[methods in molecular biology] basic cell culture protocols volume 946 || isolation and...

181

Chapter 12

Isolation and Characterization of Cancer Stem Cells In Vitro

Craig Gedye and Laurie Ailles

Abstract

The cancer stem cell hypothesis is an appealing concept to account for intratumoral heterogeneity and the observation that systemic metastasis and treatment failure are often associated with the survival of a small number of cancer cells. Whilst in vivo evidence forms the foundation of this concept, in vitro methods and reagents are attractive as they offer opportunities to perform experiments that are not possible in an animal model. While there is abundant evidence that existing cancer cell lines are not reliable models of tumor heterogeneity, recent advances based on well validated novel cancer cell lines established de novo in de fi ned serum-free media are encouraging, particularly in the study of glioblastoma multiforme. In this chapter we wish to broadly outline the process of establishing, characterizing, and managing novel cancer cell lines in de fi ned serum-free media, and discuss the limitations and potential opportunities that may arise from these model systems.

Key words: Cancer stem cell , De fi ned serum-free media , Tumor-initiating cell , Model fi delity

Manfred Eigen is quoted as saying, “A theory has only the alterna-tive of being right or wrong. A model has a third possibility: it may be right, but irrelevant” ( 1 ) . The cancer stem cell (CSC) hypoth-esis is a contentious yet intriguing theory proposed to account for the intratumoral heterogeneity seen in many cancers ( 2 ) . While it is clear that the acquisition of driver genetic mutations propels the development of most clinically detected patient tumors ( 3 ) , these are in many cases genetically monoclonal ( 4 ) , and thus, clonal evo-lution alone cannot account for the functional, morphological and antigenic heterogeneity observed within the malignant cell com-partment at the time of surgical excision. The CSC hypothesis competes with other epigenetic theories (e.g., epithelial–mesen-chymal transition ( 5 ) ) to account for this heterogeneity. CSC are

1. Introduction

Cheryl D. Helgason and Cindy L. Miller (eds.), Basic Cell Culture Protocols, Methods in Molecular Biology, vol. 946,DOI 10.1007/978-1-62703-128-8_12, © Springer Science+Business Media, LLC 2013

182 C. Gedye and L. Ailles

functionally de fi ned by a priori identi fi cation as a subpopulation of malignant cells within a tumor that are highly enriched for tumorigenic potential upon xenograft implantation into an immu-nocompromised mouse recipient; they are thus better de fi ned as tumor-initiating cells (TIC). There is growing experimental support for the CSC hypothesis but the ultimate relevance of the theory will be determined if these insights are effectively translated into improvements in cancer patient outcomes. The CSC hypothesis does not imply that “only stem cells are the cell-of-origin for can-cer” and there is no evidence to suggest that clonal evolution and epigenetic heterogeneity are mutually exclusive processes.

As TIC are functionally de fi ned by their in vivo behavior, from one perspective it is not possible to culture “true” CSC or TIC in vitro. Indeed there is substantial evidence that many traditional cancer cell lines are poor models for human cancer, let alone for CSC ( 6 ) . Traditional cancer cell lines, commonly grown in media supple-mented with animal serum, undergo extensive genetic change such that they develop genotypes and phenotypes that are distinct from the human tumors from which they were derived ( 7 ) . Moreover, we commonly subject these lines to cross-contamination ( 8 ) and infec-tion with Mycoplasma ( 9 ) , further diluting their utility. For example the NCI60 panel of human cancer cell lines contains three lines that are contaminated replicates of other NCI60 lines ( 10 ) and nine lines are phenotypically unlikely to be from the presumed tissue of origin ( 11 ) ; for example neither of the “prostate” cancer cell lines PC3 and DU145 appear to have arisen from prostate cancer.

Despite these limitations cancer cell lines remain appealing tools for research as they allow experiments that are impractical or not possible in vivo to be performed on large numbers of cells which can be more easily manipulated and assayed. Fortunately there is increasing evidence across many tumor types that relevant cell line models of human cancer can be generated and propagated in vitro. Perhaps the most important point we wish to make in this chapter is that it is critical to establish the relevance and fi delity of any cancer cell line model by validating in vitro fi ndings in primary xenograft and patient samples. This has been best exempli fi ed by several publications within the glioblastoma multiforme (GBM) research fi eld.

Work from the Fine lab demonstrated that establishment of GBM cell lines in bovine serum rapidly generates cells with a homogeneous, differentiated phenotype that form non-physiolog-ical tumors, and have gene expression signatures indistinguishable from GBM cell lines that have been in culture for decades ( 12 ) . In contrast matched patient samples cultured in a de fi ned serum-free media formulation originally employed to grow neural stem cells ( 13 ) generated cell lines with much richer morphological heterogeneity, which expressed markers of a more primitive stem-like phenotype, and formed tumors that were diffusely invasive as is typically seen

18312 Cancer Stem Cells Isolation and Characterization

in GBM. Most importantly these cell lines in serum-free de fi ned media closely preserved the phenotype and genotype of the original patient’s cancer. This landmark paper was associated with a practice change within the GBM research fi eld; for example these de fi ned cell lines have been employed to demonstrate the ef fi cacy of targeting Notch ( 14 ) , Shh ( 15 ) , and TGF- β ( 16, 17 ) signalling pathways, and the TIC niche ( 18 ) . These models have been recently extended by work that demonstrates that cells grown in similar media but with the support of a laminin substrate to encourage adherent growth further improves the viability and phenotype of these novel GBM cell lines ( 19 ) .

These culture conditions have been applied in many tumor types and detailed instructions for establishing novel cell lines from a variety of tumors such as glioblastoma multiforme ( 20 ) , prostate ( 21, 22 ) , and colon ( 23, 24 ) cancers have been published else-where. In this chapter we wish to focus on optimizing this process more generally while discussing some of the challenges we continue to face in attempting to translate the stringent validation that has been demonstrated in GBM into other cancer models. We also highlight opportunities that are available in the isolation and char-acterisation of “cancer stem cells” in vitro.

1. Instruments: number 25 (straight-edge, 45° angle) sterile scalpel blades (Swann-Morton, Shef fi eld, UK), No. 4 scalpel blade handles, sterile forceps.

2. Disposable labware: 100 mm diameter × 20 mm deep plastic dishes, 50 mL plastic tubes, 70 μ m cell strainer, 5 mL syringe.

3. Frozen primary specimen: Cryomolds (TissueTek II, Sakura Finetek), O.C.T. Compound (Sakura Finetek), 2-methyl-butane, dry ice, insulated bath.

4. Digestion enzymes: collagenase/hyaluronidase 10× (Stem Cell Technologies), DNase I (Invitrogen), ammonium chloride red blood cell lysis buffer (Gibco Invitrogen) ( see Note 1 ).

5. Typical cell culture laboratory equipment, e.g., laminar fl ow biosafety cabinet (BSC), pipet-aid, micropipettes (10 μ L, 100 μ L, and 1,000 μ L), fi ltered serological pipettes and fi ltered micropipette tips, centrifuge, hemocytometer, inverted micro-scope, 37°C humidi fi ed cell culture air/CO 2 incubator, pref-erably nitrogen fed hypoxic incubator ( see Note 2 ), various sized tissue culture fl asks ( see Note 3 ).

6. De fi ned serum-free culture medium (D-SFCM): DMEM/F12 1:1 media, B27 serum-free supplement (50×), penicillin

2. Materials

2.1. Sample Dissociation

2.2. Cell Culture


(10,000 IU/mL), and streptomycin (10,000 μ g/mL) 100× stock, recombinant human fi broblast growth factor, recombi-nant human epidermal growth factor (all Invitrogen), HEPES 1 M solution, cell culture tested heparin (both Sigma-Aldrich), 500 mL 22 μ m sterile fi ltration systems (Stericup, Millipore) ( see Note 4 ).

7. Cell culture fl ask surface coatings: laminin (L2020), collagen type I (C3867; both Sigma-Aldrich).

8. Cell line passaging: trypsin 0.25% solution (Invitrogen), 2 mM EDTA in PBS pH 7.4, dimethyl sulfoxide (sterile fi ltered, fro-zen in 1 mL aliquots), cryopreservation vials, 1°C/min freez-ing container, isopropanol.

9. Mycoplasma testing: MycoAlert Mycoplasma detection kit (Lonza), luminescence plate reader (e.g., SpectraMax microplate reader, Molecular Devices) ( see Note 5 ).

10. Cell line identity by short-tandem repeat (STR) pro fi ling: Kit for DNA extraction and puri fi cation (e.g., DNeasy Mini Kit, QIAgen).

11. Flow cytometry: Hanks’ buffered saline solution, heat inactivated fetal bovine serum (many suppliers), BD CompBeads (Becton Dickinson), puri fi ed mouse, rat, goat, etc. IgG from pooled normal serum (Cedarlane or Sigma-Aldrich), DAPI (4 ¢ ,6-diami-dino-2-phenylindole dihydrochloride; Sigma-Aldrich).

12. Limiting dilution analysis: D-SFCM, 96-well and 6-well fl at bottom tissue culture treated plates.

13. Tumorigenicity: NOD/SCID or NOD/SCID/IL2 γ R −/− (NSG) immunocompromised mice, basement membrane matrix solution (Matrigel, standard growth factor ( see Note 6 ), BD Biosciences or BME Cultrex, Trevigen), 96-well round bottom non-treated microplates, 29G 300 μ L insulin syringes (Becton Dickinson).

Studying intratumoral heterogeneity requires reliable access to freshly excised human cancer specimens. As such the success of the project relies on generous patient donation of tissue in excess of that required for pathological diagnosis, and close collaboration with the appropriate surgical, oncology, and pathology team mem-bers. Our experience has been that all parties are enthusiastic, and that with appropriate consultation and institutional human ethics review board approval we can routinely collect adequate tissue. At

2.3. Cell Line Model alidation

2.4. Identi fi cation of Clonogenic and Tumor-Initiating Cells

3. Methods

3.1. Patient Specimen Acquisition


the time of asking consent for excess cancer tissues, we would nor-mally request a blood sample from the patient to collect peripheral blood mononuclear cells (which can be stored directly or used to generate a lymphoblastoid B-cell line ( 25 ) ). This provides a source of normal genomic DNA from the patient, providing a normal control for genomic studies, and a gold-standard for identi fi cation of derived cell lines by short-tandem repeat (STR) pro fi ling. Collection of the cancer sample should occur as soon as possible after removal from the operating room, and in direct consultation with the responsible pathologist. Saving directly adjacent samples for immediate RNA/DNA extraction and immunohistochemistry for later comparison is crucial, as many cancers can have variable histology in different parts of the same tumor. The sample is trans-ported in an aliquot of de fi ned serum-free culture media on ice. For some cancers we have found that samples are stable at room temperature, or can be left for processing overnight if refrigerated. This may allow for collection of samples from consenting patients at geographically distant sites.

1. All procedures should take place within an appropriate BSC. Treat all specimens as potentially infected carriers of blood-borne pathogens and use Universal Precautions. Save a frag-ment of the donated tumor for later histological characterisation by freezing in optimal cutting temperature (O.C.T.) solution. Place a thin layer of O.C.T. into a pre-labelled cryomold, place the piece of tissue into the cryomold, and cover completely with more O.C.T. In a fume hood, place the cryomold into a bath of 2-methyl-butane cooled by dry ice, being careful not to allow the liquid to come over the top of the cryomold. Once the O.C.T. is solid white, store the cryomold in a −80°C freezer.

2. Place remaining tissue into a 100 mm × 20 mm deep plastic dish with sterile forceps, and using the No. 25 scalpel blades cut the tissue into small pieces, in a “crossed-blades,” shearing fashion ( see Note 7 ).

3. Continue to gently mince tumor into a slurry until fragments are small enough to pass through the tip of a 5 mL pipette.

4. Add the D-SFCM used to transport the sample (9 mL; which may contain cells in suspension), collagenase/hyaluronidase (1 mL of 10×, fi nal concentration 1×) and DNase (100 μ L, fi nal concentration 125 U/mL) and incubate at 37°C in a 5% CO 2 incubator.

5. Every 10–15 min, return digesting tumor fragments to the BSC and pipette up and down with a 5 mL, then a 1,000 μ L pipette until the tumor is well dissociated (determined by ease of pipetting, and microscopic evaluation of presence of single cells; see Note 8 ). The specimen should not be left in the

3.2. Sample Dissociation


solution for longer than necessary to achieve cellular dissociation. Depending on the tumor, this should take anywhere from 30 min to 2 h.

6. Pass the suspension through a sterile 70 μ m fi lter into a 50 mL tube, and gently break up any remaining fragments by squeez-ing against the top of the fi lter with the rubber end of a sterile 5 mL syringe plunger; rinse fi lter thoroughly with PBS.

7. Centrifuge and resuspend the pellet in a small volume of cold ammonium chloride red blood cell lysis buffer. Incubate on ice for 5 min, then wash with a 10× volume of PBS; centrifuge and wash again in PBS.

8. Resuspend cells in D-SFCM and perform a cell count with a hemocytometer, then use cells for culture, cryopreservation, clonogenic or xenotransplantation assays as appropriate. For immediate ex vivo clonogenicity and tumorigenicity assays ensure a single cell suspension by passing through a 40 μ m cell fi lter before preparation of dilutions.

Preparation of de fi ned serum-free culture medium (D-SFCM)

1. To a 500 mL bottle of DMEM/F12, add antibiotics, HEPES, heparin, and the B27 supplement. Thaw and add FGF and EGF aliquots ( see Table 1 ).

Adjust pH to 7.4 with 1 M NaOH, then sterile fi lter and refrigerate.

2. Aliquots (40 mL in a 50 mL tube) may be frozen and thawed once for later use. Stock solutions can be prepared and frozen for later use. EGF, FGF: Reconstitute 50 μ g in 500 μ L PBS, sterile fi lter and freeze as 5 μ g/50 μ L aliquots. Heparin: prepare a 50 mg/mL stock solution in PBS and sterile fi lter. Store at 4°C for up to 2 years.

1. Plate the primary cell suspension at a density of at least 10,000 viable cells per cm 2 in standard tissue culture fl asks some of which may be coated with various substrates ( see Note 9 ). We typically use smaller T25 fl asks or multiwell plates depending on how many cells are available. Keep a stock of refrigerated pre-coated fl asks or plates on hand.

2. Culture cells in a 37°C humidi fi ed incubator with 5% CO 2 , and if available, in a hypoxic incubator (O 2 tension of 2–5%) ( see Note 10 ).

3. Inspect daily by inverted microscope to monitor growth and con fl uency. Cells may require feeding with a half volume media change at intervals (e.g., weekly) if slow-growing.

4. Passage fl asks when cells are 70–90% con fl uent. Collect culture supernatant and centrifuge at 450 × g for 5 min to collect

3.3. Cell Culture Medium

3.4. Cell Culture Work fl ow


non-adherent viable cells ( see Note 11 ). Aspirate and save some conditioned media at this point.

5. Wash any non-adherent cell pellet with PBS, centrifuge again at 450 × g for 5 min, aspirate and discard the supernatant, and resuspend in an appropriate volume of 0.05% trypsin in 2 mM EDTA (0.25% trypsin (Gibco), diluted 1:5 with 2 mM EDTA in PBS) to dissociate spheres or aggregates and incubate at 37°C for 5–10 min.

6. In parallel, wash fl ask with PBS, then add an appropriate volume of 0.05% trypsin in 2 mM EDTA and incubate at 37°C for 5–10 min until all adherent cells detach.

7. When all cells are detached, inactivate the trypsin in both the fl ask and non-adherent cell pellet with an equal volume of the saved conditioned media. Combine and wash the fl ask twice with PBS to collect all detached cells.

8. Centrifuge at 450 × g for 5 min, resuspend in fresh D-SFCM and perform a cell count.

9. Replate cells at a minimum density of 10,000 cells/cm 2 . Choose a fl ask that allows this to be approximately a 1:2–1:4 split.

10. Continue growing in culture for up to three passages, or when at least 10 million cells are available.

11. Cryopreserve 2–5 million cells per mL in 1 mL cryovials using 1°C/min freezing container. Freezing media consists of 45% saved conditioned media, 45% fresh media and 10% DMSO.

12. Ensure that cryopreserved cells can be successfully revived from frozen stocks before identifying a successful cell line establishment. Pellet and freeze a separate aliquot of cells at

Table 1 Basic formulation for de fi ned serum-free medium (D-SFCM)

Reagent Stock concentration Final concentration Dilution Volume/amount

DMEM/F12 – – – 500 mL

B-27 50× 1× 1:50 10 mL

Heparin 50 mg/mL 4 μ g/mL 1:12,500 40 μ L

HEPES 1 M 10 mM 1:100 5 mL

FGF 100 μ g/mL 10 ng/mL 1:10,000 50 μ L

EGF 100 μ g/mL 10 ng/mL 1:10,000 50 μ L

Pen/strep 10,000 U/mL + 10,000 μ g/mL 100 U/mL + 100 μ g/mL 1:100 5 mL

Total 520 mL


the time of initial cryopreservation for RNA and DNA extrac-tion for subsequent cell line veri fi cation and characterization.

Mycoplasma infection in cancer cell lines remains a common prob-lem despite the relative simplicity of its detection and eradication. Spread mostly due to poor laboratory technique, infections gener-ally remain super fi cially occult but have wide-ranging effects on cell biology and behavior. Mycoplasma infection is almost always spread from existing infected cell lines by laboratory workers during cell culturing (double-dipping pipettes, using the same suction pipette twice, generating aerosols in an over- fi lled BSC). Infection from the patient sample or laboratory worker themselves is very rare.

1. Detection of Mycoplasma infection: Many methods are available for effective detection and surveillance of infection ( 26 ) , but we recommend the Lonza MycoAlert luminescence kit, with a complementary PCR assay to con fi rm positive samples ( 27 ) . Though perhaps more expensive than PCR detection, the MycoAlert test is rapid, sensitive and speci fi c in our hands. False positive results may occur if absolute luminescence readings are low; use well conditioned media, centrifuge samples to remove debris and ensure that the luminometer settings (e.g., sensitivity, number and duration of reads) are optimized to minimize this possibility. 1 mL samples of centrifuged conditioned media may be stored at 4°C for up to 1 month, thus facilitating routine surveillance; we have noted samples that consistently return positive readings after over 18 months at 4°C.

2. Eradication of Mycoplasma infection: With good laboratory practice infection of novel cell lines ought not to occur, but if needed Mycoplasma can be simply and reliably eradicated. Many methods have been described by leaders in the fi eld ( 9 ) , but we favor BM Cyclin (Roche) ( 28 ) . This regimen is more time-con-suming but we have encountered quinolone-resistant Mycoplasma species where cipro fl oxacin, enro fl oxacin and Plasmocin all failed to eradicate the infection. We hypothesize that this strain had become resistant after a past ineffective treat-ment with Mycoplasma Removal Agent ( 29 ) . Use of antibiotics for “maintenance or prophylaxis” is unnecessary and indeed harmful; rather one should focus on inculcating good labora-tory technique and effective surveillance to prevent infection.

Cell line cross-contamination remains as much of a problem as Mycoplasma infection, whether cells are adherent or non-adherent. Though commonly and frequently described this problem has received increasing attention as major scienti fi c journals seek to hold researchers more accountable ( 8 ) . Best practice for cell line management is the use of a cell bank (such as the Johns Hopkins CellCenter http://cellcenter.grcf.jhmi.edu/ ) but in the absence

3.5. Cell Line Model Validation

3.5.1. Mycoplasma Detection and Eradication

3.5.2. Cell Line Identi fi cation

http://cellcenter.grcf.jhmi.edu/


of a centralized service one can still maintain identity validation of one’s cell lines by serial short-tandem repeat (STR) pro fi ling ( 30 ) on a routine basis. This testing requires a small amount of DNA from the original patient (either their tumor or peripheral blood mononuclear cells) to act as a reference sample. Testing can be requested at many academic (e.g., Johns Hopkins, SickKids Toronto) and commercial vendors (e.g., ATCC, Cell Bank Australia, DSMZ). Cross-contamination with commercial cell lines can also be disproven by routine comparison against the Online STR Analysis database available at http://www.dsmz.de/human_and_animal_cell_lines/main.php?contentleft_id=101 .

A critical initial criterion of isolating and characterizing “cancer stem cells” in vitro is to validate that the novel cell line is actually a reasonable model of the patient cancer from which it was derived. A number of modalities are appropriate including assays at the pro-tein, RNA and DNA levels.

1. Flow Cytometry: Flow cytometry represents a powerful tech-nique to rapidly interrogate the phenotype of single cells in suspension (whether ex vivo, ex xenograft or from an estab-lished cell line). For example fl ow cytometry can be used to establish the phenotype and cellular identity of a cell popula-tion (e.g., EpCAM (CD326) or MUC1 (CD227) positive epi-thelial cells) or to investigate if subpopulations of cells (e.g., CD44+ cells in HNSCC) exist within the novel cancer cell line ( 31 ) . This information can then be applied prospectively to de novo cell lines and ex vivo patient samples, e.g., for the identi fi cation of lineage markers that allow discrimination of tumor versus stroma, or for interrogating putative TIC sub-populations ( see below).

(a) Staining cells for fl ow cytometry analysis: Prepare cells as a single cell suspension from patient tumor, xenograft, or cell line. Centrifuge and resuspend cells in FACS buffer (Hanks’ balanced salt solution (HBSS) with 2% heat-inactivated fetal bovine serum) at 10 5 –10 6 cells per 100 μ L. To further block nonspeci fi c binding of antibodies, and depending on the spe-cies in which your antibodies of interest are generated, add mouse, rat, goat etc. IgG at a fi nal concentration of 20 μ g/mL and incubate on ice for 5 min. Do not wash. Ensure that ade-quate control samples are set aside ( see Note 12 ). To 100 μ L aliquots of blocked cells add 100 μ L of buffer with a 2× con-centration of desired antibodies (can be prepared while incu-bating cells with blocking IgG) to give a fi nal volume of 200 μ L with 1× antibody concentrations. The optimal antibody con-centration should be determined empirically by performing titrations in preliminary experiments ( see Note 13 ) ( 32 ) . Incubate on ice for 15 min. Wash with 10× volume of FACS

3.5.3. Validation of DSFM Cell Line Versus Primary Patient Tumor

http://www.dsmz.de/human_and_animal_cell_lines/main.php?contentleft_id=101

http://www.dsmz.de/human_and_animal_cell_lines/main.php?contentleft_id=101


buffer, centrifuge, resuspend in 100 μ L FACS buffer contain-ing any secondary reagents (e.g., fl uorophore-labelled second-ary antibodies or streptavidin) at the appropriate concentration. Wash again and resuspend in FACS buffer with DAPI ( fi nal concentration 0.1 μ g/mL), propidium iodide or 7-aminoac-tinomycin-D to identify nonviable cells. We favor DAPI due to lower spectral overlap with other fl uorophores in common practice; however, this requires access to a fl ow cytometer with the appropriate laser (Violet or UV). Record fl uorescence data on a suitable fl ow cytometer, and analyze data using FlowJo software or Cytobank ( https://www.cytobank.org/cytobank/experiments ) according to published guidelines ( 32 ) .

(b) Immunohistochemistry and immuno fl uorescence: While fl ow cytometry provides a great deal of phenotypic information, immunohistochemistry (IHC) and immuno fl uorescence (IF) provides valuable additional information, such as the intracel-lular location of markers of interest, the location of marker positive cells within established patient tumors or xenografts (e.g., CD44+ cells adjacent to tumor and xenograft fi broblast stromal cells ( 33 ) ) or the co-expression of functional markers (e.g., CD44+/BMI1+ in HNSCC CSCs). Cultured monolay-ers on suitable matrices (chamber slides), cell pellets embedded in thrombin/plasma clots as cell blocks ( 34 ) , cell line derived xenografts and non-adherent clusters or spheroids ( 35 ) can all be examined by IHC and IF and compared to tissue sections cut from the piece of primary patient tissue that was saved in O.C.T. ( see above). IF on paraf fi n-embedded samples is also now more easily accomplished ( 36 ) and samples pre-pared in this way may better preserve cellular morphology and antigen location.

(c) Transcriptome, methylome and epigenome analysis: Global transcriptome analysis by cDNA microarray is now widely available, and represents a convenient and cost-effective method for rapidly phenotyping bulk or separated cell popula-tions. While comparison of TIC marker-positive and marker-negative subpopulations is an obvious application, we would suggest fi rst validating the phenotype of novel serum-free cancer cell line versus the patient’s tumor. Again, many studies have demonstrated that traditional serum cell lines poorly replicate the transcriptomic phenotype of the tumors of origin ( 6, 37 ) . Various platforms are available, and we have found the Illumina BeadChip technology gives reliable results at a reasonable cost. A criticism of studies investigating the cancer stem cell hypoth-esis has been a lack of detail on the mechanisms controlling the postulated irreversible epigenetic hierarchy. Some recent stud-ies are beginning to address epigenetic regulation in cancer stem cell biology ( 38 ) , and we would suggest that routine

https://www.cytobank.org/cytobank/experiments

https://www.cytobank.org/cytobank/experiments


interrogation of epigenetic mechanisms such as methylation, microRNA expression, and acetylation within de novo serum-free cancer cell lines as compared to the original patient’s tumor would add to a robust understanding of the validity and limita-tions of this cancer model. This is particularly important as there is evolving evidence that the cell culture process itself causes signi fi cant changes in methylation ( 39 ) , in particular in developmentally regulated genes ( 40 ) .

(d) Genotype analysis: Cancer cells in culture on average appear to carry a more extensive complement of mutations than the tumors from which they are derived ( 41– 44 ) . There is increas-ing evidence that the acquisition of these mutations may be a function of the culture process itself ( 7 ) , while there is compel-ling evidence in GBM that this evolution away from the geno-type of the original patient’s tumor can be prevented for some time by the use of DSFM culture conditions. To examine the genotypic fi delity in your novel cell cultures, collect genomic DNA from a patient blood sample (requested at the time of surgery), from an adjacent fragment of the original tumor spec-imen, from cells cultured for multiple passages in vitro and xenografts established from these cell lines. A variety of whole genome methods are available (array CGH, SNP arrays or sequencing) but given the quantity and quality of genomic DNA that can be expected to be collected from patient samples, we favor SNP arrays such as the Affymetrix ® Genome-Wide Human SNP Array 6.0. This platform gives a balance of density versus cost and with suf fi cient resolution for genotypic validity.

Clonogenic frequency may be a useful surrogate for tumorigenic frequency, but while useful for rapid screening of subpopulations, any marker or relevant pathway de fi ned by in vitro techniques must be promptly investigated in vivo. Clonogenic frequency can be estimated in adherent or non-adherent conditions, using limiting dilution analysis.

1. Limiting dilution assay: To assess clonogenic frequency by lim-iting dilution, multiple replicates in a 96-well format is a con-venient and reproducible technique. With a fi nal volume of 200 μ L per well, and using 16 wells per concentration, a pre-plating volume of 4 mL gives 20 replicates and enough for losses. This also gives six cell concentrations per plate, allowing a useful range of dilutions. Pre-plating dilutions can be per-formed in 6-well plates; add media fi rst, then add cells, and mix gently. A manual eight-channel multi-pipette (with only four tips attached) can then be used four times to aliquot 4 × 200 μ L of cells into 4 × 4 = 16 wells of a 96-well fl at bottom plate. Thus six doses can be tested per plate. We suggest “range- fi nding” of cell concentrations initially (e.g., 10× dilutions ranging from

3.5.4. Clonogenicity


1 to 10,000 cells per well) and once an approximate range has been identi fi ed, more closely spaced (e.g., if 10× dilu-tions indicated a clonogenic frequency of approximately 1 in 1,000, then perform 2× dilutions ranging from 200 to 6,400 cells per well). To assess clonogenicity under non-adher-ent conditions, polyHEMA-coated ( 45 ) or commercially avail-able ultra-low attachment plates can be used. If adherent colony formation is of interest, tissue culture treated plates, or plates coated with laminin, collagen or other substrates can be used. After an appropriate interval in culture (2–4 weeks), count wells that contain colonies and plot log 10 (percentage of wells without colonies) versus number of input cells/well. Limiting dilution theory states that the mean number of stem/progenitor cells per well can be calculated from the formula: u = −ln F 0 , where u represents the mean number of clonogenic cells per well and F 0 is the fraction of negative wells ( 46 ) . On the line of best fi t by linear regression analysis, the value at which this line intercepts 37% estimates the minimum clono-genic frequency ( 47– 49 ) . Analysis can also be performed using L-Calc Software (StemCell Technologies Inc.), or online using the extreme limiting dilution analysis (ELDA ( 50 ) ) website; http://bioinf.wehi.edu.au/software/elda/index.html .

A de fi ning feature of “cancer stem cells” is their ability to form tumors as xenografts in immunocompromised mice. This property must therefore be rigorously tested to prove that the chosen cul-ture conditions maintain TIC in vitro.

1. Absolute Tumorigenicity: Initial experiments should be per-formed at high doses (e.g., 10 6 ) to establish if the cultured cell lines are in fact tumorigenic. Resulting xenograft tumors should then be compared histologically to the original patient tumor ( see above). Passaged, washed and 40 μ m fi ltered single cell suspensions should be counted and resuspended in the desired volume of PBS (ranging from 10 to 50 μ L); the vol-ume will depend upon the site of injection, with sites such as the renal capsule or the brain requiring small volumes, while sites such as subcutaneous or mammary fat pad are able to accommodate higher, more manageable volumes. Allowing excess cells for losses, mix equivalent volumes of cells and Matrigel in a round-bottomed 96 well plate that is resting on ice. Aspirate 20–100 μ L per injection into cooled insulin syringes for injection and keep fi lled syringes on ice until injec-tion into mice. The appropriate strain of mouse (e.g., NOD/SCID, NSG, or Rag2 γ DKO) should be selected and xenograft techniques should be optimized carefully, as should the most relevant injection site. For many tumor types an orthotopic site is obvious (e.g., GBM into brain, hepatocellular carcinoma

3.5.5. Tumorigenicity

http://bioinf.wehi.edu.au/software/elda/index.html


into liver), but the best determinant of the appropriate niche for xenotransplantation is reproducibility of patient tumor phenotype. In some cases additional manipulations may be necessary, such as preconditioning the mice or implanting estrogen pellets subcutaneously ( 51 ) . Once xenografts are established, at a minimum, it should be veri fi ed that they his-tologically resemble the primary patient sample by hematoxy-lin and eosin staining, ideally with the help of a quali fi ed pathologist who is expert in the particular type of cancer being studied. If other markers are routinely in clinical use (e.g., ER and PR staining in breast cancer), then similar markers should be investigated on the xenograft.

2. TIC frequency by limiting dilution analysis: The frequency of TIC in the newly established cell line should be determined by limiting dilution analysis (LDA) in immunocompromised mice. Cells should be serially diluted in cold PBS and mixed 1:1 with Matrigel as above. Initial experiments should be based on a broad range of cell doses (e.g., 10 2 –10 6 ) to fi nd the most appropriate range of tumorigenicity as this is known to vary from tumor to tumor and from cell line to cell line in the same histological subtype ( 33 ) . A minimum of fi ve mice should be injected with each cell dose. Once an approximate median tumorigenic frequency is established then tighter dilutions can be used to de fi ne the minimum tumorigenic dose. A compari-son of in vivo TIC frequency and clonogenicity can then be performed to determine whether clonogenicity correlates with tumorigenicity.

3. Does clonogenicity re fl ect tumorigenicity? Wells from LDA experiments in vitro that were plated at the minimum dose and contain a single colony should be further assessed for their “stem cell” characteristics. First, they should be replated to determine whether secondary colonies can be formed, demon-strating self-renewal; and second, they should be expanded and injected into mice to demonstrate that a single colony-forming cell can ultimately generate a tumor in vivo. Again, histological veri fi cation, in consultation with a pathologist, should be done by comparing the xenograft tumor histology to that of the pri-mary patient sample.

1. Selection of candidate TIC markers: Once your novel de fi ned serum-free cell line is validated as a useful model of the patient’s cancer in vitro, it can then be investigated for the presence of an intercellular heterogeneity and hierarchy. Candidate TIC marker selection is informed by a number of criteria. For example, pub-lished CSC markers can be informative ( 51 ) , as can markers that have functional signi fi cance ( 52 ) or prognostic relevance ( 53 ) within the tumor type, or markers that are expressed in

3.5.6. Identi fi cation of TIC in De fi ned Serum-Free Cancer Cell Lines


presumptive CSC niches within tumors ( 33 ) . In the fi rst instance novel cell lines should be pro fi led by IHC and/or fl ow cytometry to validate that the marker is present, and that it is also expressed on a subset of primary patient cells before embarking upon sorting experiments. Cell surface markers are often employed, and some functional markers (e.g., ALDEFLUOR ® ) are showing promising results in some tumor systems ( 53, 54 ) . Our experience and literature ( 55, 56 ) reports of the “side-population” method suggests that results obtained with this modality must be rigorously validated, i.e., just because a cell population contains a side population does not mean that these are “cancer stem cells.” Detailed methods for fl ow cytom-etry analysis have been discussed above and elsewhere ( 32 ) .

2. TIC assays of puri fi ed cell subsets: Once candidate TIC mark-ers have been selected, their functional signi fi cance can be assayed by purifying subsets from the novel cell line. For cell surface markers, stain samples as described above for fl ow cytometry. Cell sorting of positive and negative populations can be done on any available fl uorescence activated cell sorting machine at your institute, such as a FACSAria (BD), MoFlo (Coulter), or In fl ux (Cytopia). The number of cells recovered from the sort should be veri fi ed using a hemocytometer, and clonogenic or TIC assays set up as described above. Flow cytometry cell sorting is a reliable and reproducible method, but where this is unavailable magnetic bead cell sorting (e.g., MACS from Miltenyi, EasySep or StemSep from STEMCELL Technologies) can generate cell populations with high purity ( 57 ) . Separated cell populations can be assayed initially using in vitro clonogenicity assays, and if a functional difference in colony formation is noted, this will provide useful preliminary evidence for exploring this marker in vivo, fi rstly using the novel cell line itself, and then validating back on fresh ex vivo patient cancer samples. Once a TIC marker has been validated on several novel serum-free cell lines and in several ex vivo patient samples, separated cell populations can be explored by bioinformatic ( 58, 59 ) and functional assays to investigate their mechanisms of self-renewal ( 15, 16 ) , differentiation ( 17, 60 ) , microenvironmental niche interaction ( 18, 61 ) , and treatment resistance ( 62, 63 ) .

While the validity of the cancer stem cell hypothesis can only be conclusively determined in a particular tumor type by in vivo stud-ies, there are many in vitro studies that are attractive as they may corroborate in vivo data or generate novel mechanistic hypotheses and therapeutic candidates. For example GBM cell lines generated de novo in serum-free media and grown on laminin coated plates have been employed to identify that targeting serotonin signalling may be relevant in this disease ( 19 ) . Novel de fi ned serum-free cancer cell lines can also be applied in genetic screens; for example in a

3.5.7. Data Interpretation


recent publication that identi fi ed TRRAP as a regulator of TIC in GBM ( 64 ) . Finally if a novel serum-free cell line is established as a valid model of a patient’s cancer in vitro, these cultures can be labelled with fl uorescent proteins that allow clonal tracking in vitro and in vivo, which in turn could provide further evidence for ( 65 ) , or against ( 66 ) the cancer stem cell hypothesis. George E.P. Box cautioned us to “remember that all models are wrong; the practical question is how wrong do they have to be to not be useful” ( 67 ) . The key point we wish to emphasize when isolating and character-izing “cancer stem cells” in vitro is to ruthlessly validate the novel cell line and one’s fi ndings against real human cancers, both in vivo and ex vivo. If we can honestly re fl ect on “how wrong” our novel de fi ned serum-free cancer cell lines are as models of cancer, then we can more con fi dently employ them to interrogate tumor het-erogeneity and therapeutic hypotheses, which will more rapidly lead us to more useful outcomes for patients with cancer.

1. The combination of enzymes for tissue dissociation should be optimized on a tissue-speci fi c basis. For example some tumors are so friable (e.g., melanoma) that enzyme digestion is hardly necessary, whereas others are tough and fi brous and require signi fi cant digestion (e.g., head and neck squamous carci-noma). A useful online tissue dissociation guide has been pro-duced by one manufacturer of digestion enzymes ( http://www.worthington-biochem.com/tissuedissociation/default.html ). There will also be variability between different patients’ tumors within the same tumor type, necessitating careful observation of the tumor digestion in every case.

2. Many commercial hypoxic incubators are available, some of which use premixed gas at the appropriate concentrations (e.g., 93%N 2 /5%CO 2 /2%O 2 ). A large airtight silicone-sealed plastic food container modi fi ed with ports to admit premixed gas and incubated in a standard incubator may be a useful alternative to pilot these conditions.

3. There is evolving evidence that laboratory plasticware leaches a combination of soluble contaminants that can have signi fi cant biological effects (summarized by Nature News, 26 April 2010, doi: 10.1038/news.2010.200 ). We are not aware of any speci fi c manufacturer that is free of these problems, but we would greatly appreciate if such materials became available.

4. A variety of media formulations have been applied in different tumor types; a summary of several of these formulations is pre-sented in Table 2 . The evolution of early formulations employed in the neural stem cell fi eld is also presented.

4. Notes

http://www.worthington-biochem.com/tissuedissociation/default.html



http://dx.doi.org/10.1038/news.2010.200


5. If one does not have access to a luminescence plate reader, PCR-based detection methods for Mycoplasma as described by Uphoff and Drexler ( 27 ) (and Chapters 1 and 2 in this edi-tion) are highly sensitive and reliable.

6. We have noted considerable batch-to-batch variability with Matrigel, which has made it challenging to use. When thawed on ice at 4°C overnight, the Matrigel should be liquid and barely viscous. With mixing and warming it will thicken and irreversibly gel rapidly.

7. At this point you may also wish to set aside small (1–3 mm square) fragments of tumor for direct implantation into mice for establishment of primary tumor xenografts.

8. Whilst enzymatically digesting the mechanically dissociated tumor slurry, gentle agitation may be helpful. For example one can use a sterile stir-bar and magnetic stir-plate, a rocking mixer, or a tube rotator that can be used in a 37°C incubator.

9. In our experience many de novo cell lines established in de fi ned serum-free culture media are adherent and grow well in stan-dard tissue culture treated fl asks. Some lines have grown poorly under such conditions, and we are exploring the use of addi-tional substrates that may perhaps offer a better microenviron-mental stimulus ( 19 ) . Collagen and laminin have shown promising early results. These are prepared as solutions and coated onto plates according to the manufacturer’s instruc-tions (Sigma-Aldrich). Fibronectin, Matrigel, poly- l -lysine, or gelatin are other possible substrates to consider.

10. Culture under hypoxic conditions has been very successful in several tumor types in our hands. There is a well established literature on the bene fi ts of culturing stem cell populations under hypoxic conditions ( 88, 89 ) , and we have noted that proliferation is generally equivalent if not faster, and some cell lines that failed to grow when cultured in ambient air have grown robustly and reproducibly at 2% O 2 .

11. A repeated theme in the literature is that placing an immortal-ized cancer cell line into serum-free media and non-adherent conditions leads to formation of spheres so they must be cancer stem cells. “(Insert name of your cancer here)-spheres” are not cancer stem cells. The use of ultra-low attachment plates, reduced serum conditions, or roller bottles can generate these non-adherent clusters in the presence of any culture media including serum ( 90 ) . While there is clearly a difference in gene expression and phenotype upon this microenvironmental alter-ation, this does not mean that these “tumor-spheres” replicate the phenotype or hierarchy that existed in the original patient’s cancer. Indeed at concentrations of cells above 10 cells per μ L clustering is a common occurrence and observed “spheres” are unlikely to have come from a clonal event ( 91 ) .

http://dx.doi.org/10.1007/978-1-62703-128-8_1

http://dx.doi.org/10.1007/978-1-62703-128-8_2


Tabl

e 2

Sele

cted

ser

um-f

ree

med

ia fo

rmul

atio

ns re

port

ed in

the

esta

blis

hmen

t of d

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vo c

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r cel

l lin

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Tum

or

Refe

renc

es

Year

Ba

sal m

edia

Su

pple

men

t EG

F a bF

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Othe

r

Tra

nsiti

onal

car

cino

ma

of

blad

der

Ben

tiveg

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2010

D

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20

–

Ew

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s sa

rcom

a Su

va (

69 )

2009

D

ME

M/

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Kno

ckou

t SR

b 10

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–

Acu

te ly

mph

obla

stic

leuk

emia

N

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( 70 )

20

09

IMD

M

Insu

lin, a

lbum

in,

tran

sfer

rin

– –

Cho

lest

erol

, β -M

E

End

omet

rial

car

cino

ma

Rut

ella

( 71

) 20

09

DM

EM

/F1

2 B

SA, I

TS c

20

10

Prog

este

rone

, put

resc

ine

Pros

tate

car

cino

ma

Guz

mán

-Ram

írez

( 72

) 20

09

CnT

-52

BPE

–

– –

Hea

d an

d ne

ck s

quam

ous

carc

inom

a C

hen

( 73 )

20

09

DM

EM

/F1

2 N

2 10

10

–

Pitu

itary

ade

nom

a X

u ( 7

4 )

2009

D

ME

M/

F12

B27

20

20

–

Bre

ast

carc

inom

a G

rim

shaw

( 75

) 20

08

DM

EM

/F1

2 B

27

20

– In

sulin

, hyd

roco

rtis

one

Pros

tate

car

cino

ma

Van

der

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end

( 76 )

20

08

PrE

GM

–

– –

–

Col

orec

tal

Dyl

la (

77 )

2008

D

ME

M/

F12

B27

, IT

S 20

20

LI

F, h

epar

in, h

ydro

cort

isone

Col

orec

tal

Ric

ci-V

itian

i, T

odar

o,

Cam

mar

eri e

t al

. ( 24

, 78–

81 )

2008

D

ME

M

BSA

, IT

S 20

10

Pr

oges

tero

ne, h

epar

in

Non

-sm

all-

cell

lung

car

cino

ma

Era

mo

( 82 )

20

07

DM

EM

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2 B

SA, I

TS

20

10

Prog

este

rone

, put

resc

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Pros

tate

car

cino

ma

Col

lins

( 83 )

20

05

K-S

FM

BPE

, EG

F –

– L

IF, S

CF,

cho

lera

tox

in

Glio

blas

tom

a m

ultif

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( 58

) 20

07

DM

EM

/F1

2 B

27

20

20

LIF

Glio

blas

tom

a m

ultif

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e Pe

llega

tta

( 84 )

20

06

DM

EM

/F1

2 B

27

20

20

–

Glio

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tom

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ultif

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12 )

2006

N

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basa

l d B

27:N

2 50

50

–

(con

tinue

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Tabl

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(con

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F a bF

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20

03

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20

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ultif

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( 20 )

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DM

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1992

D

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12. Controls for fl ow cytometry should include compensation controls, unstained controls and controls for background stain-ing despite compensation, so as to allow for appropriate gat-ing. While isotype controls can be used, we prefer “ fl uorescence-minus-one” (FMO) controls, where control samples containing antibodies labelled with fl uorophores for all channels bar the channel of interest are included. For exam-ple in an experiment using FITC, PE, APC, and PECy7 labelled antibodies, the FITC FMO control contains PE, APC, PECy7 antibodies, etc. For compensation controls we use BD CompBeads (Becton Dickinson) in the appropriate species, as this spares the use of potentially limited number of cells from patient tissues or xenografts.

13. Titrating antibodies for fl ow cytometry is essential prepara-tion, particularly when attempting to detect small subpop-ulations, which may often be penumbra rather a distinct binary population. A number of laboratories have kindly shared their protocols for this process online including the Altman lab at Emory ( http://www.microbiology.emory.edu/altman/f_protocols/f_ fl owCytometry/p_titering_Abs.htm ), the Herzenberg lab at Stanford ( http://herzenberg.stanford.edu/Protocols/default.htm ), and the University of Chicago Flow Cytometry Core Facility ( http://uc fl ow.blog-spot.com/2009/06/antibody-titrations.html ).

14. Cell culture conditions will need to be optimized for each and every cancer studied. The microenvironment of in vitro con-ditions is obviously markedly different from where the tumor was removed. In some cases feeder cells such as mouse embryonic fi broblasts, cancer associated fi broblasts or endothelial cells may be necessary to adequately establish cell lines in the absence of serum. Alternatively, cell lines may be dif fi cult to establish directly from human specimens but may be more easily generated from passaged xenografts. The same caveats apply however, and the validity of the cell line as a model must be based upon comparison to the original patient’s tumor.

Acknowledgments

This research was supported by the Ontario Institute for Cancer Research and the Ontario Ministry of Health and Long Term Care. The views expressed do not necessarily re fl ect those of the OMOHLTC. C.G. is supported by a Royal Australasian College of Physicians CSL Fellowship and a National Health and Medical Research Council Postdoctoral Training Fellowship. L.E.A. is

http://www.microbiology.emory.edu/altman/f_protocols/f_flowCytometry/p_titering_Abs.htm



http://herzenberg.stanford.edu/Protocols/default.htm

http://herzenberg.stanford.edu/Protocols/default.htm

http://ucflow.blogspot.com/2009/06/antibody-titrations.html

http://ucflow.blogspot.com/2009/06/antibody-titrations.html


supported by a New Investigator Award from the Ontario Institute for Cancer Research.

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[methods in molecular biology] basic cell culture protocols volume 946 || isolation and...

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