Precoated matrigel

Migration and Matrigel invasion assays were performed using 24-well insert, 8 μm pore size with or without pre-coated matrigel from Corning Inc., according to the manufacturer’s directions. Briefly, 1 × 10⁵ cells in 200 μL FBS-free DMEM medium were seeded into the upper portion of the chamber, while 900 μL DMEM medium plus 30% FBS was loaded into the lower side, which served as a chemo-attractant. After 48 h, the non-invasive cells were removed from the upper surface of the membrane with a cotton swab, cells penetrated to the underside of the membrane were fixed and stained with 0.1% crystal violet, and further counted in four random fields under a microscope.

Human iPSCs (kindly provided by Guangzhou Stem cell and Regenerative Laboratory, China) were cultured in Essential 8 (E8) medium (Stem Cell, Vancouver, BC, Canada) with pre-coated Matrigel (Corning, New York, NY, USA) as previously described [45 (link)]. COs were prepared as described by Lancaster et al. [46 (link)]. Briefly, iPSCs were in EB formation medium [46 (link)] for 7 days, and then transferred to the induction medium [46 (link)] for another 7 days. On the 15th day, organoids were started to expand by culturing in the expansion medium [46 (link)] in Matrigel, followed by culture in the maturation medium [46 (link)] on an orbital shaker (Wiggens, Straubenhardt, Germany; WOS-101SRC) from the 20th day. The medium was changed every 7 days afterwards. COs were assayed for immunostaining on the 60th, 80th, 100th, 120th, and 140th after iPSCs differentiation. The culture supernatant from mature COs, which exhibited stable neuronal phenotypes between 60 and 140 days [46 (link)], was collected for the preparation of OExo.

Transwell assay and invasion assay were applied using 24-well insert, 8 μm pore size with or without pre-coated matrigel from Corning Inc., according to the manufacturer’s directions. In brief, 500 μL RPMI 1640 medium supplemented with 30% FBS was loaded into the lower side of the Transwell chamber, while 1 × 10⁵ cells in 250 μL FBS-free RPMI 1640 medium were loaded into the upper side. Then 24 h later, cells penetrated to the underside of the membrane were fixed and stained, and further counted in 5 random fields under a microscope.
In order to screen out CRC subgroups with different metastatic ability, we recycled cells distributed in or out of the well, respectively. After five generations of migration assay and three generations of invasion assay subsequently, HM subtype derived from DLD-1 and HCT116 (DLD-1-HM, HCT116-HM, respectively), and LM subtype derived from DLD-1 (DLD-1-LM), were built.

Cells (5 × 10⁵/well) were maintained in six-well plates until they reached 100% confluence. A 10-µl pipette tip was applied to generate a wound, and the cell layer was then washed to remove detached cells. Next, the cells were incubated in serum-free RPMI 1640 at 37 °C for 24 h. The wound was photographed under a microscope at 0 h and 24 h, and the rate of closure was calculated with ImageJ software.
For the transwell assay, miR-375 -overexpressing or miR-375-silenced DU145 and PC-3 cells (5 × 10⁴) in serum-free RPMI 1640 were seeded into inner chambers with or without precoated Matrigel (Corning, New York, USA). RPMI 1640 containing 10% FBS was added to the outer chambers. After 16–24 h, the cells remaining in the inner chamber were removed with a cotton swab, and the cells migrated through the pores were fixed with 4% paraformaldehyde for 1 h. After the cells were stained with crystal violet for 1 hour, microscopic photographs were taken, and the number of migrated cells was counted.

Cell migration and invasion assays were conducted on 24-well Transwell cell culture chambers with 8-μm sized pores with or without precoated Matrigel (Corning, USA). GC cells were trypsinized and washed three times with PBS, and then 5 × 10⁴ cells were suspended in 500 μl of medium and added to the upper inserts; M1 or M2 macrophages were added to the lower inserts. For the control, 750 µl of medium with 10% FBS was placed in the lower chamber. In addition, GC cells were harvested after 24 h of coculture with supernatant or exosomes of M2 macrophages, suspended in 500 μl of FBS-free medium and added to the upper inserts, and 750 µl of medium with 10% FBS was placed in the lower chamber. After 24 h of incubation, the cells remaining in the upper chamber were removed, and the cells on the lower surface of the chamber were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet. At least five random microscopic fields (magnification ×200) were photographed, and the cells were counted. Three independent experiments were performed. For inhibition of exosome generation, macrophages were treated with culture media containing 10 μM GW4869 (Sigma).

The transwell invasion assay was performed in 24-well plates with a 6.5 mm insert transwell chamber with 8 μm polycarbonate membrane (Corning) pre-coated Matrigel (Corning). The single cell suspension was added into upper chamber with 5×10⁴ cells in 200 μL culture medium with 2% FBS, and 500 μL culture medium with 20% FBS were added into the lower chamber. After incubating in a hypoxic incubator with 1% O₂ and 5% CO₂ for 48 to 72 h, discarded the solution in the upper chamber and wiped the upper layer of the membrane. Move the chamber into 4% PFA to fix for 5 min. Stain the membrane with crystal violet for 5 min. Finally, we obtained the photographs on the microscope. All experiments were performed in triplicate.