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HomeStem Cell CultureCancer Stem Cells: Targets for Cancer Therapy

Cancer Stem Cells: Targets for Cancer Therapy

The Cancer Stem Cell Hypothesis

Are stem cells involved in cancer? Cancer stem cells (CSC’s) are subpopulations of cancer cells that can self-renew, generate diverse cells in the tumor mass, and sustain tumorigenesis1. Cancer researchers hypothesizes that tumors arises from CSCs that originate as a result of mutational hits on normal stem cells, by the transformation of restricted progenitor cells or even the differentiated cells that acquire self-renewing capacity2. These CSCs drive tumor progression and recurrence after chemotherapy treatments and have recently become targets for cancer stem cell therapies3. Since both normal stem cells and cancer cells possess ability to self-renew, many pathways that are classically associated with cancer are also involved in the regulation of normal stem cell development including Notch, Wnt, Shh and classical pluripotency transcription factor pathways Oct-4, Sox-2 and Nanog4.

An Illustration of the Cancer Stem Cell Hypothesis

An Illustration of the Cancer Stem Cell Hypothesis

3D Spheroid Culture of CSC
s

Cancer stem cells (CSCs) seek out a niche created within the tumor 3D microenvironment5. A miniscule number of cells within an in vitro 3D tumor spheroid or an in vivo tumor exhibit “stem cell-like” characteristics including 1) slowed proliferation rate 2) self-renewal capabilities and 3) undifferentiated phenotype that can undergo multilineage differentiation. CSCs, which can reconstitute tumors following drug treatment, have been described in both 3D spheroid models and in patients undergoing chemotherapy6. Increased expression of “stemness-related genes” was observed when comparing solid tumor cell lines grown as 3D spheroids to monolayers7. While clearly not definitive proof of the presence of CSCs, these types of results illustrate the tendency for the in vitro 3D microenvironment to create a “stem cell-like” population similar to the in vivo population believed to be responsible for tumor drug resistance.

Cancer Stem Cell Media

Traditionally, CSCs have been isolated and grown from cancer cell lines and tumor biopsies in undefined serum containing media using 3D tumorsphere aggregates. However, using undefined culture conditions has led to experimental variability and suboptimal cell growth. 3dGRO™ Spheroid Medium is a new chemically defined and serum-free CSCs media that supports the expansion of CSC’s in 3D tumorsphere cell cultures. The media supports serial passaging of cancer cell lines as tumorspheres leading to an overall enrichment of CSC population overtime.

Features and Benefits

  • Chemically defined, Serum-free formulation.
  • High tumorsphere formation efficiency.
  • Allows extended serial tumorsphere passaging (>10).
  • Proven to enrich CSCs from numerous cancer cell lines.

Cell Lines tested with 3dGRO™ Spheroid Medium

MCF-7, A-431, Panc-1, LNCaP, HT-29, A-549, U-87 MG, HT1080, E006AA

3D Tumorsphere formation of MCF7

Figure 1.3D Tumorsphere formation of MCF7 breast cancer and E006AA prostate cancer cells at Passage 5 cultured in 3dGRO™ Spheroid Medium.

3D Spheroid Formation Plates

Corning® Costar® Ultra-Low attachment multi-well plates are designed to inhibit cell attachment to efficiently form 3D spheroids, including cancer stem cells. The surface is coated with a highly hydrophilic and neutrally charged coating that binds to polystyrene forcing seeded cells into a suspended floating state. The coating is stable, nontoxic, biologically inert and non-degradable.

3D Spheroid Formation Plates

Cancer Stem Cell Markers

Small populations of stem-like cells have been isolated from most leukemias as well as from many solid tumors such as brain glioblastomas, medulloblastomas, breast, cervical, colorectal, gastrointestinal, hepatocellular, lung, pancreatic, prostate, and skin carcinomas8. Cancer stem cells display the same cell surface markers as their normal counterparts, but demonstrate uncontrolled proliferation, perhaps due to a reduced responsiveness to negative growth regulators or to the loss of contact inhibition and gap junction intercellular communication (GJIC).

MarkersNormal Stem CellsCancer Stem Cells
Oct3/4, Sox2,
Nanog
Maintenance of pluripotency and self-renewal of ES and iPS cellsHighly expressed in poorly differentiated tumors of breast cancer, glioblastomas and bladder cancers. Up regulation of transcription factors in stem-like cells
CD133
(Prominin-1)
Marker for hematopoietic stem cells, neural stem cells and somatic stem cells found in many tissuesHigher clongenicity, invasiveness and tumorigenesis in human oral squamous cell carcinomas.
CD44Self-renewal and differentiationUp-regulation in solid tumors and HNSCC cancers
ALDHStem like properties including colony formation, self-renewal and tumorigenesisMarker for normal and malignant mammary stem cells. Prognostic marker for breast cancer and predictor of metastasis.
Side Populations (Hoechst)Enriched in hematopoietic stem cellsIdentified in solid tumors such as hepatocellular carcinoma, glioma, ovarian and breast cancers

Table 1.Cancer Stem Cell Markers
CD133CD44CD24CD29CD90Nestin
SCA-1BMI-1ALDH1OCT3/4
Table 2.Cancer Stem Cell Antibodies

Cancer Stem Cell ALDH Detection

CSCs are unique as they express elevated levels of aldehyde dehydrogenase (ALDH) 9. Historically, the ALDEFLUOR™ assay has been used to measure ALDH levels and has been influential to study rare CSCs populations. The ALDEFLUOR assay emits in the green region of the electromagnetic spectrum (512 nm). Thus, the reagent cannot be simultaneously utilized in cells or mice expressing green-fluorescent proteins emitting in the green fluorescent spectrum. AldeRed is a red-shifted fluorescent substrate for ALDH used for labelling viable ALDH positive cells leaving the green emission channel available for further experimentation10. The AldeRed ALDH Detection Assay addresses current limitations of the ALDEFLUOR assay.

AldeRed Features and Benefits:

  • Red-shifted assay leaves green channel available for further experimentation
  • Live stem cell identification enables flow sorting of rare cell populations
  • Rapid enzymatic assay protocol
AldeRed 588-A Deab Control

Figure 2.AldeRed 588-A is a fluorescent and non-toxic ALDH substrate that diffuses freely into live intact and viable cells, but remains trapped inside the cells once converted by ALDH into the corresponding acid.

Materials
製品番号製品名詳細価格
SCR005Disolución Accutase para separación de célulasA cell detachment solution of proteolytic & collagenolytic enzymes. The reagent is useful for creating single cell suspensions from clumped cell cultures for accurate cell counting, detachment of cells from primary tissue.
MAB5326Anticuerpo anti-nestina, clon 10C2clone 10C2, Chemicon®, from mouse
MAB4401Anti-Oct-4 Antibody, clone 10H11.2clone 10H11.2, Chemicon®, from mouse
MAB4423Anti-SOX-2 Antibody, clone 10H9.1clone 10H9.1, from mouse
MAB4360Anticuerpo anti-TRA-1-60, clon TRA-1-60clone TRA-1-60, Chemicon®, from mouse
SCM004ENStem-A Neural Expansion MediumThe ENStem-A Neural Expansion Medium is a defined serum-free formulation that has been optimized for the culture & expansion of ENStem-A Human Neural Progenitor Cells.
SCC058FibroGRO® Xeno-Free Human Foreskin FibroblastsThe FibroGRO Xeno-Free Human Foreskin Fibroblasts are derived from normal human foreskin & have been isolated & manufactured under xeno-free conditions.
GF446HumanKine® Thermostable bFGF,
Human Recombinant
Engineered bFGF growth factor with enchanced thermostability compared to the wild-type bFGF protein. Allows a less vigorous feeding schedule for human es/ips and neural stem cell culture.
SCR544Human STEMCCA Constitutive Polycistronic (OKSM) Lentivirus Reprogramming KitThe Human STEMCCA Constitutive Polycistronic Lentivirus Kit contains high titer polycistronic lentivirus & Polybrene transfection reagent that have been validated for the generation of human iPS cells from human foreskin fibroblasts.
SCM133Solución PluriSTEM® Dispasa-IIDispase-II has proven to be a rapid and effective, yet gentle, agent for separating many tissues and cells grown in vitro.
SCM130PluriSTEM® Human ES/iPS Cell MediumDefined small molecule based serum-free medium that enables feeder-free culture of human ES/iPS cells and allows for media exchanges every other day without compromising the morphology or long term functionality of human pluripotent stem cells.
SCM132PluriSTEM-XF® Human ES/iPS Cell MediumPluriSTEM-XF Human ES/iPS Medium is a complete xeno-free medium formulation for the feeder-free culture of human ES and iPS cells.
CC130PluriSTEM-XF® Recombinant VitronectinHuman pluripotent stem cell culture.
SCR550Simplicon® RNA Reprogramming Kit (OKSG)
SCR508TAT-recombinasa CreTAT-CRE Recombinase is a recombinant cell-permeant fusion cre-recombinase protein consisting of TAT sequence, a nuclear localization sequence (NLS) and it is known to catalyze the site specific recombination event between two loxP DNA sites.

References

1.
Kreso A, Dick J. 2014. Evolution of the Cancer Stem Cell Model. Cell Stem Cell. 14(3):275-291. https://doi.org/10.1016/j.stem.2014.02.006
2.
Adams P, Jasper H, Rudolph K. 2015. Aging-Induced Stem Cell Mutations as Drivers for Disease and Cancer. Cell Stem Cell. 16(6):601-612. https://doi.org/10.1016/j.stem.2015.05.002
3.
Vinogradov S, Wei X. 2012. Cancer stem cells and drug resistance: the potential of nanomedicine. Nanomedicine. 7(4):597-615. https://doi.org/10.2217/nnm.12.22
4.
Karamboulas C, Ailles L. 2013. Developmental signaling pathways in cancer stem cells of solid tumors. Biochimica et Biophysica Acta (BBA) - General Subjects. 1830(2):2481-2495. https://doi.org/10.1016/j.bbagen.2012.11.008
5.
Plaks V, Kong N, Werb Z. 2015. The Cancer Stem Cell Niche: How Essential Is the Niche in Regulating Stemness of Tumor Cells?. Cell Stem Cell. 16(3):225-238. https://doi.org/10.1016/j.stem.2015.02.015
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Vidal SJ, Rodriguez-Bravo V, Galsky M, Cordon-Cardo C, Domingo-Domenech J. 2014. Targeting cancer stem cells to suppress acquired chemotherapy resistance. Oncogene. 33(36):4451-4463. https://doi.org/10.1038/onc.2013.411
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Wong DJ, Segal E, Chang HY. 2008. Stemness, cancer, and cancer stem cells. Cell Cycle. 7(23):3622-3624. https://doi.org/10.4161/cc.7.23.7104
8.
Nguyen LV, Vanner R, Dirks P, Eaves CJ. 2012. Cancer stem cells: an evolving concept. Nat Rev Cancer. 12(2):133-143. https://doi.org/10.1038/nrc3184
9.
Marcato P, Dean CA, Giacomantonio CA, Lee PW. 2011. Aldehyde dehydrogenase: Its role as a cancer stem cell marker comes down to the specific isoform. Cell Cycle. 10(9):1378-1384. https://doi.org/10.4161/cc.10.9.15486
10.
Minn I, Wang H, Mease RC, Byun Y, Yang X, Wang J, Leach SD, Pomper MG. 2014. A red-shifted fluorescent substrate for aldehyde dehydrogenase. Nat Commun. 5(1): https://doi.org/10.1038/ncomms4662
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