BACKGROUND: Biomarkers play critical roles in personalized medicine and are used to screen patients who may respond substantially to target therapies. The expression levels of folate receptor 1 (FOLR1) and transient receptor potential vanilloid 6 (TRPV6) in normal tissues are low and do not overlap. Elevated expression levels of these genes have been reported in certain cancer types. However, it is unknown whether both genes are upregulated spontaneously. We conducted a study to evaluate the expression levels of both FOLR1 and TRPV6 across different tumor types in The Cancer Genome Atlas (TCGA) and CrownBio patient-derived xenograft (PDX) databases. METHODS: Bioinformatics analyses of the CrownBio PDX and TCGA gene expression databases were performed using R software and the UAB online bioinformatics tool. RESULTS: We found the expression of both FOLR1 and TRPV6 in 24/45 (53.33%) cases of cholangial carcinoma (CCA), 650/1212 (53.63%) cases of breast cancer (BRCA), 43/196 (21.94%) cases of esophageal cancer (EC), and 237/381 (62.20%) cases of uterine corpus endometrial carcinoma (UCEC) in the database from TCGA. Based on our studies using PDX models, the co-expression of both FOLR1 and TRPV6 occurred in 90% of CCA (18/20) cases, 89.29% of BRCA (25/28) cases, 70.83% of EC (34/48) cases, and 92.86% in UCEC (13/14) cases in the CrownBio database. An in-depth analysis further revealed that tumors expressing both FOLR1 and TRPV6 were enriched in CCA, triple-negative breast cancer (TNBC), high-grade EC (HGEC), and uterine serous carcinoma (USC). PDXs that had a FOLR1+/TRPV6+ status responded well to novel investigational drugs targeting these receptors. CONCLUSION: CCA, TNBC, HGEC, and USC express high levels of both FOLR1 and TRPV6. A bispecific agent targeting FOLR1/TRPV6 with a companion diagnostic test detecting FORL1/TRPV6 expression might be an efficient and practical way to treat these tumors. (Am J Transl Med 2020. 4:95-108).