Methods for co-cultivating genotoxic bacteria with organoids

Abstract

The present invention relates to a method for co-cultivating bacteria and organoids, in particular genotoxic bacteria and organoids.The present invention also relates to a method for generating organoids with mutation signatures.The present invention also relates to the use of co-cultures and organoids, for example in methods for testing the effect of candidate compounds, for modeling host-pathogen interactions, or for identifying compounds suitable for treating bacterial infections and / or cancer. TIFF2025510180000002.tif98170

Description

[Technical field]

[0001] The present invention is in the field of co-cultivation of genotoxic bacteria with organoids, and in particular, in the field of generating organoids with cancer mutation signatures by co-cultivation of genotoxic bacteria with organoids. [Background technology]

[0002] Organoids are a promising tool for studying human physiology in vitro. Organoids are self-organized epithelial cell structures with physiological properties similar to those of their in vivo tissues. They are widely used to model aspects of cancer initiation and progression. Thus, stem cell-derived organoids provide an advanced model for studying human development and disease.

[0003] The gut microbiome has long been implicated in colorectal cancer (CRC) tumorigenesis. Various bacterial species have been reported to be enriched in feces and biopsy samples from CRC patients, including genotoxic strains of Escherichia coli (E. coli). The genomes of these genotoxic bacteria harbor a polyketide nonribosomal peptide synthase operon (pks) responsible for the production of the genotoxic agent colibactin, which induces DNA double-strand breaks, chromosomal aberrations, and cell cycle arrest at the G2 / M phase [1].

[0004] Microinjection of colibactin-producing pks+ E. coli into the lumen of human intestinal organoids resulted in the emergence of two simultaneous mutational signatures identified in a subset of CRC patients, demonstrating that pks+ E. coli plays a causative role in the development of CRC [2]. However, the scalability of the microinjection model is limited, creating a bottleneck in screening preventive therapies for these patients. Thus, there is a need in the art for improved methods for co-culturing bacteria and organoids that can produce organoids with mutational signatures, such as cancer mutational signatures. Summary of the Invention

[0005] The present invention provides a method for co-cultivating genotoxic bacteria with organoids, the method comprising a first step comprising culturing organoids or organoid fragments with genotoxic bacteria, a second step comprising culturing organoids or organoid fragments with genotoxic bacteria, where bacterial growth is restricted with an antibiotic, and a third step comprising killing the genotoxic bacteria and allowing further growth of the organoids or organoid fragments.

[0006] The present invention also provides a method for generating organoids having a mutational signature, the method comprising a first step comprising culturing the organoids or organoid fragments with genotoxic bacteria, a second step comprising culturing the organoids or organoid fragments with genotoxic bacteria, in which bacterial growth is restricted with an antibiotic, and a third step comprising killing the genotoxic bacteria and allowing further growth of the organoids or organoid fragments, in which organoids or organoid fragments having a mutational signature are generated.

[0007] The present invention also provides a method for testing the effect of a candidate compound, comprising exposing a co-culture of genotoxic bacteria from the method of the present invention with an organoid to one of a library of candidate compounds, evaluating the organoids in the co-culture for any effect, and identifying the candidate molecule that causes the effect as a potential drug.

[0008] The invention also provides organoids having mutational signatures generated by the methods of the invention and the use of these organoids in modeling host-pathogen interactions and / or to identify compounds suitable for treating bacterial infections and / or cancer. [Brief description of the drawings]

[0009] [Figure 1A] Schematic diagram of the method for co-cultivating genotoxic bacteria with organoids, consisting of three stages: first stage (no antibiotics), second stage (low antibiotic concentration), and third stage (high antibiotic concentration). [Figure 1B] Immunofluorescence of organoid fragments after 4 h co-culture with pks E. coli MOI 100 in the first step. Stained with DAPI (blue, stains nuclei), phalloidin (red, stains actin filaments) and bacteria (green). DAPI staining shows the presence of nuclei in the organoids. Phalloidin dye stains the apical side of the organoid fragment. The apical surface of the organoid fragment is shown to be in contact with bacteria stained in green. [Figure 1C] Distribution of organoid single cells derived from co-culture of genotoxic bacteria with organoids as a function of yH2AX levels at different time points obtained by flow cytometry. [Figure 1D] Representative images of organoids at the end of the recovery period. MOI0=[Bact]1, MOI10=[Bact]2, MOI50=[Bact]3, MOI100=[Bact]4. [Figure 2A] Schematic representation of the method for co-culturing genotoxic bacteria with organoids, consisting of two phases - the first phase (co-culture I) and the third phase (recovery). 0% MG = zero Matrigel in the culture. [Figure 2B] Organoids after recovery periods in the absence of bacteria (MOI 0), in the presence of colibactin-producing bacteria (WT MOI 500) and in the presence of colibactin deficiency (ΔClbP MOI 500) were stained with DAPI (blue, recognizing cell nuclei) and yH2AX stain (top row, red), as well as yH2AX stain only (bottom row, red) and imaged using immunofluorescence. yH2AX staining indicates where DNA damage has occurred in the organoid. The bottom panel shows the presence of DNA damage in the organoid and the top panel shows the location of this DNA damage relative to the nucleus of the organoid. [Figure 2C]Example immunofluorescence measurements of yH2AX and DAPI signals in nuclear regions to obtain the average yH2AX / DAPI nuclear intensity. [Figure 2D] Mean yH2AX / DAPI nuclear intensity of organoids in the absence of bacteria (MOI0), in the presence of colibactin-producing bacteria (WT MOI500), and in the presence of colibactin-deficient bacteria (ΔClbP MOI500) as measured by immunofluorescence after the recovery period. [Figure 3-1] (A) Schematic diagram of the method for co-culturing genotoxic bacteria with organoids, consisting of two stages: a first stage (co-culture I) in antibiotic-free suspension and a second stage (co-culture II) on 70% Matrigel (MG) with low antibiotic concentration (50 μg / ml gentamicin). [Figure 3-2] (B) In this co-culture (I) of Figure 3A, organoid fragments surviving after 3 h of co-culture with pks+ E. coli (WT and ΔClbP) in suspension (first stage). [Figure 3-3] (C) Organoids after recovery periods in the absence of bacteria (MOI 0), in the presence of colibactin-producing bacteria (WT MOI 300), and in the presence of colibactin-deficient bacteria (ΔClbP MOI 300) were stained with DAPI (recognizing cell nuclei, blue) and yH2AX stain (top row, red), as well as yH2AX stain only (bottom row, red) and imaged using immunofluorescence (IF). yH2AX staining indicates where DNA damage has occurred in the organoid. The right panel shows the presence of DNA damage in the organoid, and the left panel shows the location of this DNA damage relative to the nucleus of the organoid. [Diagram 3-4] (D) Nuclear intensity of yH2AX / DAPI after treatment with colibactin-producing bacteria (WT MOI50, WT MOI100, WT MOI200, and WT MOI300) or colibactin-deficient (ΔClbP MOI50, MOI100, MOI200, and MOI300). [Figure 3-5] See description of Figure 3-4.

[0010] Methods for co-cultivating bacteria and organoids The present invention provides a method for co-cultivating bacteria and organoids, comprising a first step comprising culturing organoids or organoid fragments with genotoxic bacteria, a second step comprising culturing organoids or organoid fragments with genotoxic bacteria, wherein bacterial growth is restricted with antibiotics, and a third step comprising killing genotoxic bacteria and allowing further growth of organoids or organoid fragments.In some embodiments, the method produces organoids or organoid fragments with mutation signatures.Each step can be carried out on organoids, organoid fragments, or a combination of organoids and organoid fragments.During any step, particularly in the second or third step, the organoid fragments can naturally form one or more organoids.The organoids or organoid fragments produced by these methods can be useful as screening platforms for identifying new drugs or testing drug candidates, or as models for identifying new biomarkers, particularly in the context of cancer. Additionally, the method of co-culturing bacteria with organoids can be used to test the effects of candidate compounds.

[0011] The first stage of co-culture involves culturing organoid or organoid fragment with genotoxic bacteria.In this first stage, the bacterial growth in the medium is typically unlimited.Without wishing to be bound by any particular theory, this unlimited bacterial growth causes "acute damage" to the DNA of organoid or organoid fragment.

[0012] In the second stage of co-culture, the growth of genotoxic bacteria in co-culture is restricted by using antibiotics.Therefore, the amount of bacteria in the second stage of co-culture is reduced compared with the first stage.In the second stage, organoid or organoid fragment is exposed to a lower rate of DNA damage, which is referred to herein as " persistent damage ".

[0013] In the third step, the bacteria in the co-culture are killed, for example using a high concentration of antibiotics, and the organoids or organoid fragments are further cultured. This step allows the recovery of the organoids or organoid fragments in the absence of genotoxic bacteria.

[0014] In some embodiments, the present invention provides a method for co-cultivating bacteria and organoids, which includes only the first and second steps. In this embodiment, the method for co-cultivating genotoxic bacteria and organoids includes (a) culturing organoids or organoid fragments with genotoxic bacteria (first step); and (b) culturing organoids or organoid fragments with genotoxic bacteria (second step), where bacterial growth is restricted with antibiotics. Without wishing to be bound by any particular theory, it is believed that the combination of an acute stage of DNA damage followed by a sustained period of DNA damage produces cancer mutation signature. The acute damage stage introduces a large number of mutations into organoids, but cannot be sustained for a long period of time, otherwise the organoids will die. It is believed that by co-cultivating organoids in two stages, a short stage of acute DNA damage and a longer stage of sustained DNA damage, this method can produce organoids with cancer mutation signature without killing the organoids. Exposing organoids to genotoxic compounds for extended periods of time may be useful to ensure that the organoids produced have a cancer mutation signature that faithfully represents, for example, a mutation signature that may arise in vivo.

[0015] In some embodiments, the present invention provides a method for co-cultivating bacteria and organoids (known as a rapid assay), which includes only the first and third steps. In some embodiments, the method for co-cultivating genotoxic bacteria and organoids includes (a) culturing organoids or organoid fragments with genotoxic bacteria (first step), and (b) killing the genotoxic bacteria and allowing further growth of the organoids (third step). The above method is particularly useful as a screening platform for drugs, such as anticancer drugs, because its rapid speed is suitable for medium to high throughput screening methods.

[0016] In some embodiments, the present invention provides a method for co-culturing bacteria with organoids, which includes only the second and third steps. In some embodiments, the method for co-culturing genotoxic bacteria with organoids includes (a) culturing organoids or organoid fragments with genotoxic bacteria (bacterial growth is limited by antibiotics) (second step), and (b) killing the genotoxic bacteria and allowing further growth of the organoids (third step). Without wishing to be bound by any particular theory, it is believed that cancer mutation signatures can be acquired after a long period of low-level DNA damage. In patients, the concentration of genotoxic compounds produced by bacteria in their microbiome is low. Thus, the patient's cells, such as cells of the gastrointestinal tract or lungs, are exposed to low concentrations of genotoxic compounds for a long period of time. This version of the method aims to reproduce this long-term, low-level exposure to genotoxic compounds that can give rise to certain types of cancer, such as colorectal cancer. This method is particularly useful for screening novel biomarkers, such as cancer biomarkers.

[0017] In some embodiments, the present invention provides a method for co-cultivating bacteria and organoids, which includes only the first step, the second step, and the third step.In some embodiments, the method for co-cultivating genotoxic bacteria and organoids includes: (a) culturing organoids or organoid fragments with genotoxic bacteria (first step); (b) culturing organoids or organoid fragments with genotoxic bacteria (bacterial growth is restricted by antibiotics) (second step); and (c) killing genotoxic bacteria and allowing further growth of organoids (third step).Without wishing to be bound by any particular theory, it is believed that the combination of an acute stage of DNA damage, followed by a stage of persistent DNA damage, and then a recovery stage increases the production of organoids with mutation signatures.The recovery stage ensures that organoids can recover from the DNA damage that has occurred and repair any of the DNA breaks that have occurred. During this process of repair and restoration, the organoids generate mutational signatures that may resemble those observed in cancer patients, making this method particularly useful for screening novel biomarkers, such as cancer biomarkers.

[0018] In some embodiments, the steps in the method are repeated at least two times, at least three times, at least four times, or at least five times. In a preferred embodiment, the steps in the method are repeated at least five times. Repetition is particularly useful in long-term exposure models (or any method that includes a second step of "persistent damage"), since the purpose of long-term exposure models is to faithfully replicate DNA damage that may occur in vivo. Without wishing to be bound by any particular theory, patients who develop cancer are usually exposed to genotoxic compounds over a period of time, thereby allowing the system to generate organoids with mutation signatures that resemble cancer mutation signatures, such as colorectal cancer, by repeating steps. For example, Pleguezuelos-Manzano, et al. demonstrated that repeated exposure of human intestinal organoids to genotoxic E. coli by repeated luminal injections over a period of five months results in mutation signatures that are detected in a subset of cancer genomes [2]. In addition, patients are usually exposed to genotoxic compounds produced by the microbiome for long periods of time. Therefore, organoids repeatedly exposed to genotoxic bacteria are more likely to result in mutational signatures seen in cancer patients.

[0019] The first, second, and / or third steps may include culturing in the presence of an extracellular matrix, such as Matrigel or a basement membrane extract, hi some embodiments, the second and / or third steps include culturing in the presence of an extracellular matrix, such as Matrigel or a basement membrane extract.

[0020] The first, second, and / or third steps can be performed in suspension culture. This allows the cultured organoids to be rapidly used in downstream processes such as immunofluorescence or flow cytometry assays. Furthermore, it is known in the art that colibactin genotoxicity is inhibited when bacteria and mammalian cells are separated by a porous membrane, indicating that colibactin genotoxicity is contact-dependent [3]. Without wishing to be bound by any particular theory, it is believed that Matrigel may act as a physical barrier between both cell types and partially prevent colibactin-dependent genotoxicity. Therefore, performing the first and second steps in suspension is preferred as it allows direct contact between bacteria and organoids or organoid fragments. Thus, in some embodiments, the first step is performed in suspension culture. In some embodiments, the second step is performed in suspension culture.

[0021] In some embodiments, the method for co-cultivating genotoxic bacteria with organoids does not include injecting genotoxic bacteria into the lumen of organoids.The first step, the second step, and / or the third step may not include injecting bacteria into the lumen of organoids.As detailed below, the method of the present invention includes co-cultivating genotoxic bacteria with organoids so that genotoxic bacteria can interact with either the apical surface of organoid fragments or the basal surface of organoids.In some embodiments, the first step, the second step, and / or the third step does not include culturing genotoxic bacteria with the apical surface of organoids.

[0022] The present invention also provides a method for generating the organoid with mutation signature by using the steps described herein.The present invention also provides a method for generating the organoid with mutation signature, comprising the first step, comprising culturing organoid or organoid fragment with genotoxic bacteria, the second step, comprising culturing organoid or organoid fragment with genotoxic bacteria, and bacterial growth is restricted by antibiotics, and the third step, comprising killing genotoxic bacteria and allowing organoid or organoid fragment to grow further, and generating the organoid or organoid fragment with mutation signature.

[0023] First step The first step includes culturing the organoid or organoid fragment with genotoxic bacteria. The purpose of the first step is to bring bacteria and organoid cells into close proximity in culture and / or to generate a large amount of DNA damage ("acute damage"). For example, in a long-term exposure co-culture strategy scheme including a first step, a second step, and a third step (an example of this scheme is provided in Figure 1A), the purpose of the first step is to generate a large amount of DNA damage in the organoid or organoid fragment. In an alternative embodiment, in a rapid co-culture strategy scheme including a first step and a third step (an example of this scheme is provided in Figures 2A and 3A), the purpose of the first step is to bring bacteria and organoid cells into close proximity in culture.

[0024] Acute injury can be defined as injury that causes significant loss of cell viability.For example, acute injury can cause at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% loss of cell viability.In some embodiments, after the first stage, at least 50% of organoid and organoid fragment are viable.For example, after the first stage, at least 50%, at least 60%, or at least 70% of organoid and organoid fragment are viable.

[0025] A variety of assays are known in the art for measuring cells, including assays that determine viability based on cell membrane integrity (e.g., using nucleic acid dyes, propidium iodide, TO-PRO-3 iodide, or 7-AAD), cell function, e.g., enzyme activity (e.g., using Calcein or CyQUANT cytotoxicity assay kits), or metabolic activity (e.g., using alarmarBlue cell viability reagent or yQUANT MTT cell viability assay). In some embodiments, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 7.5%, at least 10%, at least 20%, at least 30%, or at least 40% of the DNA in the organoid or organoid fragment is damaged, for example, as measured by the Comet assay [4] or yH2AX assay [5] (e.g., as shown in Figure 2C).

[0026] The first step may involve culturing organoids, organoid fragments, or a combination of organoid fragments and organoids.

[0027] The apical surface of intestinal epithelial cells faces the lumen and regulates interactions with luminal contents, including mediating nutrient absorption, detects microbial products, and secretes molecules that protect the epithelium from potentially harmful agents in the lumen. The basolateral surface anchors the epithelial cells to the underlying basement membrane, delivers nutrients from the lumen to the bloodstream, and communicates with nearby cells [6]. As discussed in detail below, polarized organoids have an apical surface on the inside of the organoid and a basal surface on the outside of the organoid. It is generally believed that genotoxic bacteria must interact with the apical surface of the organoid to damage DNA. By fragmenting the organoid, the apical surface can be exposed to the genotoxic bacteria in the co-culture. Thus, in some embodiments, the first stage includes culturing the organoid fragments. Organoid fragments for use in the first stage can be generated by shearing one or more organoids, for example with a glass pipette. This can include first enzymatically digesting one or more organoids and then shearing them. In some embodiments, the method comprises obtaining organoid fragments by shearing one or more organoids, and then culturing the organoid fragments with genotoxic bacteria.In some embodiments, the sheared organoids are grown in 60-80% matrigel, optionally about 70% matrigel, for 2-4 days.

[0028] The organoid fragments naturally reconstitute into organoids in culture, which can occur at any stage of the method, but is primarily performed in the second or third stage of the method.

[0029] The present inventors have hypothesized for the first time that apical exposure to genotoxic bacteria is not necessary for DNA damage, and exposure (especially long-term exposure) of the basal surface of organoid (optionally without fragmentation) can cause DNA damage.Therefore, in other embodiments, the first step (and subsequent steps) comprises culturing organoid (without fragmentation) with genotoxic bacteria that cause DNA damage.

[0030] It is well known in the art that bacterial growth in batch cultures can have four phases: lag phase, exponential growth phase (also known as log phase), stationary phase, and death phase. During the lag phase, bacteria adapt themselves to the growth conditions and there is little or no cell division. The exponential growth phase is a period characterized by cell doubling. The number of new bacteria that appear per unit time is proportional to the current population. If growth is not limited, doubling continues at a constant rate, so that both the cell number and the population rate double every successive period. The stationary phase is often due to growth-limiting factors such as the depletion of essential nutrients and / or the formation of inhibitory products such as organic acids. The stationary phase results from a situation where the growth rate and death rate are equal. In the death phase (declining phase), bacteria die, which can be caused by lack of nutrients, environmental temperatures above or below the tolerance range of the species. Those skilled in the art are aware of different techniques for determining bacterial growth rate, including measuring the optical density (OD) at 600 nm (OD600). Optical density measures the degree of light scattering caused by the bacteria in a culture; the more bacteria there are, the more light is scattered. By monitoring the rate of increase in OD600, it is possible to distinguish between lag, logarithmic, and stationary phases of a bacterial culture. Assays for measuring bacterial growth rates using OD600 are well known in the art and are described in references

[27] ,

[30] , and

[31] .

[0031] In some embodiments, the genotoxic bacteria is in exponential growth phase when it is incorporated into the co-culture. In some embodiments, the genotoxic bacteria in exponential growth phase may have an optional concentration measured at a wavelength of 600 nm (OD600) of greater than 0.4 and less than 1. It is preferred to incorporate the genotoxic bacteria in exponential growth phase into the co-culture, as this ensures fitness of the bacteria introduced into the system, which improves consistency between replicates of the claimed method.

[0032] Multiplicity of infection (MOI) is the ratio of genotoxic bacteria to organoids or organoid fragments. The MOI of bacteria can be determined using colony forming unit assays as described in references

[27] ,

[30] , and

[31] . In some embodiments, the genotoxic bacteria has a multiplicity of infection (MOI) of 0.1, 0.5, 1, 2, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, or 800 when it is incorporated into the co-culture in the first stage. In a preferred embodiment, the genotoxic bacteria has a multiplicity of infection of 0.1 to 800, 10 to 100, 50 to 100, or about 100 when it is incorporated into the co-culture. In a preferred embodiment, the genotoxic bacteria has a MOI of 10, 50, or 100 when it is incorporated into the co-culture in the first stage. In a preferred embodiment, in the first stage, the genotoxic bacteria has an MOI of 10, 50 or 100, and the genotoxic bacteria is in exponential growth phase and may have any concentration, measured at a wavelength of 600 nm (OD600), greater than 0.4 and less than 1. In a preferred embodiment, in the first stage, the genotoxic bacteria has an MOI of 10, 50 or 100 and is cultured for 3 to 4 hours. In a preferred embodiment, in the first stage, the genotoxic bacteria has an MOI of 500 and is cultured for 4 hours.

[0033] The length of the first stage can vary depending on, for example, the growth rate of the bacteria, the length of the second stage, and the amount of acute damage desired. For example, the first stage can be performed for between 15 minutes and 5 hours. In some embodiments, the first stage can be performed for between 30 minutes and 18 hours. In some embodiments, the first stage is performed for between 30 minutes and 5 hours, between 1 hour and 4 hours, between 1 hour and 3 hours, between 2 hours and 4 hours, between 2 hours and 4 hours, or between 3 hours and 4 hours. In some embodiments, the first stage is performed for at least 30 minutes, at least 1 hour, at least 1.5 hours, at least 2 hours, at least 2.5 hours, at least 3 hours, at least 3.5 hours, at least 4 hours, at least 4.5 hours, at least 5 hours. In a preferred embodiment, the first stage is performed for at least 3 hours. In a preferred embodiment, the first stage is performed for 3 to 4 hours.

[0034] The amount of bacteria added to the culture in the first stage (i.e., multiplicity of infection) and the length of the first stage may be correlated. In general, if a higher MOI is used when the bacteria is incorporated into the co-culture, the length of the first stage may be shorter. If a low MOI is used when the bacteria is incorporated into the co-culture, the first stage may be carried out for a longer period of time. For example, if an MOI of less than 50 is used, the first stage may be carried out overnight (e.g., at least 12 hours). In some embodiments, the first stage is carried out for between 15 minutes and 5 hours, and the genotoxic bacteria has a multiplicity of infection of 100 to 500 when it is incorporated into the co-culture. In some embodiments, the first stage is carried out for between 3 minutes and 6 hours, and the genotoxic bacteria has a multiplicity of infection of 50 to 200 when it is incorporated into the co-culture. In some embodiments, the first stage is carried out for between 6 and 8 hours, and the genotoxic bacteria has a multiplicity of infection of 0.1 to 50 when it is incorporated into the co-culture.

[0035] The first stage may be performed in the absence of antibiotics and / or bacterial growth may be unrestricted.

[0036] Second Phase The second stage may occur after the first stage, or in an alternative embodiment, the second stage is the first step in the method, e.g., a method comprising a second stage and a third stage.

[0037] In the second stage, the growth of the genotoxic bacteria is restricted with one or more antibiotics. In this stage, the growth of the organoids or organoid fragments is not restricted. Many antibiotics are well known in the art. Exemplary antibiotics for use in the methods of the invention include ampicillin, carbenicillin, fosmidomycin, gentamicin, amphotericin, kanamycin, neomycin, primocin, penicillin and streptomycin (PenStrep), polymyxin B, tetracycline, and cationic antimicrobial peptides (CAMPs) such as chloramphenicol, and streptomycin. In a preferred embodiment, the antibiotic is gentamicin. The purpose of the antibiotic in the second stage is not to kill the bacteria, but to restrict the growth of the bacteria. The amount of antibiotic used in the second stage will depend on the antibiotic used in the method, the bacterial strain, and the bacterial MOI used. For example, the second stage may contain 0.1 to 100 μg / ml of gentamicin. In some embodiments, the second stage contains about 50 μg / ml of gentamicin. Suitable antibiotic concentrations for limiting the growth of genotoxic bacteria can be readily identified by one of skill in the art by testing the growth rate of the genotoxic bacteria with various concentrations of the antibiotic.

[0038] Limited genotoxicity bacteria no longer have a classical growth phase. In the presence of low concentrations of antibiotics, the bacteria are expected to follow a "bistable growth" pattern described in [7]. Bistable growth of bacteria is characterized by the coexistence of growing and non-growing populations.

[0039] The limited genotoxic bacteria have a reduced doubling time or continue to grow bistable in a plateau-like fitness environment. In some embodiments, the doubling time of the genotoxic bacteria in the second stage can be shortened by at least 10%, 20%, 30%, 40%, or 50%. When the method includes a first stage and a second stage, the growth rate of the genotoxic bacteria in the second stage follows bistable growth and / or the growth rate is lower than the growth rate of the genotoxic bacteria in the first stage. For example, the growth rate of the genotoxic bacteria in the second stage can be reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% compared to the growth rate of the genotoxic bacteria in the first stage. When the second stage is the first stage in the method, the growth rate of the genotoxic bacteria in the second stage is lower and / or the growth of the bacteria is not limited compared to the culture medium without antibiotics. In some embodiments, the growth rate of the genotoxic bacteria in the second stage is lower than the growth of the genotoxic bacteria in a medium without antibiotics, and / or the growth of the bacteria is not restricted. Methods for comparing the growth rates of different bacteria are well known in the art.

[0040] The length of the second stage may vary depending on, for example, the length of the first stage, the amount of DNA damage induced in the first stage, the bacterial load at the end of the first stage, the required organoid cell viability, and the amount of sustained damage desired. For example, the second stage may be performed for 0.5 to 3 days. In some embodiments, the second stage is performed for at least 0.5 days, at least 1 day, at least 1.5 days, at least 2 days, at least 2.5 days, or at least 3 days. In some embodiments, the second stage may be performed for 1 to 3 days, 1 to 2 days, or 2 to 3 days. In a preferred embodiment, the second stage is 2 days. If the first stage is short, for example 15 minutes to 1 hour, the second stage is longer, preferably for example 2 to 3 days.

[0041] The second step can be performed after the first step, or in an alternative embodiment, the second step is the first step of the method. If the second step is performed after the first step, the organoids or organoid fragments can be washed between the first and second steps. This washing step can include washing the organoids or organoid fragments with saline (i.e., PBS) or mammalian cell medium (i.e., AdDMEM) at a speed of less than 500G for 1-5 minutes. Mammalian cells and organoids are sedimented at a slower centrifugation speed, such as less than 500G, while bacterial cells require a higher centrifugation speed, such as about 3000G, to be sedimented. This difference in centrifugation speed allows the separation of the organoids from the majority of bacteria. This washing step allows the reduction and control of the bacterial load when transferring from the first to the second step. In other embodiments, the first stage organoids are treated to remove all bacteria in the culture, for example by treating the first stage with a high concentration of antibiotic (e.g., at least 150 μg / ml, at least 180 μg / ml, at least 190 μg / ml, at least 200 μg / ml gentamicin, optionally about 200 μg / ml gentamicin), and then supplementing the culture with additional genotoxic bacteria.

[0042] If the second stage is the first step of the method, the genotoxic bacteria must be incorporated into the co-culture. In some embodiments, the genotoxic bacteria is in exponential growth phase when it is incorporated into the co-culture. In some embodiments, the genotoxic bacteria in exponential growth phase may have an optional concentration greater than 0.4 and less than 1, as measured at a wavelength of 600 nm (OD600). It is preferred to incorporate the genotoxic bacteria in exponential growth phase into the co-culture. This ensures that bacterial growth remains essentially constant throughout the stages, improving consistency between replicates of the claimed method. The multiplicity of infection (MOI) is the ratio of genotoxic bacteria to organoids or organoid fragments. The MOI of bacteria can be determined using a colony-forming unit assay, as described in references

[27] ,

[30] , and

[31] . In some embodiments, the genotoxic bacterium, when incorporated into the co-culture in the first stage, has a multiplicity of infection (MOI) of 0.1, 0.5, 1, 2, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, or 800. In preferred embodiments, the genotoxic bacterium, when incorporated into the co-culture, has a multiplicity of infection of 0.1-800, 10-100, 50-100, or about 100.

[0043] During the second stage, the organoid fragments can form one or more organoids, for example, in some embodiments, during the second stage, at least 60%, at least 70%, at least 80%, or at least 90% of the organoid fragments can form one or more organoids. In some embodiments, the organoids formed can be polarized. As discussed in detail below, polarized organoids have an apical surface on the inside of the organoid and a basal surface on the outside of the organoid. Without wishing to be bound by any particular theory, it is believed that the apical surface does not necessarily need to be exposed in order for DNA in the organoid to be damaged. Thus, during the second stage, when the organoid fragments can form one or more organoids, genotoxic bacteria are exposed to the basal surface of the organoid. The inventors hypothesize that DNA damage caused by genotoxic compounds on the basal surface can produce mutation signatures similar to human cancer signatures.

[0044] When the method includes only the first and second steps, the method can generate organoids or organoid fragments having a mutational signature.

[0045] After the second step, at least 20% of the organoids and organoid fragments are expected to be viable, for example, at least 20%, at least 25%, or at least 30% of the organoids and organoid fragments are viable after the second step.

[0046] Third Phase The third stage may be performed after the first or second stage, for example, the third stage may be performed only after the first stage, after the second stage which is followed by the first stage, or only after the second stage.

[0047] In the third stage, the genotoxic bacteria in the culture are removed, for example, by killing the bacteria with a high concentration of antibiotic. In this stage, the growth of the organoids or organoid fragments is not restricted. Exemplary antibiotics for use in the third stage include ampicillin, carbenicillin, fosmidomycin, gentamicin, amphotericin, kanamycin, neomycin, primocin, penicillin and streptomycin (PenStrep), polymyxin B, tetracycline, and cationic antimicrobial peptides (CAMPs) such as chloramphenicol, and streptomycin. In a preferred embodiment, the antibiotic is gentamicin. The amount of antibiotic used in the third stage will depend on the antibiotic used in the method, the bacterial strain, and the bacterial MOI used. In some embodiments, the genotoxic bacteria are killed with 100-300 μg / ml of gentamicin, and optionally, the genotoxic bacteria are killed with 200 μg / ml of gentamicin. In some embodiments, the genotoxic bacteria are killed with 20-100 μg / ml primocin, and optionally, the genotoxic bacteria are killed with 50 μg / ml primocin.

[0048] After DNA is damaged by genotoxic bacteria, the organoid or organoid fragment can repair the damage, which may result in mutational errors in the DNA. Following DNA damage, cells may enter G2 / M cell cycle arrest. Only cells that have re-entered proliferation are expected to have resolved the DNA damage by incorporating mutations. This repair can occur at any time during the method, mainly by taking place in the third stage. During the third stage, the organoid or organoid fragment can recover from DNA damage and / or expand. The purpose of the third stage is to obtain enough material for downstream processes and / or ensure that the cells are healthy enough to withstand any further rounds of DNA damage.

[0049] At the end of the recovery period (i.e., at the end of the third stage), less than 60% of the organoids or organoid fragments are expected to be arrested in G2 / M. Alternatively, more than 5% of the organoids or organoid fragments are positive for ki67 staining (a marker of cell proliferation) after the third stage.

[0050] The length of the third stage may vary depending on, for example, the recovery time required, the amount of genotoxic bacterial exposure, or the type of organoids being cultured. For example, the third stage may be performed for between 2 and 20 days. In some embodiments, the third stage is performed for at least 0.5 days, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 12 days, at least 14 days, at least 16 days, at least 18 days, or at least 20 days. In some embodiments, the third stage may be performed for between 2 and 15 days, 5 and 15 days, or 10 and 15 days. For some organoid cultures, such as lung organoids, which take longer to passage, the third stage may be longer, for example, the third stage may be 15 to 25 days or 15 to 20 days. In some embodiments, the third stage is performed for 2 to 5 days.

[0051] In some embodiments, the third step is performed in suspension, rather than on a substrate such as Matrigel, which allows the organoids cultured in culture medium to be rapidly used in downstream processes such as immunofluorescence or flow cytometry assays.

[0052] During the third stage, the organoid fragments may form one or more organoids, for example, in some embodiments, during the third stage, at least 60%, at least 70%, at least 80%, or at least 90% of the organoid fragments may form one or more organoids.

[0053] After the third stage, the method can generate organoids or organoid fragments that have a mutational signature.

[0054] Exemplary methods for co-culturing genotoxic bacteria with organoids include: A method for co-culturing genotoxic bacteria with organoids, comprising a first step comprising culturing the organoids with the genotoxic bacteria, and a second step comprising culturing the organoids with the genotoxic bacteria, wherein bacterial growth is restricted by an antibiotic.

[0055] The method for co-cultivating genotoxic bacteria and organoid fragments, comprising: a first step, comprising culturing organoid fragments with genotoxic bacteria; and a second step, comprising culturing organoid fragments with genotoxic bacteria, wherein bacterial growth is restricted by antibiotics.In this embodiment, organoid fragments can naturally re-form the organoid during culture at any stage, but this mainly occurs during the second step of the method.

[0056] A method for co-culturing genotoxic bacteria with organoids, comprising culturing the organoids with the genotoxic bacteria, a second step in which bacterial growth is restricted by an antibiotic, and a third step in which the genotoxic bacteria are killed, allowing further growth of the organoids.

[0057] The method for co-cultivating genotoxic bacteria and organoid fragments, comprising: culturing organoid fragments with genotoxic bacteria, the second stage of bacterial growth is restricted by antibiotics, and the third stage of killing genotoxic bacteria allows further growth of organoid fragments.In this embodiment, organoid fragments can naturally re-form organoids in both the second stage and the third stage.

[0058] A method for co-culturing genotoxic bacteria with organoids, comprising a first step comprising culturing the organoids with the genotoxic bacteria, and a third step comprising killing the genotoxic bacteria and allowing further growth of the organoids.

[0059] The method for co-cultivating genotoxic bacteria and organoid fragments, comprising: a first step, comprising culturing organoid fragments with genotoxic bacteria; and a third step, comprising killing genotoxic bacteria, allowing further growth of organoid fragments.In this embodiment, organoid fragments can naturally re-form organoids during culture at any stage, but this mainly occurs during the third step of the method.

[0060] A method for co-culturing genotoxic bacteria with organoids, comprising a first step comprising culturing the organoids with the genotoxic bacteria, a second step comprising culturing the organoids with the genotoxic bacteria, in which bacterial growth is restricted by an antibiotic, and a third step comprising killing the genotoxic bacteria, allowing further growth of the organoids.

[0061] A method for co-cultivating organoid fragments with genotoxic bacteria, comprising: a first step, comprising culturing organoid fragments with genotoxic bacteria; a second step, comprising culturing organoid fragments with genotoxic bacteria, in which bacterial growth is restricted by antibiotics; and a third step, comprising killing genotoxic bacteria, in which organoid fragments can be allowed to grow further.In this embodiment, organoid fragments can naturally re-form organoid at any step, but this mainly occurs during the second and third steps.

[0062] Although the above embodiments show how genotoxic bacteria are co-cultured with organoids or organoid fragments, all stages may include a mixture of organoids or organoid fragments, since organoid fragments can spontaneously re-form organoids at any time during culture.

[0063] bacteria The human microbiome is the collection of all microbiota present on or in human tissues and biological fluids, including skin, mammary gland, semen, uterus, ovarian follicles, lungs, saliva, oral mucosa, lacrimal tissue, conjunctiva, biliary tract, retinal tissue, and gastrointestinal tract. The gastrointestinal tract includes the mouth, pharynx (throat), esophagus, stomach, small intestine, large intestine, colon, rectum, and anus. Some of the bacteria in the microbiome are genotoxic bacteria. Genotoxic bacteria are any bacteria that secrete bacterial genotoxins. Bacterial genotoxins are a class of molecules that have the ability to enter the nucleus of host cells and cause DNA damage by introducing single-stranded and double-stranded DNA breaks, resulting in a variety of effects, including the activation of DNA damage response, aging, apoptosis, and genetic abnormalities [8]. The bacteria used in the method of the present invention are genotoxic bacteria and are used to produce organoids or organoid fragments with mutational signatures.

[0064] In some embodiments, the genotoxic bacterium is a colibactin-producing bacterium. Colibactin is a natural genotoxic compound synthesized by a hybrid enzyme encoded by polyketide synthase, nonribosomal peptide synthase, and pks, specified in a 54 kb genomic island. The pks island is found in members of the Enterobacteriaceae family, including Escherichia coli, Klebsiella pneumoniae, Enterobacter aerogenes, and Citrobacter koseri. Colibactin induces DNA double-strand breaks, chromosomal aberrations, and cell cycle arrest at the G2 / M phase [1]. In some embodiments, the colibactin-producing bacterium is from the Enterobacteriaceae family. In some embodiments, the colibactin-producing bacteria is from the species Escherichia coli, Klebsiella pneumoniae, Enterobacter aerogenes, and Citrobacter koseri. In a preferred embodiment, the colibactin-producing bacteria is from the species Escherichia coli. In a further preferred embodiment, the colibactin-producing bacteria is pks+ Escherichia coli.

[0065] The genotoxic bacterium may also be a bacterium that has been genetically engineered to express a genotoxic compound, such as a pks genomic island.

[0066] Alternative genotoxic bacteria include non-typhoidal Salmonellae, Campylobacter jejuni, Fusobacterium nucleatum, Helicobacter pylori, Shigella, E. faecalis https: / / pubmed.ncbi.nlm.nih.gov / 23661025 / , or Akkermansia muciniphila https: / / pubmed.ncbi.nlm.nih.gov / 23661025 / .

[0067] Organoids The present invention relates to a method for co-culturing genotoxic bacteria with organoids. Organoids can be obtained by culturing epithelial stem cells. Epithelial stem cells are obtained from adult tissue. That is, epithelial stem cells are adult epithelial stem cells. In this context, "adult" refers to mature tissue, that is, including newborn or child, but excluding embryo or fetus. Alternatively, epithelial stem cells are not derived from embryonic stem cells or embryonic stem cell lines, for example, differentiated in vitro. Adult tissues can be healthy adult tissues. In other embodiments, adult tissues can be derived from patients with genotypes or phenotypes of diseases, such as cancer or inflammatory diseases.

[0068] The epithelial stem cells can be derived from colon, small intestine, stomach, pancreas, liver, lung, breast, prostate, kidney, mouth, nasopharynx, throat, hypopharynx, larynx, trachea, skin, fallopian tube, ovary, salivary gland, esophagus, hair follicle tissue, and / or cochlear tissue. In some embodiments, the epithelial stem cells are colorectal cells. Methods for culturing epithelial stem cells to obtain organoids from various epithelial tissues have been previously described (e.g., WO2009 / 022907, WO2010 / 090513, WO2012 / 014076, WO2012 / 168930, WO2015 / 173425, WO2016 / 083613, and WO2016 / 083612, WO2017 / 149025, WO2020 / 234250, and [9]). Cells taken directly from tissue, i.e., freshly isolated cells, are also called primary cells. In some embodiments, the epithelial stem cells are primary epithelial stem cells.

[0069] As mentioned above, bacteria can be present on or in various human tissues and biological fluids. These tissues are particularly relevant to the co-culture method of the present invention. Thus, in some embodiments, the organoid fragment or organoid for use in the method can be derived from the gastrointestinal tract, pancreatic tissue, lung tissue, trachea, skin, and / or vaginal tissue. In a preferred embodiment, the organoid fragment or organoid is derived from the gastrointestinal tract. The gastrointestinal tract includes the mouth, pharynx (throat), esophagus, stomach, small intestine, large intestine, colon, rectum, and anus. Thus, in some embodiments, the organoid fragment or organoid is derived from the mouth, pharynx (throat), esophagus, stomach, small intestine, large intestine, colon, rectum, and anus. In a preferred embodiment, the organoid fragment or organoid is derived from the stomach, small intestine, large intestine, colon, or rectum. In a preferred embodiment, the organoid fragment or organoid is derived from colon tissue.

[0070] The organoids having a mutational signature generated by the claimed methods can be gastrointestinal organoids, pancreatic organoids, pulmonary organoids, tracheal organoids, skin organoids, or vaginal organoids.

[0071] The organoid used in the present invention is preferably obtained using epithelial cells from adult tissue, optionally using epithelial stem cells from adult tissue that express Lgr5. In some embodiments, the organoid is derived from a single cell that optionally expresses Lgr5. Advantageously, this allows the formation of a homogeneous cell population. The single cell suspension that comprises epithelial stem cells can be mechanically generated. In some embodiments, the single cell suspension that comprises epithelial stem cells is generated using mechanical process and / or enzyme digestion. Mechanical process includes, but is not limited to, dissection, micro-dissection, and filtering.

[0072] Organoids obtained using culture medium suitable for expansion may also be referred to as "expanded organoids". Expanded organoids contain at least one epithelial stem cell, which can divide to give rise to epithelial stem cells or give rise to differentiated progeny. It will be understood that in preferred expanded organoids, the majority of cells are expanded cells (i.e., dividing cells) that retain an undifferentiated phenotype. Although some spontaneous differentiation may occur, the cell population is generally an expanded population. The length of time that an organoid can continue to expand while maintaining the presence of a core of epithelial stem cells, as well as the integrity of the genotype and phenotype of the cells, is an important feature of organoids. Organoids typically express Lgr5. Organoids also have a unique structure that occurs rapidly as the cells expand and self-organize in vitro. These features are described in detail below.

[0073] Image analysis can be used to evaluate the characteristics of cells in culture, such as cell morphology; cell structure; evidence of apoptosis or cell lysis; and the composition and structure of organoid.Many types of image analysis are well known in the art, such as electron microscopy, including scanning electron microscopy and transmission electron microscopy, confocal microscopy, stereomicroscopy, and fluorescence microscopy.Histological analysis can reveal basic structure and cell type.

[0074] In some embodiments, organoid has three-dimensional structure, i.e., organoid is three-dimensional organoid.In some embodiments, organoid comprises epithelial cells.In some embodiments, organoid comprises only epithelial cells, i.e., non-epithelial cells are not present in organoid.Even if other cell types are transiently present in culture medium, for example in starting tissue fragment, these cells are unlikely to survive, and are instead replaced by long-term expansion of stem cells, which generates a population of pure epithelial cells.

[0075] In some embodiments, the epithelial cells in the organoid surround a lumen. In some embodiments, the organoid does not contain a lumen (particularly tumor organoids generally do not have a lumen). In some embodiments, the epithelial cells are polarized (meaning that proteins are differentially expressed at the apical or basolateral side of the epithelial cells). In some embodiments, the lumen is a sealed lumen (meaning that a continuous cell barrier separates the contents of the lumen from the medium surrounding the organoid). In some embodiments, the organoid contains stem cells, which can actively divide and preferably differentiate into all major differentiated cell lineages present in the corresponding in vivo tissue (e.g., when the organoid or cells are transferred to a differentiation medium). In some embodiments, the organoid contains basic cells on the outside and more differentiated cells in the center.

[0076] In some embodiments, organoid comprises stratified epithelium. "Stratified" means that there are multiple (more than one) layers of cells. Such cells often tend to have their nucleus in the more central part of the cell, i.e., they are not polarized. Cells in the multi-layered part can be organized to include gaps or lumens between the cells themselves.

[0077] In some embodiments, the organoid comprises a single monolayer that is folded (or invaginated) to form two or more layers. It may be difficult to distinguish between a folded (or invaginated) monolayer and a region of stratified cells. In some embodiments, the organoid comprises both a region of stratified cells and a region of folded monolayer. In some embodiments, the organoid has a portion that is formed from multiple layers and a portion that comprises a single monolayer of cells. In some embodiments, the organoid comprises or consists of a single monolayer of cells. In some embodiments, the organoid does not comprise a monolayer. The organoid may have at least one layer of cells with budding and a central lumen.

[0078] In some embodiments, the organoid used in the claimed method comprises or consists of epithelial cells.In some embodiments, the organoid comprises or consists of a single monolayer of epithelial cells.In some embodiments, non-epithelial cells are not present in the organoid.

[0079] In some embodiments, the organoids used in the claimed methods are cultured or can be cultured in expansion medium for at least 2 months, for example, at least 10 weeks, at least 12 weeks, at least 14 weeks, at least 16 weeks, at least 4 months, at least 5 months, at least 6 months, at least 9 months, at least 1 year.In some embodiments, the organoids with mutation signatures generated in the claimed methods are cultured or can be cultured in expansion medium for at least 2 months, for example, at least 10 weeks, at least 12 weeks, at least 14 weeks, at least 16 weeks, at least 4 months, at least 5 months, at least 6 months, at least 9 months, at least 1 year.

[0080] In some embodiments, the organoids used in the claimed methods or the organoids with mutation signatures generated by the claimed methods can be cultured or cultivated for at least 5 passages, at least 10 passages, at least 15 passages, or at least 20 passages.In some embodiments, the organoids or epithelial stem cell populations are cultured for at least 10 passages.In some embodiments, the organoids with mutation signatures generated by the claimed methods can be cultured or cultivated for at least 5 passages, at least 10 passages, at least 15 passages, or at least 20 passages.In some embodiments, the organoids or epithelial stem cell populations are cultured for at least 10 passages.

[0081] In some embodiments, the cell number of organoid increases exponentially over 5 passages, 10 passages, 15 passages or 20 passages.In a preferred embodiment, the cell number of organoid or epithelial stem cell population increases exponentially over 5 passages.

[0082] In some embodiments, the organoids have a diameter at their widest point of at least 50 μm, at least 60 μm, at least 70 μm, at least 80 μm, at least 90 μm, at least 100 μm, at least 125 μm, at least 150 μm, at least 175 μm, at least 200 μm, at least 250 μm or more.

[0083] In the context of the present invention, tissue fragments are parts of adult tissue, preferably human adult tissue.In contrast, organoids are distinct from tissue fragments because they develop structural characteristics through in vitro expansion.

[0084] In preferred embodiments, the organoids used in the claimed methods or the organoids with mutation signatures generated by the claimed methods can be cultured for at least 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 months or more.In some embodiments, the organoids are expanded or maintained in culture for at least 3 months, preferably at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 9 months, or at least 12 months or more.

[0085] Mutation signatures The DNA damage caused by genotoxic bacteria leads to mutation signature in organoid.Therefore, the method of the present invention can generate organoid with mutation signature, such as cancer mutation signature.After being exposed to genotoxic bacteria in the method of the present invention, the organoid can be subcloned and analyzed (e.g., by whole genome sequencing or whole exome sequencing (WES)) to identify the resulting mutation signature.Cancer mutation signature can be identified by comparing the signature generated by the method of the present invention with whole genome sequencing data from cancer.

[0086] As mentioned above, the human microbiome can be present in various human organs, including the skin, mammary gland, semen, uterus, ovarian follicles, lungs, saliva, oral mucosa, conjunctiva, biliary tract, and gastrointestinal tract. Many studies have shown that dysfunction of the intestinal epithelial barrier can result in the translocation of gut microbes to other organs in the human body. These translocated bacteria have been linked to the pathogenesis of many cancers. For example, bacteria from the genera Bacteroides, Romboutsia, and Lachnospiraceae have been identified in liver tumors

[10] . Other studies have shown that Enterobacteriaceae and Pseudomonas bacteria are present in the tumor microenvironment of pancreatic cancer

[11] . Thus, bacteria in the microbiome and translocated bacteria can produce cancer mutation signatures in relevant tissues.

[0087] In some embodiments, the method of the present invention generates organoids with gastrointestinal tract mutation signature, such as colorectal cancer mutation signature.In other embodiments, the method of the present invention generates organoids with gastric cancer mutation signature, pancreatic cancer mutation signature, liver cancer mutation signature, lung cancer mutation signature, oral cavity cancer mutation signature, nasopharyngeal cancer mutation signature, tracheal cancer mutation signature, skin cancer mutation signature, vaginal cancer mutation signature, or esophageal tissue cancer mutation signature.

[0088] For example, Pleguezuelos-Manzano, et al. demonstrated that repeated exposure of human intestinal organoids to genotoxic E. coli by repeated luminal injections over a 5-month period resulted in two mutational signatures. The first was a pks-specific single base substitution signature (SBS-pks), in which organoids displayed an increased number of single base substitutions with a bias towards T to N substitutions. These T to N substitutions occurred preferentially at ATA, ATT, and TTT (where the middle base is mutated). The second was a small indel signature (ID-pks), which is characterized by a single T deletion in a T homopolymer. Characterization of mutations in these two signatures identified the presence of an adenine residue 3 bp upstream of the mutated SBS-pks T to N site, and in the ID-pks signature, poly-T stretches showed enrichment of adenines immediately upstream of the affected poly-T stretch. The length of the adenine stretch and T-homopolymers was inversely correlated, consistently resulting in a total length of 5 or more A / T nucleotides. Pleguezuelos-Manzano, et al. found that the SBS-pks and ID-pks signatures were highly enriched in CRC-derived metastases [2].

[0089] In some embodiments, the method of the present invention generates organoids with mutation signatures of SBS-pks and ID-pks.In some embodiments, the method of the present invention generates organoids with single base substitution signatures.In some embodiments, the single base substitution signature is characterized by an increase in the number of single base substitutions that are biased toward T to N substitutions.In some embodiments, the method of the present invention generates organoids with small indel signatures.In some embodiments, the small indel signatures result in organoids with an increase in the number of single base substitutions that are biased toward T to N substitutions.

[0090] Culture medium The medium suitable for culturing organoid and organoid fragment is well known in the art, and described in, for example, WO2009 / 022907, WO2010 / 090513, WO2012 / 014076, WO2012 / 168930, WO2015 / 173425, WO2016 / 083613, WO2016 / 083612, WO2017 / 149025 and WO2020 / 234250.The medium described in these documents is incorporated herein by reference, and any of them can be used in the present invention.

[0091] In some embodiments, the culture medium suitable for culturing organoid and organoid fragment can comprise one or more of Wnt agonist, BMP inhibitor, mitogenic growth factor, and TGF-beta inhibitor.For example, the culture medium suitable for organoid and organoid fragment comprises Wnt agonist.The culture medium suitable for organoid and organoid fragment can further comprise mitogenic growth factor and / or BMP inhibitor.The same culture medium can be used in the first stage, the second stage, and the third stage.

[0092] In some embodiments, the culture medium suitable for organoid and organoid fragment comprises Wnt agonist, BMP inhibitor, mitogenic growth factor and TGF-beta inhibitor.The culture medium suitable for organoid and organoid fragment can further comprise one or more of p38 inhibitor, cAMP agonist, prostaglandin pathway activator, nicotinamide, gastrin, B27 and N-acetylcysteine.

[0093] In some embodiments, the culture medium comprises a basal medium for human or animal cells (e.g., DMEM / F12 (GultaMax, Advanced Dulbecco's Modified Eagle / F12 Medium supplemented with 1M HEPES) optionally containing B27 or Ad-DF+++), an R-spondin family protein, a mitogenic growth factor (such as EGF), a BMP inhibitor (such as noggin), a TGF-beta inhibitor (such as A83-01), a p38 inhibitor (such as SB202190), and optionally nicotinamide and N-acetylcysteine.

[0094] In some embodiments, the culture medium comprises Advanced DMEM / F12 medium containing B27, nicotinamide, N-acetylcysteine, noggin, R-spondin 1-4, EGF, Wnt (WNT culture supernatant (50%, produced using stably transfected L cells) or NGS Wnt), TGF-b type I receptor inhibitor A83-01, and P38 inhibitor SB202190.

[0095] The examples demonstrate that the claimed method is particularly effective in generating intestinal organoids with mutational signatures, such as colorectal cancer mutational signatures.In this embodiment, the method for co-culturing genotoxic bacteria and intestinal organoids comprises culturing organoids or organoid fragments in a culture medium that includes R-spondin family proteins, mitogenic growth factors (such as EGF), BMP inhibitors (such as noggin), and TGF-beta inhibitors.In some embodiments, the method for co-culturing genotoxic bacteria and intestinal organoids comprises culturing organoids or organoid fragments in a culture medium that includes N-acetylcysteine, TGFβ receptor inhibitor A83-01, B27 supplement, EGF, gastrin, Noggin, nicotinamide, R-spondin-3, P38 inhibitor SB202190, MAPK inhibitors, and NGS Wnt. In a preferred embodiment, the method for co-culturing intestinal organoids with genotoxic bacteria comprises culturing organoids or organoid fragments in culture medium containing Ad-DF++, N-acetylcysteine ​​(0.1-1 mM), TGFβ receptor inhibitor A83-01 (100-800 nM), B27 supplement (1x-5x), EGF (20-80 ng / ml), gastrin (2-10 nM), Noggin (1-5% culture supernatant from UPE or recombinant Noggin (N) at 50-250 ng / ml), nicotinamide (2-20 mM), R-spondin 1-4 (100-500 ng / ml), SB202190 P38 MAPK inhibitor (2-20 μM), and NGS Wnt (0.1-2 nM).

[0096] Extracellular and synthetic matrices Epithelial stem cells are typically grown in culture with exogenous extracellular matrix (ECM) that is known to support cell proliferation (e.g., 12]). The culture medium suitable for culturing organoids and organoid fragments can contain ECM. The ECM is an exogenous ECM (meaning that it is added to any extracellular matrix protein that is naturally secreted by epithelial stem cells or a population of epithelial stem cells when it contacts with the expansion medium of the present invention). Any suitable ECM can be used. Cells are preferably cultured in a microenvironment that at least partially mimics the cell niche where the cell is naturally located. The cell niche is partially determined by the cell and by the ECM that the cell secretes in the niche. The cell niche can be mimicked by culturing the cell in the presence of a biomaterial or synthetic material that allows interaction with cell membrane proteins such as integrins. Thus, the ECM described herein is any biomaterial or synthetic material or combination thereof that mimics the in vivo cell niche by, for example, interacting with cell membrane proteins such as integrins.

[0097] In other embodiments, the ECM is in suspension. That is, the cells are in contact with the ECM in a suspension system. In some embodiments, the ECM is in suspension at a concentration of at least 1%, at least 2%, or at least 3%. In some embodiments, the ECM is in suspension at a concentration of 1% to about 10% or 1% to about 5%. Suspension methods may have advantages over up-scaling methods.

[0098] One type of ECM is secreted by epithelial cells, endothelial cells, parietal endoderm-like cells (e.g., Englebreth Holm Swarm parietal endoderm-like cells described in

[13] ), and connective tissue cells. This ECM is composed of various polysaccharides, water, elastin, and glycoproteins, where glycoproteins include collagen, entactin (nidogen), fibronectin, and laminin. Thus, in some embodiments, the ECM for use in the methods of the invention comprises one or more components selected from the list: polysaccharides, elastin, and glycoproteins, e.g., glycoproteins include collagen, entactin (nidogen), fibronectin, and / or laminin. For example, in some embodiments, collagen is used as the ECM. Various types of ECM are known, composed of various compositions including different types of glycoproteins and / or combinations of different glycoproteins.

[0099] ECM can be produced by culturing ECM-producing cells, such as epithelial cells, endothelial cells, parietal endoderm-like cells, or fibroblasts, in a container, then removing the cells and adding isolated tissue fragments or isolated epithelial cells. Examples of extracellular matrix-producing cells include chondrocytes, which mainly produce collagen and proteoglycans, fibroblasts, which mainly produce type IV collagen, laminin, interstitial procollagen, and fibronectin, and colon myofibroblasts, which mainly produce collagen (types I, III, and V), chondroitin sulfate proteoglycans, hyaluronic acid, fibronectin, and tenascin-C. These are "naturally produced ECMs." Naturally produced ECMs can be commercially provided. Examples of commercially available extracellular matrices include extracellular matrix proteins (Invitrogen), basement membrane preparations from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells (e.g., Cultrex® Basement Membrane Extract (Trevigen, Inc.) or Matrigel™ (BD Biosciences)).

[0100] Thus, in some embodiments, the ECM is a naturally produced ECM. In some embodiments, the ECM is a laminin-containing ECM, such as Matrigel™ (BD Biosciences). In some embodiments, the ECM is Matrigel™ (BD Biosciences) which comprises laminin, entactin, and type IV collagen. In some embodiments, the ECM comprises laminin, entactin, type IV collagen, and heparan sulfate proteoglycan (e.g., Cultrex® basement membrane extract type 2 (Trevigen, Inc.)). In some embodiments, the ECM comprises at least one glycoprotein, such as collagen and / or laminin. A preferred ECM for use in the methods of the invention comprises collagen and laminin. A more preferred ECM comprises laminin, entactin, and type IV collagen. If desired, a mixture of naturally produced or synthetic ECM materials may be used.

[0101] In another embodiment, the ECM can be a synthetic ECM. For example, a synthetic ECM such as ProNectin (Sigma Z378666) can be used. In a further example, the ECM can be a plastic (e.g., polyester, or hydrogel). In some embodiments, the synthetic matrix can be coated with a biomaterial (e.g., one or more glycoproteins such as collagen or laminin).

[0102] In some embodiments, the first stage, the second stage, and / or the third stage comprises culturing in the presence of an extracellular matrix. In some embodiments, the second stage and / or the third stage comprises culturing in the presence of an extracellular matrix. In some embodiments, the extracellular matrix is ​​at least 50%, at least 60%, or at least 70% Matrigel, optionally 50-100%, 50-80% Matrigel, optionally about 70% Matrigel. In preferred embodiments, the second and third stages comprise culturing in the presence of an extracellular matrix, which is at least 70% Matrigel.

[0103] In some embodiments, the ECM is a three-dimensional matrix. In some embodiments, the cells are embedded in the ECM. In some embodiments, the cells are attached to the ECM. The culture medium of the present invention can be dispersed in the three-dimensional ECM. In another embodiment, the ECM is in suspension. That is, the cells are in contact with the ECM in a suspension system. In some embodiments, the ECM is in suspension at a concentration of at least 1%, at least 2%, or at least 3%. In some embodiments, the ECM is in suspension at a concentration of 1% to about 10% or 1% to about 5%.

[0104] In some embodiments, the culture method of the present invention comprises culturing epithelial stem cells in contact with extracellular matrix.By "contacting" is meant physical or mechanical or chemical contact, which means that force must be used to separate the resulting organoid or epithelial cell population from the matrix.Culture medium and / or cells can be placed on, embedded in, or mixed with extracellular matrix or synthetic matrix.

[0105] In some embodiments, the culture medium is placed on top of the extracellular or synthetic matrix. The culture medium can then be removed and replenished as needed. In some embodiments, the culture medium is replenished every 1, 2, 3, 4, 5, 6, or 7 days. When a component is "added" or "removed" from the medium, this means, in some embodiments, that the medium itself is removed from the extracellular or synthetic matrix, and then new medium containing the "added" component or excluding the "removed" component is placed on top of the extracellular or synthetic matrix.

[0106] The three-dimensional matrix supports the culture of three-dimensional epithelial organoids.Thus, in some embodiments, the extracellular matrix or synthetic matrix is ​​a three-dimensional matrix.

[0107] In some embodiments, the medium further comprises an integrin agonist (e.g., as described in WO2020 / 234250). Specific examples of integrin agonists include anti-integrin antibodies, such as anti-b1 integrin antibodies (e.g., TS2 / 16, 12G10, 8A2, 15 / 7, HUTS-4, 8E3, N29, and 9EG7 antibodies). Integrin agonists can be used in place of or in addition to extracellular matrix.

[0108] Wnt agonists The culture medium suitable for culturing organoids and organoid fragments may contain a Wnt agonist. Wnt signaling pathway and small molecules that activate Wnt signaling are described in

[14] . Wnt agonists are defined herein as agents that activate or enhance TCF / LEF-mediated transcription in cells. Thus, Wnt agonists are selected from true Wnt agonists that bind and activate Wnt receptor complexes, including any and all Wnt family proteins, intracellular β-catenin degradation inhibitors, GSK inhibitors (such as CHIR9901), and TCF / LEF activators. One or more Wnt agonists in the culture medium may be selected from Wnt ligands from the Wnt family of secreted glycoproteins, inhibitors of intracellular β-catenin degradation, GSK-3 inhibitors, activators of TCF / LEF, inhibitors of RNF43 or ZNRF3, and R-spondin family proteins. In some embodiments, the Wnt agonist in the culture medium comprises an R-spondin family protein and a GSK-3 inhibitor, and optionally further comprises a Wnt ligand from the Wnt family of secreted glycoproteins. One or more Wnt agonists, for example, two, three, four or more Wnt agonists, can be used in the culture medium. In one embodiment, the medium comprises an Lgr5 agonist (e.g., Rspondin) and further comprises a further Wnt agonist. In this regard, the further Wnt agonist can be selected from the group consisting of, for example, Wnt-3a, a GSK inhibitor (such as CHIR99021), Wnt-5, Wnt-6a, Norrin, and NGS-Wnt. In one embodiment, the medium comprises Rspondin and further comprises a soluble Wnt ligand, such as Wnt3a or NGS-Wnt. The addition of soluble Wnt ligands has been shown to be particularly advantageous for the expansion of human epithelial stem cells (described in WO2012 / 168930).

[0109] The R-spondin family proteins (also referred to herein as "R-spondins") may be selected from R-spondin1, R-spondin2, R-spondin3, R-spondin4, and analogs, fragments, variants, and derivatives thereof. In this context, the fragments, variants, or derivatives are capable of preventing the action of the E3 ligase RNF43 / ZNRF3 on the Wnt receptor complex. R-spondin1, R-spondin2, R-spondin3, and R-spondin4 (also referred to herein as "R-spondin1-4") are all characterized by two amino-terminal furin-like repeats, which are necessary and sufficient for Wnt signal enhancement, and a thrombospondin domain located towards the more carboxyl-terminal members

[15] . Examples of R-spondin fragments, variants and derivatives suitable for use in the present invention are known to those skilled in the art (see, for example, Example 2 of WO2012 / 140274, which describes furin domain fragments that can enhance Wnt signaling, which is incorporated herein by reference). Examples of R-spondin family protein analogs include, for example, antibodies that interact with RNF43 / ZNRF3 / Lgr. Agonistic anti-Lgr5 antibodies that can enhance Wnt signaling are known in the art (see, for example, antibody 1D9 described in Example 3 of

[16] ).

[0110] Many GSK-3 inhibitors are known in the art (see, e.g.,

[17] and

[18] ) and commercially available (see, e.g., the list available from Santa Cruz Biotechnology here: https: / / www.scbt.com / scbt / browse / GSK-3-beta-Inhibitors / _ / N-x6oud). Any of these GSK-3 inhibitors are suitable for use in the context of the present invention, and the skilled artisan can determine suitable concentrations using IC50 values.

[0111] CHIR-99021 (CAS: 252917-06-9; 6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile; CT99021) is a potent and selective inhibitor of GSK-3. Other aminopyrimidine inhibitors with IC50 values ​​between 0.6 nM and 7 nM include CHIR98014 (Axon, catalog 1126), CHIR98023, CHIR99021 (see above), and TWS119 (Tocris, catalog 3835). Thus, in some embodiments, the GSK-3 inhibitor is an aminopyrimidine inhibitor optionally selected from CHIR98014, CHIR98023, CHIR99021, or TWS 119. In some embodiments, the GSK3 inhibitor is CHIR99021.

[0112] The Wnt ligand from the Wnt family of secreted glycoproteins may be selected from Wnt-1 / Int-1, Wnt-2 / Irp (InM-related protein), Wnt-2b / 13, Wnt-3 / Int-4, Wnt-3a (R&D systems), Wnt-4, Wnt-5a, Wnt-5b, Wnt-6 (see

[19] ), Wnt-7a (R&D systems), Wnt-7b, Wnt-8a / 8d, Wnt-8b, Wnt-9a / 14, Wnt-9b / 14b / 15, Wnt-10a, Wnt-10b / 12, WnM 1 and Wnt-16. A summary of human Wnt proteins is given in

[20] . In some embodiments, the Wnt ligand is Wnt-3a, Wnt-5 or Wnt-6a, or optionally Wnt-3a. The addition of soluble Wnt ligands has proven to be particularly advantageous for the expansion of human organoids and organoid fragments (e.g., as described in

[21] ).

[0113] In some embodiments, the Wnt agonist in the culture medium is a Wnt surrogate. The Wnt surrogate is a water-soluble Wnt agonist engineered by linking antagonistic Fzd and Lrp5 / 6 binding modules into a single polypeptide chain, thus forcing receptor heterodimerization while blocking endogenous Wnt binding. The Wnt surrogate supports the growth of a wide range of cultures. Furthermore, the Wnt surrogate is a non-lipidated Wnt agonist that can be produced in serum-free medium and maintained in a frozen state, avoiding differences in activity of Wnt culture supernatants from different laboratories

[22] . In some embodiments, the Wnt surrogate is a next-generation surrogate Wnt (NGS-Wnt), for example as described in

[23] . The NGS-Wnt can be provided at a concentration of about 0.1 nM to about 0.5 nM. In some embodiments, the medium comprises NGS-Wnt at a concentration of about 0.5 nM. In some embodiments, the medium comprises NGS-Wnt at a concentration of about 0.1 nM.

[0114] Mitogenic growth factors The culture medium suitable for culturing organoid and organoid fragment can contain mitogenic growth factor. Mitogenic growth factor typically induces cell division through the signal transduction pathway of mitogen-activated protein kinase. Many receptor tyrosine kinase ligands are mitogenic growth factors. In some embodiments, mitogenic growth factor can bind to receptor tyrosine kinase. In some embodiments, mitogenic growth factor can bind to one or more receptor tyrosine kinases. In some embodiments, one or more mitogenic growth factors bind to receptor tyrosine kinase, for example, EGFR, FGFR or HGFR, and optionally, one or more mitogenic growth factors are selected from EGF, FGF and HGF.

[0115] In some embodiments, the mitogenic growth factor binds to EGFR, HER1, HER2, HER3, or HER4. In some embodiments, the mitogenic growth factor binds to EGFR. In some embodiments, a HER2-4 ligand is included in the culture medium in addition to an EGFR ligand. For example, in some embodiments, neuregulin is included in the culture medium in addition to EGF. Neuregulin has been shown to be advantageous for the culture of lung and breast tissue (see, e.g.,

[24] and

[25] ). In some embodiments, one or more mitogenic growth factors in the culture medium is EGF. Any suitable EGF may be used, for example, EGF obtained from Peprotech.

[0116] FGF stimulates cells by interacting with cell surface tyrosine kinase receptors (FGFRs). Four closely related receptors (FGFR1-FGFR4) have been identified. Thus, in some embodiments, the mitogenic growth factor binds to a member of the FGF receptor family. Members of the FGF receptor family include (but are not limited to) FGFR1, FGFR2, FGFR3, or FGFR4. The FGFR1-FGFR3 genes have been shown to encode multiple isoforms, and these isoforms may be important in determining ligand specificity. There are several FGFs that bind to members of the FGF receptor family, including (but are not limited to) FGF2, FGF4, FGF7, and FGF10. These are commercially available. Thus, in some embodiments, the mitogenic growth factor is an FGF. In some embodiments, the FGF is selected from FGF2, FGF4, FGF7, and FGF10. In preferred embodiments, the FGF is FGF2 and / or FGF10. In a most preferred embodiment, the FGFs are FGF2 and FGF10.

[0117] Hepatocyte growth factor / scatter factor (HGF / SF) is a morphogenetic factor that regulates cell proliferation, cell motility, and morphogenesis by activating a tyrosine kinase signaling cascade after binding to the pro-tumorigenic HGFR. HGFR is also known as the c-Met receptor. HGF has been shown to be useful in the culture of epithelial stem cells. Thus, in some embodiments, the mitogenic growth factor binds to HGFR. In some embodiments, the mitogenic growth factor is HGF. Any suitable HGF may be used, for example, HGF obtained from Peprotech.

[0118] In some embodiments, multiple mitogenic growth factors, for example, two or three mitogenic growth factors, are included in the culture medium. For example, in some embodiments, the one or more mitogenic growth factors in the culture medium are EGF and FGF. In some embodiments, the one or more mitogenic growth factors in the culture medium are EGF, FGF2, and FGF10. In some embodiments, the one or more mitogenic growth factors in the culture medium are EGF, optionally at a final concentration of about 50 ng / ml, FGF2, optionally at a final concentration of about 5 ng / ml, and FGF10, optionally at a final concentration of about 10 ng / ml.

[0119] In some embodiments, hepatocyte growth factor (HGF) is also present, with or without EGF and / or FGF.

[0120] BMP inhibitors The culture medium suitable for culturing organoid and organoid fragment can contain BMP inhibitor.BMP inhibitor is defined as an agent that binds to BMP molecule and forms a complex, for example, by preventing or inhibiting the binding of BMP molecule to BMP receptor, thereby neutralizing BMP activity.Alternatively, said inhibitor is an agent that acts as antagonist or inverse agonist.This type of inhibitor binds to BMP receptor and prevents BMP from binding to said receptor.An example of the latter agent is an antibody that binds to BMP receptor and prevents BMP from binding to antibody-bound receptor.

[0121] BMP inhibitor can be added to culture medium in an amount effective to inhibit the BMP-dependent activity in cells by up to 90%, more preferably up to 80%, more preferably up to 70%, more preferably up to 50%, more preferably up to 30%, more preferably up to 10%, more preferably 0%, when evaluated in the same cell type, compared to the level of BMP activity in the absence of said inhibitor.As known to those skilled in the art, BMP activity can be determined by measuring the transcription activity of BMP, for example, as exemplified in Zilberberg et al., 2007.BMC Cell Biol. 8:41.

[0122] Several classes of natural BMP binding proteins are known, including noggin (Peprotech), Chordin and chordin-like proteins containing a chordin domain (R&D systems), follistatin and follistatin-related proteins containing a follistatin domain (R&D systems), DAN and DAN-like proteins containing a DAN cysteine ​​knot domain (R&D systems), sclerostin / SOST (R&D systems), decorin (R&D systems), and alpha-2 macroglobulin (R&D systems).

[0123] Thus, in some embodiments, the BMP inhibitor is selected from noggin, DAN, and DAN-like proteins, including Cerberus and Gremlin (R&D systems). These diffusible proteins can bind to BMP ligands with varying degrees of affinity and inhibit their access to signaling receptors. Addition of any of these BMP inhibitors to basal culture medium prevents loss of stem cells. The preferred BMP inhibitor is noggin.

[0124] TGF-beta inhibitors The culture medium suitable for culturing organoids and organoid fragments can contain TGF-beta inhibitor.The presence of TGF-beta inhibitor in the expansion medium is particularly advantageous for increasing human organoid formation efficiency.TGF-beta inhibitor is any agent that reduces the activity of TGF-beta signaling pathway, also referred to herein as ALK4, ALK5 or ALK7 signaling pathway.The TGF-beta inhibitor according to the present invention can be a protein, peptide, small molecule, small interfering RNA, antisense oligonucleotide, aptamer or antibody.The inhibitor can be naturally occurring or synthetic.

[0125] In some embodiments, the TGF-beta inhibitor is a small molecule inhibitor such as A83-01, which is a commercially available selective inhibitor of ALK4, ALK5, and ALK7 (Tocris catalog number 2939). The catalog also includes potent inhibitors (ICs) of TGF-beta type I receptor ALK5 kinase, type I activin / nodal receptor ALK4, and type I nodal receptor ALK7. 50The IC50 values ​​of these inhibitors are listed as 12, 45, and 7.5 nM, respectively, and they block phosphorylation of Smad2 and only slightly inhibit ALK-1, ALK-2, ALK-3, ALK-6, and MAPK activity. Other commercially available inhibitors with similar properties include, but are not limited to, A77-01, LY2157299, LY2109761, LY3200882, GW788388, pirfenidone, RepSox, SB431542, SB505124, SB525334, LY364947, SD-208, and bactoxib. The IC50 values ​​of these inhibitors are known in the art, and those skilled in the art can select the appropriate inhibitor at the appropriate concentration based on the teachings provided in the examples of this application.

[0126] Nicotinamide In some embodiments, the culture medium suitable for organoid and organoid fragment further comprises nicotinamide.Nicotinamide is an amide derivative of vitamin B3, a poly(ADP-ribose) polymerase (PARP) inhibitor, and represents the primary precursor of NAD+.It is commercially available (e.g., from Stemcell Technologies catalog 07154).

[0127] Prostaglandin pathway activators In some embodiments, the culture medium suitable for organoid and organoid fragment further comprises a prostaglandin pathway activator.The prostaglandin pathway activator can be any one or more of the compounds selected from the list including phospholipid, arachidonic acid (AA), prostaglandin E2 (PGE2), prostaglandin G2 (PGG2), prostaglandin F2 (PGF2), prostaglandin H2 (PGH2), prostaglandin D2 (PGD2).In some embodiments, the activator of prostaglandin signaling pathway is PGE2 and / or AA.In some embodiments, the activator of prostaglandin signaling pathway is PGE2.

[0128] cAMP activator In some embodiments, the culture medium suitable for organoids and organoid fragments further comprises a cAMP pathway activator. The cAMP pathway activator can be any suitable activator that increases the level of cAMP in cells. In some embodiments, the cAMP pathway activator is an adenylyl cyclase activator or a cAMP analog. Examples of suitable adenylyl cyclase activators include forskolin, forskolin analogs, and cholera toxin. Examples of forskolin analogs are known in the art and include NKH477 (e.g., catalog number Tocris 1603). Examples of cAMP analogs are also known in the art and include, for example, 8-bromo-cAMP. 8-bromo-cAMP is a cell-permeable cAMP analog that is more resistant to hydrolysis by phosphodiesterase than cAMP. Thus, in some embodiments, the cAMP pathway activator is selected from forskolin, cholera toxin, NKH477, and 8-bromo-cAMP. In some embodiments, the cAMP pathway activator is forskolin. In some embodiments, the cAMP pathway activator is not cholera toxin.

[0129] Additional Ingredients Basal media for cell culture typically contain a number of components necessary to support the maintenance of cultured cells. Appropriate combinations of components can be readily prepared by those skilled in the art in light of the following disclosure. Basal media for use in the present invention generally contain a nutrient solution containing standard cell culture components such as amino acids, vitamins, lipid supplements, inorganic salts, carbon energy sources, and buffers, as described in the literature and in more detail below. In some embodiments, the culture medium is further supplemented with one or more standard cell culture components selected from, for example, amino acids, vitamins, lipid supplements, inorganic salts, carbon energy sources, and buffers. Suitable basal media are known to those skilled in the art and are commercially available, for example, but not limited to, Dulbecco's Modified Eagle's Medium (DMEM), Advanced DMEM, Minimum Essential Medium (MEM), Knockout-DMEM (KO-DMEM), Glasgow Minimum Essential Medium (G-MEM), Basal Eagle's Medium (BME), DMEM / Ham's F12, Advanced DMEM / Ham's F12, Iscove's Modified Dulbecco's Medium and Minimum Essential Medium (MEM), Ham's F10, Ham's F12, 199 medium, and RPMI 1640 medium. For example, the basal medium may be preferably Advanced DMEM supplemented with glutamax, penicillin / streptomycin, and HEPES.

[0130] The culture medium suitable for organoids and organoid fragments may be supplemented with one or more of the compounds selected from the group consisting of gastrin, B27, N-acetylcysteine, and N2.Thus, in some embodiments, the above-mentioned culture medium further comprises one or more components selected from the group consisting of gastrin, B27, N2, and N-acetylcysteine.B27 (Invitrogen), N-acetylcysteine ​​(Sigma), and N2 (Invitrogen), gastrin (Sigma) are believed to control cell proliferation and support DNA stability.In some embodiments, the culture medium further comprises B27 and N-acetylcysteine.

[0131] In some embodiments, the culture medium further comprises a ROCK inhibitor (Rho kinase inhibitor). ROCK inhibitors are particularly useful for cell attachment when establishing new cultures and / or splitting cells ("passaging"). Suitable ROCK inhibitors are known in the art and commercially available (including but not limited to GSK 269962, GSK 429286, H 1152 dihydrochloride, glycyl-H 1152 dihydrochloride, SR 3677 dihydrochloride, SB 772077B dihydrochloride, and Y-27632 dihydrochloride, all available from Tocris). In some embodiments, the medium is supplemented with 5 μM to 20 μM or 8 μM to 15 μM of ROCK inhibitor, optionally about 10 μM of ROCK inhibitor.

[0132] The culture medium preferably does not contain undefined components (such as fetal bovine serum or fetal bovine serum or feeder cells). A variety of different serum replacement formulations are commercially available and known to those skilled in the art. When serum replacement is used, it may be used at about 1% to about 30% of the medium volume according to conventional techniques. In some embodiments, the culture medium is serum-free and / or feeder-free.

[0133] A preferred cell culture medium is a defined synthetic medium that is buffered with a carbonate-based buffer at pH 7.4 (preferably pH 7.2-7.6 or at least 7.2 but not greater than 7.6) while the cells are cultured in an atmosphere containing 5%-10% CO2, or at least 5% to 10% CO2, preferably 5% CO2.

[0134] Use of co-cultured organoid models The invention provides methods for drug screening, target validation, target discovery, toxicology, or toxicology screening using the co-culture methods of the invention. For example, the invention provides a method for testing the effect of a candidate compound, comprising: Exposing the co-culture of genotoxic bacteria and organoids according to the method of the present invention to one of a library of candidate compounds; Evaluating the organoids in co-culture for any effects (e.g., compared to a control not exposed to the candidate compound); and identifying candidate molecules that cause said effect as potential drugs.

[0135] Genotoxic bacteria can be pre-cultured with one of the libraries of candidate compounds and then introduced into the co-culture method. Alternatively, one of the libraries of candidate compounds can be introduced in the first, second, and / or third stages of the co-culture method. In some embodiments, one of the libraries of candidate compounds is introduced in one, two, or all three stages. These methods can be used to test libraries of chemicals, antibodies, natural products (e.g., plant extracts or microbial compounds), etc. for suitability for use as drugs, cosmetics, and / or prophylactics.

[0136] The present invention also provides the use of the organoid with the mutation signature generated by the method of the present invention in drug screening, target validation, target discovery, toxicology or toxicology screening.In some embodiments, the organoid is used to model host-pathogen interaction and / or identify the compound suitable for treating bacterial infection and / or cancer.

[0137] For example, the cells are preferably exposed to multiple concentrations of the test agent for a period of time. At the end of the exposure period, the culture is evaluated. The organoids can also be used to identify drugs that specifically target epithelial cancer cells. Those skilled in the art will understand that the organoids of the present invention are widely applicable as drug screening tools for infectious, inflammatory, and neoplastic conditions. In some embodiments, the present invention provides the use of the organoids in drug screening, target validation, target discovery, toxicology, toxicology screening, or ex vivo cell / organ model. In some embodiments, the present invention provides the use of the organoids in ex vivo methods for predicting clinical outcomes. In some embodiments, the organoids of the present invention can be used for screening cancer drugs.

[0138] In some embodiments, the organoids of the present invention can be used to test libraries of chemicals, antibodies, natural products (e.g., plant extracts or microbial compounds), etc. for suitability for use as drugs, cosmetics, and / or prophylactics.

[0139] In some embodiments, the present invention provides a method for testing the effect of a candidate compound, comprising: exposing said organoids having a mutation signature to one or a library of candidate compounds; Evaluating the expanded organoids for any effects (e.g., compared to a control not exposed to the candidate compound); and identifying candidate molecules that cause said effect as potential drugs.

[0140] In some embodiments, the method for testing the effect of candidate compound comprises exposing the organoid with mutation signature to radiation in the presence or absence of candidate compound.In some embodiments, the effect evaluated in the method for testing the effect of candidate compound is selected from the list comprising: proliferation, morphological change, cell death, or reduction or loss of gene or protein expression change.The library of candidate molecules comprises a plurality of candidate molecules.

[0141] In some embodiments, the present invention provides a method for the preparation of a exposing the organoid having the mutational signature, or a population of cells derived from the organoid having the mutational signature, to a treatment, such as radiation, and / or to one or a library of candidate molecules; Evaluating the organoid or cell population for any effect of a candidate molecule; and correlating the effect with a characteristic of the organoid, e.g., the presence of one or more genetic mutations, e.g., a mutation in the EGFR signaling pathway, including PIK3CA, KRAS, HRAS, or BRAF.

[0142] In some embodiments, the present invention provides a method for the preparation of a exposing the organoid or a population of cells derived from the organoid to a treatment, such as radiation, and / or to one or a library of candidate molecules; Evaluating the organoid or cell population for any effect of a candidate molecule; Comparing said effect to a standard and / or previous observations, and optionally and predicting a clinical outcome and / or selecting a personalized medicine.

[0143] definition As used herein, the verb "comprise" and its conjugations are used in an open-ended sense, meaning that the items following the word are included, but not excluding items not specifically mentioned. In addition, the verb "consisting of" may be replaced with "consisting essentially of" if necessary, meaning that the product defined herein may include additional component(s) other than the specifically identified components, and said additional component(s) do not modify the specific characteristics of the invention. In addition, the method defined herein may include additional step(s) other than the specifically identified steps, and said additional step(s) do not modify the specific characteristics of the invention. Furthermore, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that a plurality of the elements may be present, unless the context clearly requires that one and only one of the elements is present. Thus, the indefinite article "a" or "an" usually means "at least one".

[0144] As used herein, the term "about" or "approximately" means that the indicated value may vary by + / - 10%. The value may also be read as an exact value, and therefore the term "about" may be omitted. For example, the term "about 100" includes 90 to 110, and also includes 100.

[0145] All patent and literature references cited herein are hereby incorporated by reference in their entirety.

[0146] Any reference to a method of treatment involving the administration of an agent to a patient also includes that agent for use in said method of treatment, as well as the use of that agent in said method of treatment, and the use of that agent in the manufacture of a medicament.

[0147] The following examples are presented for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

[0148] The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology that are within the skill of the art. Such techniques are explained fully in the literature; see, for example, references [26-32].

[0149] Embodiment The present invention provides the following numbered embodiments: 1. A method for co-culturing genotoxic bacteria with organoids, comprising at least two steps selected from the following: A first step involves culturing the organoids or organoid fragments with genotoxic bacteria; A second step comprising culturing said organoids or organoid fragments with genotoxic bacteria, wherein said bacterial growth is restricted with an antibiotic; A third step includes killing the genotoxic bacteria to allow further growth of the organoids or the organoid fragments. 2. The method of embodiment 1, which generates an organoid or organoid fragment having a mutation signature. 3. The method of any of the preceding embodiments, wherein the genotoxic bacteria are introduced in the first stage and allowed to infest the system for further cultivation in the second stage. 4. The method of any of the preceding embodiments, wherein the organoid fragments are obtained by shearing one or more organoids. 5. The method of any of the preceding embodiments, wherein during the second stage, at least 60%, at least 70%, at least 80%, or at least 90% of the organoid fragments form one or more organoids. 6. (a) the first and second stages only; (b) the first and third stages only; (c) the second and third stages only; or (d) The first, second, and third stages 4. The method of any preceding embodiment, comprising: 7. The method of any of the preceding embodiments, wherein the steps of the method are repeated at least 2 times, at least 3 times, at least 4 times, or at least 5 times, and optionally, the steps of the method are repeated at least 5 times. 8. The method of any of the preceding embodiments, wherein the genotoxic bacteria are in exponential growth phase when incorporated into the co-culture. 9. The method of any of the preceding embodiments, wherein the genotoxic bacteria have a multiplicity of infection of 0.1 to 800, 10 to 100, 50 to 100, or about 100 when incorporated into the co-culture. 10. The method of any of the preceding embodiments, wherein the rate of growth of the genotoxic bacteria in the second stage is lower than the rate of growth of the genotoxic bacteria in the first stage, and optionally, the growth rate of the genotoxic bacteria in the second stage is reduced by at least 10%, 20%, 30%, 40%, or 50% compared to the growth rate of the genotoxic bacteria in the first stage. 11. The method of any of the preceding embodiments, wherein the rate of growth of the genotoxic bacteria in the second stage is limited using an antibiotic. 12. The method of any of the preceding embodiments, wherein the organoid fragment or organoid is derived from the gastrointestinal tract, stomach, pancreas, lung, mouth, nasopharynx, throat, hypopharynx, larynx, trachea, skin, vagina, and / or esophageal tissue, and optionally, the organoid fragment or organoid is derived from the gastrointestinal tract. 13. The method of any of the preceding embodiments, wherein the first stage is carried out for a period of between 15 minutes and 5 hours. 14. A method according to any of the preceding embodiments, wherein the second stage is carried out over a period of between 2 and 3 days. 15. A method according to any of the preceding embodiments, wherein the third stage is carried out for between 1 and 20 days. 16. The method of any of the preceding embodiments, wherein the genotoxic bacterium is a colibactin-producing bacterium. 17. The method of embodiment 16, wherein the colibactin-producing bacterium is from the family Enterobacteriaceae. 18. The method of embodiment 17, wherein the colibactin-producing bacteria is derived from the species Escherichia coli. 19. The method of embodiment 18, wherein the colibactin-producing bacterium is a pks+ E. coli. 20. The method of any of the preceding embodiments, wherein the first step is carried out in suspension culture. 21. The method of any of the preceding embodiments, wherein the second step and / or the third step comprises culturing in the presence of an extracellular matrix, optionally wherein the extracellular matrix is ​​matrigel or a basement membrane extract. 22. The method of any preceding embodiment, generating an organoid having a mutational signature. 23. The method of embodiment 22, which generates an organoid having a cancer mutation signature. 24. A method for generating organoids with a mutational signature, comprising at least two steps selected from the following: A first step involves culturing the organoids or organoid fragments with genotoxic bacteria; A second step comprising culturing the organoids or organoid fragments with genotoxic bacteria, wherein the bacterial growth is restricted with an antibiotic; A third step includes killing the genotoxic bacteria and allowing further growth of the organoid or organoid fragment, whereby an organoid or organoid fragment having a mutation signature is generated. 25. The method of embodiment 24, wherein the mutation signature is a cancer mutation signature. 26. The method of embodiment 24 or 25, further comprising detecting DNA damage and / or determining the mutational signature. 27. The method of any one of embodiments 24 to 26, further comprising treating the organoids produced by the method with one or more test compounds to identify compounds suitable for treating bacterial infections and / or cancer. 28. A method according to any one of embodiments 22 to 27, wherein the organoids generated by the method are a medium- or high-throughput platform suitable for modeling host (organoid)-pathogen (bacteria) interactions. 29. A method for testing the effect of a candidate compound, comprising: Exposing the co-culture of genotoxic bacteria and organoids from the method according to any of the preceding embodiments to one of a library of candidate compounds; Evaluating the organoids in the co-culture for any effects; identifying candidate molecules that cause said effect as potential drugs; The method comprising: 30. The method of embodiment 29, wherein the genotoxic bacteria are pre-cultured with one of a library of candidate compounds and then introduced into the co-culture method described in any of the preceding embodiments. 31. An organoid having a mutational signature generated by the method of any one of the preceding embodiments. 32. The organoid of embodiment 31, which is a gastrointestinal organoid having a colorectal cancer mutation signature. 33. Use of the organoid described in embodiment 31 for modeling host-pathogen interactions and / or for identifying compounds suitable for treating bacterial infections and / or cancer. EXAMPLES

[0150] Example 1 - A method for co-cultivating genotoxic bacteria with organoids, comprising three steps In this example, intestinal organoids were co-cultured with colibactin-producing pks+ E. coli to model aspects of cancer initiation and progression. The co-culture strategy scheme tested consists of three steps, as shown in Figure 1A: · First phase (co-culture I) - a phase of "acute" DNA damage where bacteria and organoid fragments interact in suspension and bacterial growth is not restricted; · The second phase (co-culture II) - a phase of “sustained” DNA damage in which organoid fragments and bacteria are cultured in hydrogels and bacterial growth is controlled by the addition of low concentrations of antibiotics; The third phase (recovery phase) - the bacteria are killed using antibiotics and the organoids or organoid fragments continue to grow in the hydrogel.

[0151] First step Processing of colon tissue resections for the establishment of organoid cultures was performed according to the method described by Sato et al.

[33] . Organoids derived from healthy donors were grown in 70% Matrigel for 2–4 days, sheared with a glass pipette, and incubated for 3–4 h in suspension with bacteria (MOI0[Bact]1, MOI10[Bact]2, MOI50[Bact]3, MOI100[Bact]4, MOI500[Bact]5, and MOI1000[Bact]6) in exponential growth phase (OD600 >0.4 and <1) in 0.5 medium (composition below) supplemented with 10 μM Rhoki and in the absence of antibiotics. MOI (multiplicity of infection) refers to the ratio of bacteria to organoid cells at the start of coculture I. Organoids exposed to MOI500 and MOI1000 did not survive.

[0152] The composition of CSM medium was Ad-DF++ (GultaMax, Advanced Dulbecco's Modified Eagle's / F12 medium supplemented with 1M HEPES) containing 0.25 mM N-acetylcysteine, 500 nM TGFβ receptor inhibitor A83-01, 1× B27 supplement, 50 ng / ml EGF, 5 nM gastrin, 2% Noggin culture supernatant made with UPE or 100 ng / ml recombinant Noggin (N), 10 mM nicotinamide, 250 ng / ml R-spondin-3, 10 μM SB202190 p38 MAPK inhibitor, and 0.5 nM NGS Wnt.

[0153] Figure 1B shows organoid fragments after 4 h of co-culture with E. coli (green) MOI100 in CSM medium supplemented with 10 μM Rhoki, 10% Matrigel, and in suspension 0.5 in the absence of antibiotics. Prior to co-culture, bacteria were stained with CFSE in PBS for 30 min. Nuclei of cells of the organoids (blue) were stained with DAPI. Phalloidin (orange) stained the apical side of the organoid fragments. The addition of 10% Matrigel in this experiment allowed the immobilization and subsequent visualization of bacteria in the vicinity of the organoid cells.

[0154] Second Phase After co-culture I, the organoids were washed with AdDMEM medium and seeded in 70% Matrigel. After Matrigel solidification, the organoids were cultured for 2 days in 0.5 CSM medium supplemented with 50 μg / ml gentamicin.

[0155] Third Phase We then cultured the organoids in 0.5 CSM containing 50 µg / ml primocin and allowed the organoids to recover for 2–5 days. Organoids that survived after co-culture with colibactin-producing bacteria are shown in Figure 1D.

[0156] Co-culture of organoid fragments with pks+ E. coli resulted in DNA damage, as shown by an increase in the levels of yH2AX in live single cells, which is a measure of the extent of DNA damage, as analyzed by flow cytometry. Figure 1C shows the distribution of organoid single cells derived from the co-culture as a function of yH2AX levels and at different time points, obtained by flow cytometry. In this experiment, the first phase lasted 3 h, the second phase 2 days, and the third phase 3 days. yH2AX levels in live single cells were analyzed after 3 h of co-culture in suspension (first phase), after 16 h of co-culture in 70% Matrigel in the presence of 50 μg / ml gentamicin (second phase), and after 3 days of recovery in the presence of primocin (third phase). [Bact]1=MOI1, [Bact]2=MOI10, [Bact]3=MOI100, [Bact]4=MOI500. DNA damage was detected in a dose-dependent manner after stage 1. After 16 h of co-culture II, DNA damage was only detected at MOI 100. No DNA damage was detected after recovery.

[0157] Example 2: DNA damage detected in co-cultures of genotoxic bacteria with organoids depends on colibactin production The co-culture scheme used in this example is shown in Figure 2A. The co-culture strategy scheme tested consists of two steps, the first step and the third step as outlined above.

[0158] This is the first step that involves shearing the organoid fragments that were cultured for 3 days with a glass pipette. They were then incubated for 4 hours in suspension with exponentially growing (OD600 > 0.4 and < 1) bacteria (control without bacteria = MOI 0; colibactin producing and colibactin deficient (ΔClbP) = MOI 500) in 0.5 CSM medium supplemented with 10 μM Rhoki and without antibiotics.

[0159] The organoid fragments were then washed with ADMEM and cultured in suspension in 0.5 CSM medium supplemented with 10 μM Rhoki and 100 μg / ml gentamicin for the recovery phase (third phase). After this 4 h recovery phase, the organoids were washed and processed for staining (immunofluorescence (IF)). Figures 2B, 2C, and 2D demonstrate that colibactin-producing bacteria induced higher levels of DNA damage (levels of yH2AX) in organoid fragments than colibactin-deficient (ΔClbP) bacteria.

[0160] Example 3: A method for co-cultivating genotoxic bacteria with organoids comprising two steps The co-culture scheme used in this example is shown in Figure 3 A. The co-culture strategy scheme tested consisted of two steps, a first step and a second step as outlined above.

[0161] This is the first step that involves shearing the organoid fragments cultured for 4 days using a glass pipette. They were then incubated with bacteria: control without bacteria = MOI 0; exponentially growing (OD600 > 0.4 and < 1) MOI 50, MOI 100, MOI 200, and WT MOI 300 colibactin-producing (WT:KanR) and colibactin-deficient (ΔClbP:KanR) bacteria for 3 h in suspension in 0.5 CSM medium supplemented with 10 μM Rhoki and in the absence of antibiotics.

[0162] Organoids and organoid fragments were washed with AdDMEM medium and seeded in 70% Matrigel in medium containing 50ug / ml gentamicin for 12 hours. Organoids were washed and processed for immunofluorescence staining. Figure 3B shows that organoids or organoid fragments exposed to bacteria at MOI 50-300 survive to the stage of acute DNA damage. Figure 3C and 3D show that organoid fragments exposed to colibactin-producing bacteria have enhanced nuclear levels of yH2AX, a proxy for DNA damage, than organoid fragments exposed to colibactin-deficient bacteria, thus demonstrating that colibactin-producing bacteria induced higher levels of DNA damage (levels of yH2AX) than colibactin-deficient (ΔClbP) bacteria in organoid fragments.

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Claims

1. A method for co-cultivating genotoxic bacteria and organoids, comprising at least two steps selected from the following: A first step comprising culturing organoids or organoid fragments with genotoxic bacteria, A second step comprising culturing the organoids or organoid fragments with genotoxic bacteria, wherein the bacterial growth is restricted with an antibiotic; and A third step includes killing the genotoxic bacteria to allow further growth of the organoids or organoid fragments.

2. The method of claim 1, which does not include injecting the genotoxic bacteria into the lumen of the organoid.

3. The first step comprises culturing the organoid fragments with genotoxic bacteria; A second step comprising culturing the organoid fragments with genotoxic bacteria, wherein the bacterial growth is restricted with an antibiotic; and A third step, comprising killing the genotoxic bacteria to allow further growth of the organoid fragments; Optionally, the organoid fragments are obtained by shearing one or more organoids. The method of claim 1.

4. The method of claim 1 or claim 3, wherein during the second stage, at least 60%, at least 70%, at least 80%, or at least 90% of the organoid fragments form one or more organoids.

5. 2. A method according to any preceding claim, generating an organoid or organoid fragment having a mutational signature.

6. 10. The method according to any of the preceding claims, wherein said genotoxic bacteria are introduced in said first stage and allowed to infest the system for further cultivation in said second stage.

7. (a) the first step and the second step only; (b) the first and third steps only; (c) only the second and third steps; or (d) the first stage, the second stage, and the third stage 13. A method according to any preceding claim, comprising:

8. 10. The method of any preceding claim, wherein said steps of said method are repeated at least 2 times, at least 3 times, at least 4 times, or at least 5 times, optionally wherein said steps of said method are repeated at least 5 times.

9. 10. The method of any of the preceding claims, wherein the genotoxic bacteria are in exponential growth phase when incorporated into the co-culture.

10. 10. The method of any of the preceding claims, wherein the genotoxic bacteria have a multiplicity of infection of 0.1-800, 10-100, 50-100, or about 100 when incorporated into the co-culture.

11. 2. The method of any preceding claim, wherein the rate of growth of the genotoxic bacteria in the second stage is lower than the rate of growth of the genotoxic bacteria in the first stage, and optionally the growth rate of the genotoxic bacteria in the second stage is reduced by at least 10%, 20%, 30%, 40%, or 50% compared to the growth rate of the genotoxic bacteria in the first stage.

12. 10. The method according to any of the preceding claims, wherein the rate of growth of the genotoxic bacteria in the second stage is limited using an antibiotic.

13. The method of any of the preceding claims, wherein the organoid fragment or organoid is derived from gastrointestinal tract, stomach, pancreas, lung, mouth, nasopharynx, throat, hypopharynx, larynx, trachea, skin, vagina, and / or esophageal tissue, and optionally, the organoid fragment or organoid is derived from the gastrointestinal tract.

14. 10. The method of any of the preceding claims, wherein the first stage is carried out for a period of between 15 minutes and 5 hours.

15. 10. The method according to any of the preceding claims, wherein the second stage is carried out over a period of between 2 and 3 days.

16. 13. The method of any of the preceding claims, wherein the third step is carried out for between 1 and 20 days.

17. 10. The method of any of the preceding claims, wherein said genotoxic bacterium is a colibactin-producing bacterium, optionally wherein said colibactin-producing bacterium is from the family Enterobacteriaceae.

18. 18. The method of claim 17, wherein the colibactin-producing bacterium is pKs+ E. coli.

19. 2. The method according to any of the preceding claims, wherein the first step is carried out in a suspension culture.

20. 2. The method of any of the preceding claims, wherein the second step and / or the third step comprises culturing in the presence of an extracellular matrix, optionally wherein the extracellular matrix is ​​matrigel or a basement membrane extract.

21. 10. The method of any preceding claim, comprising generating an organoid having a mutational signature, and optionally generating an organoid having a cancer mutational signature.

22. 1. A method for testing the effect of a candidate compound, comprising: Exposing a co-culture of genotoxic bacteria and organoids from the method according to any of the preceding claims to one of a library of candidate compounds; Evaluating the organoids in the co-culture for any effects. identifying candidate molecules that cause said effect as potential drugs; The method comprising:

23. 23. The method of claim 22, wherein the genotoxic bacteria are pre-cultured with one of a library of candidate compounds and then introduced into the co-culture method according to any of the preceding claims.

24. 2. An organoid having a mutational signature generated by the method of any one of the preceding claims, said organoid being optionally a gastrointestinal organoid having a colorectal cancer mutational signature.

25. Use of the organoid of claim 24 for modeling host-pathogen interactions and / or for identifying compounds suitable for the treatment of bacterial infections and / or cancer.

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