Treatment was conducted in accordance with the standard protocol and regulations (The Association for Assessment and Accreditation of Laboratory Animal Care International) approval, and mice were routinely monitored for any effects of tumor growth and treatments on normal behavior such as mobility, visual estimation of food and water consumption, body weight gain/loss, eye/hair matting, and other abnormal effects. Tumor size and volume (V = 0.5ab2, where a and b are the long and short diameters of the tumor, respectively) were recorded every other day in two dimensions via caliper measurements. The T/C percent value was calculated by taking the ratio of the mean volume in treated and control groups on any given day.

Colo320DM colon cancer cells (ATCC) were cultured according to the standard protocol (5) at Viva Biotech and harvested for s.c. inoculation (5 × 106 cells in PBS) in the right flank region of BALB/c female nude mice (SLAC, SCXK2013-0018, certification 20130018040083) at 6 to 8 weeks of age. Mice without developing tumors were excluded before grouping. A total of 16 mice were separated into four randomized cohorts (n = 4), with treatment beginning 14 days after inoculation (mean tumor size of 156.78 mm3). These xenograft murine models were treated by i.v. injection with vehicle control (2.5% DMSO in sterile water) or hsBCL9CT-24 peptide (5, 10, and 15 mg/kg) with daily doses over 14 days. Tumor weight was measured at the end of study.

PDX murine models were created in NOD/SCID male mice according to the standard protocol (SLAC, SCXK2013-0018, certification 2013001812168) in two randomized arms (n = 8; Viva Biotech). CRC patient tumors selected for the xenograft model demonstrated high expression levels of β-cat, BCL9, c-Myc, and CD44 in IHC staining and were sectioned into 3 mm × 3 mm × 3 mm fragments before s.c. implantation in the right flank region. Treatment was administered 18 days following transplantation (mean tumor size of 134.77 mm3) via i.v. injection with vehicle control (4% ethanol, 8% Tween 80 in PBS) or hsBCL9CT-24 peptide (15 mg/kg), with i.p. daily doses over 31 days. Tumor mass was collected and fixed in 10% formalin at the conclusion of study for further analyses. The investigator was blinded to the group allocation during the experiment. Statistical analysis was determined by one-way analysis of variance (ANOVA) (GraphPad Prism), and differences between groups were considered significant when the P value was less than 0.05.

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