PRODUCTS FOR CANCER RESEARCH

PRODUCTS FOR CANCER RESEARCH

TABLE OF CONTENTS 3 4 6 10 11 12 Introduction ALDEFLUOR A Powerful Tool to Study Cancer Stem Cells Culture Media for Cancer Research Culture Media Products for Breast Cancer Research Culture Media Products for Leukemia Research Culture Media Products for Brain Tumor Research Cell Separation Solutions for Cancer Research Enrichment of Human Epithelial Tumor Cells Enrichment of Multiple Myeloma Cells Enrichment of B Cells From Chronic Lymphocytic Leukemia (CLL) Samples Contract Assay Services References Standardizing cancer stem and progenitor cell isolation, identification, evaluation and culture STEMCELL Technologies offers standardized tools and solutions that are highly appropriate for functional studies on cancer precursor cells, including research into the mechanismsof malignant transformation and tumor progression, and evaluating the responsiveness of patients cells to cytotoxic compounds. 2

PRODUCTS FOR CANCER RESEARCH PRODUCTS FOR CANCER RESEARCH INTRODUCTION Cancer stem cells (CSCs) are transformed cells (found within tumors or hematological cancers) that are thought to share some of the same characteristics associated with normal stem cells. They represent a relatively small proportion of the cells within cancers and have the ability to form tumors through the classic stem cell processes of self-renewal and differentiation into all cell types found in a particular cancer sample. 1,2 CSCs are thought to persist in tumors as a distinct population and cause relapse and metastasis by giving rise to new tumors. 1,2 Conventional chemotherapies are not always effective at preventing cancer from recurring. This is believed to be due to inability of some of the interventions to effectively target CSCs. 8 day-old MCF7 breast cancer cell line tumorspheres cultured in MammoCult The first conclusive evidence for the existence of CSCs was published in 1997. 3 In this study a subpopulation of leukemic cells, distinguished by the expression of CD34 and the absence of CD38 expression, proved to be capable of initiating cancers in immunocompromised mice that were histologically similar to the donor. Since then a number of studies identifi ed CSCs in several solid tumors, including cancers of the brain, 4 breast, 5 colon, 6 ovary, 7 pancreas 8 and prostate. 9 STEMCELL Technologies offers a variety of tools and reagents to study leukemias as well as breast and brain cancers. 3

ALDEFLUOR A POWERFUL TOOL TO IDENTIFY CANCER PRECURSOR CELLS HOW IS ALDEFLUOR USED IN CANCER RESEARCH? Various studies have shown that ALDH may have a role in both normal and cancer stem cell differentiation. Human hematopoietic and mouse neural stem and progenitor cells have been shown to have high ALDH activity. 12 Increased ALDH expression has been found in multiple myeloma and ALDEFLUOR Kit acute myeloid leukemia (AML), 32-34 prostate, 37 colon, 38 and lung 39 cancers. ALDH activity may thus serve as a common marker for both normal and malignant precursor cells. In a study conducted by Ginestier et al., it was shown that ALDH may not only be a good identifi er of cancer stem cells, but also a good predictor of patient outcome. Out of 577 human breast cancer tissue samples studied, those that expressed ALDH had the worst overall outcome. 15 WHAT IS ALDEFLUOR? ALDEFLUOR is a kit used for the non-immunological identification and characterization of a broad spectrum of normal and cancer stem and progenitor cells based on their expression of aldehyde dehydrogenase (ALDH). ALDEFLUOR has been shown to identify a unique population of human hematopoietic cells, that includes lineage-antigen negative (Lin - ) cells, colony-forming cells, long-term cultureinitiating cells, and NOD/SCID repopulating cells. 10-13 Human endothelial, 13 mammary 14-22 and mesenchymal progenitors (CFU- F) 13, 23-24 can also be sorted with ALDEFLUOR. ALDH br human cord blood cells engraft a variety of non-hematopoietic tissues in immunodeficient mice. 25 ALDEFLUOR has been used to identify rat embryonic neural stem cells, 26 mouse embryonic and adult neural stem cells 27-29 and lung cancer cell lines. 29 Recent publications have also shown ALDEFLUOR to be WHY USE ALDEFLUOR? NO ANTIBODIES. Enzyme-based detection method of ALDH expression. Staining with antibodies is not required. FLEXIBILITY. Reported to work with cryopreserved or fresh samples. Optimized for use with human hematopoietic cells, but can be adapted for use with other species (mouse, rat) and cell types (neural, mesenchymal, endothelial, tumor). INFORMATIVE. Identifies only viable cells with an intact cellular membrane. Expression of cell surface markers can be detected simultaneously, as cells can be counterstained with standard fl uorescently-labeled antibodies. EASY. The process is simple and highly reproducible. No specialized equipment is necessary, as cells can be detected by most cell sorters and analyzers already in the lab. useful in the identifi cation of malignant cell subpopulations. 30-34 ALDEFLUOR has been developed by Aldagen Inc. and is distributed by STEMCELL Technologies Inc. 4

PRODUCTS FOR CANCER RESEARCH IDENTIFICATION OF ALDH BR CELLS FROM HUMAN BREAST CANCER CELL LINES FIGURE 1: SKBR3 breast cancer cells stained with ALDEFLUOR for 45 minutes. FACS profi les of DEAB control (A) and ALDH staining (B). IDENTIFICATION OF ALDH BR CELLS FROM HUMAN MAMMARY EPITHELIAL SAMPLES FIGURE 2: Primary normal human mammary epithelial samples stained with ALDEFLUOR. FACS profi les of DEAB control (A) and ALDH staining (B). A ALDEFLUOR + DEAB (negative control) B ALDEFLUOR A ALDEFLUOR + DEAB (negative control) B ALDEFLUOR 0 10 3 10 4 10 5 0 50 100 150 200 250 0 10 3 10 4 10 5 IDENTIFICATION OF ALDH BR CELLS FROM MOUSE EMBRYONIC BRAIN SAMPLES FIGURE 4: E14 SVZ cells stained with ALDEFLUOR. FACS profi les of DEAB control (A) and ALDH staining (B). A ALDEFLUOR + DEAB (negative control) B 0 200 400 600 800 1000 IDENTIFICATION OF ALDH BR SSC LO CELLS FROM HUMAN HEMATOPOIETIC SAMPLES FIGURE 3: Bone marrow low density cells (A, B), peripheral blood mononuclear cells (C, D) and umbilical cord blood cells (E, F) stained with ALDEFLUOR. FACS profi les of DEAB control (A, C, E) and ALDH staining (B, D, F). A ALDEFLUOR + DEAB (negative control) B ALDEFLUOR 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 0 50 100 150 200 250 0 50 100 150 200 250 ALDEFLUOR C D E F 0 50 100 150 200 250 64K 128K 192K 256K 64K 128K 192K 256K 5

CULTURE MEDIA PRODUCTS FOR BREAST CANCER RESEARCH Cancers are speculated to contain stem cell and non-stem cell components, but only the relatively rare stem cell fraction can generate new tumors upon transplantation into immune-defi cient hosts. 39-41 Precursor cancer cells at various stages along their differentiation continuum can be studied using a variety of products for culture and in vitro evaluation of progenitors and stem cells. MAMMARY EPITHELIAL COLONY AND SPHERE IMAGES Examples of Colonies Derived From Human and Mouse Mammary Epithelial Progenitors Products for Mammary Progenitor Identification and Culture PRODUCT APPLICATION CATALOG # EpiCult -B (human) kit Colony-forming cell assay allowing for differential assessment of progenitor content 05601 Mixed Human Mammary Epithelial Cell Colony Luminal Human Mammary Epithelial Cell Colony EpiCult -B (mouse) kit Colony-forming cell assay allowing for assessment of progenitor content 05610 MammoCult kit Generation of robust human mammospheres and tumorspheres in optimized and defi ned culture conditions. 05620 EpiCult -C (human) kit Short-term culture of primary human mammary epithelial cells. 05630 NEW! Myoepithelial Human Mammary Epithelial Cell Colony Mouse Mammary Epithelial Cell Colony STEMCELL Technologies offers products for in vitro identifi cation and culture of mammary precursor cells. Bilineage and unilineage progenitors can be differentially assessed using the Colony- Forming Cell Assay with EpiCult -B Medium. EpiCult -C Medium is appropriate for short-term culture of human mammary luminal epithelial and myoepithelial cells. MammoCult is a rich and defi ned medium that supports the culture of mammospheres from normal primary human mammary samples and tumorspheres from transformed breast epithelial cells or breast cancer cell lines. Evaluation of mammospheres and tumorspheres is becoming one of the key tools for the study of mammary cell differentiation and transformation. Mammospheres From Primary Human Mammary Epithelial Cells Tumorspheres From MCF7 Human Breast Cancer Cell Line DID YOU KNOW? MammoCult can be used to culture tumorspheres from a variety of breast cancer cell lines including MCF7, SKBR3, MDA-MB-231, AU565, SUM149 and BT474. For more information, see our Technical Bulletin on Tumorsphere Culture of Human Breast Cancer Cell Lines (Catalog #29936). 6

PRODUCTS FOR CANCER RESEARCH CULTURE MEDIA PRODUCTS FOR LEUKEMIA RESEARCH Leukemic cells have the capacity for clonogenic growth in vitro. 42 Therefore, culture methods and media used for the study of normal hematopoiesis are also useful for functional studies of leukemic cells. These include colony assays in MethoCult media, long-term culture in MyeloCult media and expansion cultures in StemSpan serum-free expansion media. Applications include research of mechanisms underlying malignant transformation and cancer progression or evaluation of responsiveness of patients cells to chemotherapeutic agents such as specifi c inhibitors of the BCR-ABL tyrosine kinase in HEMATOPOIETIC COLONY IMAGES Examples of Colonies Derived From Human Hematopoietic Progenitors Chronic Myeloid Leukemia (CML). 43 Products for Identification and Culture of Hematopoietic Stem and Progenitor Cells Human CFU-GM Human CFU-GEMM PRODUCT APPLICATION CATALOG # MethoCult for Human Cells Base media allowing for the addition of desired growth factors Detection of CFU-E, BFU-E, CFU-GM, CFU-GEMM in bone marrow, cord blood, peripheral blood and mobilized peripheral blood 04330 04236 04230 04100 04034/04044 04434/04444 04435/04445 04436/04446 Human BFU-E Human CFU-GM & BFU-E Examples of Colonies Derived From Mouse Hematopoietic Progenitors Detection of CFU-GM (including CFU-G and CFU-M) in bone marrow, cord blood, peripheral blood and mobilized peripheral blood 04035/04045 04534/04544 04535/04545 04536/04546 Detection of BFU-E, CFU- GM and CFU-GEMM in bone marrow, spleen, peripheral blood and fetal liver 03434/03444 Mouse CFU-M Mouse BFU-E MethoCult for Mouse Cells Detection of CFU-GM in bone marrow, spleen, peripheral blood and fetal liver Base media allowing for the addition of desired growth factors 03534 03334 03234 03231 StemSpan Serum-Free Expansion Medium MyeloCult Serum-free medium for culture and expansion of hematopoietic cells Myeloid long-term culture medium for primitive hematopoietic cells 09600/09650 05100/05150 05300/05350 7

CULTURE MEDIA PRODUCTS FOR BRAIN TUMOR RESEARCH Multipotent neural stem-like cells or brain tumor stem cells (BTSCs), also known as cancer stem cells (CSCs), have recently been identifi ed and isolated from different grades (low and high) and types of brain cancers, including gliomas, medulloblastomas, astrocytomas, and ependymomas. 44 Similar to neural stem cells, these brain tumor stem cells exhibit self-renewal, high proliferative capacity and multilineage differentiation potential in vitro. The Neurosphere Assay is based on serum-free culture conditions containing specifi c mitogens that selectively allow neural stem cells (or BTSCs) and their progeny to proliferate and self-renew while maintaining their multi-lineage potential. Originally used to isolate, identify and expand neural stem cells, the Neurosphere Assay has recently been applied to the study of BTSCs. Importantly, the Neurosphere Assay may be a clinically relevant functional read-out for the study of BTSCs, with emerging evidence suggesting that renewable neurosphere formation is a signifi cant predictor of increased risk of patient death and rapid tumor progression in cultured human glioma samples. 45 Products to Assess Self-Renewal and Proliferation Potential PRODUCT APPLICATION CATALOG # NeuroCult NS-A Proliferation Kit (Human)* NeuroCult -XF Proliferation Medium* NeuroCult Proliferation Kit (Mouse)* NeuroCult NS-A Proliferation Kit (Rat)* NeuroCult Neural Colony-Forming Cell (NCFC) Assay Kit (Mouse)* NeuroCult Neural Colony-Forming Cell (NCFC) Assay Kit (Rat)* Human Proliferation Media 05751 05761 Mouse Proliferation Medium 05702 Rat Proliferation Medium 05771 Colony Assay to Quantify Cells with High Proliferative Potential 05740 05742 *Requires supplementation with rh EGF (Cat #02633). When culturing cells obtained from adult mouse, rh bfgf (Cat #02634) and Heparin (Cat #07980) are also required. STEMCELL Technologies offers standardized NeuroCult media for human, mouse and rat neural stem cells. Components for all media and supplements are rigorously prescreened to ensure that you are using consistently high quality reagents for every experiment. Products to Assess Multi-Lineage Differentiation Potential PRODUCT APPLICATION CATALOG # NeuroCult NS-A Differentiation Kit (Human) Human Differentiation Medium 05752 NeuroCult Differentiation Kit (Mouse) NeuroCult NS-A Differentiation Kit (Rat) Mouse Differentiation Medium 05704 Rat Differentiation Medium 05772 8

PRODUCTS FOR CANCER RESEARCH FIGURE 5: Proliferation and differentiation of mouse neural stem cells using NeuroCult. Precursor Cell NeuroCult Proliferation Kit (Mouse) NEUROCULT MEDIA AND DISSOCIATION REAGENTS HAVE BEEN USED TO: Dissociate human glioblastoma and oligodendroglioma samples 46 A Neurosphere NeuroCult Differentiation Kit (Mouse) Culture human glioblastoma-derived 47-49 and oligodendroglioma-derived 50 tumorspheres Culture cells obtained from mouse models of medulloblastoma 51 and glioma 52 as tumorspheres Differentiate brain tumor stem cells into neurons, astrocytes and oligodendrocytes 51-52 Passage/dissociate tumorspheres 53 B C Neurons Astrocytes and Neurons D E Oligodendrocytes GABA-nergic Neurons A. Mouse neurosphere cultured with the NeuroCult Proliferation Kit (Mouse). B. Immunofluorescent staining of neural cell body and processes (red) with mouse monoclonal ß-Tubulin III antibody (Catalog #01409). C. Immunofluorescent staining of astrocytes (green) with rabbit polyclonal GFAP antibody (Catalog #01415) and neurons (red) with mouse monoclonal MAP2 antibody (Catalog #01410). D. Immunofluorescent staining of oligodendrocytes (green) with mouse monoclonal O4 Oligodendrocyte Marker antibody (Catalog #01416). E. Immunofluorescent staining of GABA-nergic neurons (green) with rabbit polyclonal GABA antibody (Catalog #01411). 9

CELL ISOLATION SOLUTIONS FOR CANCER RESEARCH STEMCELL Technologies offers a wide range of optimized cell separation products for the isolation of cancer cells. Our innovative cell separation platforms (RoboSep, RosetteSep, EasySep, and StemSep ) are particularly suitable for the isolation of cancer cells as they provide a simple and effective method of isolating rare cells with high purity and recovery. APPLICATION PRODUCT CATALOG # DESCRIPTION RECOMMENDED FOR: RosetteSep Human Circulating Epithelial Tumor Cell Enrichment Cocktail 15127 15167 (5 x 15127) The non-magnetic enrichment of circulating epithelial tumor cells from fresh whole blood by negative selection. The enriched cells have not been labeled with antibody. The enrichment of untouched circulating epithelial tumor cells from whole blood Circulating Epithelial Tumor Cells StemSep Human Epithelial Tumor Cell Enrichment Kit 14152 14162 (5 x 14152) The immunomagnetic, column-based enrichment of tumor cells from peripheral blood mononuclear cells (PBMC) by negative selection. The enriched cells have not been labeled with antibody. The enrichment of untouched circulating epithelial tumor cells from PBMC CD45 Depletion RosetteSep Human CD45 Depletion Kit 15122 15162 (5 x 15122) The non-magnetic depletion of CD45 + cells from whole blood. The enriched cells have not been labeled with antibody. The enrichment of circulating tumor cells from whole blood EasySep Human CD45 Depletion Kit* 18259 (PBMC) 18289 (WB) The immunomagnetic, column-free depletion of CD45 + cells from peripheral blood mononuclear cells (PBMC) or whole blood (WB). The enriched cells have not been labeled with antibody. The enrichment of circulating tumor cells from PBMC or whole blood Multiple Myeloma (CD138) Cells RosetteSep Multiple Myeloma Cell Enrichment Cocktail 15129 15169 (5 x 15129) The non-magnetic enrichment of multiple myeloma cells from bone marrow aspirates. The enriched cells have not been labeled with antibody. The enrichment of untouched multiple myeloma cells from whole blood EasySep Human CD138 Positive Selection Kit* 18357 (PBMC) The immunomagnetic, column-free selection of CD138 + cells from PBMC or whole blood by positive selection. Desired cells are targeted for selection by antibodies 18387 (WB) recognizing CD138. The selection of highly purified CD138 + cells from PBMC or whole blood B Cells From Chronic Lymphocytic Leukemia (CLL) Samples EasySep Human B Cell Enrichment Kit without CD43 Depletion* 19154 The immunomagnetic, column-free enrichment of B cells expressing CD43 from peripheral blood mononuclear cells (PBMC) from CLL patients. The enriched cells have not been labeled with antibody. The enrichment of untouched B cells expressing CD43 from PBMC *EasySep can be fully automated using RoboSep technology. 10

PRODUCTS FOR CANCER RESEARCH CONTRACT ASSAY SERVICES STEMCELL Technologies Contract Assay Service works with you to design and execute customized experiments to meet your research needs. Our primary cell assays provide clinically relevant screening of the effects of small molecule compounds, including chemotherapeutic agents. These assays can assess a compound s effects on the proliferation and differentiation of hematopoietic, mesenchymal and neural stem and progenitor cells. Join the more than 80 organizations worldwide that have trusted our experts to conduct more than 400 studies in our GLP-compliant facility, saving both time and money. WEBSITE www.contractassay.com EMAIL contractassay@stemcell.com REFERENCES 1. T. Lapidot et al., Nature 367, 645 (1994) 2. J.E. Dick, Blood 112, 4793 (2008) 3. D. Bonnet, J.E. Dick, Nat Med, 3, 730 (1997) 4. S.K. Singh et al., Nature 432, 396 (2004) 5. M. l Hajj et al., Proc. Natl. Acad. Sci. U.S.A. 100, 3983 (2003) 6. CA O Brien, et al., Nature 445, 7123 (2007) 7. S. Zhang et al., Cancer research 68, 11 (2008) 8. C Li et al., Cancer research 67, 3 (2007) 9. NJ Maitland et al., J. Clin. Oncol. 26, 17 (2008) 10. R. W. Storms et al., Proc Natl Acad Sci. 96, 9118 (1999) 11. P. Fallon et al., Br J Haematol 122, 93 (2003) 12. D.A. Hess et al., Blood 104: 1648 1655 (2004) 13. T. Gentry et al., Cytotherapy 9: 259 274 (2007) 14. E. Charafe-Jauffret et al., Cancer Res. 69: 1302 1313 (2009) 15. C. Ginestier C et al., Cell Stem Cell. 1: 555 567 (2007) 16. J.D. Graham JD et al., Endocrinology. 150: 3318 3326 (2009) 17. I. Ibarra et al., Genes Dev. 21: 3238 3243, 2007 [Mouse] 18. M. Luo M et al., Caner Res. 69: 466 474 (2009) 19. A. Magnifi co A et al., Clin Can. Res. 15: 2010 2021 (2009) 20. E. Charafe-Jauffret et al., Clin Cancer Res. 2009 Dec 22. [Epub ahead of print] 21. E. Charafe-Jauffret et al., Cell Cycle. 8(20): 3297 302. 2009 Oct 10 [Epub ahead of print] 22. Pan Q et al., Clin. Can Res. 15, 7441 (2009) 23. Gentry et al., Cytotherapy 9: 259 274 (2007) 24. Capoccia et al., Blood 113: 5340 5351 (2009) 25. F. Zeng et al., Proc Natl Acad Sci U S A. 2006 May 16;103 (20): 7801 7806 26. Cai et al., J Neurochem 88: 212-226 (2004) 27. Corti et al., stem cells 24: 9750985 (2006) 28. Corti et al., Hum Mol Genet 15: 167 187 (2006) 29. Moreb JS, et al., Cytometry B Clin Cytom. 72: 281 289 (2007) 30. Jiang F, et al., Mol Cancer Res. 7: 330 338 (2009) 31. Jiang T, et al., Cancer Res. 69: 845 854 (2009) 32. W. Matsui et al., Blood. Mar 15;103(6): 2332 6 (2004) 33. D. J. Pierce et al., Stem Cells. Jun-Jul;23(6): 752 60 (2005) 34. Ran et al., Exp Hematol, published online 12 October 2009 35. D. Bonnet, J.E. Dick, Nat Med, 3, 730 (1997) 36. S.K. Singh et al., Nature 432, 396 (2004) 37. M. Yao et al., Cells Tiss. Org. 191: 203 212 (2010) 38. Li T et al., Laboratory Investigation 90, 234 244 (2010) 39. Huang E. H. et al., Cancer Res. 69(8): 3382 9 (2009) 40. Jiang F. et al., Mol Cancer Res. 27(3): 330 8 (2009) 41. Visus C. et al., Cancer Res. 67(21): 10538 45 (2007) 42. A Carral, et al., Bone Mar. Tran. 29: 10, 825 832 (2002) 43. X Jiang et al., J Natl Cancer Inst. 99(9): 680 93 (2007) 44. A.L. Vescovi, et al., Nat Rev Cancer 6: 425 436 (2006) 45. D.R. Laks, et al., Stem Cells. 27: 980 987 (2009) 46. N. Platet et al., Cancer Lett 258: 286 290 (2007) 47. S.G. Piccirillo et al., Nature 444: 761 765 (2006) 48. G.L. Gallia et al., Mol Cancer Ther 8: 386 393 (2009) 49. R.B. Jenkins et al., Cancer Res 66: 9852 9861 (2006) 50. L.T. McGillicuddy et al., Cancer Cell 16: 44 54 (2009) 51. Z.J. Yang et al., Cancer Cell 14: 135 145 (2008) 52. M.A. Harris et al., Cancer Res 68: 10051 10059 (2008) 53. H. Wakimoto et al., Cancer Res 69: 3472 3481 (2009) 11

FOR RESEARCH USE ONLY. NOT FOR THERAPEUTIC OR DIAGNOSTIC USE. CATALOG #29189 VERSION 1.2.0 MAY 2010