Murine Slap inactivation induces colonic epithelial hyperplasia.

(A) Constitutive Slap deletion. Top: Hematoxylin and eosin (H&E) staining of transverse colon sections. Crypt thickness was measured and quantified as mean ± SEM from 25-40 crypts per mouse (n = 6 female mice per group). Middle: Immunohistochemistry (IHC) for the proliferation marker Ki67, with quantification of Ki67-positive colonic epithelial cells (CECs). (mean ± SEM from 20-40 crypts per mouse; n = 5-6 mice per group). Bottom: RNAscope analysis of Lgr5 to identify colonic stem cells, combined with E-cadherin immunofluorescence to delineate crypt architecture. Lgr5 RNA particles were quantified and are shown as mean ± SEM from 20-40 crypts per mouse (n = 5-6 mice per group). ***p<0.001, ****p<0.0001 Mann–Whitney test. (B) Inducible epithelial-specific Slap deletion (villin-CreERT2). Top: H&E staining of transverse colon sections with quantification of crypt thickness (mean ± SEM from 25-40 crypts per mouse, n = 6 mice/group). Middle: IHC for Ki67 with quantification of Ki67-positive CECs (mean ± SEM from 20-40 crypts per mouse; n = 5-6 mice per group). Bottom: RNAscope analysis of Lgr5 combined with E-cadherin immunofluorescence; Lgr5 RNA particles were quantified (mean ± SEM from 20-40 crypts per mouse, n = 5-7 mice per group). Mice were analyzed at 3 months of age, 10 days after tamoxifen induction. ***p<0.001, ****p<0.0001 Mann–Whitney test.

Intestinal Slap inactivation enhances colonic organoid development through SFK activation.

(A) Slap-dependent colonic organoid development. Representative images of colon-derived organoids from Slap f/f and Slap f/f Villin-CreERT2 mice cultured in Matrigel for 2 days. Quantification of organoid number and area is shown as mean ± SEM from 100-200 organoids per mouse (n = 8 mice per group). ****p<0.0001 Mann–Whitney test. (B) Intestinal Slap deletion increases SFK activity and global protein tyrosine phosphorylation in colonic crypts. Representative immunoblots (right) and quantification (left) of phospho-SFK (pSRC) and total phospho-tyrosine levels in lysates from isolated colonic crypts of the indicated genotypes. Data are presented as mean ± SEM from n = 4-5 mice per group. *p<0.05; **p<0.001 t test. (C) Slap-dependent colonic organoid expansion requires SFK activity. Representative images and quantification of colon-derived organoids from Slap f/f and Slap f/f Villin-CreERT2 mice cultured for 2 days in the presence or absence of the SRC-family kinase inhibitor eCF506 (100 nM). Data are shown as mean ± SEM from 100-200 organoids per mouse, n = 4 mice per group. ***p<0.001, ****p<0.0001 Mann–Whitney test.

Inducible Slap intestinal inactivation drives colon tumorigenesis and promotes SFK-dependent tumoroid growth.

(A) Experimental workflow of AOM/DSS-induced colon tumorigenesis. Mice received tamoxifen, followed 10 days later by a single azoxymethane (AOM) injection to induce mutagenesis and one cycle of dextran sodium sulfate (DSS) administered for 7 consecutive days. A second AOM injection was then performed, followed by two additional DSS cycles (7 days each). Mice were sacrificed at day 80. (B) Epithelial Slap loss accelerates colon tumor development. Left, representative H&E-stained sections showing transformed regions. Right, quantification of epithelial lesion area (mean ± SEM; n = 11-15 mice per group. Statistical analysis is shown (Mann–Whitney test). (C) Slap depletion increases SFK activation in colonic tumors. Left, representative immunohistochemistry for phosphorylated SFK (pSRC) in transformed epithelium. Right, quantification of p-SFK–positive cells per mm² (mean ± SEM; n = 7-9 mice per group; ****p < 0.0001, Mann–Whitney test). (D) Tumoroids derived from Slap f/f and Slap f/f Villin-CreERT2 mice were cultured in 3D Matrigel in the presence or absence of SRC inhibitor (SRCi) and imaged at day 3. Tumoroid area (µm²) was quantified following SRCi treatment (mean ± SEM; 150–250 organoids per mouse, n = 4 mice per group; ns: p>0.05; ****p < 0.0001, Mann–Whitney test).

Slap targets EphB2 to restrict SFK proliferative signaling in CEC.

(A) Intestinal Slap deletion increases EphB2 protein levels in colonic crypts. Left, quantification and right, representative immunoblot of EphB2 protein levels in isolated colonic crypt lysates from the indicated mouse strains (mean ± SEM; n = 4-5 mice; *p < 0.05, t test). (B) SLAP–EPHB2 interaction in HEK293T cells. Co-immunoprecipitation of the indicated SLAP-FLAG constructs (wild-type, WT; or SH3*SH2* mutant, SLAP mut) and EPHB2-MYC constructs (wild-type, WT; or Y596F/Y602F mutant, YF) transfected into HEK293T cells. Expression levels of SLAP and EPHB2 and relative quantification of SLAP-EPHB2 interaction are shown (representative example of three independent experiments). (C) Slap-dependent colon organoid expansion requires EphB2 activity. Representative images and quantification of colon-derived organoids from Slap f/f and Slap f/f Villin-CreERT2 mice treated with the indicated EPH inhibitors (EPHB2i: 200 nM, pan-EPH2i: 100 nM, or DMSO control) and analyzed at day 2 (mean ± SEM; 150-200 organoids per mouse; n = 4 mice per group; ns: p>0.05; ****p < 0.0001, Mann–Whitney test).

Slap inactivation increases goblet cell number and SFK activity in the colon.

(A) X-Gal staining of Slap expression (left) and IF for Slap (red), nuclei (blue), and β-catenin (green) (right). (B) Slap loss increases goblet cell number and SFK activity. Top: Alcian blue staining and quantification. Bottom: IHC for pSRC and quantification (mean ± SEM; 25-40 crypts from 5-7 female mice; ****p<0.0001, Mann–Whitney). (C) Inducible Slap inactivation increases goblet cells. Top: IF after 10 days of tamoxifen. Bottom: Alcian blue staining and quantification (mean ± SEM; 25-40 crypts from 6 female mice; ****p<0.0001, Mann–Whitney).

SLAP suppresses human CRC stem cell properties.

(A) SLAP overexpression reduces ALDH activity in SLAP-low HT29 and SW620 cells (representative FACS plots and quantification; mean ± SEM; n=3; **p<0.01, ****p<0.0001; unpaired t-test).(B) SLAP overexpression decreases tumoroid formation in HT29 and SW620 cells (representative images and quantification 25-50 organoids/condition; mean ± SEM; n=3; ****p<0.0001; Mann–Whitney).

SLAP interacts with EPHB2 and reduces its levels in HT29 cells.

(A) EPHB2 expression and association with SLAP in CRC cells (transcript and protein; mean ± SEM; n=3-5; ns>0.05; *p<0.05; t-test). (B) EPHB2i (400 nM) reduces EPHB2 activity (i.e. pTyr level) in HEK293T cells. (C) Model of SLAP regulation of SFK signaling in CSCs to maintain colonic epithelial homeostasis by limiting RTK activity.