Cancer-Associated Fibroblasts from Mouse Mammary Tumors as Tools for Molecular and Computational Studies

Our research investigates how cancer-associated fibroblasts shape breast cancer progression using molecular, in vitro, in vivo, and computational approaches to dissect molecular mechanisms and identify potential therapeutic targets. Recent tools, such as tumor organoids, single cell, and spatial transcriptomics, are changing the way we can model tumor microenvironment, allowing for a precise and dynamic understanding of tumor stroma or strokes. Cancer research faces microenvironment heterogeneity, and stromal-epithelial complexity.

The resection of which both in vitro and in a mouse model can reveal key molecular mechanisms amiable to therapeutic interventions. We have established a key role for the transcription factor STAT3 and its target genes in mediating the bronchogenic functions of cancer-associated fibroblasts in breast tumors. We'll take advantage of patient-derived scaffolds in order to validate the biological and clinical relevance of our findings in a human system, including the induction of drug resistance.

To begin, obtain the reagents and labware required for the experiment. Pipette 700 microliters of supplemented DMEM into each well of a 24-well plate. Place an eight-micrometer transwell insert into each well coated with a biologically active matrix for invasion assays or uncoated for migration assays.

Then, transfer the plate to a 37 degrees Celsius incubator to equilibrate. Now, obtain a 100-millimeter dish containing 4T1 cells pretreated for 48 hours with immortalized cancer-associated fibroblast conditioned medium. Pipette one milliliter of five-millimolar EDTA into the dish to detach the cells.

Then, add five milliliters of complete DMEM to the dish. Transfer the cell suspension into a 15-milliliter conical tube. Centrifuge the tube to pellet the cells.

Resuspend the pellet in supplemented DMEM. Count the cells using a Neubauer chamber. Pipette 100 microliters of the prepared cell suspension into the upper chamber of each transwell insert.

As a plating control, seed 100 microliters of the same cell suspension into a 24-well plate containing 500 microliters of complete medium. Label the upper side of each insert using a thin marker. Using tweezers, submerge the inserts in a 50-milliliter conical tube filled with PBS for washing.

Then, use a cotton swab to carefully clean the internal part of the transwell. Under a fume hood, place each insert into 700 microliters of 4%paraformaldehyde and incubate. Then, wash the inserts by placing them three times into 700 microliters of PBS.

After letting them dry for 10 minutes, incubate the inserts in 700 microliters of 0.1%crystal violet for 10 minutes at room temperature. Using tweezers, submerge the inserts three times in a 50-milliliter conical tube containing double distilled water. Dry the internal surface using a cotton swab.

Store samples at room temperature for up to three days before imaging. Using a phase contrast microscope. Capture one image with a 10X objective to visualize most of the well surface.

Acquire images from five independent areas of the membrane using a 20X objective. Add acetic acid to dissolve the crystal violet. For wells, add 100 microliters of 10%acetic acid.

Mix on an orbital shaker for 10 minutes at room temperature. For transwells, lay them with the lower surface facing up. Deposit 50 microliters of acetic acid on the surface and mix by pipetting until the dye dissolves.

Transfer 50 microliters of the dissolved crystal violet from each sample to a 96-well plate for spectrometric analysis At day 21 post-injection, in a fume hood, open the chest cavity of a euthanized tumor-injected BALCB/C mouse using surgical scissors. Remove the skin from the submandibular area and the salivary glands to expose the trachea clearly. Prepare a 22-gauge needle syringe filled with 4%paraformaldehyde.

Insert the needle into the trachea and direct it toward the lungs while clamping the trachea toward the head. Inject the solution slowly until the lungs swell, typically using around two milliliters of solution. Dissect the lungs and transfer them into a 15-milliliter tube containing 4%paraformaldehyde.

Then, gently invert the tube a few times. Incubate the lungs at four degrees Celsius for 24 hours to allow fixation before sectioning and staining. Access the MetaLCM application for the analysis of transcriptomic datasets of laser capture microdissected breast tumors.

To begin the analysis, select the features representing the conditions to be compared. Choose the comparison direction by selecting either upregulated or downregulated genes. When upregulated genes are selected, click Run to show genes with higher expression in condition one.

Now, select the p-value threshold for differential expressions. Press the Run button to execute the analysis each time a parameter is adjusted. To limit the analysis to a specific gene list, type gene symbols separated by commas or upload a CSV file with gene symbols in a single column.

Press Submit gene list to load the gene symbols into the analysis. Then, press Reset to analyze all genes. Save the resulting table of differentially expressed genes, which includes average log fold change, collapsed p-value, and detailed values for each dataset.

Treatment with conditioned medium from superactivated immortalized cancer-associated fibroblasts, or ICAFs, significantly enhanced 4T1 cell proliferation compared to control medium. ICAFs-conditioned medium doubled the migration capacity of 4T1 cells relative to control. A moderate, but significant, increase in invasion was observed in 4T1 cells treated with ICAFs-conditioned medium compared to control.

Co-injection of ICAFs with 4T1 cells in mice significantly increased tumor volume by day 10 compared to injection of 4T1 cells alone. Tumor weights were significantly higher in mice co-injected with ICAFs and 4T1 cells compared to those injected with 4T1 cells alone. The presence of ICAFs led to a substantial increase in lung metastases in mice, as shown by a higher percentage of nodule area per lung section.

Four STAT3 regulated genes, MMP13, MMP3, LGI2, and TIMP1 were consistently upregulated in the tumor stroma of human breast cancer samples compared to normal stroma.