cancer research

Cancer Stem Cell ResearchStandardized Tools and Reagents

CanCeR ReSeaRCh PRoduCTS

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PRODUCTS FOR CANCER RESEARCH

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Introduction

ALDEFLUORTM: Detection of a Potential Biomarker in Cancer Research

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 for Cancer Research

Enrichment of Human Epithelial Tumor CellsEnrichment of Multiple Myeloma Cells Enrichment of B Cells From Chronic Lymphocytic Leukemia (CLL) Samples

Contract Assay Services

References

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Table of Contents

Standardizing Cancer Stem and Progenitor Cell Isolation, Identification, evaluation and Culture

STEMCELL Technologies offers standardized tools and solutions for functional studies on cancer precursor cells, including research into the mechanisms of malignant transformation and tumor progression, and evaluating the responsiveness of patients’ cells to cytotoxic compounds.

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Front cover: MCF7 tumorspheres cultured for 8 days in MammoCultTM. Learn more on page 6.

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Cancer stem cells (CSCs) are transformed cells (found within tumors or hematological cancers) that are thought to share several characteristics with normal stem cells.They represent a relatively small proportion of the cells within cancers and, through the classic stem cell processes of self-renewal and multi-lineage differentiation, can form new tumors containing all cell types found in the parent cancer sample.1,2 CSCs are thought to persist in tumors as a distinct population and cause relapse and metastasis.1,2 The present inability to effectively target and eliminate CSCs is thought to be one of the reasons why conventional chemotherapies are not always able to prevent cancer recurrence.

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 capable of initiating cancers in immunocompromised mice that were histologically similar to the donor. Since then a number of studies have identified CSCs in several solid tumors, including cancers of the brain,4 breast,5 colon,6 ovary,7 pancreas8 and prostate.9

STEMCELL Technologies offers a variety of tools and reagents to study leukemias as well as breast and brain cancers. Learn more from relevant webinars, videos and tutorials at www.stemcell.com.

Introduction

Products For

Cancer Research

Figure 1. Over 200 publications have used ALDEFLUORTM to detect ALDHbr precursor cells in the following tissue types.

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ALDEFLUORTM is a trademark of Aldagen Inc.

ALDEFLUORTM Detection of ALDH, a Potential Biomarker in Cancer Research

aldefluoRTM and aldhbr CellsThe ALDEFLUORTM fluorescent reagent system has supported more than 200 publications by detecting Aldehyde Dehydrogenase bright (ALDHbr) cells in over 80 distinct cell types.

ALDEFLUORTM has been shown to identify a unique population of human hematopoietic cells, that includes lineage-antigen negative (Lin-) cells, colony-forming cells, long-term culture-initiating cells, and NOD/SCID repopulating cells.10-13 Human endothelial,13 mammary14-22 and mesenchymal precursor cells (CFU-F)13, 23-24 can also be sorted with ALDEFLUORTM, while ALDHbr human cord blood cells engraft a variety of non-hematopoietic tissues in immunodeficient mice.25 In other species, ALDEFLUORTM has been used to identify rat embryonic neural stem cells,26 mouse embryonic and adult neural stem cells.27-29

A complete list of publications using ALDEFLUORTM is available at www.stemcell.com/references.

aldefluoRTM in Cancer ResearchVarious studies have shown that ALDH may play a role in both normal and cancer stem cell differentiation. Increased ALDH expression has been found in multiple myeloma and acute myeloid leukemia (AML),30-32 prostate,35 colon,36 head and neck, thyroid gland, breast, liver, ovarian, cervical, bladder and lung37-38 cancers. ALDH activity may thus serve as a common marker for both normal and malignant precursor cells. A 2007 study conducted by Ginestier et al., showed that the presence of ALDH-expressing breast cancer cells was correlated with poor patient outcome.15 Since then similar studies have emerged that correlate ALDH phenotype with poor prognosis in AML, lung, pancreas, prostate and head and neck cancers.

Video:Optimizing ALDeFLuOrTM Protocols for Your Tissue Typewww.stemcell.com/OptALDEFLUOR

Why Use ALDEFLUORTM?

NO ANTIBODIES. Detect viable normal or cancer cells based on ALDH activity. No antibody staining required.

VERSATILE. Optimized for use with human hematopoietic cells, and can be adapted for use with other species (mouse, rat) and cell types (mammary, neural, mesenchymal, endothelial and many more).

INFORMATIVE. Identifies only viable cells with an intact cellular membrane. Compatible with immunophenotyping.

WIDELy-PUBLISHED. Has supported more than 200 peer-reviewed articles.

EASy. The process is s imple and highly reproducible. No specialized equipment aside from standard cell sorters or analyzers is needed.

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ALDHbr Cells: Human Mammary Epithelial Samples

Figure 3. Primary normal human mammary epithelial samples stained with ALDEFLUORTM. FACS profiles of DEAB control (A) and ALDH-stained cells (B).

Intracellular Fluorescence

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Figure 5. E14 SVZ cells stained with ALDEFLUORTM. FACS profiles of DEAB control (A) and ALDH-stained cells (B).

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ALDHbr Cells: Human Breast Cancer Cell Lines

Figure 2. SKBR3 breast cancer cells stained with ALDEFLUORTM for 45 minutes. FACS profiles of DEAB control (A) and ALDH-stained cells (B).

ALDHbr SSClo Cells: Human Hematopoietic Samples

Figure 4. Bone marrow low density cells (A, B), peripheral blood mononuclear cells (C, D) and umbilical cord blood cells (E, F) stained with ALDEFLUORTM. FACS profiles of DEAB control (A, C, E) and ALDH-stained cells (B, D, F).

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Video:The Basic FACS About ALDeFLuOrTM www.stemcell.com/BasicFACS

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Cancers are speculated to contain stem cell and non-stem cell components, of which only the relatively rare stem cell fraction can generate new tumors upon transplantation into immuno-deficient hosts.37-38 Culture and evaluate breast cancer precursor cells at various stages along the differentiation continuum, using the defined culture media listed below.

Culture Media Products For Breast Cancer Research

EpiCultTM-C is optimized for the routine culture of primary human mammary epithelial cells, while species-specific EpiCultTM-B media can be used to differentially assess bi-lineage and unilineage progenitors in the mammary colony-forming cell (Ma-CFC) assay.

Mammosphere and tumorsphere evaluation is becoming a key tool for the study of mammary cell differentiation and transformation. MammoCultTM supports robust mammosphere formation from primary mammary tissues, as well as tumorsphere culture from multiple breast cancer cell lines.

PRODUCT APPLICATION CATALOG #

EpiCultTM-B (Human)

Colony-forming cell assay for differential assessment of progenitor content

05601

EpiCultTM-B (Mouse) Colony-forming cell assay for assessment of progenitor content

05610

EpiCultTM-C (Human)

Short-term culture of primary human mammary epithelial cells

05630

MammoCultTM

Generation of robust human mammospheres and tumorspheres in optimized and defined culture conditions

05620

Assay and Culture Primary Mammary Precursor Cells

Tumorsphere Culture with MammoCultTM

MammoCultTM can be used to culture tumorspheres from primary breast cancer tissue and a variety of breast cancer cell

lines including MCF7, MCF10A, SKBR3, MDA-MB-231, AU565, SUM149 and BT474. Learn more from our Technical Bulletin

Tumorsphere Culture of Human Breast Cancer Cell Lines (Document #29936) or www.stemcell.com/mammocultrefs.

Mammary epithelial Colony and Sphere Images

Myoepithelial Human Mammary Epithelial Cell Colony

Mixed Human Mammary Epithelial Cell Colony

Luminal Human Mammary Epithelial Cell Colony

Examples of Colonies Derived From Human and Mouse Mammary Epithelial Progenitors

Mouse Mammary Epithelial Cell Colony

Mammospheres from Primary Human Mammary Epithelial Cells

Tumorspheres from MCF7 Human Breast Cancer Cell Line

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Leukemic cells have the capacity for clonogenic growth in vitro.39-40 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 MethoCultTM media, long-term culture in MyeloCultTM media and expansion cultures in StemSpanTM 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 specific inhibitors of the BCR-ABL tyrosine kinase in Chronic Myeloid Leukemia (CML).40

Culture Media Products For Leukemia Research


MethoCultTM for Human Cells

Base media allowing for the addition of desired growth factors

04330042360423004100

Detection of CFU-E, BFU-E, CFU-GM, CFU-GEMM in bone marrow, cord blood, peripheral blood and mobilized peripheral blood

04034/0404404434/0444404435/0444504436/04446

Detection of CFU-GM (including CFU-G and CFU-M) in bone marrow, cord blood, peripheral blood and mobilized peripheral blood

04035/0404504534/0454404535/0454504536/04546

MethoCultTM for Mouse Cells

Detection of BFU-E, CFU-GM and CFU-GEMM in bone marrow, spleen, peripheral blood and fetal liver

03434/03444

Detection of CFU-GM in bone marrow, spleen, peripheral blood and fetal liver

03534

Base media allowing for the addition of desired growth factors

033340323403231

StemSpanTM Serum-Free Expansion Medium

Serum-free medium for culture and expansion of hematopoietic cells

09600/09650

MyeloCultTM Myeloid long-term culture medium for primitive hematopoietic cells

05100/05150 05300/05350

Assay and Culture Hematopoietic Stem and Progenitor Cells

hematopoietic Colony Images

Examples of Colonies Derived From Mouse Hematopoietic Progenitors

Examples of Colonies Derived From Human Hematopoietic Progenitors

Human BFU-E

Human CFU-GM

Human CFU-GM & BFU-E

Human CFU-GEMM

Mouse CFU-M Mouse BFU-E

Wallchart:Mouse Hematopoietic Progenitors Wallchart

www.stemcell.com/MouseHemaWallchart

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Multipotent neural stem-like cells or brain tumor stem cells (BTSCs), have recently been identified and isolated from different grades (low and high) and types of brain cancers, including gliomas, medulloblastomas, astrocytomas, and ependymomas.41 As with neural stem cells, these brain tumor stem cells exhibit self-renewal, high proliferative capacity and multi-lineage differentiation potential in vitro.

BTSCs can either be cultured as free-floating aggregates in the neurosphere culture system, or as an adherent monolayer of cells. In the neurosphere culture method, neural precursor cells proliferate in response to specific mitogens, forming clusters of cells called neurospheres. In contrast, the adherent monolayer culture method uses cultureware coated with an appropriate substrate to enable cell-surface attachment instead of cell-cell aggregation. 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 significant predictor of increased risk of patient death and rapid tumor progression in cultured human glioma samples.42

Use standardized NeuroCultTM media to culture human, mouse and rat neural stem and progenitor cells from normal and tumor CNS tissues. Components for all NeuroCultTM media and supplements are rigorously prescreened to ensure consistently high quality, and optimized protocols for both neurosphere and adherent monolayer culture methods are available.

Assess Self-Renewal and Proliferation Potential


NeuroCultTM NS-A Proliferation Kit (Human)* Human Proliferation

Media

05751

NeuroCultTM-XF Proliferation Medium*

05761

NeuroCultTM Proliferation Kit (Mouse)*

Mouse Proliferation Medium

05702

NeuroCultTM NS-A Proliferation Kit (Rat)*

Rat Proliferation Medium

05771

NeuroCultTM Neural Colony-Forming Cell (NCFC) Assay Kit (Mouse)*

Colony Assay to Quantify Cells with High Proliferative Potential

05740

NeuroCultTM Neural Colony-Forming Cell (NCFC) Assay Kit (Rat)*

05742

*Requires supplementation with rh EGF (Catalog #02633). When culturing cells obtained from adult mouse, rh bFGF (Catalog #02634) and Heparin (Catalog #07980) are also required.


NeuroCultTM NS-A Differentiation Kit (Human)

Human Differentiation Medium

05752

NeuroCultTM Differentiation Kit (Mouse)

Mouse Differentiation Medium

05704

NeuroCultTM NS-A Differentiation Kit (Rat)

Rat Differentiation Medium

05772

Assess Multi-Lineage Differentiation Potential

Culture Media Products For Brain Tumor Research

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NeuroCultTM media and dissociation reagents have been used to:

• Dissociate human glioblastoma and oligodendroglioma samples.43

• Culture human glioblastoma-derived44-46 and oligodendroglioma-derived47 tumorspheres.

• Culture cells obtained from mouse models of medulloblastoma48 and glioma49 as tumorspheres.

• Differentiate brain tumor stem cells into neurons, astrocytes and oligodendrocytes.48-49

• Passage/dissociate tumorspheres.50

A. Mouse neurosphere cultured with the NeuroCultTM 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).

NeuroCultTM Proliferation Kit (Mouse)

Precursor Cell

NeuroCultTM Differentiation Kit (Mouse)

Neurosphere or adherent monolayer cultures

Figure 6. Proliferation and differentiation of mouse neural stem cells using NeuroCultTM.

Neurons Astrocytes and Neurons

Oligodendrocytes GABA-nergic Neurons

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Webinar:Standardized Media & reagents for Neural Stell Cell research

www.stemcell.com/NeuroCultWebinar


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APPLICATION PRODUCT CATALOG # DESCRIPTION RECOMMENDED FOR:

Circulating Epithelial Tumor Cells

RosetteSepTM Human Circulating Epithelial Tumor Cell Enrichment Cocktail

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

15167 (5 x 15127)

StemSepTM Human Epithelial Tumor Cell Enrichment Kit

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

14162 (5 x 14152)

CD45 Depletion

RosetteSepTM Human CD45 Depletion Kit

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 blood15162

(5 x 15122)

EasySepTM Human CD45 Depletion Kit*

18259 (PBMC)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 blood18289 (WB)

Multiple Myeloma (CD138) Cells

RosetteSepTM Multiple Myeloma Cell Enrichment Cocktail

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

15169 (5 x 15129)

EasySepTM 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 recognizing CD138.

The selection of highly purified CD138+ cells from PBMC or whole blood18387 (WB)

B Cells From Chronic Lymphocytic Leukemia (CLL) Samples

EasySepTM 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

STEMCELL Technologies offers a wide range of fast and easy cell separation products for the isolation of cancer cells. Our innovative cell separation platforms (RoboSepTM, RosetteSepTM, EasySepTM, and StemSepTM) provide a simple and effective method of isolating rare cells with high purity and recovery.

Cell Isolation Platforms For Cancer Research

*EasySepTM can be fully automated with RoboSepTM (WWW.READYSEPGO.COM).

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References

Cancer Stem Cells: Background References1. Lapidot T, et al., Nature 367:645, 1994

2. Dick JE., Blood 112:4793, 2008

3. Bonnet D, Dick JE., Nat Med 3:730, 2007

4. Singh SK, et al., Nature 432:396, 2004.

5. Hajj MI, et al., Proc Natl Acad Sci U.S.A. 100:3983, 2003

6. O’Brien CA, et al., Nature 445:7123, 2007

7. Zhang S, et al., Cancer Res 68:11, 2008

8. Li C, et al., Cancer Res 67:3, 2007

9. Maitland NJ, et al., J Clin Oncol 26:17, 2008

ALDEFLUORTM

10. Storms PW, et al., Proc Natl Acad Sci 96:9118, 1999

11. Fallon P, et al., Br J Haematol 122:93, 2003

12. Hess DA et al., Blood 104:1648–1655, 2004

13. Gentry T, et al., Cytotherapy 9:259–274, 2007

14. Charafe-Jauffret E, et al., Cancer Res 69:1302–1313, 2009

15. Ginestier C, et al., Cell Stem Cell 1: 555–567, 2007

16. Graham JD et al., Endocrinology. 150: 3318–3326, 2009

17. Ibarra I, et al., Genes Dev. 21: 3238–3243, 2007

18. Luo M, et al., Caner Res. 69: 466–474, 2009

19. Magnifico A, et al., Clin Can. Res. 15: 2010–2021, 2009

20. Charafe-Jauffret E, et al., Clin Cancer Res 69(4):1302-13, 2009

21. Charafe-Jauffret E, et al., Cell Cycle 8(20): 3297–3302, 2009

22. Pan Q et al., Clin Can Res. 15:7441, 2009

23. Gentry T, et al., Cytotherapy 9: 259–27, 2007

24. Capoccia et al., Blood 113: 5340–5351, 2009

25. Zeng F, et al., Proc Natl Acad Sci USA 103(20):7801–6, 2006

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 [email protected]

Contract Assay Services

26. Cai J, et al., J Neurochem 88: 212-226, 2004

27. Corti S, et al., Stem Cells 24: 975–985, 2006

28. Corti S, et al., Hum Mol Genet 15: 167–187, 2006

29. Pearce DJ, et al., Exp Hematol 35:1437-46, 2007

30. Boonyaratanakornkit JB, et al., J Invest Dermatol 130(12):2799-808, 2010

31. Matsui W, et al., Blood. 103(6):2332–6, 2004

32. Pierce DJ, et al., Stem Cells. 23(6):752–60, 2005

33. Ran D, et al., Exp Hematol 37(12):1423-1434, 2009

34. Bonnet D, Dick JE., Nat Med 3:730, 1997

35. Singh SK, et al., Nature 432: 396, 2004

36. Yao M, et al., Cells Tiss. Org. 191: 203–212, 2010

Tissue-Specific Product References37. Li T, et al., Laboratory Investigation 90, 234–244, 2010

38. Huang EH, et al., Cancer Res. 69(8): 3382–9, 2009

39. Jiang F, et al., Mol Cancer Res. 27(3): 330–8, 2009

40. Carral A, et al., Bone Mar. Tran. 29: 10, 825–832, 2002

41. Jiang X, et al., J Natl Cancer Inst 99(9): 680–93, 2007

42. Vescovi AL, et al., Nat Rev Cancer 6: 425–436, 2006

43. Laks DR, et al., Stem Cells. 27: 980–987, 2009

44. Platet N, et al., Cancer Lett 258: 286–290, 2007

45. Piccirillo SG, et al., Nature 444: 761–765, 2006

46. Gallia GL, et al., Mol Cancer Ther 8: 386–393, 2009

47. Jenkins RB, et al., Cancer Res 66: 9852–9861, 2006

48. McGillicuddy LT, et al., Cancer Cell 16: 44–54, 2009

49. Yang ZJ, et al., Cancer Cell 14: 135–145, 2008

50. Harris MA, et al., Cancer Res 68: 10051–10059, 2008

51. Wakimoto H, et al., Cancer Res 69: 3472–3481, 2009

52. Jiang T, et al., Cancer Res. 69: 845–854, 2009

Copyright © 2012 by STEMCELL Technologies Inc. All rights reserved including graphics and images. STEMCELL Technologies & Design, STEMCELL Shield Design, Scientists Helping Scientists, EpiCult, MammoCult, MethoCult, StemSpan, MyeloCult, Neurocult, RosetteSep, and EasySep are trademarks of STEMCELL Technologies Inc. ALDEFLUOR is a trademark of Aldagen Inc.

FOR RESEARCH USE ONLY. NOT FOR THERAPEUTIC OR DIAGNOSTIC USE. CATALOG #29189 VERSION 1.3.1 JUNE 2012

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