IHC staining was performed on FFPE (formalin-fixed, paraffin-embedded) tissue samples under the standard protocol (ChemPartner). Samples were collected during passage of 10 CRC PDX models in Colo320DM BALB/c nude mice, administered hsBCL9CT-24 treatment over 21 days. Additional analyses were also performed on tumor samples from CT26 BALB/c syngeneic mice administered hsBCL9CT-24. Patient samples were collected from 10 adenocarcinoma patients (moderate, metastatic, and poor differentiation) according to the standard protocol for optimal selection of candidates tested in PDX mouse models.

Sections at 4 μm in thickness were prepared and dried in thermotanks before staining, followed by deparaffinization and rehydration via sequential washing with xylene, graded ethanol, and PBS. Samples were then subjected to high temperature–induced epitope retrieval in target retrieval solution [10 mM citrate buffer (pH 6.0)], 3% hydrogen peroxide blocking buffer (in PBS), and Ultra V Block. During protein blocking, slides were treated with 5% goat serum in 1× tris-buffered saline, 0.05% Tween 20 for 30 min at room temperature. Slides were incubated overnight at 4°C with 200 μl of the following primary Abs: β-cat (Abcam, ab32572, 1:1000), BCL9 (Abcam, ab37305, 1:200), c-Myc (Abcam, ab86356, 1:50), CD4 (Affymetrix eBioscience,149766, 1:100), CD8α (Affymetrix eBioscience, 140808, 1:200), CD44 (Abcam, ab157107, 1:500), FOXP3 (Spring Bioscience, M3974, 1:25), Ki67 (Abcam, ab15580, 1:200), vascular endothelial growth factor A (VEGFA) (5 μg/ml, LS-B10263, LS Biosciences), PD-1 (5.69 μg/ml, LS-B2519, LS Biosciences), or rabbit immunoglobulin G (IgG; Beyotime, A7016) as negative control. Slides were then incubated with HRP-conjugated secondary Abs for 30 min at room temperature: EnVision+/HRP Rabbit Ab (Dako 4003, lot 10069185), rabbit anti-FITC Ab conjugated with HRP (Hypoxyprobe HP2-100), or peroxidase-conjugated Affinipure rabbit anti-rat Ab (Jackson ImmunoResearch 312-035-003, lot 107245). Color development was performed with DAB Chromogen (Dako), and sections were then counterstained with Harris hematoxylin (Leica), differentiated with HCl-alcohol (70%) for 1 to 2 s, dehydrated, and mounted for detection (UltraVision Quanto Detection System).

Sample scoring was determined independently by two observers blinded to clinical data. Each specimen received a composite score assessing both the percentage of stained cells (0 to 100%) and staining intensity. The size of the positively stained compartment was estimated and classified on a five-point positive range score as follows: grade 0, 0 to 5%; grade 1, 6 to 25%; grade 2, 26 to 50%; grade 3, 51 to 75%; and grade 4, >75%. The positive extent score was determined as follows: 0, no staining; 1, light yellow; 2, brown; and 3, dark brown. The positive range score and positive extent score were subsequently added, yielding a composite quantitative score: <2, negative (−); 2 to 3, slight positive (+); 4 to 5, moderately positive (++); and 6 to 7, strongly positive (+++). The investigator was blinded to the group and labeling allocation during the experiment. Representative images were selected and depicted in figures at ×40 magnification as indicated (viewed via an inverted microscope: Olympus CKX31SF).

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