Additional file 2 of Phytogalactolipids activate humoral immunity against colorectal cancer
Creators: name: Lin, Han-Huei
name: Wu, Yi-Shin
name: Jian, Ting-Yan
name: Liao, Jia-Yun
name: Chang, Meng-Ting
name: Shyur, Lie-Fen
name: Lin, Yu-Ling
Publisher: name: figshare
Year: 2023
Subjects: subject: Immunology
subject: FOS: Clinical medicine

description: Additional file 2: Table S2. Effects of CRA on cytokines and chemokines expression in splenocytes isolated from CT26.CL25 tumor-bearing mice. Fig. S1. Effects of CRA on tumor growth in immunocompetent or B cell-depleted BALB/c mice. Panel A. Schematic representation of animal study with in vivo B cell depletion and CRA treatment. Immunocompetent or B cell-depleted BALB/c mice were inoculated subcutaneously with CT26.CL25 cells. When the average tumor mass reached ~80 mm3, the animals were treated with 50 mg/kg of CRA by oral injection every day. Panel B. B cell expression in tumor from CRA-treated immunocompetent or B cell-depleted mice. After treating with CRA, the expression of TIL-B cells (anti-B220, red) in the tumor was checked by IHC staining. Panel C. Anti-tumor effects of CRA in B cell-depleted BALB/c mice declined. *: P &lt; 0.05; **: P &lt; 0.01, compared to the control group. Mice with small tumors (&lt;1000 mm3) or without tumors were counted, respectively. Fig. S2. Effects of CRA on B cell differentiation in splenic B cells in vitro. Panel A. CRA did not affect IL-21 and IFN-γ expression in splenic B cells. Panel B. Cell population of plasmablasts (CD19+CD138+) and plasma cells (CD19- or low CD138+) in CRA-treated mouse splenic B cells were analyzed by flow cytometry. Panel C. The proportion of plasmablasts (CD19+CD138+) and plasma cells (CD19- or low CD138+). Mouse splenic B cells were treated with serial dilution of CRA for 72h. ns: no significant difference, compared to the control group (n=6). M: cell culture medium. Panel D. CRA did not affect IL-21 and IFN-γ expression in anti-CD40/IL-4 stimulated splenic B cells. Panel E. Cell population of plasmablasts (CD19+CD138+) and plasma cells (CD19- or low CD138+) in CRA and/or anti-CD40/IL-4-treated splenic B cells were analyzed by flow cytometry. Panel F. The proportion of plasmablasts (CD19+CD138+) and plasma cells (CD19- or low CD138+) in anti-CD40/IL-4 stimulated splenic B cells. Splenic B cells were pre-stimulated with 1 µg/mL of anti-CD40 antibody and 100 U/mL of mouse IL-4. *: P&lt;0.05; **: P&lt;0.01, compared to the control group. NC: splenic B cells without anti-CD40/IL-4 and CRA treatment. Fig. S3. Effects of IL-21 neutralization on tumor growth in CRA-treated CT26.CL25 tumor mice. Panel A. Schematic representation of animal studies of CRA treatment followed by serum IL-21 neutralization. Panel B. Serum IL-21 concentrations in CRA-treated mice after IL-21 neutralization. Panel C. Blockade of serum IL-21 in CT26.CL25 tumor mice declined CRA-induced tumor suppressive effect. CT26.CL25 tumor-bearing mice were orally administrated with 50 mg/kg of CRA every day and iv injected with 800 ng of neutralizing anti-IL21 antibody weekly. Tumor volumes were determined every 3 days. *: P &lt; 0.05, compared to the CRA group (n=5/group). Mice with small tumors (&lt;1000 mm3) or without tumors were counted, respectively. Fig. S4. Chemical profile of CRA analyzed by reverse phase HPLC. Two major compounds CRDG (retention time = 10.2) and CRG (retention time = 12.9) are observed in CRA. A reverse phase C18 analytical column and diode array detector (DAD) set at 210 nm, 254 nm, and 360 nm were used. Fig. S5. Effects of CRA on STAT3/cMaf/BCL6 pathway in colorectal cancer cells. Panel A. Effects of low dose of CRA on gene expression of STAT3, cMaf and BCL6 in mouse or human CRCcells. Mouse CT26.CL25 (while bars) and human HT-29 cells (gray bars) were treated with 1.5, 3, 6, or 12 μg/ml CRA (effective concentration for T cells) for 72 h. The gene expression of STAT3, cMaf and BCL6 was determined by qPCR. Panel B. The STAT3 expression and phosphorylation did not change after CRA treatment below 12 mg/ml. Panel C. Human HT-29 and COLO205 cells were treated high doses of CRA for 72 h. The protein expression of Stat3were determined by western blot.