Generation and Characterization of Patient-Derived Head and Neck, Oral, and Esophageal Cancer Organoids

Abstract

Esophageal cancers comprise adenocarcinoma and squamous cell carcinoma, two distinct histologic subtypes. Both are difficult to treat and among the deadliest human malignancies. We describe protocols to initiate, grow, passage, and characterize patient-derived organoids (PDO) of esophageal cancers, as well as squamous cell carcinomas of oral/head-and-neck and anal origin. Formed rapidly (<14 days) from a single-cell suspension embedded in basement membrane matrix, esophageal cancer PDO recapitulate the histology of the original tumors. Additionally, we provide guidelines for morphological analyses and drug testing coupled with functional assessment of cell response to conventional chemotherapeutics and other pharmacological agents in concert with emerging automated imaging platforms. Predicting drug sensitivity and potential therapy resistance mechanisms in a moderate-to-high throughput manner, esophageal cancer PDO are highly translatable in personalized medicine for customized esophageal cancer treatments. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Generation of esophageal cancer PDO Basic Protocol 2: Propagation and cryopreservation of esophageal cancer PDO Basic Protocol 3: Imaged-based monitoring of organoid size and growth kinetics Basic Protocol 4: Harvesting esophageal cancer PDO for histological analyses Basic Protocol 5: PDO content analysis by flow cytometry Basic Protocol 6: Evaluation of drug response with determination of the half-inhibitory concentration (IC₅₀ ) Support Protocol: Production of RN in HEK293T cell conditioned medium.

Keywords: esophageal cancer; patient-derived organoids; personalized medicine.

Figure 1.. Workflow of PDO generation and characterizationc

An esophageal tumor fragment, procured via either endoscopy or surgery, is dissociated and filtered into single cell suspension. Cells are seeded into Matrigel and grown with tumor type-specific organoid culture medium. Resulting PDO are processed for subculture or cryopreservation, and subjected to morphological and functional assays coupled with pharmacological drug treatments.

Figure 2.. ESCC and EAC PDO morphological characteristicsc

Representative ESCC (A) and EAC (B) PDO images under phase contrast microscope and histologic characterization of PDO as well as corresponding original primary tumor tissues by Hematoxylin-Eosin (H&E) staining and immunohistochemistry. ESCC PDO comprise poorly differentiated squamous cell carcinoma cells featuring increased cell proliferation (Ki67), stabilization of tumor suppressor TP53 protein, and overexpression of SOX2, an oncogene essential in ESCC. EAC PDO feature high chromatin density along with focal luminal formations reminiscent of glandular structures compatible with adenocarcinoma as corroborated by nuclear expression (arrows) of caudal type homeobox 2 protein (CDX2). Note that TP53 was negative in the representative EAC POD and original primary tumor. Cancer cells within PDO recapitulate those in original tumors. Scale bar, 100 μm.

Figure 3.. IC₅₀ curve from drug-treated PDO

EAC PDO size were evaluated by Celigo Imaging Cytometer measuring the mean organoid size following 72 h-exposure to Cisplatin and Paclitaxel at indicated final concentrations. Organoid size was normalized by vehicle-treated control as 100%. IC₅₀ for Cisplatin and Paclitaxel was determined as 7.1 and 2.3 μM (with R squares of 0.5874 and 0.8652), respectively.

Figure 4.. Tools used for embedding of paraformaldehyde-fixed PDO

200-μL pipet tips are modified to make embedding tips and embedding bottom-less barrels (A). A polypropylene 1.7-mL tube rack, covered by a sheet of Parafilm M, is used as a scaffold to place embedding bottom-less barrel where fixed organoids will be cast in along with embedding gel (pre-heated Bacto-agar) (B). To liquefy embedding gel, an aliquot of 5-mL Bacto-agar will be microwaved for 1 min in a 150-mL beaker containing 100-mL water.