In this study, we demonstrate that MCs are enriched in BRAF mutant human CRC and mouse colon tumors, and secretory cells in human BRAF mutant CRC promote the migration of MCs in vitro. Additionally, we demonstrate that MCs promote EMT in CRC cells in an integrin-mediated, and contact- and calcium-dependent manner. We further find that AKT activation in both cell types is necessary for the promotion of EMT in CRC cells. Lastly, we demonstrate that molecules encoded by MCs are transferred into CRC cells. Altogether this work improves our understanding of how EMT can be induced in CRC cells while describing novel interactions between MC and CRC cells.

All cell lines were maintained in a humidified atmosphere with 5% CO2. HT-29 and SW403 cells were cultured in McCoys 5A media (Corning, #10-050-CV) and RPMI 1640 media (Corning, #10-040-CV), respectively, supplemented with 10% FBS (Corning, #35-015-CV). Both cell lines were purchased from the ATCC and tested for Mycoplasma using the Universal mycoplasma detection kit (ATCC, 30-1012 K) in March 2024. The human MC line LAD2 (generously provided by Dr. Dean Metcalfe at NIH/NIAID, MD, USA) was cultured as described previously [17, 18] in StemPro medium supplemented with 4 mM l-glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin and 100 ng/ml SCF (300-07, Peprotech). BMMCs were generated by differentiation of human bone marrow CD34+ cells (70002.2, StemCell Technologies). For differentiation, CD34+ cells were supplemented with 100 µg/ml recombinant SCF, 100 µg/ml recombinant IL6 (200-06, Peprotech), and 30 µg/ml recombinant IL3 (200-03, Peprotech) for the first week only. BMMCs were used after 4 weeks of differentiation. 817 BRAF mutant colon cancer organoids were derived from a patient-derived xenograft model obtained from the NCI Patient-Derived Models Repository and passaged and differentiated as described previously [7, 19] and as described in the Supplementary Methods. All cells used in experiments were passaged fewer than 15 times.

For the coculture system, cancer cells were seeded in media 24 h prior to the coculture. On the day of the coculture, cancer cells were washed once with PBS, and LAD2 cells were added in a 1:1 ratio. The coculture was incubated in complete StemPro media (free from SCF) for indicated time points. At the end of the incubation, media containing the LAD2 cells was carefully removed and centrifuged at 500 × g at 4 °C for 5 min and pellets were saved for downstream applications. Cancer cells were washed vigorously 3 times with PBS, scraped and centrifuged at 500 × g at 4 °C for 5 min. Pellets were saved for downstream applications. LAD2 cells were passaged one day prior to each experiment. For migration experiments, BMMCs and cancer cells were starved overnight in media lacking SCF or FBS, respectively. Reparixin (MedChemExpress, HY-15251), Calcium Ionophore (Sigma-Aldrich, C7522), BAPTA-AM (MedChemExpress, HY-100545), LY294002 (MedChemExpress, HY-10108), Borussertib (MedChemExpress, HY-122913), BIRT-377 (MedChemExpress, HY-110117), Lifitegrast (MedChemExpress, HY-19344) were solubilized in DMSO prior to treatment. Treatment dosages and durations are defined in the figure legends. For LY294002, BIRT-377, and Lifitegrast treatments, MCs were pretreated for 1 h prior to the direct coculture. Because these drugs are reversible, treatments were kept during the duration of the coculture.

Deidentified archived human samples were obtained after Institutional Review Board approval (5 BRAF mutant and 5 sex, age, and primary vs. metastatic matched non-BRAF-mutant CRC adenocarcinomas). Min and BLM mouse tumor tissue samples were obtained from a previous study [6]. All mouse experiments were covered under a protocol approved by the Indiana University Bloomington Animal Care and Use Committee in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care International. IHC was performed and scored as indicated in the Supplementary Methods. Sample size used for IHC analysis was based on available samples, not a power analysis.

Following tissue processing, single-cell suspensions were stained with a fixable viability dye, surface markers, and intracellular markers. For surface staining, cells were stained in FACS buffer containing CD117 (c-Kit) (Biolegend, 105808) and CD129 (IL9R) (Millipore/Sigma-Aldrich, MABF2304F) antibodies for 30-60 min at 4 °C. Following surface staining, cells were fixed using an IC fixation buffer (Invitrogen) for 20 min at 4 °C in the dark. For intracellular staining, cells were washed and stained in permeabilization buffer for 5 min and 60 min, respectively. Stained cells were washed 3×with FACS buffer and analyzed using an Attune flow cytometer. Data was processed and analyzed in Flowjo.

To produce cancer cell conditioned medium, empty vector (EV) or knockdown (KD) cells were grown to 100% confluency in standard medium. Medium was replaced with serum-free medium for 48 h. The conditioned medium was then collected, centrifuged at 500 × g for 5 min at 4 °C and used for migration assays.

Cancer cells were seeded on the membrane of the upper chamber of the transwell (8 µm PET membrane; Corning, 3422) in a 24-well culture plate. 24 h later, LAD2 cells were incubated with cancer cells in a 1:1 ratio for 12 h. At the end of the incubation, LAD2 cells were removed and cancer cells were washed 3 times with PBS. 20% FBS was then added to the lower chamber. 24 h later transwell inserts were stained using Hema 3 Stat Pack (Thermo Fisher Scientific, #123-869). Migration inserts were randomized before manual quantification and the outer 5% of the inserts were not included during quantification to reduce edge-effect bias. All images were taken on an EVOS FL Auto microscope.

All MC migration experiments were carried out in 24-well culture plates using transwells with 5 µm PET membranes (Corning, 3421). EV or KD cancer cell conditioned media was used as the chemoattractant with unconditioned media serving as control. BMMCs (5 ×10 cells/ml) were starved overnight, resuspended in control medium, and migrated overnight. Quantification of the migration was done as described previously (CytoSelect™ 24-Well Cell Migration Assay from Cell BioLabs, Inc protocol). Briefly, transwells were removed, migrated cells were lysed, and DNA was labeled using CyQUANT™ NF Cell Proliferation Assay kit. Fluorescence was read with a fluorescence plate reader at 480 nm/520 nm. Experiments were performed in triplicates and repeated at least 3 times. To generate the standard curve of relative fluorescence units (RFUs) as a function of cell number, known numbers of cells were added to separate wells and lysed. DNA was labeled using CyQUANT™ NF Cell Proliferation Assay kit, and the resulting fluorescence intensity values were plotted against cell number.

817 organoids were seeded in 50% Matrigel (Corning, 356234). 5 days later, LAD2 cells labeled with DiO (10 µg/ml) (Invitrogen, D275) for 20 min were washed and added to 817 organoids. The migration assay was stopped 24 h later. Wells were washed twice with PBS to remove LAD2 cells not in the organoid containing Matrigel domes. Images were acquired on an Olympus OSR spinning disk confocal system with IX83 inverted microscope with CellSense acquisition software. LAD2 cells present in the 817 organoid Matrigel dome were counted from different biological replicates in each condition (z-stack (20 µm step)).

Conditioned media from EV and ATOH1 KD cancer cells were collected (N = 3 per condition), centrifuged at 500 × g for 5 min at 4 °C, and analyzed for cytokine and chemokine levels using the human cytokine/chemokine 96-Plex Discovery Assay® Array (HD96) (Eve Technologies, Calgary, AB).

For Vimentin immunofluorescence, cancer cells were seeded on No 1.5 coverslip. The next day, cancer cells were labeled with Vybrant™ DiI Cell-Labeling Solution (Invitrogen, V22885) per manufacturer's instructions and incubated with LAD2 cells in 1:1 ratio for 3 h. Then, cancer cells were washed vigorously 3 times with PBS and stained with anti-Vimentin (CST #5741, 1:50) according to manufacturer's protocol. Images were acquired on a Leica STERLLARIS 8 Falcon scanning confocal system with MDi8-inverted microscope with LASX software (Leica Microsystems). All the images were taken at 63X magnification, 1.2 A water immersion at room temperature, and processed using ImageJ to remove background signal. To quantify green fluorescence, ROIs of each cell were measured in both conditions. The integrated density (IntDen) values were used as a readout of intensity. At least 6 pictures per condition were used. The quantification was repeated with an independent replicate.

Cancer cells were seeded on Poly-D-lysine coated Nunc™ Glass Bottom Dishes (Thermo Fischer Scientific, 150680). The next day, unlabeled or DiO (10 µg/ml) (Invitrogen, D275) labeled LAD2 cells or BMMCs were incubated with cancer cells in HEPES Tyrode's buffer immediately upon imaging. Images were acquired on an Olympus OSR spinning disk confocal system with IX83 inverted microscope with CellSense acquisition software. All the images were taken at 40X or 60X magnification, 0.95 A water immersion and 1.3 A silicon immersion, respectively, at 37 °C and 5% CO, and processed using ImageJ.

Total RNA was isolated from cell pellets using the RNeasy mini kit (Qiagen #74104) according to the manufacturer's protocol. The Maxima first strand cDNA synthesis kit (Thermo Fisher #K1642) was used to synthesize cDNA. For quantitative reverse transcription PCR, cDNA was amplified using gene-specific primers and FastStart Essential DNA Green Master (Roche #06402712001) [19]. Cq values of genes of interest were normalized to housekeeping gene RHOA expression. Primer sequences are listed in the Supplementary Methods.

See the Supplementary Methods for details on shRNAs and plasmids used. Lentivirus was generated as described previously [19].

5 × 10 MCs were resuspended in 200 µl of fresh SP34 media and 50 µl of Vimentin-Flag (Vim-Flag) viral suspension with 8 µg/ml polybrene in 12-well plates. Cells were spun at 1500 × g for 90 min at 30 °C. Fresh media was added to transduced cells. The next day, cells were resuspended in fresh media and were used for the experiments.

Statistical analyses for qPCR and functional assays were performed using Graphpad Prism 10. All variances were tested for similarity and found to be similar between groups. Specific statistical tests used are indicated in the figure legends. In general, experiments were performed with three independent biological replicates and repeated multiple times, with most experiments being repeated three times. A sample size of three biological replicates was chosen to assure adequate experimental power based on preliminary experiments to determine variability. All samples were included in the analyses. qRT-PCR data from HT-29 cells in coculture with LAD2 cells was performed N = 3 independent times (with 3 independent biological replicates each). qRT-PCR data from SW403 in coculture with LAD2 cells and HT-29 cells in coculture with BMMCs were performed N = 1 or N = 2 independent times (with 3 independent biological replicates each). For western blots, representative images are presented with the number of independent biological replicates indicated in the figure legend. All bands for a given experiment were obtained from the same membrane unless otherwise stated.