Identifying compounds modifying a cellular phenotype

Abstract

The present invention relates to a method and tools for extracting information on a compounds influence on a cellular phenotype. The method of the invention may be used as a very efficient procedure for testing the efficacy or resistance of single drugs or combinations of drugs on cells from individual patients. Thus, the methods may be useful for predicting efficacy of a drug on a given patient. The methods are also useful for testing of compounds for toxicity, identifying drug targets for known or novel compounds.

1 . A method of identifying a compound or a combination of compounds that modify a least one cellular phenotype, said method comprising the steps of:
 i) providing a library containing a plurality of library members, wherein each library member is a compound or a combinations of compounds;   ii) providing a suspension of cells which may acquire said cellular phenotype;   iii) providing a cell-compatible support, wherein the support reversible can change between a sol-state and a gel-state   iv) providing an array containing a plurality of spaces   v) adding said support, said library members and said suspension of cells to spaces of said array, wherein at least two different library members are added to two different spaces, and each space comprises a specific library member,   wherein addition of said support, said library members and said suspension of cells may be performed simultaneously or sequentially,   vi) incubating the array under conditions allowing maintenance and/or growth of the cells   vii) detecting the cellular phenotype in the cells,   viii) identifying library members modifying the cellular phenotype,   thereby identifying a compound or a combination of compounds modifying said cellular phenotype.   
     
     
         2 . A method of identifying a compound or a combination of compounds that modify a least one cellular phenotype, said method comprising the steps of:
 i) providing a library containing a plurality of library members, wherein each library member is a compound or a combinations of compounds;   ii) providing a suspension of cells which may acquire said cellular phenotype;   iii) providing a cell-compatible support, wherein the support reversible can change between a sol-state and a gel-state   iv) providing an array containing a plurality of spaces   v) adding said support in sol-state to the spaces of said array   vi) adding library members to the spaces of said array, wherein at least two different library members are added to two different spaces,   wherein steps v) and vi) may be performed simultaneously or sequentially in any order,   vii) bringing the support to the gel-state;   viii) contacting the spaces of said array with the suspension of cells, and   ix) bringing the support into the sol-state   wherein steps viii) and ix) may be performed simultaneously or sequentially in any order; and   x) bringing the support to the gel-state thereby entrapping cells in the support; and   xi) incubating the array under conditions allowing maintenance and/or growth of the cells   xii) detecting the cellular phenotype in the cells,   xiii) identifying library members modifying the cellular phenotype,   thereby identifying a compound or a combination of compounds modifying said cellular phenotype.   
     
     
         3 . The method according o any one of  claims 1  to  2 , wherein the support is capable of supporting growth of cells. 
     
     
         4 . The method according to any one of  claims 1  to  3 , wherein said support is a temperature reversible gel. 
     
     
         5 . The method according to any one of  claims 1  to  4 , wherein said support is a sol-gel with a sol-gel transition temperature in the range of 0 to 35° C. 
     
     
         6 . The method according to any one of the preceding claims wherein said support is a hydrogel. 
     
     
         7 . The method according to any one of the preceding claims, wherein the support is a hydrogel selected from the group consisting of gelatinous gels and, copolymers, wherein the copolymer for example may be pluronic lecithin organogels or alginate hydrogels. 
     
     
         8 . The method according to any one of the preceding claims, wherein the array is a container comprising a plurality of compartments, which are separated by physical barriers. 
     
     
         9 . The method according to any one of the preceding claims, wherein the array comprises a plurality of wells. 
     
     
         10 . The method according to any one of the preceding claims, wherein the array is a device containing wells, wherein the device can be mass-produced by injection moulding. 
     
     
         11 . The method according to any one of  claims 8  to  10 , wherein steps v) and vi) comprises adding the support and the library members to said compartments or wells, wherein at least two different library members are added to two different compartments or wells. 
     
     
         12 . The method according to any one of  claims 8  to  11 , wherein each compartment or well has a volume of at the most 50 μL, such as at the most 20 μL, for example in the range of 10 to 25 μL. 
     
     
         13 . The method according to any one of  claims 8  to  12 , wherein at least two compartments or wells are separated by a physical barrier having a thickness of at the most 2 mm. 
     
     
         14 . The method according to any one of  claims 1  to  7 , wherein said spaces are discrete spots of regions on said array. 
     
     
         15 . The method according to any one of the preceding claims, wherein the cells are mammalian cells. 
     
     
         16 . The method according to any one of the preceding claims, wherein the cells are cells resected from a human being. 
     
     
         17 . The method according to any one of the preceding claims, wherein the cell are primary cancer cells. 
     
     
         18 . The method according to any one of the preceding claims, wherein the cells are cancer cells provided in the form of spheroids. 
     
     
         19 . The method according to any one of the preceding claims, wherein the cellular phenotype is selected from the group consisting of cell proliferation and cell death. 
     
     
         20 . The method according to any one of  claims 16  to  19 , wherein the cells are cancer cells provided at least on part in the form of spheroids and the cellular phenotype is in vitro growth of said spheroids. 
     
     
         21 . The method according to any one of the preceding claims, wherein at least 10, such as at least 50, for example at least 90 library members are provided. 
     
     
         22 . The method according to any one of the preceding claims, wherein one or more library members are drug(s) useful in cancer treatment or a combination of drugs useful in cancer treatment. 
     
     
         23 . The method according to any one of the preceding claims, wherein all library members are drugs useful in cancer treatment or a combination of drugs useful in cancer treatment. 
     
     
         24 . An array comprising a plurality of wells,
 wherein one reservoir is connected to each of the wells; and   wherein each well comprises a cell-compatible support, wherein the support reversible can change between a sol-state and a gel-state; and   wherein at least 10 wells further comprises different library members; and   wherein each library member is a drug useful in treatment of cancer or a combination of drugs useful in treatment of cancer.   
     
     
         25 . The array according to  claim 24 , wherein at least 50, for example at least 90 wells comprises different library members. 
     
     
         26 . The array according to any one of  claims 24  to  25 , wherein each well has a volume of at the most 50 μL, such as at the most 20 μL, for example in the range of 10 to 25 μL. 
     
     
         27 . The array according to any one of  claims 24  to  26 , wherein the wells are separated from each other by a physical barrier having a thickness of at the most 2 mm. 
     
     
         28 . A method of identifying a compound or a combination of compounds that modify a least one cellular phenotype, said method comprising the steps of:
 i) providing an array comprising a plurality of spaces, wherein each space comprises a cell-compatible support, wherein the support reversible can change between a sol-state and a gel-state; and   wherein at least 2 spaces further comprises different library members; and   wherein each library member is a compound or a combination of compounds,   and wherein the support is in the gel-state   ii) providing a suspension of cells which may acquire said cellular phenotype;   iii) contacting the spaces of said array with the suspension of cells; and   iv) bringing the support into the sol-state   wherein steps iii) and iv) may be performed simultaneously or sequentially in any order; and   v) bringing the support to the gel-state thereby entrapping cells in the support; and   vi) incubating the array under conditions allowing maintenance and/or growth of the cells   vii) detecting the cellular phenotype in the cells,   viii) identifying library members modifying the cellular phenotype,   thereby identifying a compound or a combination of compounds modifying said cellular phenotype.   
     
     
         29 . The method according to  claim 28 , wherein the array is the array according to any one of  claims 24  to  27 . 
     
     
         30 . The method according to  claim 28 , wherein the method is performed according to any one of  claims 3  to  23 . 
     
     
         31 . A method of treatment of a clinical condition characterized by at least one cellular phenotype in an individual in need thereof, said method comprising the steps of:
 i) obtaining cells associated with said clinical condition from an individual suffering from said clinical condition;   ii) identifying a compound or a combination of compounds modifying said at least one cellular phenotype characterizing said clinical condition by the method according to any one of  claims 1  to  23  and  28  to  30 ,   iii) Administering a therapeutically effective amount of said compound or combination of compounds to said individual,   thereby treating said clinical condition.   
     
     
         32 . A method for predicting the efficacy of treatment of a clinical condition with each of a plurality of library members in an individual suffering from said clinical condition, wherein the clinical condition is characterized by at least one cellular phenotype, and wherein each library member is a compound or a combinations of compounds said method comprising the steps of:
 i) providing a sample comprising cells associated with said clinical condition from an individual suffering from said clinical condition,   ii) determining whether said library members modify said cellular phenotype by performing the method according to any one of  claims 1  to  23  and  28  to  30 ,   wherein modification of the cellular phenotype by the library members is indicative of efficacy of treatment of the clinical condition in said individual.   
     
     
         33 . The method according to any one of  claims 31  to  32 , wherein the cells are causative of said clinical condition. 
     
     
         34 . The method according to any one of  claims 31  to  33 , wherein the clinical condition is a neoplastic condition, and said cells are neoplastic cells. 
     
     
         35 . The method according to  claim 34 , wherein the clinical condition is cancer. 
     
     
         36 . The method according to  claim 35 , wherein said cells are primary cancer cells. 
     
     
         37 . The method according to any one of  claims 34  to  36 , wherein the cellular phenotype is in vitro cancer cell proliferation. 
     
     
         38 . The method according to any one of  claims 4  to  37 , wherein one or more library members are drugs useful in cancer treatment or a combination of drugs useful in cancer treatment. 
     
     
         39 . The method according to  claim 35 , wherein the method is a method for predicting the efficacy of treatment of a cancer in an individual in need thereof, the method comprising the steps of:
 i) providing an array comprising a plurality of spaces, wherein each space comprises a cell-compatible support, wherein the support reversible can change between a sol-state and a gel-state; and   wherein at least 2 spaces further comprises different library members; and   wherein each library member is a drug or a combination of drugs useful in the treatment of cancer,   and wherein the support is in the gel-state   ii) providing a suspension of cancer cells or cancer cells in spheroids obtained from said individual;   iii) bringing the support to the gel-state;   iv) contacting the spaces of said array with the suspension of cells; and   v) bringing the support into the sol-state   wherein steps iv) and v) may be performed simultaneously or sequentially in any order; and   vi) bringing the support to the gel-state thereby entrapping cells in the support; and   vii) incubating the array under conditions allowing growth of the cells   viii) detecting cancer cell proliferation and/or growth of cancer cell spheroids,   ix) identifying library members inhibiting proliferation of cancer cells and/or growth of cancer cell spheroids   wherein library members inhibiting proliferation of cancer cells and/or growth of cancer cell spheroids are predicted to be effective in treatment of cancer in said individual.   
     
     
         40 . A compound or a combination of compounds for treatment of a clinical condition in an individual in need thereof, wherein the clinical condition is associated with at least one cellular phenotype, and wherein the individual comprises cells associated with the clinical condition, in which said compound or combination of compounds are capable of modifying said cellular phenotype, wherein the compound or combination of compounds have been identified by the method according to any one of  claims 1  to  23  and  28  to  30 . 
     
     
         41 . The compound of combination of compounds according to  claim 40 , wherein the compound or combination of compounds have been identified by the method according to any one of  claims 1  to  23  and  28  to  30  using cells obtained from said individual. 
     
     
         42 . The compound of combination of compounds according to any one of  claims 40  to  41 , wherein the cells are causative of said clinical condition. 
     
     
         43 . The compound of combination of compounds according to any one of  claims 40  to  42 , wherein the clinical condition is a neoplastic condition, and said cells are neoplastic cells. 
     
     
         44 . The compound or combination of compounds according to any one of  claims 40  to  43 , wherein the clinical condition is cancer. 
     
     
         45 . The compound or combination of compounds according to  claim 44 , wherein said cells are primary cancer cells. 
     
     
         46 . The compound or combination of compounds according to any one of  claims 44  to  45 , wherein the cellular phenotype is in vitro cancer cell proliferation. 
     
     
         47 . The compound or combination of compounds according to any one of  claims 44  to  46 , wherein one or more library members are drug(s) useful in cancer treatment or a combination of drugs useful in cancer treatment. 
     
     
         48 . A kit-of-parts comprising the array according to any one of  claims 24  to  27  and information for performing the method according to any one of  claims 1  to  23  and  28  to  30 .