Creating Matched In vivo/In vitro Patient-Derived Model Pairs of PDX and PDX-Derived Organoids for Cancer Pharmacology Research

This protocol can be used to establish a matched library of paired patient-derived xenografts, or PDX, and corresponding patient-derived organoid lines. The advantage of this method is that it can be used to create organoids using PDX for in vitro screening, resulting in matched pairs of in vivo/in vitro models. Begin by harvesting the tumors and processing the tissue as described in the text manuscript.

After tissue digestion, pipette the tissue up and down with a five milliliter sterile plastic pipette. Then add 20 milliliters of AD+to the homogenate and filter it with a 100 micrometer cell strainer. Wash the filtrate twice with AD+then transfer it to a plastic tube and centrifuge it at 450 times G for five minutes.

Resuspend the cells in BME and keep them on ice. To prepare the organoid culture, transfer 200 microliters of the cell suspension to each well of a six-well plate and incubate the plate at 37 degrees Celsius for 30 minutes. Add two milliliters of organoid media to each well and image the representative drops under a microscope.

Maintain the organoid cultures at 37 degrees Celsius and 5%carbon dioxide with medium changes every three to four days and passage at a one-to-two ratio every seven days. After dissociating the organoids, run them through a 70 micrometer filter into a 50 milliliter plastic tube. Count the organoids under a microscope and resuspend them in BME on ice for a final concentration of 5%Add 50 microliters of the organoid suspension into each 384-well plate with a liquid dispenser for a seeding density of 200 CR2110 PDXOs per well.

Use the digital dispenser to add SN38 to each well in serial dilution. Create a plate map using the digital dispenser software tool. The treatments should include a negative control vehicle with 100%viability and positive control of five micromolar Staurosporine with 0%viability.

When finished, place the drug-treated 384-well plates back into 37 degree Celsius incubator. Under light microscopy, PDXO CR2110 shows typical cystic morphology demonstrating the similarity between PDX-derived organoids and patient-derived organoids under the same culture conditions. Histopathological examination by H&E staining reveals that the tissue structures and cell types of PDXO CR2110 are similar to the original PDX CR2110.

Genomic profile comparisons of PDX and PDXO demonstrate a 94.92%correlation of transcriptome expression and a 97.67%concordance of DNA mutations, suggesting an overall genomic similarity between this pair of models. Drug sensitivity assays were performed on PDXO CR2110 in 384-well plates. PDXO CR2110 was sensitive to irinotecan and resistant to cisplatin, consistent with PDX treatment results.

The matched pair of in vitro and in vivo models can compliment each other for in vitro screening and validation in vivo, improving the success rate of drug discovery and potentially reducing attrition rates in clinical development.