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. 2021 Dec 20;13(1):16. doi: 10.1038/s41419-021-04461-6

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — a Migratory ability of DLD-1 and HCT-116 upon co-culture with CM from CAFs or not was assessed by scratch wound healing assay. Left: Representative images of CM-treated DLD-1 and HCT-116 taken at the indicated time intervals. Right: Plot showing wound healing rate of DLD-1 and HCT-116 upon co-culture with CM from CAFs or not at 24 h. b Invasive capability of DLD-1 and HCT-116 with or without CM treatment was assessed by Boyden chamber trans-well assay. Up: Representative images of CM-treated DLD-1 and HCT-116 taken at the indicated time intervals (Scale bar, 50 μm). Down: Plot showing the percentage of invasive cells area upon co-culture with CM or not at 24 h. c Migratory capability of DLD1 and HCT-116 induced by CM in presence or absence of LRG1 was assessed by scratch wound healing assay. Hereafter, CM represents conditioned medium from CAF1 unless stated otherwise. Left: Representative images of CM-treated DLD-1 and HCT-116 with or without silencing of LRG1. Right: Plot showing statistical analysis of wound healing rate at 24 h. d Invasive capability of DLD1 and HCT-116 induced by CM in the presence or absence of LRG1 was assessed by Boyden chamber assay. Up: Representative images of CM-treated DLD-1 and HCT-116 with or without silencing of LRG1 (Scale bar, 50 μm). Down: Plot showing statistical analysis of the percentage of invasive cells area at 24 h. e Migrative ability of DLD-1 and HCT-116 with ectopic expression of LRG1 or not was analyzed by scratch wound healing assay. Up: Representative images of DLD-1 and HCT-116 with or without ectopic expression of LRG1. Down: Plot showing rate of wound healing in DLD1 and HCT-116 with or without LRG1 overexpression at 24 h. f Invasive capability of DLD-1 and HCT-116 with ectopic expression of LRG1 was assessed by Boyden chamber assay. Up: Representative images showing invaded DLD-1 and HCT-116 cells (Scale bar, 50 μm). Down: Graphs showing statistical analysis of the percentage of invasive cells area at 24 h. g Tumorsphere growth of DLD1 and HCT-116 induced by CM in the presence or absence of LRG1 was assessed by tumorsphere formation assay after 7 days culture. Up: Representative images showing spheroids of DLD-1 and HCT-116 cells (Scale bar, 50 μm). Down: Graphs showing statistical analysis of the number of spheroids with diameter >0.1 cm. h Left: Images showing the liver metastasis of DLD-1 cells which expressed indicated shRNAs and were cultured with control medium or conditioned medium from CAFs before transsplenic injection into nude mice. (The red arrows indicate metastatic nodules). Right: Plot showing the liver metastatic nodules per mouse in three groups. i Representative images showing H&E staining of liver tissue samples from the three groups as indicated in h (Scale bar, 50μm). Error bars represent SD; n = 3. **p < 0.01, ***p < 0.001, ****p < 0.0001.