Laser Microdissection: A Technique to Isolate Invading Colorectal Cancer cells from a 3D Organotypic Culture Model

- 3D organotypic models of colorectal cancer are in-vitro cultures engineered to mimic in-vivo tumor microenvironments. The culture often contains stromal cells, like fibroblasts, co-cultured with colorectal cancer epithelial cells, in a suitable extracellular matrix microenvironment.

In established cultures, tumor cells with metastatic potential invade the extracellular matrix. To isolate these invading cells, first, treat the organotypic model with formaldehyde to fix cells and extracellular matrix. Incubate with alcohol to dehydrate the sample.

Next, embed in liquid paraffin wax to generate a tissue block. Use an ultramicrotome to obtain thinly sliced sections. Mount the sections onto an appropriate glass slide.

Add Xylene to deparaffinize the sections. Sequentially, incubate the sections in decreasing concentrations of ethanol to rehydrate samples.

Treat with Cresyl Violet to stain and differentiate colorectal cancer cells from the microenvironment. Place the stained glass slide face down on the microscope stage. Using appropriate software, identify the cells to be microdissected and delineate the borders. Fire the laser to cut the highlighted section and collect the microdissected portion.

In the following protocol, we will show the isolation of colorectal cancer cells from a 3D organotypic co-culture model by laser microdissection.

- Remove the whole organotypic, including nylon sheet, from the well and place it on cling film. Bisect the organotypic and nylon sheet using a clean disposable scalpel and fix both halves in formaldehyde for 24 hours at room temperature. Replace formaldehyde with 70% ethanol after 24 hours and leave overnight prior to embedding in paraffin, sectioning, and staining.

To perform laser microdissection, first, section the organotypic to 10-micron thickness onto membrane-mounted slides. After treating the sections as described in the text protocol, prepare to conduct laser microdissection using the chosen platform.

Place the glass slide with the stained section to be microdissected face down on the microscope stage. Mount a 0.5 milliliter microcentrifuge tube into the collection cassette and add 50 microliters of cell lysis buffer to the cap into which microdissected tissue will collect. Under direct vision, use the joystick to identify the tissue of interest, and using the software interface, annotate the stained organic section, highlighting cells to be microdissected at the tumor invasion front.

Instruct the laser to fire, which should both cut out the highlighted section and propel the microdissected tissue into the cap of the microcentrifuge tube. Proceed to process samples by an appropriate method to extract the analyte of interest.