An organoid library of salivary gland tumors reveals subtype-specific characteristics and biomarkers

Abstract

Background Salivary gland tumors (SGTs) include a large group of rare neoplasms in the head and neck region, and the heterogeneous and overlapping features among the subtypes frequently make diagnostic difficulties. There is an urgent need to understand the cellular mechanisms underlying the heterogeneity and overlap among the subtypes, and explore the subtype-specific diagnostic biomarkers. Methods The tumor tissue and the adjacent normal tissue from the 6 most common types of SGTs were processed for organoid culture which only maintained tumor epithelial cells. Organoids were histologically evaluated based on phenotype markers, followed by transcriptional profiling using RNA-sequencing. The transcriptomic similarities and differences among the subtypes were analyzed by subtype consensus clustering and hierarchical clustering. Furthermore, by comparative transcriptional analysis for these 6 types of SGTs and the matched organoids, the potential diagnostic biomarkers from tumor epithelium were identified, in which two selected biomarkers were evaluated by qPCR and confirmed by immunohistochemistry staining using a tissue microarray. Results We generated a biobank of patient-derived organoids (PDOs) with 6 subtypes of SGTs, including 21 benign and 24 malignant SGTs. The PDOs recapitulated the morphological and transcriptional characteristics of the parental tumors. The overlap in the cell types and the heterogenous growth patterns were observed in the different subtypes of organoids. Comparing the bulk tissues, the cluster analysis of the PDOs remarkably revealed the epithelial characteristics, and visualized the intrinsic relationship among these subtypes. Finally, the exclusive biomarkers for the 6 most common types of SGTs were uncovered by comparative analysis, and PTP4A1 was demonstrated as a useful diagnostic biomarker for mucoepidermoid carcinoma. Conclusions We established the first organoid biobank with multiple subtypes of SGTs. PDOs of SGTs recapitulate the morphological and transcriptional characteristics of the original tumors, which uncovers subtype-specific biomarkers and reveals the molecular distance among the subtype of SGTs.

TGFβ‐induced EN1 promotes tumor budding of adenoid cystic carcinoma in patient‐derived organoid model

“…Because the protein level of EN1 in SACC‐83 and SACC‐LM was very low and hardly detected, we established organoids from the surgically resected specimens, including ACC, BCA and NSG tissues (Figure S2A). The organoids of ACC and BCA grew slightly slower in the Matrigel, compared to the organoids established from the adjacent NSG (Figure S2B), which is consistent with our recent report and other report 7,26 . The ductal and basal cell markers CK5 and p63, and the luminal cell markers CK8 and AQP5 were stained by co‐IF (Figure S2C).…”

“…The organoid culture system was established following the protocol 7 . Briefly, the tissues were trimmed into 1–3 mm 3 pieces and incubated with 1× Dispase (Roche, IN) at 37°C for 0.5–1 h with intermittent agitation.…”

“…After dissociation, the cell pellets were passed through 100 μm cell strainers (BD Falcon, CA), centrifuged at 1000 rpm/min for 5 min and then the pellet was embedded in the Matrigel (Corning, NY) and cultured at 37°C, 5% CO 2 humidity environment. The medium was changed every 2–3 days, and the detailed information was written in our recent article 7 …”

“…The histological staining of organoids followed the methods 7,19 . Shortly, organoids were fixed with 4% paraformaldehyde for 2 min and then removed into the mold.…”

Effect of HER2‐targeted therapy on PDX and PDX‐derived organoids generated from HER2‐positive salivary duct carcinoma

“…Despite these limitations, we believe that our results demonstrate its significance as a preclinical model for evaluating the therapeutic efficacy both in vitro and in vivo for patients with HER2‐positive SDC. Furthermore, as Wang et al recently reported their organoid library of salivary gland tumor PDOs including 21 benign tumors and 24 SGCs, 34 larger SGC PDO biobanks would contribute to the establishment of an effective preclinical model for personalized medicine in patients with SGC.…”