Effect of immunosuppression in miRNAs from extracellular vesicles of colorectal cancer and their influence on the pre-metastatic niche

Colorectal cancer (CRC) occurs with more aggressiveness in kidney transplant recipients compared to the general population. Immunosuppressive therapy plays a crucial role in the development of post-transplant malignancy. Concretely, cyclosporine A (CsA) has intrinsic pro-oncologic properties, while several studies report a regression of cancer after the introduction of rapamycin (RAPA). However, their effect on the extracellular vesicle (EV) content from CRC cell lines and their relevance in the pre-metastatic niche have not yet been studied. Here, we investigated the effect of RAPA and CsA in EV-miRNAs from metastatic and non-metastatic CRC cell lines and the role of relevant miRNAs transferred into a pre-metastatic niche model. EV-miRNA profiles showed a significant upregulation of miR-6127, miR-6746-5p, and miR-6787-5p under RAPA treatment compared to CsA and untreated conditions in metastatic cell lines that were not observed in non-metastatic cells. From gene expression analysis of transfected lung fibroblasts, we identified 22 shared downregulated genes mostly represented by the histone family involved in chromatin organization, DNA packaging, and cell cycle. These results suggest that EV-miR-6127, miR-6746-5p and miR-6787-5p could be a potential epigenetic mechanism induced by RAPA therapy in the regulation of the pre-metastatic niche of post-transplant colorectal cancer.

• Supplemental methods Viability and proliferation assays SW480 and HCT116 cells were seeded in a 96-well plate and treated with CsA (2, 5 and 10 µM) and RAPA (10,20,50 nM) or untreated for 24h. Viability and proliferation assays were performed with MTT assay (Sigma-Aldrich) and CyQuant assay kit (Molecular Probes, Invitrogen), respectively according to the manufacturer's instructions. (n=3 per group).
Subsequently, cells were analyzed by flow cytometry, using CANTO II (BD Biosciences). A total of 10,000 beads/events were acquired for each sample. All data were analyzed with FlowJo software (Tree Star).

Mandatory
• Generic term extracellular vesicle (EV): With demonstration of extracellular (no intact cells) and vesicular nature per these characterization (Section 4) and function (Section 5) guidelines OR • Generic term, e.g., extracellular particle (EP): no intact cells but MISEV guidelines not satisfied Encouraged (choose one) • Generic term extracellular vesicle (EV) + specification (size, density, other) • Specific term for subcellular origin: e.g., ectosome, microparticle, microvesicle (from plasma membrane), exosome (from endosomes), with demonstration of the subcellular origin • Other specific term: with definition of specific criteria

Tissue Culture Conditioned medium (CCM, Section 2-a)
General cell characterization (identity, passage, mycoplasma check…). Medium used before and during collection (additives, serum, other) • exact protocol for depletion of EVs/EPs from additives in collection medium • Nature and size of culture vessels, and volume of medium during conditioning A T175 flask with 15ml of medium was used during conditioning • specific culture conditions (treatment, % O2, coating, polarization…) before and during collection • Number of cells/ml or /surface area and % of live/dead cells at time of collection (or at time of seeding with estimation at time of collection) 5x10 6 cells/15ml were seeded in a T175 per condition with estimation at time of collection of ± 25x10 6 cells and ±97% of live cells. • Frequency and interval of CM harvest 24 h.

Storage and recovery (Section 2-d)
• Storage and recovery (e.g., thawing) of CCM, biofluid, or tissue before EV isolation (storage temperature, vessel, time; method of thawing or other sample preparation) The CCM was stored at 4˚C before starting the experiments. After 24 h, the recovered CCM was used at 4°C during sequential centrifugations. • Storage and recovery of EVs after isolation (temperature, vessel, time, additive(s)…) After EVs isolation, samples were used immediately or stored during only one night at 4°C for the following applications.

Experimental details of the method
• Centrifugation: reference number of tube(s), rotor(s), adjusted k factor(s) of each centrifugation step (= time+ speed+ rotor, volume/density of centrifugation conditions), temperature, brake settings Reference number of tubes: Polypropylene Centrifuge Tubes, Beckman Coulter 337986. Each tube contained 30ml of CCM. Rotor: SW32Ti Centrifugation steps: -800 g for 7 min at 4˚C -2,000 g for 12 min at 4˚C -Supernatants filtered through 0.1 μm pore filter -Samples ultracentrifuged (Optima L100XP, Beckman) at 100,000 g for 2 h at 4˚C -PBS washing step -Samples ultracentrifuged (Optima L100XP, Beckman) at 100,000 g for 2 h at 4˚C

Quantification (Table 2a, Section 4-a)
• Volume of fluid, and/or cell number, and/or tissue mass used to isolate EVs NTA 30 ml of CCM were used to isolate EVs for NTA • Global quantification by at least 2 methods: protein amount, particle number, lipid amount, expressed per volume of initial fluid or number of producing cells/mass of tissue • Ratio of the 2 quantification figures It has already been shown in the Figure 1 (f,g).

Global characterization (Section 4-b, Table 3)Citometria y los marcadores
• Transmembrane or GPI-anchored protein localized in cells at plasma membrane or endosomes The CD63 marker was observed by Flow Cytometry • Cytosolic protein with membrane-binding or -association capacity The CD9 and CD81 markers were observed by Flow Cytometry • Assessment of presence/absence of expected contaminants A total absence of contaminants was observed by Electron Microscopy (At least one each of the three categories above) • Presence of proteins associated with compartments other than plasma membrane or endosomes No presence of proteins was observed. • Presence of soluble secreted proteins and their likely transmembrane ligands • Topology of the relevant functional components (Section 4-d)

Single EV characterization (Section 4-c)
• Images of single EVs by wide-field and close-up: e.g. electron microscopy, scanning probe microscopy, super-resolution fluorescence microscopy