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

Summary

A method is described to create organoids using patient-derived xenografts (PDX) for in vitro screening, resulting in matched pairs of in vivo/in vitro models. PDX tumors were harvested/processed into small pieces mechanically or enzymatically, followed by the Clevers’ method to grow tumor organoids that were passaged, cryopreserved and characterized against the original PDX.

Abstract

Patient-derived tumor xenografts (PDXs) are considered the most predictive preclinical models, largely believed to be driven by cancer stem cells (CSC) for conventional cancer drug evaluation. A large library of PDXs is reflective of the diversity of patient populations and thus enables population based preclinical trials (“Phase II-like mouse clinical trials”); however, PDX have practical limitations of low throughput, high costs and long duration. Tumor organoids, also being patient-derived CSC-driven models, can be considered as the in vitro equivalent of PDX, overcoming certain PDX limitations for dealing with large libraries of organoids or compounds. This study describes a method to create PDX-derived organoids (PDXO), thus resulting in paired models for in vitro and in vivo pharmacology research. Subcutaneously-transplanted PDX-CR2110 tumors were collected from tumor-bearing mice when the tumors reached 200-800 mm³, per an approved autopsy procedure, followed by removal of the adjacent non-tumor tissues and dissociation into small tumor fragments. The small tumor fragments were washed and passed through a 100 µm cell strainer to remove the debris. Cell clusters were collected and suspended in basement membrane extract (BME) solution and plated in a 6-well plate as a solid droplet with surrounding liquid media for growth in a CO₂ incubator. Organoid growth was monitored twice weekly under light microscopy and recorded by photography, followed by liquid medium change 2 or 3 times a week. The grown organoids were further passaged (7 days later) at a 1:2 ratio by disrupting the BME embedded organoids using mechanical shearing, aided by addition of trypsin and the addition of 10 µM Y-27632. Organoids were cryopreserved in cryo-tubes for long-term storage, after release from BME by centrifugation, and also sampled (e.g., DNA, RNA and FFPE block) for further characterization.

Introduction

Cancers are a collection of diverse genetic and immunological disorders. Successful development of effective treatments is highly dependent on experimental models that effectively predict clinical outcomes. Large libraries of well-characterized patient-derived xenografts (PDXs) have long been viewed as the translational in vivo system of choice to test chemo- and/or targeted therapies due to their ability to recapitulate patient tumor characteristics, heterogeneity and patient drug response¹, thus enabling Phase II-like mouse clinical trials to improve clinical success^2,3. PDXs....

Protocol

All the protocols and amendment(s) or procedures involving the care and use of animals were reviewed and approved by the Crown Bioscience Institutional Animal Care and Use Committee (IACUC) prior to conducting the studies. The care and use of animals was conducted in accordance with AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care) International guidelines as reported in the Guide for the Care and Use of Laboratory Animals, National Research Council (2011). All animal experimental procedures.......

Discussion

The preliminary data for PDX-/PDXO-CR2110 in this report supports the biological equivalence between PDX and its derivative, PDXO, with regards to genomics, histopathology and pharmacology, since both models represent the disease forms from the original CSC of patient. Both models are patient-derived disease models, potentially predictive of the clinical response of patients^10,11,12,21. The mat.......

Materials

Name	Company	Catalog Number	Comments
Advanced DMEM/F12	Life Technologies	12634028	Base medium
DMEM	Hyclone	SH30243.01	Washing medium
Collagenese type II	Invitrogen	17101015	Digest tumor
Matrigel	Corning	356231	Organoid culture matrix (Basement Membrane Extract, growth factor reduced)
N-Ac	Sigma	A9165	Organoid culture medium
A83-01	Tocris	2939	Organoid culture medium
B27	Life Technologies	17504044	Organoid culture medium
EGF	Peprotech	AF-100-15	Organoid culture medium
Noggin	Peprotech	120-10C	Organoid culture medium
Nicotinamide	Sigma	N0636	Organoid culture medium
SB202190	Sigma	S7076	Organoid culture medium
Gastrin	Sigma	G9145	Organoid culture medium
Rspondin	Peprotech	120-38-1000	Organoid culture medium
L-glutamine	Life Technologies	35050038	Organoid culture medium
Hepes	Life Technologies	15630056	Organoid culture medium
penicillin-streptomycin	Life Technologies	15140122	Organoid culture medium
Y-27632	Abmole	M1817	Organoid culture medium
Dispase	Life Technologies	17105041	Screening assay
CellTiter-Glo 3D	Promega	G9683	Screening assay (luminescent ATP indicator)
Multidrop dispenser	Thermo Fisher	Multidrop combi	Plating organoids/CellTiter-Glo 3D addition
Digital dispener	Tecan	D300e	Compound addition
Envision Plate reader	Perkin Elmer	2104	Luminescence reading
Balb/c nude mice	Beijing HFK Bio-Technology Co
RNAeasy Mini kit	Qiagen	74104	tRNA purification kit
DNAeasy Blood & Tissue Kit	Qiagen	69506	DNA purification kit
Histogel	Thermo Fisher	HG-4000-012	Organoid embedding