Fig 2: Histopathological characterization of patient-derived organoids and cell lines. See also Figures S1 and S2 in the Supporting Information. A) Overall study design. We successfully generated 43 PDOs and 23 PDCs (15 PDCs and 8 PDO culture-derived PDC cultures) corresponding to 12 different patients. We compared multiomics data from all subregional derivatives, and analyzed heterogenetic factors that might affect drug responses. B) Organoid architecture resembles primary tumor epithelium. Most organoids had one or more lumens, resembling the tubular structures of the primary tumor. Tumors devoid of a lumen give rise to compact organoids without the lumen. Hematoxylin-eosin (H&E) staining of formalin-fixed paraffin-embedded (FFPE) organoid sections revealed that tumor-derived organoids presented patient-specific heterogeneous morphologies. Organoid (n = 43) bright-field and H&E scale bar = 250 × 10-6 m. Tissue (n = 12) scale bar = 105 × 10-6 m. Cell line (n = 21) bright-field scale bar = 70 × 10-6 m. C) Cytokeratin 20 (CK20) and caudal type homeobox 2 (CDX2), as well as nuclear ß-catenin (CTNNB1) and KI-67 (MKI67), were compared between PDOs and parental tumors using immunohistochemistry. Also, the expressional patterns of MMR genes such as MutL homolog 1 (MLH1), MutS Homolog 2 (MSH2), MutS Homolog 6 (MSH6), and PMS1 Homolog 2 (PMS2) were compared between PDOs and matched tumors. PDOs retained similar levels of these markers as the parental tumors. Scale bar = 200 × 10-6 m.

Fig 3: Histopathological characterization of SIC patient-derived organoids and cell lines.See also Supplementary Fig. 1 and Table 1. a Overall study design. The cartoons representing gut, exome-seq, RNA-seq, methyl-seq, organoid engraftment, drug screening, and arrows were illustrated by BioRender. The H&E slides of multifocal tumor tissues and organoids as well as the images representing multi-omics layers were original pictures created by the authors. We successfully generated 6 PDOs and 12 PDCs (6 PDCs and 6 PDO culture-derived PDC cultures) corresponding to one synchronous intestinal cancer (SIC) patient. We compared multi-omics data from all sub-regional derivatives and analyzed heterogeneous factors that might affect drug responses, Scale bar = 200 µm. b The morphology of SIC organoids and cell lines derived from the patient SNU-6685. All PDOs had mixed morphology and retained both the inter-tumor and intra-patient heterogeneity. Established cell lines were composed of relatively uniformed clones compared to organoids, cellular structures were comprised of mono-layered epithelial cells displaying polygonal morphology and cell aggregates were formed in all of PDCs and PDOCs. Scale bar = 500 µm and insets are ×200. c Organoids resemble primary tumor epithelium in histological features. The protein expressions of MMR genes include MutL Homolog 1 (MLH1), MutS homolog 2 (MSH2), MutS Homolog 6 (MSH6), and PMS1 Homolog 2 (PMS2) in PDOs resembled their corresponding tumors, Scale bar = 200 µm.

Fig 4: The FAN1 N-terminal region (p.73-349) mediates its interaction with MLH1 and its effect on CAG stabilization activity(A) CoIP extracts from human HD iPSCs showing FAN1 interacts with MutLa components MLH1 and PMS2. Note that MSH3 is absent from the anti-FAN1 IP fraction (n = 3 biological replicates).(B) CoIP extracts from human HD lymphoblasts confirming FAN1 interacts with MLH1 (n = 3 biological replicates).(C) Pull-down assays using GFP-Trap beads in U2OS cells showing FAN1 interacts with MutL components, but not MutS components or PCNA (proliferating cell nuclear antigen). FAN1-/- cells act as a negative control, demonstrating specificity of the pull-down (n = 4 biological replicates).(D) CoIP of cortical extracts from mouse zQ175 at 6 months of age confirming FAN1 interacts with MLH1. Observations were also confirmed in R6/2 HD mice at 12 weeks of age (zQ175, n = 3 biological replicates; R6/2, n = 2 biological replicates).(E) Crosslinks identified between FAN1, MLH1, and PMS2 in unstimulated HEK293T cells and HD lymphoblasts. Grey parts on the proteins are structurally unsolved (no PDB structure available). Turquoise line, interprotein; purple line, intraprotein; green line, crosslinks close to the SPYF motif. See also Figure S1A and Table S1.(F) Schematic illustrating FAN1 constructs cloned into U2OS system. Locations of UBZ null (C44A/C47A) and nuclease null (D960A) mutations are also outlined. UBZ, ubiquitin-binding zinc-finger domain; SAP, SAF-A/B, Acinus and PIAS domain; TPR, tetratricopeptide repeat domain; VRR_NUC, virus-type replication-repair nuclease domain.(G) Pull-down using GFP-Trap beads in U2OS cells expressing GFP-FAN1 deletion constructs. FAN1?73–349 (highlighted in bold) did not interact with MLH1. Note that inactivation of UBZ or VRR_NUC domains (C44A/C47A and D960A mutants, respectively) does not affect FAN1-MLH1 interaction (n = 3 biological replicates).(H) CoIP extracts using aMLH1 antibody in U2OS cells showing N-terminal FAN11–349 is sufficient to interact with MLH1 (n = 4 biological replicates).(I) MMC viability curves in U2OS cells expressing FAN1 variants (mean ± SD) showing lower viability when FAN1 lacks an intact nuclease domain (n = 5–8 biological replicates; n = 3 technical replicates). See also Figure S1E.(J) CAG expansion rates in U2OS cells expressing truncated FAN1 constructs, including mutations within key functional domains (UBZ, C44A/C47A; VRR_NUC, D960A). Note that only FAN1?73–349 shows a higher expansion rate than cells expressing FAN1FL but does not equate to FAN1-/- (mean ± SEM, n = 2–5 biological replicates, n = 3–6 technical replicates, F(5,97) = 40.8, p < 0.001 by one-way ANOVA with false discovery rate [FDR] correction of 5%). ***p < 0.001; ns, non-significant.(K–M) Fragment analysis traces illustrating expansion of the exogenous HTT 118 CAG repeat in U2OS cells expressing FAN1 constructs over 6 weeks in culture (K) with time courses plotted (L and M). Note that cells expressing FAN1?73–349 (individual data points shown) expand at a rate between that of FAN1FL or FAN11–349 (individual data points shown) and FAN1-/- cells (mean ± SD, 95% confidence interval [CI] in shaded areas, n = 2–5 biological replicates, n = 3–6 technical replicates).