Figure 5. Leflunomide treatment enhances survival and function of TTC7A-deficiency patient-colonoids.

(A) Personalized medicine approach to evaluate drug efficacy in human TTC7A-deficiency using patient-derived colonoids. Endoscopic biopsies were obtained from healthy control and TTC7A-deficiency (E71K and L304R compound heterozygous, TTC7A^mut) patients and cultured into colonoids, which were then tested with leflunomide. (B) Leflunomide improves the survival of human colonoids derived from TTC7A-deficiency patients (TTC7A^mut). Example images of control (TTC7A^+/+) and TTC7A-deficient (TTC7A^mut) colonoids with and without leflunomide (10 μM) treatment at 2 and 6 days and grown in the absence of ROCK inhibitor (Y27632). Red arrows indicate examples of dead colonoids. (C) Summary graph showing the percentage of dead TTC7A^mut colonoids +/− leflunomide treatment. TTC7A^mut and TTC7A^+/+ colonoids were cultured +/− ROCK inhibitor (Y27632) or with leflunomide and viability was assessed at day 2 and 6. TTC7A^mut colonoids grown without ROCK inhibitor have significantly increased death, which is reversed following leflunomide treatment. One-way ANOVA with post-hoc test (Tukey-Kramer), *p<0.05. **p<0.01. (D) Immunofluorescence staining of TTC7A^+/+ and TTC7A^mut colonoids with villin (green) marking the apical brush border and cytokeratin 20 (CK20, red) marking basal epithelial structure and DAPI (blue, nuclei). TTC7A^+/+ colonoids show normal polarity with defined basal CK20 staining. TTC7A^mut colonoids treated with ROCK inhibitor show grossly normal staining with mild cytological atypia. TTC7A^mut colonoids without ROCK inhibitor show normal sidedness but abnormal polarity with cytological atypia and basal structural abnormalities which is improved in leflunomide treated colonoids. Quantification of abnormal polarity as assessed by the presence of multiple lumens in colonoids. Percentage of colonoids with multiple lumens in TTC7A-deficient colonoids with and without leflunomide (10 μM). Healthy control colonoids did not have multiple lumens (data not shown). Two tailed t-test, *p<0.05. (E-F) TTC7A-deficient colonoids (TTC7A^mut) have a reduced swelling response to forskolin stimulation. Example images showing reduced swelling in TTC7A^mut compared to TTC7A^mut treated with leflunomide (10 μM). Inset panel show magnified images with red arrows indicating example colonoids pre and post swelling. Summary data showing significantly increased swelling in leflunomide (10 μM) treated TTC7A^mut colonoids. Two tailed t-test, **p<0.01.