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

Do your overall goal of the following experiment is to use the DOT scan antibody microarray to molecularly profile surface antigens on colorectal cancer tissue. In addition, fluorescent multiplexing is employed to distinguish cell types of specific profiles within the mixed population. Cluster of differentiation or CD antigens have been identified as potential prognostic or metastatic biomarkers in colorectal cancer.

These antigens make ideal biomarkers as their expression often changes with tumor progression or interaction with other cell types. In this video, we employ the DOT scan antibody microarray to capture live cells via the expressed surface antigens. The DOT scan antibody microarray consists of a glass slide with a layer of nitrocellulose.

The DOT scan antibody microray panel is boarded by alignment dots consisting of CD 29 and CD 44. Duplicate arrays are also dotted on each side of the array. The first panel consists of the original 82 antibodies of the DOT scan leukemia microarray and additional 40 antibodies were selected based on a prognostic potential as the colorectal cancer satellite microarray.

And the last panel consists of ice type control antibodies. Here, colorectal cancer tissue and normal intestinal mucosa are digested and filtered to get a viable single cell suspension. The suspension can be frozen or stored for later.

The cell suspension is used for cell capture on a DOT scan antibody microarray antigens expressed on different cell subtypes are profiled by fluorescence multiplexing. The resulting expression profiles show a significant change in antigen expression between the colorectal cancer cells and a normal intestinal mucosa. This difference is also reflected in the tumor associated leukocyte population.

Hierarchial clustering of several tumor profiles show clustering of disease signatures with colorectal cancer stages. Hi, I'm Jerry from the School of Molecular Bioscience at the University of Sydney Australia, and today I'll be showing you an experiment for the profiling of surface antigens on colorectal cancer tissue. Using a dot scan antibody microarray and fluorescence multiplexing Dot scan microarrays enable profiling of approximately 140 surface antigens on live cancer cells with the associated inflammatory leukocytes.

Subsets of cells captured on microarrays may be distinguished using fluorescently labeled antibodies that bind to a highly expressed marker antigen on the cells of interest. Up to three different subsets of cells can be profiled on a single microarray using appropriate fluorescent labels on antibodies with three different specificities. The DOT scan microarray assay is a relatively simple method and has already been used for cell surface profiling of several cancers.

A key feature of DOT scan is its ability to capture live cells via their surface antigens, so the binding pattern we get is a reflection of the cell's surface profile. Surface antigens play an important role in tumor development and metastatics and in the inflammatory response to fight the cancer. Extensive surface profiles or immuno phenotypes can be used as disease signatures to improve cancer classification, enabling optimal treatment after surgery.

Okay, let's get started. In the experiment shown here, clinical colorectal cancer tumor samples, grades A to D are collected in Hank's buffer. To begin using two scalpels carefully sliced the tumor into one millimeter thin slices.

Place the slices into 2%collagenase type four and 10%DNA solution for digestion. Sections of normal intestinal mucosa from the same patient are collected 10 centimeters away from the primary tumor site. This acts as our control and is processed identically to the tumor sample.

Tumor digestion occurs at 37 degrees Celsius for one hour to promote the breakdown of connective tissue, the D ns will clean up DNA from any damaged cells. Following incubation, the tissue suspension is passed through a wire mesh wash called Hanks buffer over the sample while plunger from a 10 mil syringe is used to help push cells through. This process is repeated several times until most of the cells have passed through the wire mesh.

The mesh separates cells from any undigested connective tissue. Next, further filter the cell suspension by taking it up in a 10 M syringe and filtering through a fcon 200 micrometer filter followed by a fil con 50 micrometer filter. These finer filters should ensure the removal of excess mucus or DNA as well as breakup cell clusters to obtain a viable single cell suspension.

The same treatment is done for the normal mucosa. We found that the normal mucosa tends to contain more connective tissue and as a result, takes some more washes to pass through to mesh centrifuge cell suspension at 410 G at 20 degrees Celsius for five minutes. In preparation for cold storage, we suspend the cell pallet with 750 microliters of fetal calf serum, followed by eco volumes of fetal calf serum with DMS.

So solution at this DMS. So solution dropwise with constant mixing aliquot the supinate into cryo tube and placed in an insulator container. These samples can be stored in minus 80 for up to one year.

The insulation and DMSO should ensure the cells free slowly without bursting. A side effect of the storage is the lysis of red blood cell population. Fortunately for us, they're not needed.

For the experiment. Take the samples out of storage and throw them quickly. In a 37 degree Celsius water bath resuspend cell suspension in 10 mils of Hank's buffer, the buffer should help dilute out the 10%DMSO and fetal calf serum central feature cells for five minutes at 410 GS at 20 degrees Celsius.

Next, decant the snat and resuspend in 500 microliters of Hank's buffer, a few cells may be disrupted during the freezing and thawing process causing a release of DNA into a suspension. To counter this problem, incubate the sample with 0.1%DNAs at broom temperature. During this time, a cell viability count can be conducted.

Mix equal amounts of triam blue with 10 microliters of cell suspension and pipette 10 microliter of the mixture into a cell counter. Under the microscope, healthy and viable cells appear clear. On the other hand, non-viable cells with their membranes disrupted will appear blue.

A minimum of 4 million viable cells are required for cell capture on the DOT scan antibody. MICRORAY falling. DNA treatment washed the cells for a second time with 10 mils of Hank's buffer and centrifuge at 410 G for five minutes and 20 degrees Celsius.

We suspend the final cell palette with 200 microliters of RPMI media. This media contains relatively more nutrients than the Hanks buffer and is used to facilitate cell capture on the antibody microarray while maintaining cell viability. The colorectal cancer dot scan antibody microarray consists of 140 different antibody dots duplicated on the nitrocellulose.

Each dot on the array contains 10 nanoliters of a monoclonal antibody against the cell surface antigen. To prepare the antibody microarray, dip it into a tray of PBS to moisten the nitrocellulose wipe the glass edge of the array. With Kim wipes care is taken not to touch the nitrocellulose section, place the microarray in an tray and pipette the cell suspension onto the nitrocellulose.

A even spread of the cell suspension will ensure all antibodies have the potential to capture cells. Incubate the microarray at 37 degrees Celsius for one hour. During the incubation, different cells would drift down and come in contact with antibodies on the array cells.

Presenting surface antigens corresponding to its antibody will bind and capture the cell following incubation. Wash up the unbound cells by gently dipping the arrays into three changes of fresh PPS. Fix the bounce cells in 10%from outer height and PPS solution.

Gently pipet a generous amounts of fixative to cover the nitro cell load section. Incubate for 20 minutes at room temperature. Next, dip microarrays three times in fresh PBS.

Leave the arrays in PBS to soak for 30 seconds on each wash as excess fromer height can interfere with fluorescence multiplexing. We can check now to see if we have cell binding by holding a microarray under some light for a far more precise analysis. The DOT scan scanner and analysis software allows us to scan the binding pattern and produce quantified results of antigen expression in a mixed cell population.

The software compares the intensity of cell binding against the background noise on a eight bit grainer scale. The intensity units are normalized to a maximum value of 256. For ease of comparison between a array, any positive ice type control binding is deducted from antibodies with the corresponding IG isotypes.

Add 200 microliters of blocking buffer to the microarray and incubate for 20 minutes at room temperature. In preparation for fluorescence multiplexing in 118 microliters of blocking buffer add 10 microliters of fiery and conjugated CD three and 20 microliters of Alexa conjugated epam. In addition, two microliters of he inactivated serum is also added to the mixture to prevent non-specific binding.

Once blocking is complete, the blocking buffer is replaced with multiplexing solution and incubated in a dark for 30 minutes. At room temperature during the incubation CD three conjugated antibodies will bind to CD three expressing T cells bound to the array while EPAM conjugated antibody will bind to epithelial cells or colorectal cancer cells on the array. Next, watch the micro array three times in fresh PBS allow them to dry in the dark before scanning for fluorescence.

Fluorescence is detected with a typhoon FLA 9, 000 scanner cd. FICO ery profile is scanned using a 5 2 3 nanometer laser and 5 8 0 BP 30 emission filter EP camm Alexa 6 4 7 is scanned using a 6 3 3 nanometer laser and a 6 7 0 BP 30 emission filter. The resulting fluorescent images are converted to tip format for analysis.

Using a DOT scan software. Fluorescent multiplexing allows us to distinguish antigen profiles of different cell populations within the same tissue sample. The images shown here are from a colorectal cancer sample.

These three images are of an optical scan, fluorescent scan for CD three expressing cells and fluorescent scan for EPAM expressing cells. Results from the DOT scan microray should show consistent cell binding patterns between the two duplicate slides. Intense alignment dot binding along the edge of the slide indicates adequate amounts of cells were captured.

A PDF report can be generated to give quantitative values to cell binding. The bar graph. Representation of cell binding shows a difference in antigen expression between normal mucosa and the tumor sample.

The optical results given overall representation of antigen expression on all cell types within the sample, the AAM multiplexing results show a clearer picture of the difference in antigen expression between normal epithelial and cancer cells. While many of the differentially expressed antigens are known colorectal cancer biomarkers as a group, they provide a far more accurate disease signature for stratification that any single biomarker can ever achieve. CD three multiplexing results show more T cells in the cancer sample compared to the normal.

Often the cancer T cells are expressing activation markers giving rise to the possibility that these T cells are tumor infiltrating lymphocytes or inflammatory associated T cells. Large scale analysis can be conducted by multi experiment viewer version 4.4, a open source heat map and data analysis software. Hierarchial clustering of colorectal cancer samples can be used to correlate with disease stage or patient prognosis.

An extensive disease signature library is a valuable tool in the prognosis prediction In colorectal cancer patients or in a determination of the course of postoperative treatment, we've just shown your procedure for profiling surface antigen expression in a mixed cell population in colorectal cancer tissue using the DOT scan antibody microarray and fluorescence multipley. One important aspect of this experiment to remember is that always use fresh tissue and at least 4 million viable cells are required for this experiment to work. Thank you for watching this video and good luck with your experiments.