Additional file 2 of Regulating lactate-related immunometabolism and EMT reversal for colorectal cancer liver metastases using shikonin targeted delivery
Creators: name: Long, Li
name: Xiong, Wei
name: Lin, Fenwang
name: Hou, Jiazhen
name: Chen, Guihua
name: Peng, Taoxing
name: He, Yihao
name: Wang, Rui
name: Xu, Qin
name: Huang, Yongzhuo
Publisher: name: figshare
Year: 2023
Subjects: subject: Immunology
subject: FOS: Clinical medicine

description: Supplementary Material 2: Fig. S1. The confocal images of CT26 cells and NCM460 cells. Fig. S2. Preliminary evaluation regulating EMT of SHK in a CRLM model. (A) and (B) The expression and grayscale analysis of E-cadherin of epithelial or mesenchymal markers in CT26 and NCM460 cells. (C) Expression of epithelial or mesenchymal markers in CT26 cells after treatment with free SHK. (D) The grayscale analysis of (C). (E) Therapeutic schedule. (F) The expression of E-cadherin of liver tissues in normal and tumor mice. (G) and (H) Expression and grayscale analysis of epithelial or mesenchymal markers of normal or tumor mice. (I) and (J) Western blot analyzed free SHK-regulated EMT in a CRLM model and grayscale analysis of EMT-related makers. (K) Grayscale analysis of Fig. 6G. (L) Grayscale analysis of Fig. 6H. Fig. S3. Flow cytometry gate diagram of CTLs (A) and MDSCs (B) in vitro. Fig. S4. LA inducing EMT depending on TGF/EGF in an EMT model of SW480 cells. (A) and (B) Expression and grayscale analysis of E-cadherin in colorectal cancer cells. (C) and (D) Western blot analysis of epithelial or mesenchymal markers in mesenchymal-like cells with or without TGFβ (10 ng/mL) and SB431542 (5 µM). (E) The LA production of mesenchymal-like cells and epithelial-like cells (TGFβ: 10 ng/mL). (F) and Western blot analysis of LA depended on pH-stimulated TGF/EGF inducing EMT in SW480 cells (LA: 20 mM; SB431542: 5 µM). (G) Grayscale analysis of (F). Fig. S5. The profiles of SHK@MPDA@HA nanosystem. (A–D) The photothermal performance of MPDA in vitro. The temperature changes curve with 808 NIR irradiation of CT26 subcutaneous tumor after treatment with SHK@MPDA (E) and SHK@MPDA@HA (F). The temperature changes curve (G) and image (H) of CT26 subcutaneous tumor with 808 NIR irradiation at 30 h. Fig. S6. Reversion of EMT and remodeling tumor immunodepression microenvironment in early liver metastasis of colorectal cancer. (A) Western blot analysis of epithelial or mesenchymal markers of liver tissues. (B) Grayscale analysis of (A). (C) The relative fold change of the MDSCs subset (CD11b+Gr-1+). (D) and (E)The relative fold change of the NK subset (CD49b+NK1.1+, CD49b+NK1.1+IFNγ+). (F) The relative fold change of the CT8+T cells (CD8+Ki67+) and (G and H) cytotoxicity CD8+ T cells (CD8+Granzyme B+ and CD8+IFNγ+). Fig. S7. Flow cytometry gate diagram of MDSCs (A), NK cells (B), and CTLs (C) in a mouse model with early liver metastasis of colorectal cancer. Fig. S8 The isolated tumor volume in a subcutaneous excision model at end point of experiment. Fig. S9. Flow cytometry gate diagram of MDSCs (A), NK cells (B), and CTLs (C) of CT26 subcutaneous tumor in situ recurrence model. Fig. S10 In vivo therapeutic efficacy of CT26 subcutaneous excision model. (A) Therapeutic schedule. (B) The tumor growth curve. (C) Statistical analysis of tumor weight after surgical of primary tumors. (D) The image of tumor volume of groups. (E) Statistical analysis of the isolated tumor volume of subcutaneous excision model at end point of experiment. (F–J) Individual tumor growth curves of PBS, MPDA, Anti-PD1, and SHK@HA-MPDA, and SHK@HA-MPDA&amp;Anti-PD1 groups. Fig. S11. (A–C) Preliminary safety evaluation