The overall goal of this protocol is to study the direct and indirect communication between primary breast cancer cells and monocytes in a three-dimensional co-culture system. This method can help answer key questions in inflammatory microenvironment of breast cancer field such as how immune cells are regraded and activated, how these cells help to sustain the tumor inflammatory microenvironment and how these microenvironment facilitates tumor cell invasion. The main advantage of this technique is that it provides an affordable alternative to study intra-tumor communication and illustrates the potential of in vitro, 3-D cell systems for interrogating specific features of tumor biology.
Particularly those related to tumor aggression. To begin, cultivate two times 10 to the sixth U937 and THP1 monocytes in 10 milliliters of RPMI1640 medium, supplemented with 10 percent FBS and one percent antibiotic antimycotic. Also cultivate fresh PMs in supplemented DMEMF12 medium.
Incubate the cells at 37 degrees Celsius in a humidified five percent carbon dioxide environment. Plate four time 10 to the fifth monocytes in one milliliter per well of their corresponding medium. Place an insert in each well and add 900 microliters of the four times 10 to the fifth BrC cell suspension in the corresponding medium.
Incubate the cultures at 37 degrees Celsius in a humidified five percent carbon dioxide environment for five days and recover the supernatants. Retrieve the cells by trypsinization if subsequent analysis is performed. Include controls of individual cell cultures with the respective media.
To label BrC cells and monocytes with commercially available cumerine and rhodamine before cell culture, prepare a monolayer of two times 10 to the sixth BrC cells of interest and replace the standard medium with sufficient, pre-warmed working solution of the fluorescent dye. Incubate the cells at 37 degrees Celsius in a humidified five percent carbon dioxide environment for 30 minutes. Then aspirate the dye solution and use sufficient one XPBS to gently rinse the cells.
Aspirate the PBS and add the standard medium. To trypsinize the cells, add two milliliters of 05 percent trypsin and 0.48 millimolar EDTA to the flask and incubate the cells at 37 degrees Celsius for five to 10 minutes. Add 200 microliters of FBS to stop the trypsinization and seven milliliters of the corresponding medium without supplements.
Re-suspend the cells by pipetting, then transfer 10 microliters of the cells to a Nuebauer chamber and count them. Next, pellet the cells at 430 times g and room temperature for five minutes, then discard the supernatant and use three milliliters of pre-warmed fluorescent dye working solution to re-suspend the cells. Incubate the cell suspension at 37 degrees Celsius in a humidified five percent carbon dioxide environment for 30 minutes.
After pelleting the cells again, discard the dye working solution and use five to seven milliliters of one XPBS to gently re-suspend the cells. Then, after pelleting the cells once more and discarding the PBS, re-suspend the cells in five to seven milliliters of standard medium. Count 10 microliters of the cell suspension.
Set up a co-culture by spreading enough ECME at the bottom of the well to form an even layer in each well of a four well chamber slide system. Plate 20 microliters of a single cell suspension containing five time 10 to the fifth labeled BrC cells per well. After 15 to 20 minutes at 37 degrees celsius, add a suspension of 2.5 times 10 to the fifth labeled monocytes in 80 microliters of assay medium supplemented with 60 percent ECME.
Allow the ECME to solidify at 37 degrees Celsius for 15 to 20 minutes. Now add one milliliter of a one to one mixture of the BrC cells and monocyte culture medium. Incubate the co-cultures at 37 degrees Celsius in a humidified five percent carbon dioxide environment for 24 hours, 48 hours, or five days to track changes at different time points.
To degrade the ECM proteins and recover the cells from the cultures, aspirate and discard the medium, then add 0.5 milliliters of one XPBS with 0.1 percent trypsin and 0.25 percent EDTA to the culture. Incubate the cells at 37 degrees Celsius for three hours. Following the incubation, add 0.5 milliliters of one XPBS with 10 percent FBS to neutralize the trypsin and re-suspend the cells by vigorously pipetting to attain a single cell suspension.
After pelleting the cells, discard the supernatant and re-suspend the cells in five milliliters of one XPBS with 10 percent FBS. Repeat this step once. Subject the cell suspension to fax with the appropriate instrument.
Ensure that the final populations are at least 95 percent pure. In each well of an eight well chamber slide system spread a 40 microliter base of ECME and let it solidify at 37 degrees Celsius for 30 minutes. Seed 800 MCF10 A cells per well in 400 microliters of supplemented DMEM F12 culture medium according to the text protocol.
Then add 400 microliters of conditioned medium or supplemented DMEM F12 culture medium with 20 nanograms per milliliter of human recombinant IL1 beta. Place the chamber slide into a glass petri dish containing a 35 milliliter petri dish filled with two milliliters of PBS. The MCF 10A cells should be seeded in each well very slowly to avoid damage of the base of ECME and to prevent cells from settling down and forming a monoly.
Using an optical microscope record glandular acini formation every 24 hours for 14 days. After 14 days of culture, add 100 microliters of 100 nanomolar dapy in one XPBS and incubate the samples at room temperature with constant stirring for 25 minutes. Rinse the samples with one XPBS at room temperature, three times for five minutes each with constant stirring.
Use mounting medium specific for fluorescent staining to mount the preparations. Then seal the mounted samples with transparent polish and maintain the slides, protected from light at four degrees Celsius. Demonstrating the procedure in the confocal microscope will be Vadim Perez, a researcher from The National Laboratory of Advanced Microscoping.
Analyze the acini with a confocal microscope taking images of transversal stacks at different depths of the acini. Visualize different cellular proteins in the acini with proper staining protocols. For example, caheren was stained here to assess cell to cell adhesion, cell polarity, and oralimen formation.
The morphology of primary BrC cells growing in 3D cultures at low and high densities was studied over five days. During the first 48 hours, cells adhere to the ECME with an elongated spindle shape and form knit-like structures at five days. After five days in 3D co-cultures, both MCF7 and MDA-MB-231 commercial BrC cells formed disorganized aggregates.
However, aggressive MDA-MB-231 cells for aggregates with elongated forms, while non-aggressive MCF7 cells for aggregates with rounded shapes that are more densely compacted then MDA-MB-231 cells. The supernatants from indirect interaction 3D primary BrC cultures and their five day co-cultures with primary monocytes were harvested. Significantly increased levels of IL1 beta and IL8 were observed in the primary BrC cell monocyte co-cultures.
Both crucial and flammatory cytokines that have been previously associated with malignant progression. MCF10A cells were cultivated with two different supernatants from two primary BrC cell lines to observe lumen formation as a measure of resistance to anoykis. Unlike MCF10A cells grown under standard conditions, bi-confocal microscopy at day 14, transversal cuts of the spheroids show that MCF10A cells evaded anoykis as measured by counting the number of central cells in the acini.
Following this procedure, other methods like medical analysis can be performed in order to answer additional questions. Test the signaling pathways in both cancer migration. After its development this technique paved the way for researchers in the field of tumor micro-environments to explore the communications of breast cancer cells with mass cells, fibroblasts, neutrofils, or even different clones of tumor cells in 3D co-culture systems.
After watching this video, you should have a good understanding of how to establish a 3D culture system to model the tumor microenvironment.
