The authors have no acknowledgements.

The experimental procedures and ethical treatment of animals was approved by the University of Pittsburgh IACUC (protocol #1104674).
1. Preparation of G6PD Staining Mixture
NOTE: Ensure that the final concentration of each reagents is as follows; 5 mM glucose-6-phosphate (G6P); 2 mM NADP, 5 mM MgCl2, 0.35 mM 1-methoxy-5-methylphenazinium methyl sulfate (MMPMS) and 0.8 mM NBT.1,8,9 For each reagent the initial concentration was derived for a 40 ml final volume. Solutions were freshly prepared and used each day.
<ol>
	<li>Add 2 ml of 200 mM pH 7.4 phosphate buffer (PB) to 35 ml of OCT medium in a 50 ml tube and shake vigorously. Confirm with pH paper that the solution is ~pH 7.4. If not discard the solution.</li>
	<li>Add 61 mg G6P dissolved in 0.94 ml of 200 mM PB, 61 mg NADP dissolved in 0.94 ml of 200 mM PB, and 41 mg MgCl2 dissolved in 0.96 ml of 100 mM PB. Reconfirm that the pH is ~7.4 with pH paper. If not ~7.4, discard the solution.</li>
	<li>Add 5 mg of MMPMS dissolved in 0.25 ml of 200 mM PB (pH 7.4). Vigorously shake the resulting reaction mixture to generate a clear dark red homogenate. If the solution is turbid, the mixture should be discarded. The tube containing the reaction mixture is wrapped in foil to minimize exposure to light. Store the reaction mixture at RT while the final component, NBT, is prepared.</li>
	<li>Prepare separately a 5 mM NBT solution by adding 0.4 ml anhydrous dimethyl formamide (DMF) and 0.4 ml anhydrous ethanol to 164 mg of NBT in a 2 ml tube with an O-ring lid. Close the lid loosely (not tightly sealed) and bring the solution to a vigorous boil in an oil bath until the NBT is in solution.8</li>
	<li>Allow the solubilized NBT to cool to RT and then add all of the solution to the OCT reaction mixture and shake vigorously to obtain a clear solution. Observe the solution color change from initial orange-red to a dark-red or purple color over time. This is normal.</li>
	<li>Place the final G6PD staining mixture in a 37&#160;&#176;C warm room for 1 hr before use.</li>
</ol>
2. Animal Treatment and Tissue Collection
<ol>
	<li>Treat 6-week-old C57Bl/6 male mice with DNA damaging agent, e.g., 10 mg/kg azoxymethane (AOM) in a volume of 200 &#181;l PBS by i.p. injection. Treat control mice with 200 &#181;l of PBS by i.p. injection.</li>
	<li>At 90 days, euthanize mice by CO2 asphyxiation followed by cervical dislocation as approved by IACUC protocol.</li>
	<li>Surgically open the abdominal cavity from just above the anus to the base of the sternum. Move the small intestine out of the body cavity but do not excise it. Excise the lower intestine just below the cecum to the rectum.10</li>
	<li>Carefully slice along one side of the colon using a micro-dissection scissor and remove feces from the opened colon by washing with clean sterile PBS. Swiss roll the excised colon with the luminal side facing outward, and place in a tissue mold.10</li>
	<li>Completely immerse the colon in OCT medium and place mold in a Dewar containing liquid N2. Hold the mold so that the plastic base is just in contact with the liquid N2 until the OCT medium is frozen solid.</li>
	<li>Store the frozen tissue block at -80 &#160;until cutting frozen sections. Do not use any fixative, which would result in the inactivation of the G6PD enzyme.</li>
</ol>
3. Preparation of Frozen Sections
<ol>
	<li>Cut frozen tissue sections at a thickness of 7 &#181;m using a cryostat at -25 &#176;C. Cut 4 consecutive 7 &#181;m sections and place two of them on the same slide (Figure 1). Prepare 2 slides, or four consecutive 7 &#181;m sections to represent one level of the colon (Figure 1).</li>
	<li>Repeat for the next level with a minimum distance of &#62;50 &#181;m from the last section of the previous level. This distance ensured that the crypts assessed for each level did not duplicate one another.</li>
	<li>Frequently wipe the cryostat blade with 100% ethanol to remove the OCT residue that accumulates during cutting. Before sectioning is resumed, the dry blade is wiped with an antistatic dryer sheet to dissipate accumulated static charge. Store slides with the frozen sections at -80 oC.</li>
</ol>
4. Staining of Frozen Section Tissue
<ol>
	<li>Perform the enzyme histochemistry reaction in a 37 &#176;C&#160;warm room with high humidity. 
	NOTE: Attempting the enzymatic reaction in an incubation oven or on a slide warmer led to poor and inconsistent G6PD staining.</li>
	<li>Build wells for tissue staining using two 1.5 cm x 0.15 cm steel washers (interior diameter x height) that are bonded together using silicone grease (Figure 1). Cut the parafilm to fit the base of the well with the center cut out and the well placed over the tissue section on the slide. 
	NOTE: The parafilm on the bottom of the washer creates a seal between the slide and the washer that maintains the viscous G6PD staining mixture on the tissue section. This construct gives a well with a 0.4 - 0.5 ml volume so that each tissue section in the study receives the same volume of the G6PD reaction staining mixture.</li>
	<li>Incubate frozen tissue slides at 37 oC in a warm room for 10 min. At 37 &#176;C, add the G6PD staining reaction mixture to each tissue section well (2 sections per slide) for 45-50 min. Remove slides from warm room, and carefully lift the stainless steel wells off the slides.</li>
	<li>Set slides on their long edge to allow the reaction medium to drain. Place slides in 100 mM PB (pH 7.4) for 30-60 min to remove the G6PD staining reaction mixture from the tissue without disturbing the tissue staining. Submerge slides in distilled water for 5-10 min to remove PB salts</li>
	<li>Seal the tissue by adding fluoro-gel from a dropper onto the tissue and waiting ~ 30 min for it to dry. Avoid bubbles that can confound the identification of mutated crypts. 
	NOTE: If bubbles form, the procedure can be repeated after removal of the fluoro-gel by soaking in distilled water. Do not use cover slips as they tend to trap air bubbles.</li>
</ol>
5. Determination of G6PD Mutant Crypts and Mutation Frequency
<ol>
	<li>Analyze slides for G6PD mutant crypts under a light microscope. 
	NOTE: The criteria used to identify a fully mutant crypt were: (i) the entire crypt was devoid of blue color; (ii) the outer structure of the crypt was intact, (iii) the mutant crypt was observed on adjacent 7 &#181;m sections and, (iv) two observers had to identify the same crypt as a mutant. Mutants observed on adjacent 7 &#181;m sections are counted as one mutant.</li>
	<li>Image each G6PD mutant crypt at 40x magnification and catalogue the images using a 5.0 megapixel camera with imaging software.</li>
	<li>Quantify the total number of crypts examined in a mouse by selecting a representative section for each mouse from the level with the smallest surface area and image it at 10x magnification (Figure 1C). 
	NOTE: A level represents four consecutive 7 &#181;m sections (Figure 1D), which due to their proximity often show mutated crypts on more than one section. The levels are separated by &#62;50 &#181;m to visualize crypts from different areas of the colon.</li>
	<li>Open each image file and visually count the number of crypts on a level by analyzing the files (shown as boxes in Figure 1C). Images files will vary in the number of crypts observed from as low as 20 to &#62;300 crypts in one file.</li>
	<li>Multiply the total number of crypts from the selected level (Figure 1C) for each mouse by the number of levels screened for G6PD mutant crypts for each colon. For example, a total count of 1,250 crypts per selected level multiplied by 8 levels screened for mutant crypts equals a total count of 10,000 crypts. Evaluate a minimum of 10,000 crypts for each colon for statistical analysis.</li>
	<li>Combine the number of mutants observed in all of the animals by the total number of crypts screened to calculate the MF for each treatment. The background stem cell MF level in untreated mice is &#60; 0.1x10-4 (Table 1), which is close to that previously reported in C57Bl/6 mice.11</li>
</ol>

<ol>
	<li>Griffiths, D. F., Davies, S. J., Williams, S., Williams, G. T., Williams, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demonstration+of+somatic+mutation+and+colonic+crypt+clonality+by+X-linked+enzyme+histochemistry.">Demonstration of somatic mutation and colonic crypt clonality by X-linked enzyme histochemistry.</a> Nature. 333 (6172), 461-463 (1988).</li><li>Griffiths, D. F., Sacco, P., Williams, G. T., Williams, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+clonal+origin+of+experimental+large+bowel+tumours.">The clonal origin of experimental large bowel tumours.</a> Br. J. Cancer. 59 (3), 385-387 (1989).</li><li>Kuraguchi, M., Thomas, G. A., Williams, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somatic+mutation+of+the+glucose-6-phosphate+dehydrogenase+(g6pd)+gene+in+colonic+stem+cells+and+crypt+restricted+loss+of+G6PD+activity.">Somatic mutation of the glucose-6-phosphate dehydrogenase (g6pd) gene in colonic stem cells and crypt restricted loss of G6PD activity.</a> Mutat. Res. 379 (1), (1997).</li><li>Van Noorden, C. J., Vogels, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyvinyl+alcohol+and+other+tissue+protectants+in+enzyme+histochemistry:+a+consumer&#8217;s+guide.">Polyvinyl alcohol and other tissue protectants in enzyme histochemistry: a consumer&#8217;s guide.</a> Histochem. J. 21 (7), 373-379 (1989).</li><li>Giardina Rosenberg, D. W., C, T., Tanaka, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+for+the+study+of+colon+carcinogenesis.">Mouse models for the study of colon carcinogenesis.</a> Carcinogenesis. 30 (2), 183-196 (2009).</li><li>Tanaka, T., Kohno, H., Suzuki, R., Yamada, Y., Sugie, S., Mori, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+inflammation-related+mouse+colon+carcinogenesis+model+induced+by+azoxymethane+and+dextran+sodium+sulfate.">A novel inflammation-related mouse colon carcinogenesis model induced by azoxymethane and dextran sodium sulfate.</a> Cancer Sci. 94 (11), 965-973 (2003).</li><li>Suzuki, R., Kohno, H., Sugie, S., Tanaka, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dose-dependent+promoting+effect+of+dextran+sodium+sulfate+on+mouse+colon+carcinogenesis+initiated+with+azoxymethane.">Dose-dependent promoting effect of dextran sodium sulfate on mouse colon carcinogenesis initiated with azoxymethane.</a> Histol. Histopathol. 20 (2), 483-492 (2005).</li><li>Frederiks, W. M., Vreeling-Sindalarova, H., Van Noorden, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+peroxisomes+causes+oxygen+insensitivity+of+the+histochemical+assay+of+glucose-6-phosphate+dehydrogenase+activity+to+detect+cancer+cells.">Loss of peroxisomes causes oxygen insensitivity of the histochemical assay of glucose-6-phosphate dehydrogenase activity to detect cancer cells.</a> J. Histochem. Cytochem. 55 (2), 175-181 (2007).</li><li>Hisada, R., Yagi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=1-Methoxy-5-methylphenazinium+methyl+sulfate.">1-Methoxy-5-methylphenazinium methyl sulfate.</a> Biochem. 82 (5), 1469-1473 (1977).</li><li>Whittem, C. G., Williams, A. D., Williams, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+Colitis+modeling+using+Dextran+Sulfate+Sodium+(DSS).">Murine Colitis modeling using Dextran Sulfate Sodium (DSS).</a> J Vis Exp. (35), (2010).</li><li>Kuraguchi, M., Cook, H., Williams, E. D., Thomas GA, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+susceptibility+to+colonic+stem+cell+somatic+mutation+in+three+strains+of+mice.">Differences in susceptibility to colonic stem cell somatic mutation in three strains of mice.</a> J. Pathol. 193 (4), 517-521 (2001).</li><li>Whetstone, R. D., Gold, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-Cells+Enhance+Stem+Cell+Mutagenesis+in+the+Mouse+Colon.">T-Cells Enhance Stem Cell Mutagenesis in the Mouse Colon.</a> Mutat. Res. 744, 1-5 (2015).</li><li>Van Noorden, C. J., Vogel, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histochemistry+and+cytochemistry+of+glucose-6-phosphate+dehydrogenase.">Histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase.</a> Prog. Histochem. Cytochem. 15 (4), 1-85 (1985).</li><li>Van Noorden, C. J., Vogels, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+cytochemical+staining+method+for+glucose-6-phosphate+dehydrogenase+activity+in+individual+erythrocytes.+II.+Further+improvements+of+the+staining+procedure+and+some+observations+with+glucose-6-phosphate+dehydrogenase+deficiency.">A sensitive cytochemical staining method for glucose-6-phosphate dehydrogenase activity in individual erythrocytes. II. Further improvements of the staining procedure and some observations with glucose-6-phosphate dehydrogenase deficiency.</a> Br. J. Haematol. 60 (1), 57-63 (1985).</li><li>Cook, H. A., Williams, D., Thomas, G. 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The authors have nothing to disclose.

Often the genotoxic effect of a compound is determined by its ability to modify DNA. This is normally done by isolating the tissue and measuring the global level of DNA adducts. For AOM, this would involve quantifying O6-methylguanine adducts in the colon. Using this approach information on damage within specific cell types, such as in the stem cell niche, is lost. In addition, a DNA adduct is not the same as a mutation since only a small subset of adducts are eventually converted into fixed mutations.12<...

Colon cancer is thought to involve exposure to environmental agents and dietary components that can damage DNA and produce activating somatic mutations in oncogenes (e.g., &#223;-catenin) or inactivating mutations in tumor suppressor genes (e.g., APC). It is assumed that these critical mutations occur in colonic stem cells. Because of the unique crypt architecture of the colonic epithelium, it is possible to measure stem cell mutations in the colon after exposing animals to chemicals associated with colon carcinogenesis. Several X-linked enzymes can serve as indicators for mutations that occur in stem cells, as the mutations will be present in all cells within a crypt.
Previously, a procedure was published demonstrating that chemicals that induce colon tumors in mice also generated somatic stem cell mutations in the colon via analysis of mutations in the X-linked glucose-6-phosphate dehydrogenase (G6PD) gene.1-3 The method quantifies the incidence of random somatic mutations in colonic stem cells without any selection pressure. The procedure involves the production of unfixed frozen colon sections from treated and control mice and the identification of crypts devoid of cells that produce functional G6PD activity. These mutated crypts, which appear white, indicate that the mutation occurred in a stem cell that gave rise to progeny also harboring a mutated G6PD gene. In the enzymatic assay, G6PD deficient mutant crypts cannot oxidize glucose-6-phosphate, which is required for the reduction of nitro blue tetrazolium (NBT). When the G6PD enzyme is functional, the NBT in the staining mixture is reduced to insoluble formazan and precipitates, accumulating at the location of the enzyme and &#8220;staining&#8221; cells blue. Cells that are G6PD mutants remain whitish in contrast to blue stained wild type crypts. This method measures mutations that have a &#8220;null&#8221; phenotype. Because enzymes, such as G6PD, can diffuse out of the cells on an unfixed tissue section if the sections are placed in an aqueous solution, it is necessary to stabilize the enzyme in the tissue sections to be analyzed.4 The stabilization of the enzyme in the tissue must not interfere with diffusion of small reagent molecules necessary for the G6PD enzymatic reaction.
We have made a number of significant changes to the original procedure. The medium for the critical enzymatic staining has been changed from polyvinyl alcohol to Optimal Cutting Temperature Compound (OCT), which facilitates pH control and solubilization of the components used in the assay. The staining is performed using 0.4 &#8211; 0.5 ml wells made of steel washers so that each tissue section received the same volume of the G6PD reaction mixture. A procedure was developed to estimate the number of crypts in an imaged section that provides a large sampling of the colonic tissue without having to manually count &#62;105 crypts for each colon. The analysis of adjacent 7 &#181;m tissue sections affords visualization of a mutant crypt on more than one slide, which reduces potential staining artifacts. These changes make the procedure more efficient and reproducible.
Using this revised protocol, we have quantified the mutation frequency in colonic stem cells after exposure of C57Bl/6 mice to azoxymethane, a well-known colon carcinogen in rodents.5-7

<table><tbody><tr><td>reagents</td><td></td><td></td><td></td></tr><tr><td>nitroblue tetrazolium</td><td></td><td></td><td></td></tr><tr><td>NADP</td><td></td><td></td><td></td></tr><tr><td>glucose-6-phosphate</td><td></td><td></td><td></td></tr><tr><td>1-methoxy-5-methylphenazinium methyl sulfate</td><td></td><td></td><td></td></tr><tr><td>dimethyl formamide</td><td></td><td></td><td></td></tr><tr><td>phosphate buffer (pH 7.4</td><td></td><td></td><td></td></tr><tr><td>Optimal Cutting Temperature Compound (OCT)&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Equipment</td><td></td><td></td><td></td></tr><tr><td>light microscope equipped with 5 megapixel camera</td><td></td><td></td><td></td></tr><tr><td>cryostat</td><td></td><td></td><td></td></tr><tr><td>warm room&amp;nbsp;</td><td></td><td></td><td></td></tr></tbody></table>

quantification of  colonic stem cell mutations

The ability to measure stem cell mutations is a powerful tool to quantify in a critical cell population if, and to what extent, a chemical can induce mutations that potentially lead to cancer. The use of an enzymatic assay to quantify stem cell mutations in the X-linked glucose-6-phosphate dehydrogenase gene has been previously reported.1 This method requires the preparation of frozen sections and incubation of the sectioned tissue with a reaction mixture that yields a blue color if the cells produce functional glucose-6-phosphate dehydrogenase (G6PD) enzyme. If not, the cells appear whitish. We have modified the reaction mixture using Optimal Cutting Temperature Compound (OCT) medium in place of polyvinyl alcohol. This facilitates pH measurement, increases solubilization of the G6PD staining components and restricts diffusion of the G6PD enzyme. To demonstrate that a mutation occurred in a stem cell, the entire crypt must lack G6PD enzymatic activity. Only if a stem cell harbors a phenotypic G6PD mutation will all of the progeny in the crypt lack G6PD enzymatic activity. To identify crypts with a stem cell mutation, four consecutive adjacent frozen sections (a level) were cut at 7 &#181;m thicknesses. This approach of making adjacent cuts provides conformation that a crypt was fully mutated since the same mutated crypt will be observed in adjacent sections. Slides with tissue samples that were more than 50 &#181;m apart were prepared to assess a total of &#62;104 crypts per mouse. The mutation frequency is the number of observed mutated (white) crypts &#247; by the number of wild type (blue) crypts in a treatment group.

The ability to measure colonic stem cell mutations in animals provides a unique way to correlate mutations to cancer induction. Normally, it is assumed that the critical step in carcinogenesis involves activating mutations in oncogenes and/or inactivating mutations in tumor suppressor genes. We injected C57Bl/6 mice with 200 &#181;l of PBS or 10 mg/kg AOM in 200 &#181;l of PBS. AOM is a known colon carcinogen.5-7 At 90 days the colons were analyzed for stem cell mutations. This time is required to allow stem c...

Watch this Scientific Journal Video about Quantification of  Colonic Stem Cell Mutations at JoVE.com

Quantification of  Colonic Stem Cell Mutations

We report significant improvements for the reproducible measurement of somatic colonic stem cell mutations after exposure of mice to potential DNA damaging agents.

The ability to measure stem cell mutations is a powerful tool to quantify in a critical cell population if, and to what extent, a chemical can induce ...

quantification-of-colonic-stem-cell-mutations

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Developmental Biology

6.6K Views.  University of Pittsburgh. We report significant improvements for the reproducible measurement of somatic colonic stem cell mutations after exposure of mice to potential DNA damaging agents.

Video: Quantification of  Colonic Stem Cell Mutations

Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. In the clinical setting, formalin-fixed paraffin-embedded (FFPE) tissues are widely used for genetic testing. However, FFPE DNA is generally damaged and fragmented during the fixation process with formalin. Therefore, FFPE DNA is sometimes not adequate for genetic testing because of low quality and quantity of DNA. Here we present a method of touch imprint cytology (TIC) to obtain genomic DNA from cancer cells, which can be observed under a microscope. Cell morphology and cancer cell numbers can be evaluated using TIC specimens. Furthermore, the extraction of genomic DNA from TIC samples can be completed within two days. The total amount and quality of TIC DNA obtained using this method was higher than that of FFPE DNA. This rapid and simple method allows researchers to obtain high-quality DNA for genetic testing (e.g., next generation sequencing analysis, digital PCR, and quantitative real time PCR) and to shorten the turnaround time for reporting results.

Obtaining high-quality genomic DNA from tumor tissues is an essential first step for analyzing genetic alterations using next generation sequencing. In this article, we present a simple and rapid method to enrich tumor cells and obtain intact DNA from touch imprint cytology specimens.

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. ...

Cancer Research

Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing

The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority of CRC cases, cell dysfunction is the result of numerous mutations that modify protein expression and post-translational modification1.
A number of cell surface antigens, including cluster of differentiation (CD) antigens, have been identified as potential prognostic or metastatic biomarkers in CRC. These antigens make ideal biomarkers as their expression often changes with tumour progression or interactions with other cell types, such as tumour-infiltrating lymphocytes (TILs) and tumour-associated macrophages (TAMs).
The use of immunohistochemistry (IHC) for cancer sub-classification and prognostication is well established for some tumour types2,3. However, no single &#x2018;marker&#x2019; has shown prognostic significance greater than clinico-pathological staging or gained wide acceptance for use in routine pathology reporting of all CRC cases. 
A more recent approach to prognostic stratification of disease phenotypes relies on surface protein profiles using multiple 'markers'. While expression profiling of tumours using proteomic techniques such as iTRAQ is a powerful tool for the discovery of biomarkers4, it is not optimal for routine use in diagnostic laboratories and cannot distinguish different cell types in a mixed population. In addition, large amounts of tumour tissue are required for the profiling of purified plasma membrane glycoproteins by these methods. 
In this video we described a simple method for surface proteome profiling of viable cells from disaggregated CRC samples using a DotScan CRC antibody microarray. The 122-antibody microarray consists of a standard 82-antibody region recognizing a range of lineage-specific leukocyte markers, adhesion molecules, receptors and markers of inflammation and immune response5, together with a satellite region for detection of 40 potentially prognostic markers for CRC. Cells are captured only on antibodies for which they express the corresponding antigen. The cell density per dot, determined by optical scanning, reflects the proportion of cells expressing that antigen, the level of expression of the antigen and affinity of the antibody6. 
For CRC tissue or normal intestinal mucosa, optical scans reflect the immunophenotype of mixed populations of cells. Fluorescence multiplexing can then be used to profile selected sub-populations of cells of interest captured on the array. For example, Alexa 647-anti-epithelial cell adhesion molecule (EpCAM; CD326), is a pan-epithelial differentiation antigen that was used to detect CRC cells and also epithelial cells of normal intestinal mucosa, while Phycoerythrin-anti-CD3, was used to detect infiltrating T-cells7. The DotScan CRC microarray should be the prototype for a diagnostic alternative to the anatomically-based CRC staging system.

We described a procedure for the disaggregation of colorectal cancer (CRC) to produce viable single cells, which are then captured on customized antibody microarrays recognizing surface antigens (DotScan CRC microarray). Sub-populations of cells bound to the microarray can be profiled by fluorescence multiplexing using monoclonal antibodies tagged with fluorescent dyes.

The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority ...

Immunology and Infection

Evaluation of Colorectal Cancer Risk and Prevalence by Stool DNA Integrity Detection

Nowadays, stool DNA can be isolated and analyzed by several methods. The long fragments of DNA in stool can be detected by a qPCR assay, which provides a reliable probability of the presence of pre-neoplastic or neoplastic colorectal lesions. This method, called fluorescence long DNA (FL-DNA), is a fast, non-invasive procedure that is an improvement upon the primary prevention system. This method is based on evaluation of fecal DNA integrity by quantitative amplification of specific targets of genomic DNA. In particular, the evaluation of DNA fragments longer than 200 bp allows for detection of patients with colorectal lesions with very high specificity. However, this system and all currently available stool DNA tests present some general issues that need to be addressed (e.g., the frequency at which tests should be carried out and optimal number of stool samples collected at each timepoint for each individual). However, the main advantage of FL-DNA is the possibility to use it in association with a test currently used in the CRC screening program, known as the immunochemical-based fecal occult blood test (iFOBT). Indeed, both tests can be performed on the same sample, reducing costs and achieving a better prediction of the eventual presence of colorectal lesions.

The presented diagnostic FL-DNA kit is a time-saving and user-friendly method to determine the reliable probability of the presence of colorectal cancer lesions.

Nowadays, stool DNA can be isolated and analyzed by several methods. The long fragments of DNA in stool can be detected by a qPCR assay, which provides a ...

Discovery of Driver Genes in Colorectal HT29-derived Cancer Stem-Like Tumorspheres

Cancer stem cells play a vital role against clinical therapies, contributing to tumor relapse. There are many oncogenes involved in tumorigenesis and the initiation of cancer stemness properties. Since gene expression in the formation of colorectal cancer-derived tumorspheres is unclear, it takes time to discover the mechanisms working on one gene at a time. This study demonstrates a method to quickly discover the driver genes involved in the survival of the colorectal cancer stem-like cells in vitro. Colorectal HT29 cancer cells that express the LGR5 when cultured as spheroids and accompany an increase CD133 stemness markers were selected and used in this study. The protocol presented is used to perform RNAseq with available bioinformatics to quickly uncover the overexpressed driver genes in the formation of colorectal HT29-derived stem-like tumorspheres. The methodology can quickly screen and discover potential driver genes in other disease models.

Presented here is a protocol to discover the overexpressed driver genes maintaining the established cancer stem-like cells derived from colorectal HT29 cells. RNAseq with available bioinformatics was performed to investigate and screen gene expression networks for elucidating a potential mechanism involved in the survival of targeted tumor cells.

Cancer stem cells play a vital role against clinical therapies, contributing to tumor relapse. There are many oncogenes involved in tumorigenesis and the ...

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

A Three-dimensional Model of Spheroids to Study Colon Cancer Stem Cells

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for tumor development, maintenance, and resistance to drugs. A better understanding of CSC properties for their specific targeting is, therefore, a pre-requisite for effective therapy. However, there is a paucity of suitable preclinical models for in-depth investigations. Although in vitro two-dimensional (2D) cancer cell lines provide valuable insights into tumor biology, they do not replicate the phenotypic and genetic tumor heterogeneity. In contrast, three-dimensional (3D) models address and reproduce near-physiological cancer complexity and cell heterogeneity. The aim of this work was to design a robust and reproducible 3D culture system to study CSC biology. The present methodology describes the development and optimization of conditions to generate 3D spheroids, which are homogenous in size, from Caco2 colon adenocarcinoma cells, a model that can be used for long-term culture. Importantly, within the spheroids, the cells which were organized around lumen-like structures, were characterized by differential cell proliferation patterns and by the presence of CSCs expressing a panel of markers. These results provide the first proof-of-concept for the appropriateness of this 3D approach to study cell heterogeneity and CSC biology, including the response to chemotherapy.

This protocol presents a novel, robust, and reproducible culture system to generate and grow three-dimensional spheroids from Caco2 colon adenocarcinoma cells. The results provide the first proof-of-concept for the appropriateness of this approach to study cancer stem cell biology, including the response to chemotherapy.

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for ...

Comparative Lesions Analysis Through a Targeted Sequencing Approach

Assessing intra-tumoral heterogeneity (ITH) is of paramount importance to anticipate failure of targeted therapies and design accordingly effective anti-tumor strategies. Although concerns are frequently raised due to differences in sample processing and depth of coverage, next-generation sequencing of solid tumors have unraveled a highly variable degree of ITH across tumor types. Capturing the genetic relatedness between primary and metastatic lesions through the identification of clonal and subclonal populations is critical to the design of therapies for advance-stage diseases. Here, we report a method for comparative lesions analysis that allows for the identification of clonal and subclonal populations among different specimens from the same patient. The experimental approach described here integrates three well-established approaches: histological analysis, high-coverage multi-lesion sequencing, and immunophenotypic analyses. In order to minimize the effects on the detection of subclonal events by inappropriate sample processing, we subjected tissues to careful pathological examination and neoplastic cell enrichment. Quality controlled DNA from neoplastic lesions and normal tissues was then subjected to high coverage sequencing, targeting the coding regions of 409 relevant cancer genes. While only looking at a limited genomic space, our approach enables evaluating the extent of heterogeneity among somatic alterations (single-nucleotide mutations and copy-number variations) in distinct lesions from a given patient. Through comparative analysis of sequencing data, we were able to distinguish clonal vs. subclonal alterations. The majority of ITH is often ascribed to passenger mutations; therefore, we also used immunohistochemistry to predict functional consequences of mutations. While this protocol has been applied to a specific tumor type, we anticipate that the methodology described here is broadly applicable to other solid tumor types.

This article describes a method to identify clonal and subclonal alterations among different specimens from a given patient. Although the experiments described here focus on a specific tumor type, the approach is broadly applicable to other solid tumors.

Assessing intra-tumoral heterogeneity (ITH) is of paramount importance to anticipate failure of targeted therapies and design accordingly effective ...

Behavior

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.

Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.
Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions.

Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.
By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.&#160;&#160;&#160;

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Molecular profiling of laser microdissected cells and stroma from synthetic, tissue-engineered colorectal cancer models represents a novel and manipulatable approach to characterize and dissect the distinctive biology at the interface between tumor cells at the invasive front and cancer associated stromal cells. &#160;

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

Medicine

Deficient Pms2, ERCC1, Ku86, CcOI in Field Defects During Progression to Colon Cancer

In carcinogenesis, the "field defect" is recognized clinically because of the high propensity of survivors of certain cancers to develop other malignancies of the same tissue type, often in a nearby location. Such field defects have been indicated in colon cancer. The molecular abnormalities that are responsible for a field defect in the colon should be detectable at high frequency in the histologically normal tissue surrounding a colonic adenocarcinoma or surrounding an adenoma with advanced neoplasia (well on the way to a colon cancer), but at low frequency in the colonic mucosa from patients without colonic neoplasia.
Using immunohistochemistry, entire crypts within 10 cm on each side of colonic adenocarcinomas or advanced colonic neoplasias were found to be frequently reduced or absent in expression for two DNA repair proteins, Pms2 and/or ERCC1. Pms2 is a dual role protein, active in DNA mismatch repair as well as needed in apoptosis of cells with excess DNA damage. ERCC1 is active in DNA nucleotide excision repair. The reduced or absent expression of both ERCC1 and Pms2 would create cells with both increased ability to survive (apoptosis resistance) and increased level of mutability. The reduced or absent expression of both ERCC1 and Pms2 is likely an early step in progression to colon cancer.
DNA repair gene Ku86 (active in DNA non-homologous end joining) and Cytochrome c Oxidase Subunit I (involved in apoptosis) had each been reported to be decreased in expression in mucosal areas close to colon cancers. However, immunohistochemical evaluation of their levels of expression showed only low to modest frequencies of crypts to be deficient in their expression in a field defect surrounding colon cancer or surrounding advanced colonic neoplasia.
We show, here, our method of evaluation of crypts for expression of ERCC1, Pms2, Ku86 and CcOI. We show that frequency of entire crypts deficient for Pms2 and ERCC1 is often as great as 70% to 95% in 20 cm long areas surrounding a colonic neoplasia, while frequency of crypts deficient in Ku86 has a median value of 2% and frequency of crypts deficient in CcOI has a median value of 16% in these areas. The entire colon is 150 cm long (about 5 feet) and has about 10 million crypts in its mucosal layer. The defect in Pms2 and ERCC1 surrounding a colon cancer thus may include 1 million crypts. It is from a defective crypt that colon cancer arises.

Reduced/absent expression of Pms2 and/or ERCC1 in entire crypts is a frequent event within 10 cm on each side of colonic adenocarcinomas, likely the basis of a field defect with high mutability and progression to cancer. Deficiency in Ku86 or CcOI is much less frequent in these field defects.

In carcinogenesis, the "field defect" is recognized clinically because of the high propensity of survivors of certain cancers to develop other ...

Biology

MLH1 Gene Expression in Peripheral Blood in Colon Cancer Patients and Their Association with Colon Cancer

MutL homolog 1 (MLH1) is a component of the heterodimeric complex MutL&#945; that detects and fixes base-base mismatches and insertion/deletion loops caused by nucleotide misincorporation. In the absence of MLH1 protein, the frequency of non-repaired mismatches increases, resulting in organ cancer. The current study sought to quantify MLH1 gene expression and its relationship with tumor invasion (T) and lymph node invasion (N) in blood samples from patients with colorectal cancer (CRC). Blood samples were obtained from 36 CRC patients. RNA was extracted, and cDNA was synthesized using a kit. The primers were built using the exon-exon junction approach, and MLH1 and &#946;-actin genes were tested 3x using real-time polymerase chain reaction (Real-Time PCR). Gene expression analysis software was used to analyze the data, and a t-test was used to examine the expression of MLH1 and its connection with T and N variables. In this study, 36 patients with colorectal cancer, including 15 (41.6%) women and 21 (58.4%) men, with a mean age of 57.35 &plusmn; 4.22 years and in the age range of 26-87 years, were included. The results showed that the ratio of MLH1 gene expression in patients decreased compared to that in healthy individuals, and the decrease in gene expression at different stages of the disease was significant. The results of this study showed that the reduction of MLH1 gene expression has an effective role in the development of CRC.

This study examines the association between MLH1 gene expression in peripheral blood and colon cancer, utilizing a case-control approach to compare expression levels in patients and matched healthy controls.

MutL homolog 1 (MLH1) is a component of the heterodimeric complex MutL&#945; that detects and fixes base-base mismatches and insertion/deletion loops ...

Quantification of Colonic Stem Cell Mutations

Summary

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Chapters in this video

Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology

Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing

Evaluation of Colorectal Cancer Risk and Prevalence by Stool DNA Integrity Detection

Discovery of Driver Genes in Colorectal HT29-derived Cancer Stem-Like Tumorspheres

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

A Three-dimensional Model of Spheroids to Study Colon Cancer Stem Cells

Comparative Lesions Analysis Through a Targeted Sequencing Approach

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Deficient Pms2, ERCC1, Ku86, CcOI in Field Defects During Progression to Colon Cancer

MLH1 Gene Expression in Peripheral Blood in Colon Cancer Patients and Their Association with Colon Cancer