Dimitra K. Georgiou

Supplementary Table S6. Comparison of DNA damage repair gene expression between SNU-C4 and T84 CRC cell lines using tabulated information from multiple datasets.

Supplementary Figure S6. Health status of PDX-bearing mice following MUC13-targeted RPT. Overall health status monitored as a function of body weight change in B8315- (A) and B8248- (B) bearing mice in 161Tb radiopharmaceutical therapy study. Body weight data are presented as mean±SD.

Supplementary Table S4. Differentially expressed gene analysis comparing high and low MUC13-expressing CRCs derived from Integromics microarray dataset.

Supplementary Table S3. Clinicopathological parameters of overall cohort (primary + liver metastasis, n=172).

Purpose:The high mortality associated with metastatic colorectal cancer (mCRC) illuminates an unmet need for innovative therapeutic modalities. Radiopharmaceutical therapy (RPT) offers a potent, molecular-scale approach for managing and treating cancers with distant micrometastases. However, its clinical use in mCRC remains an unrealized opportunity. We have therefore identified the transmembrane glycoprotein mucin 13 (MUC13) as a promising antigen for developing a targeted RPT and have undertaken preclinical evaluation of its potential by utilizing a monoclonal antibody tool representative of a future class of translatable therapeutics. Experimental Design:The immunoreactivity and transcriptome of patients with colorectal cancer (n = 72 primary, 100 liver metastases) were characterized using annotated clinical datasets. Preclinical assessment of MUC13 as an RPT target for mCRC was then performed in mice using a monoclonal MUC13-targeted antibody C14 labeled with either zirconium-89 for positron emission tomography (PET) measurement of mCRC-associated MUC13 density or terbium-161 for targeted RPT. Results:Strong MUC13 immunoreactivity was observed in ∼70% of mCRC and was inversely correlated with overall survival (P < 0.01). MUC13 levels were visualized by PET and agreed with immunohistochemically determined antigen presence. Furthermore, MUC13-targeted RPT exhibited in vivo proof-of-concept efficacy and enhanced survival in preclinical colorectal cancer models. Resulting imaging, therapeutic, and pathologic analyses elucidated relationships between target density, therapeutic outcome, and a potential genetic signature. Conclusions:MUC13-targeted RPT response was not only associated with radiopharmaceutical accumulation but also seemed to be balanced by DNA damage repair gene expression, suggesting a potential sensitivity signature that could complement a future clinical theranostic approach in MUC13-positive mCRC.

Supplementary Figure S1. A. Distribution of MUC13 mRNA expression (z-score) among Consensus Molecular Subtypes (CMS). The Kruskal-Wallis test revealed no significant differences among the groups (P=0.16). B. MUC13 expression values of 207 patients in the Integromics data set with z-score values above the cohort median (z=0.12) grouped as 'High', and with z-score values less than or equal to the median classified as ‘Low’. C. Total mutation burden (log10-transformed) compared between MUC13 High and Low groups, showing no significant difference (unpaired two-tailed Student’s t test). D. Violin plots showing differential expression levels of APC, SMAD4, and CTNNB1 between MUC13 High and Low groups. P-values were calculated using unpaired two-tailed Student’s t tests. E. Heatmap of enrichment scores for hallmark pathways differentially enriched between MUC13 High and Low groups. Rows represent hallmark pathways, and columns represent individual samples, clustered by hierarchical clustering.

Supplementary Table S2. Clinicopathological parameters of cohort of patients with CRC liver metastases.

Supplementary Table S1. Clinicopathological parameters of cohort of patients with primary CRC.

Supplementary Figure S4. Single dose 161Tb radiopharmaceutical therapy studies in CDX mouse models of CRC. Single dose therapy studies were performed in mice bearing T84 xenografts injected with 5.92 MBq (160 μCi) of [161Tb]Tb-DOTA-C14 (A, C, E) and in mice bearing SNU-C4 xenografts with either 3.70 or 7.40 MBq (100 or 200 μCi, respectively) of [161Tb]Tb-DOTA-C14 (B, D, F). A, B. Overall health status throughout the studies was monitored as a function of weight change. C, D. Tumor volume was measured over time to determine antitumor activity of 161Tb-labeled C14. Data are presented as mean values ± SEM. # One-way ANOVA with Dunnett's multiple comparisons test vs. [161Tb]Tb-DOTA-C14 160 μCi group (C), ¶ Unpaired Student’s t test, # One-way ANOVA with Dunnett's multiple comparisons test (D). E, F. Survival probability of each cohort. Statistical significance determined via Log-rank test.

Supplementary Table S5. Differential enrichment of cancer hallmark pathways between MUC13 High and Low groups