutic approaches to circumvent the chemo- and radioresistance of tumor initiating cells
Stromal Cells:
Delineation of the mechanisms by which bone marrow-derived cells affect tumor growth, invasion, inflammation, angiogenesis and metastasis
Cancer-associated reactive stromal volume predicts progression to biochemical recurrence in human prostate cancer
Cancer-associated neurogenesis in human prostate predicts aggressive disease
Tumor microenvironment signature of metastasis (TMEM), which includes tumor cells, TAMs and endothelial cells, accurately predicts systemic metastasis from primary breast cancer samples
Examples of some of the seminal studies that have been published in high profile journals – commonality of stem cell markers across many tumor sites.
Targeting the Notch ligand Delta can reverse chemoresistant of CSCs. Phase I trial initiated.
Low ROS levels account for redioresisitance of CSC and inhibitor studies showed that reversal may make CVSC radiation sensitive. 6

7. TMEN 2006-2011: Programmatic Accomplishments
Scientific Collaborations
Identification of human tumor initiating cell fate using intravital imaging
Characterization of human prostate stromal changes, both genetic and epigenetic
Development of imaging beacons to determine role of specific metalloproteases in tumor and stroma

8. TMEN 2006-2011: Programmatic Accomplishments
Scientific Collaborations
Identification of human tumor initiating cell fate using intravital imaging
Characterization of human prostate stromal changes, both genetic and epigenetic
Development of imaging beacons to determine role of specific metalloproteases in tumor and stroma
Critical Resources Developed
Validated EHS sarcoma matrix (“Matrigel”) resource
Stromal antibodies – validation of existing antibodies and development of novel antibodies for stromal and tumor initiating cells
Transgenic mice expressing stromal TVB receptors for targeting with RCAS vectors
Bank of tumor initiating cells derived from early passage human tumors of head and neck, breast, colorectal cancers
Bank of murine bone marrow cells, for generating BMDC
A major effort in the Network has been the development of resources which are available to the general research community. A comprehensive list is provided at the end of the slide set in your books.
These resources have contributed to the rapid progress that’s been made in TME research and are available to the research community. 8

9. Proposed TMEN Reissuance Concept: Why?
Investigation of emerging novel concepts in TME will require new teams of interdisciplinary scientists to collaborate on large scale, complex problems
Network structure will:
Accelerate the development of new areas in TME, and of resources for the research community. (Investigator-initiated mechanisms are not suited for such efforts.)
Provide a focus for interacting with other NCI programs, to promote interdisciplinary efforts
Encourage established investigators to study the TME, through the collaborative U01 program
Facilitate training of junior investigators
In short, in its first 4 years, TMEN has either achieved or made significant progress, in all of the goals laid out in the original RFA.
So, why are we requesting a reissuance rather than allowing the field to develop further through investigator-initiated research mechanisms, such as RO1’s or PO1’s?
During the past 4 years, and in large part as a result of the advances made by TMEN in our understanding of the TME, a number of novel concepts have emerged that will require new approaches and expertise and new teams of researchers to collaborate to unravel the complexity of the TME.
A network structure will not only enhance the ability of interdisciplinary groups to make rapid progress, but will also provide leadership within the field and ensure the generation of the new resources and reagents that will be critical for the entire research community to develop these novel areas.
Neither of these can be achieved through classical investigator-initiated mechanisms.
expertise of a wide range of many disparate fields. Importantly, a network forms an organizational structure and focal point for interacting with other NCI funded programs, such as ICBP, MMMHC, phys-onc, nanotech, edrn, imaging.

10. TMEN Reissuance: Areas of Emphasis
Microenvironment
Initiation Progression Resistance Metastasis Recurrence
- Tumor initiating cells
- Tumor heterogeneity
- Stromal cell types and cellular processes
- Complex signaling pathways
- Genes and genetics
Stem cell niche
Metastatic niche
Tumor dormancy and metastasis
Metabolic dysregulation
Cell fusion and exosome secretion
Microbiome (bacteria; viruses)
Gradients and flow of soluble factors
reissuance will explore a range of novel concepts that emerged from studies over the past 4 years and span the cancer continuum; these now include resistance and recurrence, in addition to initiation, progression and metastasis.
The Network will provide the infrastructure to ensure that the needs of the community in these areas are met.
Emerging Areas 10

11. Proposed TMEN
Open RFA competition emphasizing emerging areas, soliciting new interdisciplinary teams
Up to 9 Research Programs (U54 Consortia)
Continued support at current total level of $9.08M/year
Five year duration
Because of the changes in emphasis, we anticipate that there will be considerable turn-over in the TMEN groups and their composition.
In conclusion, our intent in requesting this reissuance is not to simply continue the current TMEN. It is to build on and significantly extend the progress that was made by the existing TMEN.

12. Thank you!
Questions?

13. Background Information

14. TMEN Grant and Network Activities Funding FY2006-2010
TMEN Project and Network Activity
Average Total Cost per Year
Individual Grant Support
9 U54’s(5 fully funded, 4 partially funded)
$8,401,355
Network Activities
Resource development
Collaborative projects
$ ,033
Total Cost per year
$9,085,388

15. Current TMEN Principal Investigators
Mina Bissell (LBL, UC Berkeley)
Michael Clarke (Stanford)
John Condeelis (Albert Einstein)
Eric Holland (Memorial Sloan Kettering CC)
Richard Hynes (MIT)
Lynn Matrisian (Vanderbilt)
Stephen Plymate (Univ. Washington)
David Rowley (Baylor)
Timothy Wang (Columbia)

16. U of Washington/ Fred Hutch
TMEN: Collaborative Network
MIT
Vanderbilt
Albert Einstein
Columbia
Stanford
Baylor
MSKCC
U of Washington/ Fred Hutch
Lawrence Berkeley
Dana Farber Core
Collaborative U01 Dana Farber
Collaborative U01 Vanderbilt
Reagents
Models
Protocols
Collaborative U01 U Penn

17. TMEN 2010-2015: Examples of Scientific Goals
Using multi-disciplinary approaches , delineate biological mechanism(s)
governing the TME interactions which are critical for tumor development,
progression and metastasis, and therapeutic resistance and tumor recurrence
Focus on specific tumor sites, using human cancer tissues and models
Characterize component cells and matrix molecules in normal organ and tumor-associated stroma; examine alterations in the microenvironment
Identification of lineages of bone marrow-derived cells and immune cells and their effects in tumor initiation, progression and metastasis
Identify and characterize the stem cell niche; origin of stromal cells
Characterize the role of microbiome (bacteria and viruses)
Characterize the role of metabolic dysregulation in TME
Delineate the functional relevance of tumor cell-host cell fusion and exosomes
Determine the effects of cancer therapy on the TME
Identify and characterize the role of TME in therapeutic resistance
Identification and characterization of gradients and flow of soluble factors

18. TMEN Evaluation Criteria
The following broad criteria will be used to judge the success and effectiveness of the proposed program
Successful identification of novel components of the normal and tumor stroma in primary and metastatic tumors
New insights into the composition, role and origin of tumor-associated stroma during cancer initiation, progression and metastasis
Determining the effects of cancer therapy on the tumor microenvironment
Consortium participation in collaborative activities aimed at improving existing technologies and developing novel reagents
Development and effective dissemination of resources to the research community

19. Independent Program Evaluation Report
Addressed goals of the original RFA
Evaluation based on literature, NCI grants documentation, interviews and staff interaction
Separate evaluations from outside experts
Reviewers’ comments reflect their overall enthusiastic endorsement
“Overwhelmed by the accomplishments of the groups associated with TMEN- both in terms of number of critical questions being addressed, the quality of the investigators and the innovative approaches being pursued.”
“if research in this area was funded entirely through the R01 mechanism, there would only be a trickle of isolated research projects in the area “
“The coordination of resource generation has been largely driven by staff.”
“the program has exposed investigators who were working in select areas of the microenvironment to think ‘bigger’
Evaluation based on information generated from multiple sources

20. Current Portfolio Analysis
TMEN is the only funded program addressing a comprehensive approach to understand the TME, and its critical role in mediating tumor progression, metastasis and therapeutic resistance
The total TME portfolio budget in NCI is $43M. DCB’s portfolio includes the current 9 TMEN U54s, 3 U01s, and 61 investigator-initiated grants
Very few grants focus on the new areas proposed in this reissuance. Most examine cell migration, proteases, angiogenesis, hypoxia or metastasis
Investigator-initiated grants are unable to pursue comprehensive studies to address the microenvironment using “tumor-as- an-organ” approach as these mechanisms are not suited for to these types of studies

21. Supplemental Resource Information

22. BMDC bank
Bone marrow cells will be isolated from two commonly used strains of mice. These are:
(a) the Rosa26 (C57BL/6Jtrosa26) mice (b) smooth muscle a-actin promoter driving a reporter (EGFP)
RCAS Vector development targeting stromal cells
a) two RCAS(A) and two RCAS(B) vectors were made.  Just to clarify, these are the avian virus vectors that infect the respective transgenic mice that express the RCAS (A or B) receptors called TVA or TVB. Two TVB and one TVA mouse model have been generated
(i) FSP1-TVB mice (S100A4, that is expressed in many cells of mesenchymal origin including fibroblasts).
(ii)SM22-TVB mice (expressed in smooth muscle cells).
Currently the founders are being characterized at Vanderbilt – they have already been initially screened by Lionel Feigelbaum (sp?)
b) The third mouse (started late in the contract) is the "hit and run" mouse that allows for expression of TVA upon crossing with any Cre mouse.  That means one can target expressing either stromal or epithelial cell type.
c) The RCAS are avian retroviruses that can only infect cells that express the appropriate viral receptor

23. Standardization of Antibodies and generation of novel antibodies
Antibodies used for flow cytometry isolation of cells - sent to NCI repository
for distribution to other TMEN laboratories and the broader NIH research community
FLOTILLIN-2/ESA FITC MAB 29
HU CD10 PE-CY5 MAB HI10A
HU CD140B BIOTIN MAB 28D4
HU CD166(ALCAM) PE
HU CD18 PE-CY5
HU CD2 PE-CY5
HU CD20 PE-CY5 MAB 2H7
HU CD24 FITC MAB ML5
HU CD24 PE MAB ML5
HU CD3 PE-CY5 MAB UCHT1
HU CD31 BIOTIN MAB M89D3
HU CD31 BIOTIN MAB WM59
HU CD44 APC MAB G44-26
HU CD45 PE-CY5 MAB HI30
HU CD45 PE-CY7 MAB HI30
HU CD64 PE-CY5 MAB 10.1
HU CD66 FITC
HU CD66 PE
HU EGFR FITC (0.05MG)
HU EGFR PE
MS CD140A BIOTIN MAB APA5
MS CD90.1(THY1.1) APC
MS H-2KD BIOTIN MAB SF1-1.1
SAV PE-CY5
SAV PE-CY7

24. List of cell lines generated for antibody target overexpression
PC12 parental cell line
PC12 vector control cell line
PC12 Laminin A4 overexpression cell line
293T lysates from overexpression of TGFβ1,β2 and β3 as well as Cercam and Leprel 2 have been generated and used for Elisa and western blot screening of antibodies.
List of stable knockdown cell lines for antibody targets
MCF7 GFP shRNA KD cell line
MCF7 TGFβ1 KD cell lines
MCF7 TGFβ2 KD cell lines
MCF7 TGFβ3 KD cell lines
MDA435 GFP KD cell line
MDA435 Leprel 2 KD cell lines
MCF7 CerCam KD cell lines and GFP KD control line
T98G GFP KD cell line
T98G Laminin Alpha 4 KD cell lines

25. Antibodies
mAb against Cercam
mAb against Laminin Alpha 4
antibody supes against TGF β1/2/3 as well as Leprel 2
REAGENTS AND PROTOCOLS
Protocol for separation of mouse stroma from human tumor cells
Protocol for separation of human epithelial tumor cells from non-tumor components
Protocol for LCM following by high-resolution oligo array-CGH
Protocol for optimized WGA
Custom Agilent CGH 2x415K oligo microarray, optimized for resolution vs. cost
Monoclonal Antibodies against Laminin α4 and Cercam
Tumor initiating cells derived from early-passage human xenograft cancer tumors
established from cells isolated directly from patient tumors - sent to the NCI.
Colon cancer - 9 xenograft tumors
Breast cancer - 5 xenograft tumor
Head and neck cancer - 10 xenograft tumors

26. TMEN Publications: 2006-2010 Year Number of Publications 2010 18
In prep (2010) 9
Total