Large scale organoid analysis

Abstract

Methods, systems, and software are provided for using organoid cultures, e.g., patient-derived tumor organoid cultures, to improve treatment predictions and outcomes.

1 - 117 . (canceled) 
     
     
         118 . A method of evaluating an effect of a therapeutic agent composition for a subject, the method comprising:
 A) exposing a plurality of tumor organoids with a therapeutic agent composition comprising one or more therapeutic agents, wherein the plurality of tumor organoids comprises at least five tumor organoids,   wherein the plurality of tumor organoids are derived from one or more cells of a tumor biopsy from a tumor of the subject, and   wherein the plurality of tumor organoids is exposed to the therapeutic agent composition;   B) exposing each of the plurality of tumor organoids to one or more fluorescent markers;   C) imaging the plurality of tumor organoids thereby forming a two-dimensional pixelated digital fluorescent image;   D) obtaining, using a computer system, a tumor organoid profile using numeric values for individual pixels within the two-dimensional pixelated digital fluorescent image, thereby obtaining a tumor organoid profile comprising numeric cell viability values for each of the at least five tumor organoids in the plurality of organoids; and   E) assessing the effect of the therapeutic agent composition on the plurality of tumor organoids based on the numeric cell viability values.   
     
     
         119 . The method of  claim 118 , wherein the one or more fluorescent markers comprises one or more cell death detection agents and a total cell detection agent. 
     
     
         120 . The method of  claim 118 , wherein the assessing step E) comprises determining a therapeutic agent composition dosage curve from the tumor organoid profile. 
     
     
         121 . The method of  claim 120 , wherein the method further comprises:
 F) assigning a treatment dosage of the therapeutic agent composition to the subject based on the therapeutic agent dosage curve.   
     
     
         122 . The method of  claim 118 , wherein the assessing step E) comprises measuring the fitness of cells in the plurality of tumor organoids from the numeric cell viability values, and correlating the measured fitness of the cells with a sensitivity to the therapeutic agent composition. 
     
     
         123 . The method of  claim 118 , wherein the obtaining step D) comprises determining the proportion of numeric cell viability values of the plurality of tumor organoids have greater than 1% cell viability after contact with the therapeutic agent composition. 
     
     
         124 . The method of  claim 123 , wherein the assessing step E) comprises determining a probability or a likelihood that the subject's tumor is resistant to the therapeutic agent composition at a particular concentration based on the proportion of numeric cell viability values of the plurality of tumor organoids that have greater than 1% cell viability after contact with the therapeutic agent composition at the particular concentration. 
     
     
         125 . The method of  claim 124 , wherein the tumor of the subject is determined to be likely resistant to the therapeutic agent composition at a particular concentration when 50% or more of the numeric cell viability values of the plurality of tumor organoids contacted with the therapeutic agent composition at the particular concentration are 50% or more viable. 
     
     
         126 . The method of  claim 124 , wherein the tumor of the subject is determined to be likely to be resistant to the therapeutic agent composition at a particular concentration when 1% or more of the numeric cell viability values of the plurality of tumor organoids contacted with the therapeutic agent composition at the particular concentration are 100% viable. 
     
     
         127 . The method of  claim 124 , wherein the tumor of the subject is designated as likely to be resistant to the therapeutic agent composition and the method further comprises recommending to the subject a monitoring frequency that is more frequent than a standard monitoring frequency. 
     
     
         128 . The method of  claim 124 , wherein the tumor of the subject is not designated as likely to be resistant to the therapeutic agent composition and the method further comprises recommending to the subject a standard monitoring frequency. 
     
     
         129 . The method of  claim 124 , the method further comprising analyzing the plurality of tumor organoids for one or more genetic variants associated with resistance to the therapeutic agent composition if the tumor of the subject is designated as likely to be resistant to the therapeutic agent composition. 
     
     
         130 . The method of  claim 124 , the method further comprising the step of isolating a set of tumor organoids within the plurality of tumor organoids having 0% cell viability and genetically sequencing the isolated set of tumor organoids to detect one or more genetic variants associated with at least 50% of the isolated tumor organoids, wherein one or more genetic variants associated with at least 50% of the isolated set of tumor organoids is determined to be associated with susceptibility to the therapeutic agent composition. 
     
     
         131 . The method of  claim 130 , wherein the isolating is performed using Ficoll-Paque isolation. 
     
     
         132 . The method of  claim 118  wherein the tumor organoid profile comprises a numeric cell viability value for each of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1×10 3 , 1×10 4 , 1×10 5 , or 1×10 6  tumor organoids in the plurality of organoids. 
     
     
         133 . The method of  claim 118 , wherein the therapeutic agent composition comprises two or more therapeutic agents. 
     
     
         134 . The method of  claim 118 , wherein the subject has a basal cell skin cancer, a squamous cancer, a breast cancer, a bladder cancer, a cervical cancer, a colon 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, prostate cancer, or a rectal cancer. 
     
     
         135 . The method of  claim 118 , further comprising administering a therapy to the subject based on step E). 
     
     
         136 . The method of  claim 118 , wherein the therapeutic agent composition consists of one of the following: a DNA damage response modulator, a cell cycle inhibitor, a metabolic inhibitor, a DNA synthesis inhibitor, an RNA synthesis inhibitor, a chemotherapy, an antimetabolite antineoplastic agent, an antimicrotubular antineoplastic agent, an antimetabolite antineoplastic agent, an antimitotic antineoplastic agent, an alkylating antineoplastic agent, a topoisomerase inhibitor, an apoptosis inducer inducers, a kinase inhibitor, a proteasome inhibitor, a PARP inhibitor, a MEK inhibitor, an Akt inhibitor, a an ATM/ATR inhibitor, a TGF-beta/Smad inhibitor, a HDAC inhibitor, or a retinoic acid receptor antagonist or agonist. 
     
     
         137 . The method of  claim 118 , wherein the assessing step E) comprises determining a sensitivity of the plurality of tumor organoids to the therapeutic agent composition, and wherein the method further comprises:
 F) administering the therapeutic agent composition to the subject when the therapeutic agent composition satisfies a sensitivity threshold, and administering a therapy that is not the therapeutic agent composition when the therapeutic agent composition does not satisfy the sensitivity threshold.   
     
     
         138 . The method of  claim 118 , wherein each pixel in the pixelated digital fluorescent image has a value between 0 and 2 16 . 
     
     
         139 . The method of  claim 118 , wherein the imaging C) comprises (i) taking a set of two or more two-dimensional pixelated digital fluorescent images of the plurality of tumor organoid, wherein each respective image in the set of two-dimensional pixelated digital fluorescent images of the plurality of tumor organoids is at a different Z-plane in a plurality of Z-planes, and
 (ii) projecting the set of two-dimensional pixelated digital fluorescent images into a single two-dimensional pixelated digital fluorescent image for the obtaining D) step.   
     
     
         140 . The method of  claim 118 , wherein the imaging C) produces a plurality of two-dimensional pixelated digital fluorescent images that are used by the obtaining D) to form the tumor organoid profile, wherein the one or more fluorescent markers is a plurality of fluorescent markers, and wherein the plurality of two-dimensional pixelated digital fluorescent images constitutes a multichannel image. 
     
     
         141 . The method of  claim 140 , wherein the exposing B) exposes each respective tumor organoid in the plurality of tumor organoids with a first fluorescent marker, a second fluorescent marker, and a third fluorescent marker, wherein the first fluorescent marker is Hoechst 33342, the second fluorescent marker is Caspase 3/7, and the third fluorescent marker is TO-PRO-3, and wherein the multichannel image comprises a first two-dimensional pixelated digital fluorescent image formed by exposure to a first channel for Hoechst 33342 fluorescence, a second two-dimensional pixelated digital fluorescent image formed by exposure to a second channel for Caspase 3/7 fluorescence, and a third two-dimensional pixelated digital fluorescent image formed by exposure to a third channel for TO-PRO-3 fluorescence. 
     
     
         142 . The method of  claim 140 , wherein the exposing B) exposes each respective tumor organoid in the plurality of tumor organoids to a first fluorescent marker, a second fluorescent marker, and a third fluorescent marker and wherein the multichannel image comprises a first two-dimensional pixelated digital fluorescent image formed by exposure to a first channel for blue fluorescence, a second two-dimensional pixelated digital fluorescent image formed by exposure to a second channel for green fluorescence, and a third two-dimensional pixelated digital fluorescent image formed by exposure to a third channel for red fluorescence.