Tumor organoid culture compositions, systems, and methods

Abstract

Provided herein are novel organoid culture media, organoid culture systems, and methods of culturing tumor organoids using the subject organoid culture media. Also provided herein are tumor organoids developed using such organoid culture systems, methods for assessing the clonal diversity of the tumor organoids, and methods for using such tumor organoids, for example, for tumor modelling and drug development applications. In particular embodiments, the tumor organoid culture media provided herein is substantially free of R-spondins (e.g., R-spondin1).

1 .- 209 . (canceled) 
     
     
         210 . A method for culturing tumor organoids, the method comprising:
 a) culturing one or more tumor organoids in an organoid culture medium having less than 500 ng/mL R-spondins, wherein each respective tumor organoid in the one or more tumor organoids is derived from one or more cells of a tumor sample;   b) determining a reference genomic variant profile, wherein the reference genomic variant profile comprises for each respective genomic variant in a plurality of genomic variants identified in the tumor sample, a variant allele fraction for the respective genomic variant;   c) determining a first organoid genomic variant profile of the one or more tumor organoids, wherein the first organoid genomic variant profile comprises, for each respective genomic variant in a plurality of genomic variants identified in the one or more tumor organoids, a variant allele fraction for the respective genomic variant; and   d) confirming at least a 65% concordance in the clonal diversity of the one or more tumor organoids and the tumor sample by comparing each variant allele fraction for each respective genomic variant of the tumor sample to the corresponding variant allele fraction for the corresponding genomic variant of the one or more tumor organoids, across a plurality of genomic variants found in both the reference genomic profile and the first organoid genomic variant profile.   
     
     
         211 . The method of  claim 210 , wherein the tumor sample comprises biopsied material from a plurality of regions of a tumor from the subject. 
     
     
         212 . The method of  claim 210 , wherein the reference genomic variant profile is determined using nucleic acids obtained from the tumor sample, and the first organoid genomic variant profile is determined using nucleic acids obtained from the one or more tumor organoids. 
     
     
         213 . The method of  claim 210 , wherein the plurality of genomic variants identified in both the tumor sample and the one or more tumor organoids comprise a plurality of somatic variants. 
     
     
         214 . The method of  claim 213 , wherein the plurality of somatic variants identified in both the tumor sample and of the one or more tumor organoids comprise a single nucleotide variant. 
     
     
         215 . The method of  claim 213 , wherein the plurality of somatic variants identified in both the tumor sample and of the one or more tumor organoids comprise a point mutation or a nonsense mutation. 
     
     
         216 . The method of  claim 213 , wherein the plurality of somatic variants identified in both the tumor sample and of the one or more tumor organoids comprise a frame shift mutation. 
     
     
         217 . The method of  claim 213 , wherein the plurality of somatic variants identified in both the tumor sample and of the one or more tumor organoids comprise a deletion mutation, an insertion mutation, or a duplication mutation. 
     
     
         218 . The method of  claim 210 , wherein the plurality of genomic variants identified in both the tumor sample and of the one or more tumor organoids each comprise at least 20 genomic variants. 
     
     
         219 . The method of  claim 210 , wherein the tumor is an anal cancer, a basal cell skin cancer, a squamous cancer, a breast cancer, a bladder cancer, a cervical cancer, an endometrial cancer, a head and neck cancer, a hepatobiliary cancer, a kidney cancer, a gastric cancer, a lung cancer, a mesothelial cancer of the pleural cavity, a mesothelial cancer of the peritoneal cavity, an ovarian cancer, or a prostate cancer 
     
     
         220 . The method of  claim 210 , wherein in d), the confirming is of at least a 70% concordance in the clonal diversity of the one or more tumor organoids and the tumor sample. 
     
     
         221 . The method of  claim 210 , wherein in d), the confirming is of at least a 80% concordance in the clonal diversity of the one or more tumor organoids and the tumor sample. 
     
     
         222 . The method of  claim 210 , wherein in d), the confirming is of at least a 90% concordance in the clonal diversity of the one or more tumor organoids and the tumor sample. 
     
     
         223 . The method of  claim 210 , wherein in d), the confirming is of at least a 95% concordance in the clonal diversity of the one or more tumor organoids and the tumor sample. 
     
     
         224 . The method of  claim 210 , wherein the organoid culture medium comprise one or more growth factors selected from the following:
 a) Noggin,   b) EGF and Noggin, and   c) EGF, Noggin, FGF7 and FGF10.   
     
     
         225 . The method of  claim 224 , wherein the organoid culture medium further comprises a plurality of molecular inhibitors comprising a Rho kinase inhibitor, a transforming growth factor-beta inhibitor and a MAP kinase inhibitor. 
     
     
         226 . The method of  claim 210 , wherein the method further comprises:
 e) exposing the one or more tumor organoids to a therapeutic agent; and   f) after exposing one or more tumor organoids to the therapeutic agent, evaluating a property of the exposed tumor organoid cell line.   
     
     
         227 . The method of  claim 226 , wherein the evaluating in f) comprises:
 determining a second organoid genomic variant profile of the one or more tumor organoids after exposure of the one or more tumor organoids to the therapeutic agent, wherein the second organoid genomic variant profile comprises, for each respective genomic variant identified in a plurality of genomic variants identified in the tumor organoid cell line after exposure to the therapeutic agent, a variant allele fraction for the respective genomic variant; and   evaluating a difference or similarity between the first organoid genomic variant profile and the second organoid genomic variant profile.   
     
     
         228 . The method of  claim 227 , wherein the second organoid genomic variant profile is determined using nucleic acids isolated from the at least one tumor organoid in the tumor organoid cell line after exposure of the tumor organoid cell line to the therapeutic agent. 
     
     
         229 . The method of  claim 227 , wherein the second organoid genomic variant profile is determined using nucleic acids isolated from the organoid culture medium after exposure of the tumor organoid cell line to the therapeutic agent. 
     
     
         230 . The method of  claim 227 , wherein the evaluating the difference or similarity includes determining which somatic variants experienced a threshold level of change between the first organoid genomic variant profile and the second organoid genomic variant profile. 
     
     
         232 . The method of  claim 226 , wherein the method further comprises assigning a therapy to the subject based on a property of the one or more tumor organoids.