Available Resources from TMEN

Grant: CA126552

PI Name: BISSELL, MINA J

Project Title: Bioengineering 3-D Models for Breast Cancer Therapy

Institution: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB

Resources Generated

·         In conjunction with Kevin Healy’s laboratory at UC-Berkeley, we have developed synthetic matrices to support a series of peptides that mimic laminin-111 function.  This model system is designed to probe the necessary motifs within laminin-111 that impart function and differentiation to mammary epithelial cells. 

·         In conjunction with Nancy Boudreau’s laboratory at UC-San Francisco, we have developed heterotypic co cultures of vascular endothelial cells and breast cells to investigate the interaction of this two cell types in breast tumor progression.  These results were published and are the basis for continuing to develop heterotypic co cultures of monocytes and epithelial cells, as well as, monocytes, epithelial cells and fibroblasts in the investigation of breast cancer progression. 

·         In conjunction with Kornelia Polyak and Jeff Pollard we have developed a new program in bringing immune, endothelial and stromal cells together with epithelial cells and brought bioengineers trainees to develop the appropriate gadgets for these interactions.  These would be available to the whole TMEN community once the results are promising.

SHARING OF REAGENTS:

The principles and techniques of microenvironment arrays (MEArray) will be listed on the TMEN website.

Grant: CA126524

PI Name: CLARKE, MICHAEL

Project Title: Molecular and Functional Characterization of Colon Tumor Cancer Stem Cells and Stroma

Institution: STANFORD UNIVERSITY

Subproject 1: Colon Cancer Stem Cells and their Stroma

Subproject 2: High Thoughput Functional Screening of Colon Cancer Stem Cells and their Stroma

CORE 1: Bioinformatics Core

Resources Generated

• Antibodies used for flow cytometry isolation of cells - sent to NCI repository to share with other TMEN laboratories as well as the broader NIH research community

o    FLOTILLIN-2/ESA FITC MAB 29

o    HU CD10 PE-CY5 MAB HI10A

o    HU CD140B BIOTIN MAB 28D4

o    HU CD166(ALCAM) PE

o    HU CD18 PE-CY5

o    HU CD2 PE-CY5

o    HU CD20 PE-CY5 MAB 2H7

o    HU CD24 FITC MAB ML5

o    HU CD24 PE MAB ML5

o    HU CD3 PE-CY5 MAB UCHT1

o    HU CD31 BIOTIN MAB M89D3

o    HU CD31 BIOTIN MAB WM59

o    HU CD44 APC MAB G44-26

o    HU CD45 PE-CY5 MAB HI30

o    HU CD45 PE-CY7 MAB HI30

o    HU CD64 PE-CY5 MAB 10.1

o    HU CD66 FITC

o    HU CD66 PE

o    HU EGFR FITC (0.05MG)

o    HU EGFR PE

o    MS CD140A BIOTIN MAB APA5

o    MS CD90.1(THY1.1) APC

o    MS H-2KD BIOTIN MAB SF1-1.1

o    SAV PE-CY5

o    SAV PE-CY7

• Early-passage human xenograft cancer tumors established from cells isolated directly from patient tumors - sent to the NCI.

o    Colon cancer - 9 xenograft tumors

o    Breast cancer - 5 xenograft tumor

o    Head and neck cancer - 10 xenograft tumors

Grant: CA126511

PI Name: CONDEELIS, JOHN S

Project Title: Novel Methods for Detecting Cell Interactions in the Tumor Microenvironment

Institution: ALBERT EINSTEIN COLLEGE OF MEDICINE OF YESHIVA UNIVERSITY

Resources Generated

·         Mammary imaging window

·         Dendra2 photo-conversion protocol for multiphoton imaging

·         Live invasive/disseminating tumor cells (and cancer stem cells) from mouse and human mammary tumors

·         The NANo IntraVital Device (NANIVID) for collection of live disseminating tumor cells

• Novel Multi-photon microscope designs that allow exploitation of the new generation of photo-convertible fluorescent proteins
• New wafer production/integration processes developed in the fabrication of the NANIVID

·         Patents

“Isolation, Gene Expression, and Chemotherapeutic Resistance Of Motile Cancer Cells”

·         U.S. Provisional Patent Application No. 60/600,697 “Isolation, Gene, Expression, and Chemotherapeutic Resistance of Motile Cancer Cells”

·         PCT/US2005/027680 “ISOLATION, GENE EXPRESSION, AND CHEMOTHERAPEUTIC RESISTANCE OF MOTILE CANCER CELLS”

·         European Patent Application No. 05807467.5 (National Phase Entry of PCT/US2005/027680)

                                                              i.      Under review by EPO

·         Canadian Patent Application No. 2,576,702 (National Phase Entry of PCT/US2005/027680)

                                                              i.      Under review by Canadian Patent Office

·         U.S. Patent Application No. 11/659,514 (National Phase Entry of PCT/US2005/027680) published as US-2008-0138805-A1

                                                              i.      Under review by USPTO

“An in vivo Quantitative Screening Test for Anti-Metastasis Treatment Efficacy”

·         U.S. Provisional Patent Application No. 61/197791

“Assay for collecting and analyzing migratory tumor cells in solid tumors for metastatic potential”

·         U.S. Provisional Patent Application No. 96700/1544

Grant: CA126518

PI Name: HOLLAND, ERIC C

Project Title: Tumor Microenvironment Interactions in Brain Tumors

Institution: SLOAN-KETTERING INSTITUTE FOR CANCER RESEARCH

Subproject 1: Infiltrating gliomas act to recruit stem cells

Subproject 2: Identify the genes and functions enabling metastatic colonization of the brain

Subproject 3: Tumor Microenvironment in Modulating the Primary & Metastatic Brain Tumors

Resources Generated

• Gli-luc mouse - This is a mouse that produces light via luciferase under control of an SHH/Gli responsive promoter.  It is used to visualize the activity of Gli in tissues non-invasively.  It is currently at the MMHCC mouse consortium in Fredrick and provided free to any academic lab that wants it.  In addition, it has been sent directly by the Holland lab to UCSF (Bill Weiss), U. Mich. (Brian Ross), and Duke University (Robert Wexler-Reya).
• Novel HuMu ‘ProtIn’ microarray produced by Affymetrix that focuses solely on proteases and their endogenous inhibitors, allowing the survey of protease regulation in detail. In addition, this chip was designed to distinguish between human (Hu) and mouse (Mu) gene expression, making it ideal for xenograft models.
• Brain metastasis project to date has almost doubled the number of available model cell lines for the study of brain metastasis by human breast cancer and lung cancer in mice. These models include the = MDA231-BrM, CN34-BrM, CN37-BrM and CN41-BrM breast adenocarcinoma cell systems and the H2030-BrM and PC9-BrM lung adenocarcinoma cell systems. The  brain metastatic model cell systems have been distributed to the following groups for their research (without collaborative obligations):

Grant: CA126515

PI Name: HYNES, RICHARD O

Project Title: Tumor-Stroma Interacions in the Tumor Microenvironment

Institution: MASSACHUSETTS INSTITUTE OF TECHNOLOGY

Subproject 1: Cellular Interactions with the Extracellular Matrix of the Tumor Microenvironment

Subproject 2: Dissecting Tumor Cell – Stromal Cell Interactions in a Mouse Model of Lung Cancer

Subproject 3: In Vivo Analysis Of The Monocyte/Macrophage Response In Tumor Microenvironment

Subproject 4: Recruitment of Stromal Cells to the Tumor Microenvironment

Resources Generated

• Mice  - deposited at Jackson lab for open distribution to academic investigators (also to industrial concerns with licensing from MIT). Also distribute many mice to other laboratories– both genetically modified mice and frequently also (with permission) mice that we obtained from other laboratories.

o    av-integrin floxed mice

o    a5-integrin-floxed mice and the null counterparts

o    GPR56-null

o    TG2-null mice

o    R26-LSL-LSIY (129S4 and C57BL/6 background) - they were sent to Christian Meyer in Jonathan Powell's lab at Johns Hopkins (Division of Tumor Immunology)

• Vectors– available from the non-profit organization Addgene

o    MSCV-P2Gm

o    MSCV-FLIP

o    MSCV-FLIPi

o    LB2-CPGm

o    LB2-FLIP

• Cells

o    Various lung tumor cells lines induced in K-ras^LSL-G12D/+;p53^fl/fl mice (129S4 and C57BL/6 backgrounds) are available by request from the Jacks lab. This includes tumors induced with Ad-Cre in R26^LSL-LSIY mice or tumors induced with Lenti-x or Lenti-LucOS lentiviral vectors.

• Plasmids

o    R26-LSL-LSIY targeting vector- available by request from the Jacks lab.

o    R26-LSL-Luc (without SIY) targeting vector - available from the non-profit organization Addgene

o    Lenti-x (PGK-Cre) - available from the non-profit organization Addgene

o    Lenti-LucOS (UbC-LucOS;PGK-Cre) - available from the non-profit organization Addgene

• Macrophages

o    AMTA-680 - targets activated tumor-associated macrophages selectively

o    CLIO - pan-macrophage targeting agent for magnetic resonance imaging

o    DNP - pan-macrophage targeting agent for positron emission tomography

Reagent Sharing

o    Hynes Lab - It has been a long-standing practice to make all reagents, recombinant vectors, cells, genetically-modified animals and other research resources available to the world-wide research community. This practice of sharing will continue through individual mailing of research materials from this Project and by depositing relevant resources to various sites and facilities that make them freely available to the scientific community. For instance, mouse strains are deposited with the Jackson Laboratory to make them freely available to the research community. Many plasmids are deposited with the non-profit organization Addgene: http://www.addgene.org/pgvec1?f=c&cmd=showcol&colid=171. Protocols are posted on the Hynes laboratory website: <http://web.mit.edu/hyneslab/>

o    Jacks Lab – It has been a long-standing practice to make all reagents, recombinant vectors, cells, genetically-modified animals and other research resources available to the world-wide research community. This practice of sharing will continue through individual mailing of research materials from this Project and by depositing relevant resources to various sites and facilities that make them freely available to the scientific community. For instance, mouse strains are deposited with the Jackson Laboratory, or with the NCI Mouse Models of Human Cancer Consortium Repository to make them freely available to the research community. These two repositories currently distribute ~ 30 strains developed in the Jacks laboratory. All plasmids are deposited with the non-profit organization Addgene: http://www.addgene.org/pgvec1?f=c&cmd=showcol&colid=171. Protocols are posted on the Jacks laboratory Website: http://web.mit.edu/jacks-lab/protocols_table.html

o    Weissleder Lab - During the funding period there has been interaction between the Weissleder lab and with other TMEN investigators, including the Tyler Jacks’ and John Condeelis’ labs. The Jacks lab was provided with different molecular imaging technologies to track tumor cell progression non invasively. The Condeelis lab is testing the AMTA680 agent in their well-established mouse models of mammary cancer and the Weissleder lab is elaborating refined approaches for multiphoton intravital microscopy of leukocytes in the tumor stroma and other relevant tissues. A set of newly generated cell targeting agents were distributed to the Lynda Stuart’s lab at MGH and to the Brian Ross’ lab at the University of Michigan.

o    Weinberg Lab - All DNA clones utilized in the Weinberg lab experiments have been made available for distribution by the non-profit entity addgene.org, which distributes them for a nominal fee. Addgene,org is currently distributing clones from the Weinberg laboratory at a rate of ~1,600 per year and has to date distributed >4,200 DNA samples from the laboratory to interested parties. Cellular reagents are distributed, in response to requests, directly from the Weinberg laboratory to the interested parties with the latter paying shipping fees, usually within two weeks of receiving a request for such cells.

Grant: CA126505

PI Name: MATRISIAN, LYNN M

Project Title: Paracrine TGF-Beta Signaling in Tumor Initiation and Progression

Institution: VANDERBILT UNIVERSITY

Subproject 1: Paracrine TGF-Beta Signaling in Breast Cancer Initiation and Progression

Subproject 2: Paracrine TGF-Beta Signaling in Prostate Cancer Initiation and Progression

Subproject 3:  Host Microenvironment and Bone Metastases

CORE 1: Protein Collection and Proteomics Integrative Shared Resource

CORE 2: Image Fusion Integrative Shared Resource

CORE 3: Biomathematics and Bioinformatics Integrative Shared Resource

CORE 4: Genomics and Bioinformatics Core (Dana-Farber Cancer Institute)

Resources Generated

·         A method for spatially integrating MRI images with protein profiles derived by three dimensional MALDI MS images of protein profiles from tissues was developed.

·         A mathematical model for the role of TGFbeta in the development of prostate cancer in the prostate microenvironment involving 3 kinds of epithelial cells and 2 kinds of fibroblasts has been developed.

·         A mathematical model that explains the need for both wildtype and TGFbeta type II receptor-deficient fibroblasts, and the optimum nature of the 50:50 ratio of these two cell types, in prostate cancer was developed.

·         Mouse models

o   Generated Tgfbr2^ColTKO, a mouse model for prostate adenocarcinoma

o   Generated RCAS mice for the TMEN

·         Cell lines

o   Human prostate epithelial cell lines BHPrE1 and NHPrE1. These lines express both androgen receptor and PSA and retain an ability to undergo prostatic differentiation in vivo in the presence of an appropriate microenvironment. A manuscript describing these cells is currently in revision in “Stem Cells”. The cells have been distributed to a small number of laboratories on a collaborative basis and will be available to the research community upon acceptance of the descriptive manuscript.

o   Murine TGFbeta type II receptor-deficient fibroblast and tumor cell lines derived from MMTV-PyMT transgenic mice have been generated in the Moses laboratory and will be available to share.

o   Normal and Cancer Associated Fibroblasts (CAFs) from human prostate and breast cancers were generated

o   Two cell lines derived from MMTV-PyMT mammary tumors and used for intratibial injections were generated and characterized

o   List of cell lines showing overexpression of antibody targets

-       TGFβ1,β2 and β3 as well as Cercam and Leprel 2 are overexpressed in MCF7 cells

-       Leprel2 is also overexpressed in MDA435 cells

-       Laminin Alpha 4 is overexpressed in T98G cells

o   List of cell lines generated for antibody target overexpression

-       PC12 parental cell line

-       PC12 vector control cell line

-       PC12 Laminin A4 overexpression cell line

-       In addition, 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.

o   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

·         Developed BioTecQC, a quality control algorithm designed to identify and eliminate from further analysis, those arrays that both have poor quality scores and are biological outliers.

·         ANTIBODIES

o   mAb against Cercam

o   mAb against Laminin Alpha 4

o   antibody supes against TGF β1/2/3 as well as Leprel 2

·         REAGENTS AND PROTOCOLS

o   Protocol for separation of mouse stroma from human tumor cells

o   Protocol for separation of human epithelial tumor cells from non-tumor components

o   Protocol for LCM following by high-resolution oligo array-CGH

o   Protocol for optimized WGA

o   Custom Agilent CGH 2x415K oligo microarray, optimized for resolution vs. cost         

o   Monoclonal Antibodies against Laminin α4 and Cercam

o   EVOC protocol

SHARING OF REAGENTS:

·         The floxed TGFbeta type II receptor mice generated in the Moses laboratory were shared with the Michael Clarke laboratory.

·         Mice genetically deficient in MMPs 2, 3, 7, 9, 11, 12, and 13 are maintained in the Matrisian laboratory and have been shared with many investigators.

·         Project 2 has shared fibroblasts from human prostate and breast cancers generated as part of the VUTMEN with other VUTMEN members.

·         Project 3 members have trained several other VUTMEN laboratories in cardiac and intratibial injections to study tumor metastasis to bone. 

·         Project 3 has shared bone metastatic cell lines with other VUTMEN laboratories and other outside laboratories.

• VUTMEN investigators have entered 12 cell lines and 15 antibodies into the TMEN Inventory on the Sharepoint site that are available to all TMEN members.

Grant: CA126540

PI Name: PLYMATE, STEPHEN R

Project Title: Significance of Microenvironment for Prostate Cancer Initiation and Progression

Institution: UNIVERSITY OF WASHINGTON

Subproject 1: The Aged Microenvironment as a Contributor to Carcinogenesis

Subproject 2: Laminin Dysregulation and Prostate Cancer

CORE: Tissue and Pathology Core

Resources Generated  – made available through TMEN website

These antibodies have been shared with the Stanford TMEN

·         Reagents/Models: The Biospecimen Core has developed 23 prostate cancer xenograft lines and established methods for their growth in several sites including subcutaneous, intraprostatic, and in bone. These various microenvironments profoundly alter tumor growth characteristics and responses to therapy. The tissue acquisition and development of a large tissue bank representing local and metastatic cancers has been constructed and formalized processes for distributing these resources to the TMEN and larger research community have been established.

·         Gene expression profiles: The Biospecimen Core has generated gene and protein expression profiles of senescent and aged components of the tumor microenvironment (fibroblasts). These profiles have been shared with TMEN members and with the greater research community through the GEO data repositories. The Core is currently generating genomic profiles of cancer-associated prostate stroma and these will be integrated with collaborative TMEN efforts to simultaneously characterize transcripts and epigenetic changes

·         Developed novel in vitro 3-dimensional matrix reconstitution and organotypic models or animal models in collaboration with the Department of Bioengineering at the University of Washington.

Grant: CA126568

PI Name: ROWLEY, DAVID R

Project Title: Co-evolution of the Reactive Microenvironment in Prostate Cancer Progression

Institution: BAYLOR COLLEGE OF MEDICINE

Subproject 1: Evolution of Reactive Stroma in Prostate Cancer Progression

Subproject 2: Neurogenesis in the Prostate Cancer Microenvironment

CORE: Expression Analysis and Pathology CORE

Resources Generated

• Developed KC transgenic mice lines with KC(IL-8) expression targeted to the prostate gland
• Development of TGF-b1 transgenic mice lines with constitutively active TGF-b1 targeted to the prostate gland
• Development of Wfdc1/ps20 null mice lines
• Development of a xenograft model with recombined engineered LNCaP cells with engineered stromal cells and adaptation of this to mice with bone marrow transplantation to trace linage of reactive stroma progenitor cells
• Development a co-culture model with engineered LNCaP spheroids and wild type or engineered human prostate stromal cells or human putative progenitor cells from peripheral blood
• Development of antibodies to ps20 for Westerns, immunohistochemistry and neutralization
• Development of antibodies against S4F for Westerns, IF and immunohistochemistry
• Development of neurogenesis and 3D multicellular co-culture models to evaluate cancer nerve interactions
• Development human pleomorphic adenoma cell lines that secrete human ECM matrix as a possible alternative to EHS Matrigel
• Gene expression profile of altered genes in human prostate cancer reactive stroma grade 3 foci as compared to normal human prostate stroma

Grant: CA 126513

PI Name: WANG, TIMOTHY CRAGIN

Project Title: The Role of Inflammation and Stroma in Digestive Cancer

Institution: COLUMBIA UNIVERSITY HEALTH SCIENCES

Subproject 1: Stromal Myofibroblasts in Gastric Carcinogenesis

Subproject 2: Stromal Myofibroblasts in Hepatic Carcinogenesis

Subproject 3: Epigenetics and Genetics of Stromal Cells in Liver and Gastric Cancer

CORE: Imaging Core

Resources Generated

• Bone marrow transplant/reconstitution-methodology shared with David Rowley’s group at Baylor, and with the NCI core lab (Gordon Whitely)
• Murine bone marrow-derived fibroblast cultures– shared with investigators at Columbia (Charles Powell, Ben Tycko) and at Cornell (Andrew Dannenberg)
• INS-GAS murine model of gastric cancer– shared with Richard Peek and Keith Wilson at Vanderbilt, Susan Watson at Nottingham UK
• Transgenic mice

• Vimentin-CreTM - Cre functionality confirmed in vivo, detailed characterization pending
• LRAT-Cre - Mouse yet to be evaluated
• LRAT-CreTM - Mouse yet to be evaluated
• Lhx2-Cre - Construct injected
• PAI1-Cre - Construct finished, not injected

• Reagents

• Ad5Gremlin - Expression confirmed by western blot, function remains to be tested

·         Developed novel bioluminescence and fluorescence tomographic imaging codes

·         Introduced a novel hyper-spectral excitation-resolved fluorescence tomography methods that promises to greatly simplify imaging of quantum dots reporter probes inside scattering tissue.

·         Continued to refine and apply the Dynamic Contrast Enhanced (DyCE) molecular imaging method developed in previous years.