Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids

The overall goal of this method is to identify, characterize, and isolate cells from a multicellular steroid with respect to their cell cycle status and position within the steroid Compared to 2D cell culture. The 3D OID model recapitulates the biology of cancer in vivo as it mimics the tumor architecture and its microenvironment By pairing spheros with flow cytometry and imaging techniques. This method can answer key questions in the cancer field, such as how the tumor microenvironment, alters drug sensitivity, cell motility, and proliferation.

The main advantage of this method is that it enables real time visualization of the cell cycle and it allows for purification of live cells from a specific steroid region demonstrating the procedure will be done A sharp, a research assistant from our laboratory Begin preparing for 3D steroid formation by microwaving 1.5%aros in 100 milliliters of Hank's balance salt solution or HBSS or three to five minutes intermittently swirl the flask to ensure that the aros is fully dissolved. Then immediately pipette 100 microliters of the aros solution into each well of a flat bottomed 96 well tissue culture plate. Incubate the plate at room temperature for one hour to allow the aros to solidify.

Next, remove an 80%confluence cell culture of melanoma cells expressing the FCI constructs from the cell culture incubator. Wash the cells with 10 milliliters of HBSS. Add 1.5 milliliters of 0.05%in EDTA and then incubate the cells at 37 degrees Celsius for approximately two minutes.

Once the cells have detached resus, suspend them in 10 milliliters of cell culture.Medium. Manually count the cells using a hemo, cytometer and inverted microscope and dilute them to 25, 000 cells per milliliter in cell culture medium. Then pipette 200 microliters of the cell suspension to overlay each well of the 96 well aros plate.

Incubate the plate at 37 degrees Celsius and 5%carbon dioxide for approximately three days to form cell spheroids. Following incubation image the spheroids on an inverted microscope using a Forex objective and a phase contrast filter. Spheroids should appear as compact, roughly spherical cell aggregates to prepare the cell steroid for sectioning and imaging.

Use a one milliliter pipette to move them to a falcon tube. Then allow the steroid to settle at the bottom of the conical. Next, remove the medium bi suction and then fix the spheroids by incubating them in 10%neutral buffered formin for at least two hours at room temperature.

SP may then be stored in HBSS at four degrees Celsius for several days following sectioning and imaging of the cell. Sphe open the image analysis software and choose the quantitation only configuration. Then create a new library and import the spheroid confocal image file by dragging the raw data file into the library.

Next, go to the measurements tab to build an image analysis protocol by dragging and dropping commands in the correct order in the protocol window. To find objects in the green channel, drag and drop the find objects command from the finding section into the protocol window and select the appropriate channel. Add an open command found under processing and then add a separate touching objects command to separate the cells.

Finally, from the filtering section, add and exclude objects by size. Command for objects less than 50 micrometers to exclude small non cellular objects. Next, drag and drop a new find objects command into the protocol window.

Following the same steps, select the red channel and apply all subsequent commands. Once objects have been found, use the hide channel or show channel commands to visually ensure that red and green objects correspond to red and green nuclei. If necessary, click on measurements and then feedback options to modify the appearance of the object masks.

Now to find yellow objects that correspond to early S-phase cells, use the intersect command found in the combining section and choose intersect red objects with green objects. Again, exclude objects by size less than 50 micrometers. To identify G one phase cells, use the subtract command found in the combining section and choose subtract yellow object from red objects to find exclusively red objects.

Similarly, subtract yellow objects from green objects in order to identify exclusively green objects that correlate to SG two and MPH phase cells for both the exclusively red and exclusively green objects. Remove non cellular objects as necessary by applying a filter population command from the filtering section with a shape factor greater than 0.25. Then find the S spheroid outline.

Using a find objects command from the finding section in either the green or red channel. Use a closed command from the processing section to join the individual cells into one object. Then add a fill holes in objects command to fill in holes in the sphe.

Next, use a fine filter to remove noise from the Sphero object. Finish finding the spheroid outline by choosing exclude objects by size to remove objects smaller than 30, 000 micrometers. Once all objects have been defined, add measure distances from the relating section to determine the distance from each OID to the edge of the S spheroid outline.

Do this in each of the yellow, exclusively red and exclusively green populations. To visualize the minimum distances from the cell to the nearest S spheroid edge, open the measurements tab and select feedback options followed by relationships. Then choose show distances.

Finally, save the protocol to save data created by the protocol. Choose the make measurement item from the measurement section. Then to interpret the data, select the measurement item, go to the analysis tab in the measurement section and choose Analyze under the analysis menu to count the cells found at a certain distance from the steroid edge.

Create a filter by selecting filter from the analysis tab. For example, show only data where distance from sphere edge is less than 100 microns. To export the raw data, select export from the file tab and then save the data as comma separated or tab limited text.

This data may be opened in a spreadsheet program for further analysis. Cell sphe generated from C 8 1 16 1. Human melanoma cells that had been transduced with the Fuji system were fixed and sectioned.

Confocal imaging for AZA green, which mark cells in the SG two M phase of the cell cycle and Berra orange, which mark cells in the G one phase was performed. When these images are overlaying key features such as a necrotic core can be observed as expected. Red G one arrested cells predominate closer to this necrotic core while red and green proliferating cells increase in a gradient manner towards the outer edge of the steroid or access to oxygen and nutrients is greatest.

Following semi-automated image analysis, the fucci cell masks and S spheroid outline can be defined. Imaging software was used to quantitate red and green cells in the inner or outer region of the S spheroid. This analysis showed that G one phase arrested cells are enriched in the inner region while proliferating cells predominate closer to the steroid Edge.

Once mastered, this technique can be done in one to two weeks, a timeframe that includes growing the steroid, the imaging and analysis. Following these procedure rads can be implanted into collagen matrix and then imaged via a TimeLapse microscopy. They can then be subjected to protein or RNA analysis, and this can help us answer questions such as whether position in the steroid and access to oxygen and nutrients can alter cancer cell phenotypes.