Methods for identifying selective condensate modulators

Abstract

In some aspects, the present application provides methods for identifying stimulation of condensate modulating properties for one or more condensate types, such as for identifying selective condensate modulators. In other aspects, kits and systems are also provided herein. Traditional disease research and therapeutic drug discovery applications focus on identifying individual biomolecules (molecular targets) that cause or mediate disease-related biology, for example, via known cellular pathways.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 370,354, filed August 3, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] The present application relates to the field of biological condensates. [Background technology]

[0003] Traditional disease research and therapeutic drug discovery applications focus on identifying individual biomolecules (molecular targets) that cause or mediate disease-related biology, for example, via known cellular pathways. This "single-target" approach to disease research and therapeutic drug discovery has significant limitations because in vivo systems are highly complex and our understanding of the actions and interactions of cellular pathways and biomolecules is often incomplete. Furthermore, not all individual biomolecules are suitable drug targets when viewed alone. Such challenges contribute to the current lack of new therapeutics that address unmet medical needs. In the search for new therapeutics, biological condensates are currently being evaluated as targets and / or tools for drug discovery. Certain early studies in the field of biological condensates observed chemicals that can indiscriminately dissolve and / or prevent the formation of biological condensates. However, chemicals with such broad effects can lead to undesirable drug toxicity and off-target properties. There remains a need in the art for new methods to identify therapeutic candidates that selectively modulate condensate types and / or condensate components. Summary of the Invention [Means for solving the problem]

[0004] In certain aspects, provided herein are methods for identifying a stimulus of a condensate modulatory property for one or more condensate types, the method comprising: (a) subjecting a cellular composition comprising a cell type to the stimulus; (b) measuring a characteristic of at least one marker in at least a portion of the cellular composition subjected to the stimulus, wherein the marker associates with at least one of the one or more condensate types before and / or after the cellular composition is subjected to the stimulus; (c) determining a marker perturbation score for each marker in the cellular composition subjected to the stimulus based on the measured characteristic of each marker; and (d) identifying a stimulus of the condensate modulatory property from the marker perturbation scores.

[0005] In some embodiments, the method further includes determining a global condensate perturbation score, wherein the global condensate perturbation score is based on the at least one marker perturbation score, and wherein identifying stimulation of the condensate modulation property can be identified from the at least one marker perturbation score and / or the global condensate perturbation score.

[0006] In some embodiments, the step of measuring the characteristic measures two markers for each of at least one or more condensate types in at least a portion of the cell composition that has been subjected to the stimulus, hi some embodiments, the step of measuring the characteristic measures at least one marker for at least two or more condensate types.

[0007] In some embodiments, each marker is independently a lipid, a polypeptide, or a nucleic acid. In some embodiments, each marker is independently a polypeptide. In some embodiments, each marker (i) is within the condensate type, (ii) is partitioned into the condensate type after the cell composition is subjected to a stimulus, or (iii) is excluded from the condensate type after the cell composition is subjected to a stimulus. In some embodiments, each marker is independently a condensate scaffold polypeptide or a nucleic acid. In some embodiments, at least one marker is a condensate scaffold polypeptide. In some embodiments, each marker is independently a condensate client polypeptide or a nucleic acid.

[0008] In some embodiments, measuring the characteristic of at least one marker in at least a portion of the cellular composition that has been subjected to stimulation comprises staining at least a portion of the cellular composition for the marker. In some embodiments, the staining is immunofluorescence (IF) staining.

[0009] In some embodiments, measuring at least one marker characteristic of at least a portion of the cellular composition comprises imaging at least a portion of the cellular composition, hi some embodiments, the imaging comprises fluorescent imaging.

[0010] In some embodiments, at least one of the one or more different condensate types is formed in the cell composition after being subjected to a stimulus.

[0011] In some embodiments, the stimulus is selected from the group consisting of an exogenous compound, an exogenous peptidic agent, an exogenous genetic material, a stressor, an environmental stimulus, and combinations thereof. In some embodiments, the stimulus is an exogenous compound. In some embodiments, the compound is a small molecule therapeutic candidate or a precursor thereof.

[0012] In some embodiments, the cell composition is subjected to a compound at a known concentration.

[0013] In some embodiments, the method further includes subjecting the cellular composition to a second stimulus; measuring a characteristic of at least one marker in at least a portion of the cellular composition that has been subjected to the second stimulus; and independently determining a marker perturbation score for each marker in the cellular composition that has been subjected to the second stimulus based on the measured characteristic of each marker in the cellular composition that has been subjected to the second stimulus.

[0014] In some embodiments, the method further comprises comparing the marker perturbation score of the marker in the cell composition subjected to the stimulus with the marker perturbation score of the marker in the cell composition subjected to a second stimulus.

[0015] In some embodiments, the method further includes determining a second global condensate perturbation score and evaluating the second stimulus on the condensate modulation property, wherein the second global condensate perturbation score is based on at least one marker perturbation score of a marker in the cellular composition subjected to the second stimulus.

[0016] In some embodiments, the method further includes comparing the global condensate perturbation score to a second global condensate perturbation score.

[0017] In some embodiments, the method further includes determining at least one property associated with at least one of the one or more condensate types and / or markers. In some embodiments, the at least one property associated with at least one of the one or more condensate types and / or markers includes (i) the location of the condensate type, (ii) the distribution of the condensate type and / or marker, (iii) the number of condensate types, (iv) the size of the condensate type, (v) the ratio of the amount of the condensate type to a control condensate, (vi) a functional activity associated with the condensate type, (vii) the composition of the condensate type, (viii) co-localization of the condensate type with a biomolecule, or (ix) the diffusion coefficient of a component of the condensate type. , (x) stability of the condensate type, (xi) dissolution or size reduction of the condensate type, (xii) surface area of ​​the condensate type, (xiii) sphericity of the condensate type, (xiv) flowability of the condensate type, (xv) solidification of the condensate type, (xvi) location of the marker, (xvii) amount of the marker or its precursor, (xviii) condensate partitioning of the marker into condensate types, (xix) functional activity associated with the marker, (xx) aggregation of the marker, (xxi) post-translational modification state of the marker, and (xxii) amount of degradation products of the marker.

[0018] In some embodiments, at least one of the one or more condensate types is selected from the group consisting of cleavage bodies, p granules, histone locus bodies, multivesicular bodies, neuronal RNA granules, intranuclear gems, nuclear pore complexes, nuclear speckles, nuclear stress bodies, nucleoli, Oct1 / PTF / transcription (OPT) domains, paraspeckles, juxtanucleolar compartments, PML nucleoli, PML oncogenic domains, Polycomb bodies, processing bodies, Sam68 nucleoli, stress granules, splicing speckles, P62 bodies, Cajal bodies, heterochromatin, transcription condensates, and centrosomes.

[0019] In some embodiments, the method includes measuring a characteristic of a second marker in at least a portion of the cellular composition subjected to the stimulus, where the second marker associates with at least one of the one or more condensate types before and / or after the cellular composition is subjected to the stimulus. In some embodiments, the method further includes determining a second marker perturbation score for the second marker in the cellular composition subjected to the stimulus based on the measured characteristic of the second marker. In some embodiments, the marker perturbation score indicates whether the stimulus modulates association of the marker with at least one of the one or more condensate types in the cellular composition, and the second marker perturbation score indicates whether the stimulus modulates association of the second marker with at least one of the one or more condensate types in the cellular composition. In some embodiments, the marker and the second marker associate with the same condensate type. In some embodiments, the marker and the second marker associate with different condensate types.

[0020] In some embodiments, each marker perturbation score is independently based on at least one characteristic associated with at least one of the one or more condensate types and / or markers.

[0021] In some embodiments, each marker perturbation score is independently based on the coefficient of variation (CV) of the marker, where the CV is determined based on the standard deviation (STD) of the distribution intensity of the marker divided by the mean distribution intensity of the marker. In some embodiments, the distribution intensity of each marker is based on pixel analysis of one or more images of one or more cellular compositions or portions thereof. In some embodiments, each marker perturbation score is based on the median absolute deviation (MAD) Z-score of the CV of the marker.

[0022] In some embodiments, the method further includes obtaining the MAD Z-score of a marker. In some embodiments, (i) if the MAD Z-score of the marker is greater than 5 or less than -5, the marker perturbation score is 1; (ii) if 2.5 < MAD Z-score ≤ 5 or -5 ≤ MAD Z-score < -2.5, the marker perturbation score is 0.5; (iii) if -2.5 ≤ MAD Z-score ≤ 2.5, the marker perturbation score is 0.

[0023] In some embodiments, determining the marker perturbation score of a marker includes extracting one or more features from an image of the cell composition subjected to the stimulus. In some embodiments, the one or more features are based on one or more of one or more texture features, one or more intensity features, or one or more morphological features. In some embodiments, determining the marker perturbation score of a marker includes measuring a plurality of features associated with the marker, calculating a modified Z-score for each feature of each marker based on the plurality of measured features, calculating a feature change score for each feature of each marker based on the associated modified Z-score and a proportional scale, and then aggregating the feature change scores of the upper percentage of the feature change scores. In some embodiments, aggregating the modified Z-scores includes summing or averaging.

[0024] In some embodiments, the method further includes evaluating a reference stimulus, the evaluation including determining a reference marker perturbation score. In some embodiments, the reference marker perturbation score is 0, and a marker perturbation score greater than 0 indicates that the stimulus has condensate regulatory properties.

[0025] In some embodiments, the global condensate perturbation score indicates that the stimulus selectively regulates one or more condensate types of the cell composition subjected to the stimulus.

[0026] In some embodiments, the global condensate perturbation score indicates that the stimulus non-selectively modulates one or more condensate types of the cellular composition treated with the stimulus.

[0027] In some embodiments, the global condensate perturbation score indicates that the stimulus does not substantially modulate one or more condensate types of the cellular composition treated with the stimulus.

[0028] In some embodiments, the global condensate perturbation score is based on the marker perturbation score and the second marker perturbation score.

[0029] In some embodiments, the global condensate perturbation score is calculated by dividing the sum of all marker perturbation scores by the number of marker perturbation scores. In some embodiments, the global condensate perturbation score is calculated by summing all of the marker perturbation scores.

[0030] In some embodiments, the method includes determining a reference global condensate perturbation score. In some embodiments, the reference global condensate perturbation score is 1, where (i) a global condensate perturbation score greater than 0 and less than 1 indicates that the stimulus selectively modulates one or more condensate types in the cellular composition, and (ii) a global condensate perturbation score of 0 indicates that the stimulus does not substantially modulate one or more condensate types in the cellular composition. In some embodiments, for global condensate perturbation scores greater than 0 and less than 1, the smaller the stimulus's global condensate perturbation score, the more selective the stimulus.

[0031] Those skilled in the art will understand that changes in form and detail of the implementations described herein may be made without departing from the scope of the present disclosure. Additionally, while various advantages, aspects, and objectives have been described with respect to various implementations, the scope of the present disclosure should not be limited with respect to such advantages, aspects, and objectives.

[0032] All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety. [Brief explanation of the drawings]

[0033] [Figure 1] A and B show exemplary cell images with low coefficient of variation (CV, A) and high CV (B).

[0034] [Figure 2A] Figure 1 shows a visualization of marker perturbation scores and global condensate perturbation scores for 60 compounds at a concentration of 4 μM, evaluated across 12 condensate types using two markers for each condensate type. Condensate types are provided alphabetically, e.g., A, B, C, etc., condensate components are provided below the condensate type using Roman numerals, and compounds are provided numerically, e.g., 1, 2, 3, etc. Global condensate perturbation scores are provided following the compound identifier. [Figure 2B] Figure 1 shows a visualization of marker perturbation scores and global condensate perturbation scores for 60 compounds at a concentration of 4 μM, evaluated across 12 condensate types using two markers for each condensate type. Condensate types are provided alphabetically, e.g., A, B, C, etc., condensate components are provided below the condensate type using Roman numerals, and compounds are provided numerically, e.g., 1, 2, 3, etc. Global condensate perturbation scores are provided following the compound identifier.

[0035] [Figure 3A]Figure 1 shows a visualization of the marker perturbation scores and global condensate perturbation scores for 40 compounds evaluated across 11 condensate types using two markers for each condensate type (22 condensate markers). Condensate components (identified using the markers) are listed using Roman numerals located in the column headers. Each row represents a candidate compound, and the global condensate perturbation score is shown to the left of each row. [Figure 3B] Figure 1 shows a visualization of the marker perturbation scores and global condensate perturbation scores for 40 compounds evaluated across 11 condensate types using two markers for each condensate type (22 condensate markers). Condensate components (identified using the markers) are listed using Roman numerals located in the column headers. Each row represents a candidate compound, and the global condensate perturbation score is shown to the left of each row.

[0036] [Figure 4A] Figure 1 shows a visualization of the marker perturbation scores and global condensate perturbation scores for 40 compounds evaluated across 11 condensate types using two markers for each condensate type (22 condensate markers). Condensate components (identified using the markers) are listed using Roman numerals located in the column headers. Each row represents a candidate compound, and the global condensate perturbation score is shown to the left of each row. [Figure 4B] Figure 1 shows a visualization of the marker perturbation scores and global condensate perturbation scores for 40 compounds evaluated across 11 condensate types using two markers for each condensate type (22 condensate markers). Condensate components (identified using the markers) are listed using Roman numerals located in the column headers. Each row represents a candidate compound, and the global condensate perturbation score is shown to the left of each row. DETAILED DESCRIPTION OF THE INVENTION

[0037] In some aspects, the present application provides methods for identifying stimuli with condensate modulating properties for one or more condensate types, e.g., for identifying small molecule therapeutic candidates that are selective condensate modulators. In certain aspects, the methods provided herein are useful for identifying stimuli (e.g., small molecule therapeutic candidates comprising a set concentration or range of small molecule therapeutics) that modulate a desired condensate type (or a subset of condensate types), including modulation of the condensate(s) in a desired manner. The present disclosure is based, at least in part, on the inventors' findings regarding methodologies that enable the identification of selective condensate modulators. In certain aspects, the methods utilize marker perturbation scores. In certain aspects, the methods further utilize global condensate perturbation scores. The marker perturbation scores and global condensate perturbation scores provide robust and reproducible parameters for identifying selective condensate modulators (with selectivity assessable at the level of one or more condensate components of a condensate type and / or at the level of one or more condensate types). Furthermore, the methods taught herein are suitable for high-throughput and commercial-scale drug discovery workflows. These findings and the methods taught herein support new methods for more efficiently evaluating biological condensates and their modulation useful for therapeutic treatments.

[0038] Thus, in some aspects, provided herein are methods for identifying a stimulation of a condensate-modulating property for one or more condensate types, the methods including: (a) subjecting a cellular composition comprising a cell type to the stimulation; (b) measuring a characteristic of at least one marker in at least a portion of the cellular composition subjected to the stimulation, wherein the marker associates with at least one of the one or more condensate types before and / or after the cellular composition is subjected to the stimulation; (c) determining a marker perturbation score for each marker in the cellular composition subjected to the stimulation based on the measured characteristic of each marker; and (d) identifying a stimulation of the condensate-modulating property from the marker perturbation scores.

[0039] In some aspects, provided herein are methods for identifying a stimulus for a condensate modulatory property for one or more condensate types, the method comprising: (a) subjecting a cellular composition comprising a cell type to the stimulus; (b) measuring a characteristic of at least one marker in at least a portion of the cellular composition subjected to the stimulus, wherein the marker associates with at least one of the one or more condensate types before and / or after the cellular composition is subjected to the stimulus; (c) determining a marker perturbation score for each marker in the cellular composition subjected to the stimulus based on the measured characteristic of each marker; (d) determining a global condensate perturbation score, wherein the global condensate perturbation score is based on the at least one marker perturbation score; and (e) identifying the stimulus for the condensate modulatory property can be determined from the at least one marker perturbation score and / or the global condensate perturbation score.

[0040] In some aspects, provided herein are systems useful for performing at least certain aspects of the methods provided herein, including for computer implementation of aspects of the methods provided herein. In some embodiments, the system is configured to perform a measurement of at least one marker characteristic in at least a portion of a cell composition subjected to a stimulus. In some embodiments, the system is configured to perform a determination of a marker perturbation score. In some embodiments, the system is configured to perform a determination of a global condensate perturbation score. In some embodiments, the system includes a user interface, such as a screen, a touchpad, and / or buttons or keys for providing commands and / or reviewing information related to the computer implementation of the methods provided herein.

[0041] In some aspects, provided herein are components, e.g., kits, useful for the methods provided herein. In some embodiments, provided herein are kits that include one or more condensate components, e.g., one or more antibodies that recognize a marker panel.

[0042] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. For example, some aspects of the present disclosure are presented in a modular format, and such presentation should not be construed as limiting the possible combinations of approaches taught herein.

[0043] I. Definition For the purposes of interpreting this specification, the following definitions shall apply, and where appropriate, terms used in the singular shall include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the set forth definition shall control.

[0044] As used herein, "condensate" refers to a non-membrane encapsulated compartment formed by phase separation (including all stages of phase separation) of one or more proteins and / or other macromolecules such as nucleic acids.

[0045] As used herein, the terms "polypeptide" and "protein" can be used interchangeably to refer to polymers comprising amino acid residues and are not limited to a minimum length. Such polymers may contain natural or non-natural amino acid residues, or combinations thereof, and include, but are not limited to, peptides, polypeptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Full-length polypeptides or proteins, as well as fragments thereof, are encompassed by this definition. The terms also include modified species thereof, such as, but not limited to, post-translational modifications of one or more residues, including methylation, phosphorylation, glycosylation, sialylation, or acetylation.

[0046] The term "antibody" and its grammatical equivalents include full-length antibodies and antigen-binding fragments thereof. A full-length antibody comprises two heavy chains and two light chains. The term "antigen-binding fragment" as used herein refers to antibody fragments, including, for example, diabodies, Fab, Fab', F(ab'), Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (ds diabodies), single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies), multispecific antibodies formed from portions of an antibody comprising one or more CDRs, camelized single domain antibodies, nanobodies, domain antibodies, bivalent domain antibodies, any other antibody fragment that binds to an antigen but does not comprise the entire full-length antibody structure, or antibody mimetics (e.g., designed ankyrin repeat proteins (DARPins), affimers, or monobodies (ADNECTINS®)). An antigen-binding fragment is capable of binding to the same antigen as the parent antibody or parent antibody fragment (e.g., the parent scFv).

[0047] As used herein, the terms "comprising," "having," "containing," and "including," as well as other similar forms and grammatical equivalents thereof, are intended to be equivalent in meaning and open-ended in that the item(s) following any one of these words do not imply an exhaustive list of such item(s) or are limited only to the recited item(s). For example, an article "comprising" components A, B, and C may consist of (i.e., contain only) components A, B, and C, or may include not only components A, B, and C but one or more other components as well. As such, "comprising" and similar forms thereof, as well as grammatical equivalents thereof, are intended and understood to include disclosure of embodiments that "consist essentially of" or "consist of."

[0048] Where a range of values ​​is provided, unless the context clearly dictates otherwise, each intervening value between the upper and lower limit of that range, and at any other stated or intervening value within that stated range, to the tenth of the unit of the lower limit, is encompassed within the scope of the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0049] Reference herein to "about" a value or parameter includes (and describes) a variation that is directed to that value or parameter itself. For example, a reference to "about X" includes the description of "X."

[0050] As used in this specification, including the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise.

[0051] II. Methods for identifying and / or evaluating condensate modulators Provided herein, in certain aspects, are methods for identifying stimuli associated with condensate modulating properties, such as stimuli that produce or contribute to changes in the properties of a condensate or its components. In some embodiments, the methods taught herein can be used to evaluate one or more stimuli for one or more condensate modulating properties, including determining that the stimuli are not associated with the condensate modulating properties. In some embodiments, the methods taught herein can be used to identify one or more stimuli that have a selective effect on one or more condensate types and / or one or more condensate components.

[0052] As discussed in more detail herein, in some embodiments, the methods provided herein include a marker perturbation score. In some embodiments, exposing a cellular composition containing cells to a stimulus can result in a change in the condensate or its components, and such a change can be measured using a known marker of the condensate or its components (e.g., a polypeptide that is associated with the condensate and / or that binds or dissociates from the condensate under certain conditions, e.g., in response to a stimulus). In some embodiments, the marker is or associates with the condensate component, thereby allowing for detection of the condensate component regardless of whether the condensate component is associated with the condensate type. Thus, in some embodiments, the marker perturbation score is based on a change (or lack thereof) in a characteristic of the marker relative to the condensate type after exposure to the stimulus, indicating that the stimulus is affecting the condensate or its components. For example, after a cell composition containing a cell type is exposed to a stimulus, characteristics of condensate components known to associate with the condensate can be evaluated for at least a portion of the cell composition, e.g., a field of cells (or a portion thereof). In some embodiments, characteristics such as distribution are evaluated using a coefficient of variation (CV) calculation, which is determined, for example, by dividing the standard deviation of the signal attributable to a marker within a region by the average signal attributable to the marker within that region. In some embodiments, characteristics are evaluated using a dispersion index. As illustrated in FIG. 1A, in a scenario of uniform intensity, such as based on pixels within a region (here, cell nuclei), the coefficient of variation will be low because the standard deviation of the signal is low compared to the average signal within that region. As illustrated in FIG. 1B, in a scenario where there are bright intensity spots within a region (e.g., due to markers within the condensate) and a dark background (here, cell nuclei), the coefficient of variation will be high because the standard deviation of the signal is high compared to the average signal within that region.

[0053] In some embodiments, the methods provided herein include a global condensate score. In some embodiments, the global condensate score is a useful metric for assessing the selectivity of a stimulus across one or more condensate types. In some embodiments, the global condensate perturbation score is based on a Z-score, such as a median-based Z-score.

[0054] Accordingly, in certain aspects, a method is provided for identifying a stimulation of a condensate-modulating property for one or more condensate types, such as one or more condensate types, of a particular cell type, the method comprising: (a) subjecting a cellular composition comprising the cell type to the stimulation; (b) measuring a characteristic of at least one marker in at least a portion of the cellular composition subjected to the stimulation, wherein the marker associates with at least one of the one or more condensate types before and / or after the cellular composition is subjected to the stimulation; (c) determining a marker perturbation score for each marker in the cellular composition subjected to the stimulation based on the measured characteristic of each marker; and (d) identifying a stimulation of the condensate-modulating property from the marker perturbation score.

[0055] In certain aspects, a method is provided for identifying a stimulus for a condensate modulatory property for one or more condensate types, the method comprising: (a) subjecting a cellular composition comprising a cell type to the stimulus; (b) measuring a characteristic of at least one marker in at least a portion of the cellular composition subjected to the stimulus, where the marker associates with at least one of the one or more condensate types before and / or after the cellular composition is subjected to the stimulus; (c) determining a marker perturbation score for each marker in the cellular composition subjected to the stimulus based on the measured characteristic of each marker; (d) determining a global condensate perturbation score, where the global condensate perturbation score is based on the at least one marker perturbation score; and (e) identifying a stimulus for the condensate modulatory property from the marker perturbation scores, where identifying the stimulus for the condensate modulatory property can be determined from at least one marker perturbation score and / or the global condensate perturbation score.

[0056] Assay methodologies and elements, including marker perturbation scores and, optionally, global condensate scores, are described in more detail throughout this application, including in the following sections: The modular discussion of such methodologies and elements is not intended to limit the scope of the teachings provided herein, and one of skill in the art will readily understand their combination in light of the description provided herein.

[0057] A. Cellular Composition, Its Components, and Stimuli In certain aspects, the methods provided herein include one or more cellular compositions, components thereof, and one or more stimuli. For example, in some embodiments, the methods include subjecting a cellular composition comprising a cell type to a stimulus.

[0058] 1. Cell composition In certain embodiments, the methods described herein include one or more cell compositions. The cell compositions can include any cell type or any mixture of cell types.

[0059] In some embodiments, the cell composition comprises at least about 1,000 cells, e.g., at least about 1,000 cells of a cell type, 5,000 cells, 1 x 10 4 cells, 5 x 10 4 cells, 1 x 10 5 cells, 5 x 10 5 cells, or 1 x 10 6 Contains any one of the cells.

[0060] In some embodiments, the composition comprises a single cell type. In some embodiments, the cell composition comprises at least about 1,000 cells of a single cell type. In some embodiments, the cell composition comprises multiple cell types, such as two, three, four, or five cell types. In some embodiments, the cell composition is aliquoted for use in the methods described herein, for example, the aliquot contains at least about 100 cells, e.g., at least about 200 cells, 300 cells, 400 cells, 500 cells, 600 cells, 700 cells, 800 cells, 900 cells, 1,000 cells, 1,250 cells, 1,500 cells, 1,750 cells, or 2,000 cells.

[0061] In some embodiments, the cell composition comprises cells at or above a desired confluence, for example, in some embodiments, the cell composition comprises cells at (including growing to) at least about 60%, e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% confluence.

[0062] In some embodiments, the cell composition comprises a cell type from an animal, such as a human, rat, or mouse. In some embodiments, the cell type from the animal has one or more characteristics of a disease, such as a neurodegenerative disease, a proliferative disease, an immune disease, a cardiac disease, an infectious disease, or a metabolic disease. In some embodiments, the cell composition is useful for studying a disease or an aspect or factor thereof. In some embodiments, the cell composition comprises HeLa cells, HEK293 cells, DLD1 cells, U2OS cells, H9C2 cells, induced pluripotent stem cells (iPSC cells), cardiomyocytes, myocytes, stem cell-derived cells, neurons, cancer cells, immune cells, or adipocytes. In some embodiments, the cell model is derived from a biopsy or tissue sample, e.g., a patient sample, e.g., a healthy or diseased biopsy or tissue sample. In some embodiments, the cell composition is a tissue sample, such as a tissue sample, cell smear, or secretion. When a cell type is described, it is understood to include cells derived from that cell type unless expressly stated otherwise. For example, HeLa cells containing a heterologous transgene are considered HeLa cells unless expressly stated otherwise. In some embodiments, the cell composition comprises a cell type that has been modified, such as stably or transiently transfected.

[0063] In some embodiments, the cell composition is a cultured cell composition, e.g., a cell composition contained in a sample well or culture dish. In some embodiments, the method includes multiple aliquots of the cell composition, such as a cell composition aliquoted into two or more wells of a multi-well plate, e.g., a 1536-well plate.

[0064] In other aspects, provided herein are methods that include identifying, obtaining, and / or producing a cell composition. In some embodiments, the method includes generating a cell composition having a cell type. In some embodiments, the cell type of the cell composition is treated and / or engineered to modulate (e.g., increase or decrease) expression of one or more polypeptides. Techniques for generating cell compositions are well known in the art. In some embodiments, the cell type is generated from a progenitor cell of the cell type by modulating aspects of the precursor. For example, the cell type can be generated by subjecting the progenitor cell to stress, such as oxidative stress, or by treating the progenitor cell with a small molecule compound or hormone. In some embodiments, the cell type is obtained by subjecting the progenitor cell to infection, such as infection with a virus, bacteria, fungus, or parasite. In some embodiments, the cell type is generated via knockdown or knockout of a genetic trait or its expression product, for example, by any method known in the art, such as siRNA, RNAi, TALEN, ZFN, or CRISPR / Cas. In some embodiments, the cell type is generated via knock-in. In some embodiments, the cell type is generated via transfection. In some embodiments, the cell type is transfected with a fusion polypeptide, such as a polypeptide fused to a label, e.g., GFP. In some embodiments, the cell type is transfected with a wild-type polypeptide. In some embodiments, the cell type is transfected with a variant polypeptide, such as a mutant polypeptide. In some embodiments, the cell type is transfected to express a certain level of a gene expression product. In some embodiments, an expression level variant cell type is generated when the gene expression product reaches a predetermined level and is used in the methods described herein.

[0065] In some embodiments, a cell type is transfected to express a polypeptide, such as a wild-type or mutant polypeptide, at approximately endogenous levels. In some embodiments, the cell type does not express a polypeptide, such as a wild-type polypeptide, and the approximately endogenous levels are based on the expression level of the polypeptide in another cell type. In some embodiments, the cell type is transfected to express a polypeptide with a label, such as a labeled wild-type polypeptide, at approximately endogenous levels, and the approximately endogenous levels are based on the expression level of an unlabeled version of the respective polypeptide. In some embodiments, the cell type has reduced expression of an unlabeled polypeptide, e.g., the cell type comprises a knockout of the unlabeled polypeptide. In some embodiments, the cell type is transfected to express a variant polypeptide, e.g., a mutant polypeptide (e.g., a point mutation, a truncation mutation, a frameshift mutation, or a termination mutation), at a level substantially similar to the endogenous expression level of the wild-type polypeptide for each of the variant polypeptides. As described herein, in some embodiments, the term "approximately endogenous levels" or "substantially similar" refers to a level of expression of 1×10 6 "A" refers to a level of polypeptide expression that, when measured in a population of cells, such as a single cell, is within a two-fold difference of the measured endogenous level of the polypeptide.

[0066] 2. Stimulation and Treatment of Cellular Compositions In certain aspects, the methods provided herein include subjecting a cellular composition comprising a cell type to a stimulus. The methods provided herein allow for the evaluation of any stimulus. For ease of description of the subject matter provided herein, the cellular composition is generally described as being subjected to a single stimulus (although this is not a limitation to the description provided herein), and the single stimulus can encompass many factors. For example, the stimulus may be described as a compound or may implicitly include any light, heat, O2 concentration, and / or CO2 concentration factors to which the cellular composition is subjected. In some embodiments, the stimulus is described as a concentration of a compound, and two or more cellular compositions are subjected to the compound at different concentrations (or, for example, one concentration of the compound and one vehicle control). In some embodiments, the stimulus is configured so that the compound is evaluated in a dose-response manner, e.g., the compound is evaluated at two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more concentrations. Those skilled in the art will readily understand how to evaluate one or more factors of stimulation by establishing appropriate experimental procedures and references.

[0067] In some embodiments, the compound comprises an exogenous compound. In some embodiments, the stimulus comprises an exogenous peptidic agent, such as a hormone. In some embodiments, the stimulus comprises exogenous genetic material. In some embodiments, the stimulus comprises a stressor. In some embodiments, the stimulus comprises an environmental stimulus. In some embodiments, the stimulus comprises any combination of an exogenous compound, exogenous peptidic agent, exogenous genetic material, stressor, or environmental stimulus.

[0068] In some embodiments, the stimulus, such as an exogenous compound, is a small molecule therapeutic candidate or a precursor thereof (e.g., a prodrug). In some embodiments, the stimulus is a small molecule therapeutic candidate at a known concentration. In some embodiments, the stimulus is a mixture of small molecule therapeutic candidates (applied to the cell composition via a single or multiple compositions). In some embodiments, the stimulus is a small molecule therapeutic candidate and another agent (applied to the cell composition via a single or multiple compositions). In some embodiments, the stimulus is a polypeptide, a peptidomimetic, a lipid, a nucleic acid, or any combination thereof.

[0069] As used herein, a small molecule therapeutic candidate encompasses any small molecule being evaluated for drug discovery purposes, such as those involved in attempting to identify therapeutics for human disease. In some embodiments, the small molecule therapeutic is a member of a small molecule library, such as a compound library. In some embodiments, there may be some knowledge (obtained before or after performing the methods described herein) that a particular small molecule is not suitable for human therapeutic use, but in such cases, the small molecule is still considered a small molecule therapeutic candidate for purposes of this description.

[0070] In some embodiments, small molecule therapeutic candidates can have a molecular weight of about 5,000 Da or less, e.g., about 4,500 Da or less, 4,000 Da or less, 3,500 Da or less, 3,000 Da or less, 2,500 Da or less, 2,000 Da or less, 1,500 Da or less, 1,000 Da or less, or 500 Da or less. In some embodiments, small molecule therapeutic candidates satisfy one or more of Lipinski's Rule of Five (no more than 5 hydrogen bond donors, no more than 10 hydrogen bond acceptors, molecular weight less than 500 Da, and octanol-water partition coefficient (log P) not greater than 5). In some embodiments, small molecule therapeutic candidates have desirable partitioning properties for condensate-type structures. For example, in some embodiments, the small molecule therapeutic candidate partitions into the condensate type (e.g., the concentration of the small molecule therapeutic candidate is higher within the condensate type than outside the condensate type), such as when it is desirable to deliver the small molecule therapeutic candidate to the condensate type. In some embodiments, the small molecule therapeutic candidate does not substantially partition into the condensate type (e.g., the concentration of the small molecule therapeutic candidate is lower within the condensate type than outside the condensate type), such as when it is desirable to prevent the condensate type from acting as a sink for the small molecule therapeutic candidate.

[0071] In some embodiments, the small molecule therapeutic candidate comprises a nucleic acid. In some embodiments, the small molecule therapeutic candidate comprises RNA, e.g., siRNA, miRNA, mRNA, or lnRNA, or analogs thereof. In some embodiments, the small molecule therapeutic candidate comprises DNA or an analog thereof. In some embodiments, the small molecule therapeutic candidate is a non-naturally occurring compound. In some embodiments, the small molecule therapeutic candidate is an exogenous compound. In some embodiments, the small molecule therapeutic candidate comprises a polypeptide. In some embodiments, the small molecule therapeutic candidate is a therapeutic compound approved by a regulatory agency, such as a drug approved for medical use by the U.S. Food and Drug Administration (FDA). In some embodiments, the small molecule therapeutic candidate is a novel chemical entity.

[0072] In some embodiments, the small molecule therapeutic candidate, or a portion thereof, is charged. In some embodiments, the small molecule therapeutic candidate, or a portion thereof, is hydrophobic. In some embodiments, the small molecule therapeutic candidate, or a portion thereof, is hydrophilic. In some embodiments, the small molecule therapeutic candidate, or a portion thereof, comprises an alkaloid, a glycoside, a phenazine, a phenol, a polyketide, a terpene, or a tetrapyrrole.

[0073] In some embodiments, the small molecule therapeutic candidate comprises a label. In some embodiments, the label is a radioactive label, a colorimetric label, a luminescent label, a chemically reactive label (such as a component moiety used in click chemistry), or a fluorescent label. In some embodiments, the small molecule therapeutic candidate comprises a measurable signal, e.g., has fluorescent properties without further modification. In some embodiments, the small molecule therapeutic candidate comprises a fluorophore. In some embodiments, the small molecule therapeutic candidate is a polypeptide comprising a label. In some embodiments, the small molecule therapeutic candidate is a polypeptide comprising a fluorophore. In some embodiments, the small molecule therapeutic candidate is a nucleic acid comprising a label. In some embodiments, the small molecule therapeutic candidate is a nucleic acid comprising a fluorophore. The label can be covalently or non-covalently attached to the small molecule therapeutic candidate.

[0074] In some embodiments, the stimulus is a reference control. For example, in some embodiments, a first cell composition is subjected to a small molecule therapeutic candidate delivered to the first cell composition in a vehicle, and a second cell composition is subjected to a second stimulus, which is a reference control comprising a vehicle. In some embodiments, the reference control stimulus comprises DMSO. In some embodiments, the reference control comprises a cell composition that has not been contacted with one or more factors of the stimulus, such as a small molecule therapeutic candidate or a vehicle control.

[0075] The methods provided herein are applicable to any therapeutic regimen that involves subjecting a cellular composition to a stimulus. In some embodiments, subjecting a cellular composition to a stimulus involves subjecting the cellular composition to the stimulus in a single instance, such as heat treatment or application of a chemical composition, such as a small molecule therapeutic candidate. Such a single treatment can involve any length of duration or incubation, e.g., applying a chemical composition for a set period of time before the cellular composition medium is removed and replaced with medium without the chemical composition. In some embodiments, subjecting a cellular composition to a stimulus involves subjecting the cellular composition to the stimulus for multiple instances, e.g., to reflect a chronic exposure or chronic disease.

[0076] 3. Assay Format In certain aspects, the methods provided herein include independently subjecting any number of cell compositions to a stimulus, allowing for the evaluation of multiple stimuli across one or more cell types. In some embodiments, the methods include performing replicates, e.g., triplicates, of subjecting cell compositions comprising cell types to a stimulus. The methods described herein can be performed on arrays in a variety of formats, including formats suitable for high-throughput and commercial-scale drug discovery.

[0077] In some embodiments, the methods provided herein include aliquoting a cellular composition into a plurality of sample wells and subjecting each defined subset of the aliquoted cellular composition to a stimulus. In some embodiments, the cellular composition is aliquoted into wells of a multiwell plate, such as a multiwell plate having 6, 12, 24, 48, 96, 384, or 1,536 wells. In some embodiments, the multiwell plate is suitable for other steps of the methods described herein, such as stimuli, as well as any processing and analysis steps performed on the cell types of the cellular composition.

[0078] In some embodiments, the methods provided herein include evaluating multiple cellular compositions, each cellular composition comprising a different cell type or derivative thereof, hi some embodiments, each cellular composition may be independently subjected to multiple stimuli, such as by providing different aliquots of the cellular composition.

[0079] In some embodiments, the number of individual cell compositions (including aliquots of cell compositions) used in the methods described herein can be determined based on one or more aspects of the desired analysis, including, but not limited to, the number of stimuli to be evaluated, the number of cell types to be evaluated, the number of replicates to be evaluated, and the level of power.

[0080] 4. Processing of Cellular Compositions After Stimulation In some embodiments, the methods provided herein include processing techniques performed after subjecting the cellular composition to a stimulus, which are useful for preserving the biological context and / or preparing the cellular composition or components thereof for certain downstream analyses (e.g., staining and / or imaging) after treatment with the stimulus.

[0081] For example, in some embodiments, after a cell composition comprising a cell type is subjected to a stimulus, the cell composition or a component thereof, such as a cell type, is fixed and / or permeabilized. In some embodiments, fixation is performed to maintain the cells in place, and is achieved, for example, using a cross-linking reagent, such as formaldehyde or an analog thereof. In some embodiments, permeabilization is performed to allow markers, such as antibodies, to permeate the cells, and is achieved, for example, using a solvent (e.g., acetone) and / or a detergent (e.g., Triton, NP-40, Tween® 20, saponin, digitonin, and / or Leucoperm). In some embodiments, after a cell composition comprising a cell type is subjected to a stimulus, the cell composition or a component thereof, such as a cell type, is frozen.

[0082] 5. Condensate type and condensate adjustment characteristics As discussed herein, the provided methods are useful for identifying stimuli that have condensate-modulating properties for one or more condensate types. Thus, in some embodiments, the cellular composition comprises one or more condensate types. In some embodiments, the cellular composition comprises a condensate type before subjecting the cellular composition to a stimulus. In some embodiments, the cellular composition comprises a condensate type after subjecting the cellular composition to a stimulus. In some embodiments, the method comprises generating a condensate type, such as by gene expression and / or subjecting the cellular composition to a stress. In some embodiments, the cellular composition comprises a condensate type after subjecting the cellular composition to a stimulus, but does not comprise the condensate type (e.g., the condensate type is observed upon subjecting the cellular composition to at least a certain concentration of a small molecule therapeutic candidate).

[0083] In certain aspects, the methods described herein include characterizing one or more condensate types, such as by measuring their components. Condensates discussed herein include one or more components, such as macromolecules, e.g., polypeptides and / or nucleic acids, such as DNA or RNA. As described herein, condensate types can be characterized based on the presence of one or more components in the dense phase, such as specific macromolecules, e.g., specific polypeptides. In some embodiments, the condensate component is a condensate scaffold. A condensate scaffold is a component that contributes to the structural integrity of the condensate, is often involved in the formation of the condensate, and tends to exist more statically in the condensate. In some embodiments, the condensate component is a condensate client. A condensate client is a component that is not essential for the formation of the condensate and tends to exist in the condensate under certain conditions. In some embodiments, the condensate type is a known condensate type, such as a cleavage body, p granule, histone locus body, multivesicular body, neuronal RNA granule, intranuclear gem, nuclear pore complex, nuclear speckle, nuclear stress body, nucleolus, Oct1 / PTF / transcription (OPT) domain, paraspeckle, juxtanucleolar compartment, PML nucleolus, PML oncogenic domain, Polycomb body, processing body, Sam68 nucleolus, stress granule, splicing speckle, P62 body, Cajal body, heterochromatin, transcription condensate, or centrosome. In some embodiments, the condensate type is a centrosome. In some embodiments, the condensate type is a nuclear pore complex. In some embodiments, the condensate type is a P62 body. In some embodiments, the condensate type is a heterochromatin condensate. In some embodiments, the condensate type is a paraspeckle. In some embodiments, the condensate type is a nucleolus. In some embodiments, the condensate type is a nuclear speckle. In some embodiments, the condensate type is a Cajal body.In some embodiments, the condensate type is a PML body. In some embodiments, the condensate type is a P body. In some embodiments, the condensate type is a stress granule. In some embodiments, the condensate type is a transcription condensate.

[0084] In some embodiments, the condensate type is a cellular condensate, such as a condensate present within a cell type of the cellular composition. In some embodiments, the condensate type is an extracellular condensate, e.g., a condensate type that may be present in the extracellular space of the cellular composition. Extracellular condensates can form in biological fluids outside of cells, such as extracellular matrix or plasma, to facilitate reaction or sequestration of molecules. See Muiznieks et al., J Mol Biol, 43, 2018, 4741-4753. In some embodiments, the condensate type is a disease-associated condensate type, such as a condensate associated with a disease. In some embodiments, the condensate type is not naturally occurring. In some embodiments, the condensate type is naturally occurring.

[0085] As described herein, a condensate modulating property refers to a measurable change in one or more properties of a condensate or its components, e.g., markers, associated with a stimulus (whether or not the component is associated with the condensate at the time of measurement, e.g., a condensate component that dissociates from the condensate). In some embodiments, a condensate modulating property of a stimulus can be determined by measuring (i) the location of a condensate type, (ii) the distribution of condensate types and / or condensate components, (iii) the number of condensate types, (iv) the size of a condensate type, (v) the ratio of the amount of a condensate type to a control condensate, (vi) a functional activity associated with a condensate type, (vii) the composition of a condensate type, (viii) the co-localization of a condensate type with a biomolecule, (ix) the diffusion coefficient of a component of a condensate type, (x) the stability of a condensate type, (xi) the solubility of a condensate type, or (x) the solubility of a condensate type. The condensate modulating property of the stimulus may be one or more of: (i) a reduction in the resolution or size of the condensate type; (ii) a surface area of ​​the condensate type; (iii) a sphericity of the condensate type; (xiv) a flowability of the condensate type; (xv) a solidification of the condensate type; (xvi) a location of the condensate component; (xvii) an amount of the condensate component or a precursor thereof; (xviii) a condensate partitioning of the condensate component into condensate types; (xix) a functional activity associated with the condensate component; (xx) an aggregation of the condensate component; (xxi) a post-translational modification state of the condensate component; or (xxii) an amount of a degradation product of the condensate component. In some embodiments, the condensate modulating property of the stimulus comprises a distribution of the condensate type and / or condensate component as measured in a whole cell. In some embodiments, the methods provided herein include determining at least one property associated with one or more condensate types and / or at least one of the condensate components.

[0086] In some embodiments, the condensate-modulating properties of the stimulus are based on one or more observable or measurable characteristics of the condensate type, which in some aspects herein are referred to as a phenotype or phenotypic identifier of the condensate type. For example, an observable or measurable characteristic or phenotypic identifier associated with a condensate can be determined by imaging a cellular composition. Observable or measurable characteristics of a condensate phenotype include, but are not limited to, the presence (including absence and level / amount), location, distribution, kinetics (e.g., formation or dissolution kinetics), morphological (e.g., size, shape, sphericity), material (e.g., fluidity or rigidity), and compositional characteristics of the condensate. In some embodiments, the condensate phenotype comprises the presence of a target condensate type. In some embodiments, the condensate phenotype comprises the absence (including disappearance or dissolution) of a target condensate type. In some embodiments, the condensate phenotype comprises the amount of a target condensate type, including an amount based on the number and / or size characteristics of individual condensates. In some embodiments, the condensate phenotype includes the amount of a condensate type of interest with and / or without a component (e.g., a marker, such as a biological marker, or one or more other biomolecules that become components of the condensate under certain conditions). In some embodiments, the condensate phenotype includes the abundance (or level of association) of a component of the condensate type of interest within the condensate type of interest. In some embodiments, the condensate phenotype includes the location of the condensate type of interest or its components, such as its subcellular location. In some embodiments, the condensate phenotype includes the distribution of the condensate type of interest or its components (e.g., relative to other organelles, other condensates, or other biomolecules). In some embodiments, the condensate phenotype includes morphological characteristics of the condensate type of interest in a cellular model, such as size, shape, volume, surface area, and / or sphericity. In some embodiments, the condensate phenotype includes the number of condensate types per cell or region therein.In some embodiments, the condensate phenotype comprises the composition of a desired condensate type. In some embodiments, the condensate phenotype comprises the behavior or material properties of a desired condensate type, such as dynamic properties, flow properties, solidity, or fiber formation. In some embodiments, the condensate phenotype comprises information regarding the kinetics of condensate formation. In some embodiments, the condensate phenotype comprises information regarding the kinetics of condensate dissolution. In some embodiments, the condensate phenotype comprises a change in a phenotypic identifier, such as a formation or dissolution characteristic, in response to an external stimulus.

[0087] 6. Condensate formation As discussed herein, in some embodiments, the methods provided herein include a step of condensate formation. In some embodiments, the method includes subjecting the cellular composition to conditions that promote the formation of a condensate type. In some embodiments, the step of subjecting the cellular composition to conditions that promote the formation of a condensate type occurs before subjecting the cellular composition to a stimulus. In some embodiments, the step of subjecting the cellular composition to conditions that promote the formation of a condensate type occurs after subjecting the cellular composition to a stimulus and before measuring at least one marking characteristic in the cellular composition.

[0088] Methods for forming condensates are known. For example, cellular stress can cause the formation of stress granules. Examples of cellular stress include arsenic treatment, temperature change, or pH change. Thus, in some embodiments, causing the formation of one or more target condensates includes contacting the cellular composition with arsenic, acid, or base, or altering the temperature of the cellular composition. In some embodiments, the stimulus includes a factor that promotes the formation of a condensate type.

[0089] In some embodiments, the methods described herein do not include a separate step of forming a condensate. For example, in some embodiments, the methods include subjecting a cell composition comprising a cell type to a stimulus and measuring at least one marker characteristic in at least a portion of the cell composition subjected to the stimulus without further including another condensate-forming step, e.g., application of a stress.

[0090] B. Markers, Cell Staining, and Imaging Methods In certain aspects, the methods provided herein include measuring at least one condensate component in at least a portion of a cellular composition subjected to a stimulus, such as via a marker. In some embodiments, a characteristic of the condensate component is measured, such as via a marker. As described herein, the characteristic of the condensate component, such as via a marker, is evaluated across two or more locations comprising two or more pixels, thereby enabling a determination of a change in the location of the condensate component associated with subjecting the cellular composition to a stimulus. In some embodiments, the change in the characteristic of the condensate component, as measured via the marker, indicates a change in condensate properties. These and other characteristics related to markers and measurement methods are described in further detail in the following subsections.

[0091] In some embodiments, the characterization of condensate components is based on pixel analysis. In some embodiments, pixel analysis is performed on a region of a cell, such as the nucleus and / or cytosol, or a region thereof. In some embodiments, pixel analysis is performed without identifying individual condensates or their features (such as condensate boundaries). The pixels used in the pixel analysis described herein can be of any shape and size relative to the constraints imposed by the region being analyzed. For example, in some embodiments, the region being analyzed includes at least 2, e.g., at least 10, 100, 1,000, 5,000, 10,000, 20,000, 30,000, 40,000, or 50,000 pixels, such as within the boundaries of the nucleus or cytosol of an image of a cell. In some embodiments, the pixels are uniformly sized. In some embodiments, the pixels are uniformly shaped.

[0092] 1. Marker As discussed herein, a condensate includes one or more components, such as macromolecules, e.g., polypeptides, and / or nucleic acids, such as DNA or RNA. The methods provided herein include techniques for measuring, e.g., detecting, condensate components regardless of whether the condensate component is located in the condensate, and the term condensate component is not intended to imply that the component is within the condensate at the time of measurement. For example, in some embodiments, a cell type comprises a condensate type that includes polypeptide A, and additional polypeptides A are located within the cell type. For ease of description of the present technology, the entire population of polypeptides A may be described as a condensate component to reflect the possibility that polypeptides A located outside the condensate type are incorporated into the condensate type.

[0093] Markers are useful for determining the location and / or amount of the condensate and / or condensate components, for example, using imaging methods. In some embodiments, the marker allows for detection and / or measurement (direct or indirect) of the condensate component. For example, in some embodiments, the condensate component includes a detectable feature that functions as a marker, providing for direct detection and / or measurement of the condensate component. In some embodiments, the detectable feature of the condensate component is inherent to the condensate component, e.g., a fluorophore that is a natural feature of the condensate component. In some embodiments, the detectable feature of the condensate component is not naturally present on or with the condensate component. For example, in some embodiments, the condensate component includes a fused fluorescent label, e.g., a label such as GFP or mCherry. In some embodiments, the marker is an agent, e.g., an antibody, aptamer, or nucleic acid, with affinity for the condensate component, providing for indirect detection and / or measurement of the condensate component. In some embodiments, the agent having affinity for the condensate component comprises a detectable label. In some embodiments, the marker is capable of detecting condensate components associated with the condensate type (i.e., in the high density phase). In some embodiments, the marker is capable of detecting condensate components not associated with the condensate type (i.e., in the low density phase).

[0094] In some embodiments, the marker is specific to a condensate type. In some embodiments, the marker is specific to a subset of condensate types. For example, condensate components may be divided into a single condensate or a relatively small number of condensates, and detecting and / or measuring such condensate components serves as a useful marker for assessing the selectivity of one or more stimuli for a condensate type or its components.

[0095] In some embodiments, the marker comprises a detectable label that can be detected and / or measured using microscopy. In some embodiments, the label is a radioactive label, a colorimetric label, a luminescent label, a chemically reactive label (such as a component moiety used in click chemistry), or a fluorescent label. In some embodiments, the marker comprises a condensate component fused to a label, such as a fluorescent label. In some embodiments, the condensate component comprises a Halo, Dendra2, GFP, RFP, or mCherry moiety. In some embodiments, the marker is an antibody comprising a detectable label, such as a primary labeled antibody. In some embodiments, the marker is an antibody detection system comprising a system comprising a primary antibody that binds to the condensate component and a secondary labeled antibody that binds to the primary antibody.

[0096] In certain aspects, provided herein are methods for selecting and / or validating markers, such as antibodies, that specifically bind to condensate components. In some embodiments, the methods include obtaining one or more markers, including affinity agents, such as primary labeled antibodies and / or antibody detection systems, for one or more condensate components of a condensate type. In some embodiments, at least one of the one or more condensate components is a scaffold condensate component. One or more markers, individually or in combination(s), are used to stain a cellular composition comprising a cell type (subjected to a stimulus and / or not subjected to a stimulus) to assess the ability of each marker to label an associated condensate component, whether within and / or outside the condensate type. One or more markers, individually or in combination(s), are used to stain a cellular composition comprising a modified version of the cell type (subjected to a stimulus and / or not subjected to a stimulus) that has a knockdown (e.g., using siRNA) or knockout (e.g., using CRISPR) of the associated condensate component to assess any non-specific labeling of the marker. In some embodiments, markers are found to be suitable for use in the methods described herein if the marker detects the relevant condensate component both inside and outside the condensate and has a substantially low level of non-specific labeling. In some embodiments, markers that include affinity agents such as antibodies are obtained such that there are multiple species of origin for the affinity agent, such as a rabbit-derived antibody and a mouse-derived antibody.

[0097] In some embodiments, when multiple condensate components are being evaluated, it is useful to utilize antibodies from different species, for example, a mouse antibody specific for a first condensate component and a rabbit antibody specific for a second condensate component. Such an approach allows for co-staining. In some embodiments, different labeling techniques can be used to measure co-localization of GFP-fused condensate components with antibody markers, for example. In some embodiments, sequential staining of the cell composition can be performed to allow for co-localization measurements, for example, staining with a first antibody marker, followed by stripping of the first antibody marker, followed by staining with a second antibody marker.

[0098] In some embodiments, the method includes measuring a characteristic of a marker. In some embodiments, the method includes measuring a characteristic of a plurality of markers. In some embodiments, measuring a characteristic of a plurality of markers includes measuring a characteristic of a first marker and a second marker. In some embodiments, the first marker and the second marker label condensate components of a single condensate type. In some embodiments, the first marker and the second marker label condensate components of different condensate types.

[0099] 2. Cell Staining and Imaging Methods Provided herein are methods of cell staining to measure markers, and methods for measuring at least one characteristic of the marker, such as using imaging methods.

[0100] In some embodiments, the methods provided herein include subjecting a cell composition or derivative thereof, e.g., a fixed, stimulated cell composition, to one or more agents and detecting one or more condensate components. For example, in some embodiments, the methods include subjecting the fixed, stimulated cell composition to one or more antibodies, each of which specifically binds to a condensate component (primary antibody). In some embodiments, the antibodies comprise a detectable label, e.g., a label detectable by imaging. In some embodiments, the methods further include subjecting the fixed, stimulated cell composition to one or more secondary antibodies, each of which comprises a detectable label. In some embodiments, the methods include using primary antibodies from different organisms, e.g., to enable co-staining techniques to visualize co-localization of condensate components. In some embodiments, sequential staining of the cell composition is performed to enable co-localization measurements, e.g., staining with a first antibody marker, followed by stripping of the first antibody marker, followed by staining with a second antibody marker. In some embodiments, the detectable label is a fluorescent label or a colorimetric label. In some embodiments, the staining technique is an immunofluorescence (IF) staining technique.

[0101] In some embodiments, measuring the characteristic of at least one marker in at least a portion of the cellular composition subjected to the stimulus comprises imaging the stained cellular composition or an aspect thereof. In some embodiments, the imaging comprises a fluorescent imaging method. In some embodiments, the imaging method comprises any one or more of immunofluorescence (IF), in situ hybridization (ISH, e.g., FISH), gene fusion (e.g., GFP labeling), or a dye specific for a condensate component. In some embodiments, the imaging method comprises the use of an agent to visualize cellular features, such as the cell membrane or organelles.

[0102] In some embodiments, the imaging technique involves the use of immunofluorescence (IF) using an affinity label, such as a labeled antibody, that specifically binds to the condensate component. In some embodiments, the IF method involves subjecting the cellular composition or a derivative thereof to an affinity label, such as a labeled antibody, and imaging the cellular composition or a derivative thereof. In some embodiments, the imaging method involves the use of an in situ hybridization (ISH) method, e.g., fluorescent ISH (FISH), such as using a nucleic acid probe that specifically binds to the condensate component. In some embodiments, the FISH method involves subjecting the cellular composition or a component thereof to the nucleic acid probe and imaging the cellular composition or a derivative thereof. In some embodiments, the IF and / or FISH methods are performed in a high-throughput manner. In some embodiments, the method further involves the use of additional markers and / or dyes to identify features of the cell model, such as the cell bilayer and / or organelle boundaries.

[0103] In some embodiments, the imaging method comprises the use of microscopy (and associated microscopy equipment). In some embodiments, the microscopy method comprises confocal microscopy. In some embodiments, the microscopy method comprises fluorescence microscopy. In some embodiments, the microscopy method comprises high-resolution microscopy. In some embodiments, the microscopy method comprises stimulated emission depletion (STED) microscopy. In some embodiments, the microscopy method comprises SoRa super-resolution spinning disk microscopy. In some embodiments, the microscopy method comprises electron microscopy (e.g., cryo-EM or cryo-ET). In some embodiments, the microscopy method comprises total internal reflection fluorescence (TIRF) microscopy.

[0104] In some embodiments, imaging is performed at one or more of the following magnifications: 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, or 100x.

[0105] In some embodiments, imaging is performed such that the cellular composition is imaged using one or more fields of view, hi some embodiments, imaging is performed such that the cellular composition is imaged using one field of view.

[0106] In some embodiments, imaging is performed such that the cellular composition (including its field of view) is imaged at one or more Z-planes, including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 Z-planes.

[0107] C. Marker Characteristics and Scoring Methods In certain aspects, the methods provided herein include measuring at least one marker in at least a portion of a cell composition subjected to a stimulus. In certain aspects, the measured feature is useful for various scoring methods taught herein, such as a marker perturbation score and a global condensate perturbation score. In some embodiments described herein, the feature comprises a distribution.

[0108] In some embodiments, the method includes measuring marker features within a region of the image that contains information about the location of markers associated with the condensate and / or condensate components. In some embodiments, measuring includes extracting marker feature information within the region of the image. In some embodiments, the features are evaluated between two or more locations within the region of the image, including between two or more pixels. In some embodiments, the feature(s), such as a distribution, allows for a determination of a change in the location of the condensate components associated with subjecting the cellular composition to a stimulus. In some embodiments, the feature, such as a distribution, is evaluated using a coefficient of variation (CV) calculation, which is determined, for example, by dividing the standard deviation of the signal attributable to the marker within a region by the average signal attributable to the marker within that region. In some embodiments, the region of the image that is evaluated can be any region of a cellular composition or derivative thereof, such as a cellular composition that has been subjected to a fixed stimulus. In some embodiments, the region is based on a cellular feature, such as a cell membrane or organelle. For example, in some embodiments, the region is the nucleus of one or more cells of the cellular composition or derivative thereof. In some embodiments, the region is arbitrarily defined, such as based on a field of view or a portion thereof.

[0109] In some embodiments, the method includes measuring one or more features of two or more markers, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 markers. Based on the teachings provided herein, one of skill in the art will readily understand that there is no theoretical limit to the number of markers that can be evaluated, including the number of markers for which feature(s) are measured. In some embodiments, the step of measuring the features measures one or more features of two markers for each of at least one or more condensate types in at least a portion of the cell composition subjected to stimulation. In some embodiments, the step of measuring the features measures one or more features of at least one marker for at least two or more condensate types.

[0110] In some embodiments, the method includes analyzing one or more images obtained from the imaging method. In some embodiments, the analysis method can include automated or semi-automated analysis methods, for example, to detect condensate and / or condensate components and / or cellular features. In some embodiments, the analysis method includes in silico analysis methods, for example, to detect condensate and / or condensate components and / or cellular features. Certain methods for in silico analysis, such as those incorporating machine learning and deep learning, are known in the art, such as U.S. Pat. No. 10,303,979, which is incorporated herein by reference in its entirety. In some embodiments, the image analysis includes software, for example, Harmony High Content Imaging and Analysis Software (HCA), for determining one or more features of one or more markers.<https: / / www.perkinelmer.com / product / harmony-4-8-office-hh17000001> , accessed August 1, 2023).

[0111] 1. Marker Perturbation Score In some embodiments, exposing a cellular composition comprising a cell type to a stimulus can result in a change in the condensate, and such a change can be measured using a known marker of the condensate (e.g., a condensate component or an agent that detects the condensate component). Thus, in some embodiments, a marker perturbation score is based on a change (or lack thereof) in a marker feature(s) for a condensate type after exposure to a stimulus, indicating that the stimulus is affecting the condensate or its components. For example, after a cellular composition comprising a cell type is exposed to a stimulus, a marker feature(s) known to associate with the condensate can be assessed for at least a portion of the cellular composition, e.g., a field of view (or a portion thereof). In some embodiments, the feature is based on a distribution, and the distribution is assessed using a coefficient of variation (CV) calculation, determined, for example, by dividing the standard deviation of the signal attributable to the marker within a region by the average signal attributable to the marker within that region.

[0112] As described herein, in some embodiments, multiple markers are assessed for a cellular composition or an aliquot thereof. In some embodiments, the method further includes determining a second marker perturbation score for a second marker in the cellular composition subjected to the stimulus based on the measured characteristic(s) of the second marker. In some embodiments, the marker perturbation score indicates whether the stimulus modulates association of the marker with at least one of one or more condensate types in the cellular composition, and the second marker perturbation score indicates whether the stimulus modulates association of the second marker with at least one of one or more condensate types in the cellular composition. In some embodiments, the marker and the second marker bind to the same condensate type. In some embodiments, the marker and the second marker bind to different condensate types.

[0113] In some embodiments, each marker perturbation score is independently based on the coefficient of variation (CV) of the marker, where the CV is determined based on dividing the standard deviation (STD) of the distribution intensity of the marker by the average distribution intensity of the marker. In some embodiments, the distribution intensity of each marker is based on pixel analysis of one or more images of one or more cell compositions or portions thereof.

[0114] In some embodiments, the method is to evaluate a reference stimulus, and the evaluation further includes determining a reference marker perturbation score, where the method includes determining the reference marker perturbation score. In some embodiments, the reference marker perturbation score is 0, and a marker perturbation score greater than 0 indicates that the stimulus has condensate regulatory properties.

[0115] In some embodiments, the marker perturbation score is obtained by normalizing and / or bucketing one or more underlying distribution measurements, such as based on CV calculation. In some embodiments, the marker perturbation score is based on the median absolute deviation (MAD) Z-score of the CV of the marker. In some embodiments, the method further includes obtaining the MAD Z-score of the marker. In some embodiments, when the MAD Z-score is greater than 5 or less than -5, the marker perturbation score is 1. In some embodiments, when 2.5 < MAD Z-score ≤ 5, or -5 ≤ MAD Z-score < -2.5, the marker perturbation score is 0.5. In some embodiments, when -2.5 ≤ MAD Z-score ≤ 2.5, the marker perturbation score is 0.

[0116] In some embodiments, the marker perturbation score is based on calculating a modified Z-score, such as a MAD Z-score, for each of one or more features of the marker. For example, the one or more features are each selected from texture features, intensity features, and / or morphological features. In some embodiments, the modified Z-score is the median absolute deviation (MAD) Z-score. In some embodiments, the modified Z-score is

Number

number

[0117] In some embodiments, a modified Z-score, e.g., MAD Z-score, is calculated independently for each evaluated characteristic for each marker. The method may then include determining a feature change score for each characteristic for each marker based on the associated modified Z-score, e.g., MAD Z-score, and a proportional scale. As described herein, the proportional scale can be determined arbitrarily, and one of skill in the art will readily understand the range of such a scale. For example, in some embodiments, the proportional scale is between -5 and 5. In some embodiments, the proportional scale is between 0 and 1. Following determination of the feature change scores, the methods described herein may then include aggregating the feature change scores of the top percentage of feature change scores to determine a marker perturbation score. In some embodiments, the aggregation is performed by summing. In some embodiments, the aggregation is performed by averaging. In some embodiments, the set of feature alteration scores used to generate the marker perturbation score is based on a threshold or by taking the top percentage of feature alteration scores, for example, the top 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%. In some embodiments, all feature alteration scores are used to generate the marker perturbation score. In some embodiments, the marker perturbation score reflects how severely a treatment condition perturbed a single condensate marker compared to a reference.

[0118] In certain aspects, a method for identifying a stimulus of a condensate modulating property for one or more condensate types includes: (a) subjecting a cellular composition comprising a cell type to the stimulus; (b) measuring a characteristic (including one of a plurality) of at least one marker in at least a portion of the cellular composition subjected to the stimulus, wherein the marker associates with at least one of the one or more condensate types before and / or after the cellular composition is subjected to the stimulus; and (c) determining a marker perturbation score for each marker in the cellular composition subjected to the stimulus based on the measured characteristic of each marker, wherein determining includes: (i) determining a modified Z-score (e.g., a MAD Z-score) for each marker characteristic (or characteristics); and (ii) determining an associated modified Z-score, e.g., a MAD Z-score. (iii) determining a feature change score for each marker feature (or features) based on the Z-score and proportional scale; and (iv) aggregating (e.g., by summing or averaging) the feature change scores for each marker to determine a marker perturbation score; and (d) identifying a stimulus for the condensate regulatory property from the marker perturbation scores. In certain aspects, provided herein are methods for determining a marker perturbation score, the method comprising: (a) measuring a feature(s) of at least one marker in at least a portion of a cellular composition that has been subjected to a stimulation, wherein the marker associates with at least one of one or more condensate types before and / or after the cellular composition is subjected to the stimulation; and (b) determining a marker perturbation score for each marker in the cellular composition that has been subjected to the stimulation based on the measured feature(s) of each marker, wherein determining comprises: (i) determining a modified Z-score (e.g., a MAD Z-score) for the feature(s) of each marker; (ii) determining a feature change score for the feature(s) of each marker based on an associated modified Z-score, e.g., a MAD Z-score, and a proportional scale; and (iii) aggregating (e.g., by summing or averaging) the feature change scores for each marker to determine the marker perturbation score.In some embodiments, the method further includes determining a global condensate perturbation score, such as using a method taught herein. In some embodiments, measuring includes extracting feature information, such as by using software for evaluating features including any one or combination of one or more texture features, one or more intensity features, or one or more morphological features. In some embodiments, the one or more texture features include one or more of Gabor maximum, Gabor minimum, Haralick contrast, Haralick correlation, Haralick homogeneity, Haralick sum variance, SER bright, SER dark, SER edge, SER hole, SER ridge, SER saddle, SER spot, or SER valley. In some embodiments, the one or more intensity features include one or more of coefficient of variation, mean intensity, contrast, and standard deviation. In some embodiments, the one or more morphological features include one or more of area, circularity, perimeter, width, length, or width-to-length ratio.

[0119] In some embodiments, each marker perturbation score is independently based on at least one characteristic associated with at least one of the one or more condensate types and / or markers.

[0120] 2. Global Condensate Perturbation Score In some embodiments, the methods include a global condensate score. In some embodiments, the global condensate score is a metric useful for assessing the selectivity of a stimulus across one or more condensate types, e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty condensate types. Based on the teachings provided herein, one of ordinary skill in the art will readily appreciate that there is no theoretical limit to the number of marker perturbation scores and / or stimuli that can be assessed using the global condensate perturbation score.

[0121] In some embodiments, the global condensate perturbation score indicates that the stimulus selectively modulates one or more condensate types in a cellular composition subjected to the stimulus. In some embodiments, the global condensate perturbation score indicates that the stimulus non-selectively modulates one or more condensate types in a cellular composition treated with the stimulus. In some embodiments, the global condensate perturbation score indicates that the stimulus does not substantially modulate one or more condensate types in a cellular composition treated with the stimulus. In some embodiments, the global condensate perturbation score is based on two or more marker perturbation scores, such as a first marker perturbation score and a second marker perturbation score.

[0122] In some embodiments, the global condensate perturbation score is calculated by dividing the sum of all marker perturbation scores for all markers by the number of all markers (i.e., average). In some embodiments, the global condensate perturbation score is calculated by dividing the sum of all marker perturbation scores determined for all markers for two or more different target condensates by the number of all markers for two or more different target condensates. In some embodiments, the global condensate perturbation score is calculated by summing the marker perturbation scores of markers (e.g., markers of interest, such as those meeting a particular threshold or top % of scores).

[0123] In some embodiments, the method includes determining a reference global condensate perturbation score. In some embodiments, the global condensate perturbation score is based on a Z-score, such as a median-based Z-score. In some embodiments, the reference global condensate perturbation score is 1, where (i) a global condensate perturbation score greater than 0 and less than 1 indicates that the stimulus selectively modulates one or more condensate types in the cellular composition, and (ii) a global condensate perturbation score of 0 indicates that the stimulus does not substantially modulate one or more condensate types in the cellular composition.

[0124] In some embodiments, for global condensate perturbation scores greater than 0 and less than 1, the smaller the global condensate perturbation score of a stimulus, the more selective the stimulus.

[0125] 3. Comparison In certain aspects, the methods provided herein include comparisons between condensate components and / or condensate types, as enabled by marker perturbation scores and global condensate perturbation scores.

[0126] In some embodiments, the method includes subjecting a cellular composition to a first stimulus and a second stimulus (e.g., via two separate wells containing aliquots of the cellular composition), measuring a characteristic of at least one marker in at least a portion of the cellular composition subjected to the first and second stimuli, and independently determining a marker perturbation score for each marker in the cellular composition subjected to the first and second stimuli based on the measured characteristic of each marker in the cellular composition subjected to the first and second stimuli. In some embodiments, the method includes comparing the marker perturbation score for the marker in the cellular composition subjected to the first stimulus with the marker perturbation score for the marker in the cellular composition subjected to the second stimulus. In some embodiments, the method includes comparing global condensate perturbation scores of the cellular compositions subjected across two or more stimuli. In some embodiments, the method includes determining two or more second global condensate perturbation scores, such as to evaluate two or more stimuli.

[0127] III. Kits and Compositions In certain aspects, provided herein are kits and compositions useful for the methods described herein. In some embodiments, provided herein are kits comprising one or more agents, such as antibodies, that recognize one or more condensate components, e.g., a marker panel. In some embodiments, the marker panel comprises two or more antibodies, wherein the two or more antibodies recognize the same condensate component. In some embodiments, the marker panel comprises two or more antibodies, wherein the two or more antibodies recognize different condensate components. In some embodiments, the kit comprises one or more cell compositions useful for the methods described herein.

[0128] IV. System In certain aspects, provided herein are systems for carrying out aspects of the methods taught herein.

[0129] In some embodiments, the system comprises one or more processors and memory storing one or more programs, the one or more programs configured to be executed by the one or more processors, the one or more programs including instructions for receiving one or more image data packages including one or more images of a cellular composition subjected to a stimulus, applying one or more processes to the received image data package(s) to measure one or more features of one or more markers, and storing and / or outputting the one or more features in one or more feature data packages. In some embodiments, at least one image of the cellular composition subjected to a stimulus is stained using an agent, such as an antibody, that recognizes a condensate component. In some embodiments, the system is configured to automate at least a portion of the instructions within the system, such as identifying regions of the cellular composition or its derivatives. In some embodiments, the system includes instructions for determining a marker perturbation score for each marker in the cellular composition subjected to a stimulus based on the measured features of each marker stored in the one or more feature data packages, and storing and / or outputting the marker perturbation score in the marker perturbation data package. In some embodiments, the system includes instructions for determining a global condensate perturbation score, where the global condensate perturbation score is based on at least one marker perturbation score, and storing and / or outputting the global condensate perturbation score in a global condensate perturbation data package. In some embodiments, the system includes instructions for selecting stimuli that exhibit selectivity (for condensate types and / or condensate components), where selecting the stimuli that exhibit selectivity is based on one or more marker perturbation scores and / or one or more global condensate perturbation scores. In some embodiments, the system includes a user interface, such as a screen, a touchpad, and / or buttons or keys for providing commands and / or reviewing information related to the computer implementation of the methods provided herein.

[0130] V. Further Embodiments Enabled by the Disclosure Herein In other aspects, provided herein are additional methods and embodiments enabled by the disclosure herein.

[0131] In some embodiments, provided herein are methods for identifying a compound (e.g., a small molecule therapeutic candidate) that modulates, e.g., selectively modulates, a condensate type, e.g., modulates a first condensate type without modulating a second condensate type, the method comprising identifying a compound that stimulates a condensate modulating property for one or more condensate types described herein.

[0132] In some embodiments, provided herein are methods for identifying compounds useful for treating a disease, the methods including identifying a stimulus for a condensate modulating property for one or more condensate types described herein, wherein the stimulus causing the condensate modulating property is useful for treating the disease.

[0133] In some embodiments, provided herein are methods for identifying condensate types associated with a disease, the methods including identifying a stimulus of a condensate modulating property for one or more condensate types described herein, wherein the stimulus is a factor associated with the disease.

[0134] In some embodiments, provided herein are methods for identifying one or more interactions between a compound, or a portion thereof, and a condensate type, or a component thereof, the method including identifying stimulation of a condensate modulating property for one or more condensate types described herein.

[0135] In some embodiments, provided herein are methods for identifying a molecular target of a therapeutic agent useful for treating a disease, the method comprising identifying a condensate of interest using any one of the methods described herein, and identifying the molecular target based on its association with and / or interaction with the condensate of interest.

[0136] Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of the present disclosure. The present disclosure is further illustrated by the following examples, which should not be construed as limiting the scope or spirit of the disclosure to the specific procedures described therein.

[0137] VI. Illustrative Embodiments Embodiment 1. A method for identifying a stimulus of a condensate modulatory property for one or more condensate types, the method comprising: (a) subjecting a cellular composition comprising a cell type to the stimulus; (b) measuring a distribution of at least one marker in at least a portion of the cellular composition subjected to the stimulus, wherein the marker associates with at least one of the one or more condensate types before and / or after the cellular composition is subjected to the stimulus; (c) determining a marker perturbation score for each marker in the cellular composition subjected to the stimulus based on the measured distribution of each marker; and (d) identifying the stimulus of the condensate modulatory property from the marker perturbation score.

[0138] Embodiment 2. The method of embodiment 1, further comprising determining a global condensate perturbation score, wherein the global condensate perturbation score is based on at least one marker perturbation score, and wherein the identifying the stimulation of the condensate modulation property can be identified from at least one marker perturbation score and / or the global condensate perturbation score.

[0139] Embodiment 3. The method of any one of the preceding embodiments, wherein the step of measuring the distribution measures two markers for each of the at least one or more condensate types in at least a portion of the cell composition subjected to the stimulation.

[0140] Embodiment 4. The method of any one of the preceding embodiments, wherein said measuring said distribution measures at least one marker for at least two or more condensate types.

[0141] Embodiment 5. The method of any one of embodiments 1-4, wherein each marker is independently a lipid, a polypeptide, or a nucleic acid.

[0142] Embodiment 6. The method of any one of embodiments 1 to 4, wherein each marker is independently a polypeptide.

[0143] Embodiment 7. The method of any one of the preceding embodiments, wherein each marker is (i) within the condensate type, (ii) partitioned into the condensate type after subjecting the cellular composition to the stimulus, or (iii) excluded from the condensate type after subjecting the cellular composition to the stimulus.

[0144] Embodiment 8 The method of any one of embodiments 1 to 7, wherein each marker is independently a condensate scaffold polypeptide or nucleic acid.

[0145] Embodiment 9 The method of any one of embodiments 8, wherein at least one marker is a condensate scaffold polypeptide.

[0146] Embodiment 10. Embodiment 8. The method of any one of embodiments 1-7, wherein each marker is independently a condensed client polypeptide or nucleic acid.

[0147] Embodiment 11. The method of any one of the preceding embodiments, wherein said measuring the distribution of said at least one marker in at least a portion of the cellular composition subjected to said stimulation comprises staining at least a portion of said cellular composition for said marker.

[0148] Embodiment 12. The method of embodiment 11, wherein said staining is immunofluorescence (IF) staining.

[0149] Embodiment 13. The method of any one of the preceding embodiments, wherein measuring the distribution of the at least one marker for at least the portion of the cellular composition comprises imaging at least the portion of the cellular composition.

[0150] Embodiment 14. The method of embodiment 13, wherein said imaging comprises fluorescent imaging.

[0151] Embodiment 15. The method of any one of the preceding embodiments, wherein at least one of one or more different condensate types is formed in the cell composition after being subjected to the stimulus.

[0152] Embodiment 16. The method of any one of the preceding embodiments, wherein the stimulus is selected from the group consisting of an exogenous compound, an exogenous peptidic agent, an exogenous genetic material, a stressor, an environmental stimulus, and combinations thereof.

[0153] Embodiment 17 The method of any one of the preceding embodiments, wherein the stimulus is an exogenous compound.

[0154] Embodiment 18 The method of embodiment 17, wherein the compound is a small molecule therapeutic candidate or precursor thereof.

[0155] Embodiment 19. The method of embodiment 17 or 18, wherein the cell composition is subjected to the compound at a known concentration.

[0156] Embodiment 20. The method of any one of the preceding embodiments, further comprising subjecting the cellular composition to a second stimulus; measuring a distribution of the at least one marker in at least a portion of the cellular composition that has been subjected to the second stimulus; and independently determining a marker perturbation score for each marker in the cellular composition that has been subjected to the second stimulus based on the measured distribution of each marker in the cellular composition that has been subjected to the second stimulus.

[0157] Embodiment 21 The method of embodiment 20, further comprising comparing a marker perturbation score for a marker in a cell composition subjected to the stimulus with a marker perturbation score for a marker in a cell composition subjected to the second stimulus.

[0158] Embodiment 22. The method of embodiment 20 or 21, further comprising determining a second global condensate perturbation score to evaluate the second stimulation of the condensate modulatory property, wherein the second global condensate perturbation score is based on at least one marker perturbation score of a marker in a cellular composition subjected to the second stimulation.

[0159] Embodiment 23. The method of embodiment 22, further comprising comparing the global condensate perturbation score to the second global condensate perturbation score.

[0160] Embodiment 24. The method of any one of the preceding embodiments, further comprising determining at least one characteristic associated with at least one of the one or more condensate types and / or markers.

[0161] Embodiment 25. The at least one property associated with the at least one of the one or more condensate types and / or markers is (i) the location of the condensate type, (ii) the distribution of the condensate type and / or the marker, (iii) the number of the condensate types, (iv) the size of the condensate type, (v) the ratio of the amount of the condensate type to a control condensate, (vi) a functional activity associated with the condensate type, (vii) the composition of the condensate type, (viii) co-localization of the condensate type with a biomolecule, (ix) the diffusion coefficient of a component of the condensate type, (x) the amount of the condensate type. (xi) the stability of the condensate type, (xi) the dissolution or size reduction of the condensate type, (xii) the surface area of ​​the condensate type, (xiii) the sphericity of the condensate type, (xiv) the flowability of the condensate type, (xv) the solidification of the condensate type, (xvi) the location of the marker, (xvii) the amount of the marker or a precursor thereof, (xviii) the condensate partitioning of the marker into the condensate types, (xix) a functional activity associated with the marker, (xx) the aggregation of the marker, (xxi) the post-translational modification state of the marker, and (xxii) the amount of a degradation product of the marker.

[0162] Embodiment 26. The method of any one of the preceding embodiments, wherein at least one of the one or more condensate types is selected from the group consisting of cleavage bodies, p-granules, histone locus bodies, multivesicular bodies, neuronal RNA granules, intranuclear gems, nuclear pore complexes, nuclear speckles, nuclear stress bodies, nucleoli, Oct1 / PTF / transcription (OPT) domains, paraspeckles, juxtanucleolar compartments, PML nucleoli, PML oncogenic domains, Polycomb bodies, processing bodies, Sam68 nucleoli, stress granules, splicing speckles, P62 bodies, Cajal bodies, heterochromatin, transcription condensates, and centrosomes.

[0163] Embodiment 27. The method of any one of the preceding embodiments, comprising measuring the distribution of a second marker in at least a portion of the cellular composition that has been subjected to the stimulus, wherein the second marker is associated with at least one of the one or more condensate types before and / or after the cellular composition is subjected to the stimulus.

[0164] Embodiment 28. The method of embodiment 27, further comprising determining a second marker perturbation score for the second marker in the cell composition subjected to the stimulation based on the measured distribution of the second marker.

[0165] Embodiment 29. The method of embodiment 27 or 28, wherein the marker perturbation score indicates whether the stimulus modulates the association between the marker and at least one of the one or more condensate types in the cellular composition, and the second marker perturbation score indicates whether the stimulus modulates the association between the second marker and at least one of the one or more condensate types in the cellular composition.

[0166] Embodiment 30. The method of any one of embodiments 27 to 29, wherein the marker and the second marker are associated with the same condensate type.

[0167] Embodiment 31. The method of any one of embodiments 27 to 29, wherein the marker and the second marker are associated with different condensate types.

[0168] Embodiment 32. The method of any one of the preceding embodiments, wherein each marker perturbation score is independently based on at least one characteristic associated with at least one of the one or more condensate types and / or markers.

[0169] Embodiment 33. The method according to any one of the preceding embodiments, wherein each marker perturbation score is independently based on the coefficient of variation (CV) of the marker, and the CV is determined based on dividing the standard deviation (STD) of the distribution intensity of the marker by the average distribution intensity of the marker.

[0170] Embodiment 34. The method according to Embodiment 33, wherein the distribution intensity of each marker is based on pixel analysis of one or more images of the one or more cell compositions or portions thereof.

[0171] Embodiment 35. The method according to Embodiment 33 or 34, wherein each marker perturbation score is based on the median absolute deviation (MAD) Z-score of the CV of the marker.

[0172] Embodiment 36. The method according to Embodiment 35, further comprising obtaining the MAD Z-score of the marker.

[0173] Embodiment 37. (i) When the MAD Z-score is greater than 5 or less than -5, the marker perturbation score is 1; (ii) when 2.5 < MAD Z-score ≤ 5 or -5 ≤ MAD Z-score < -2.5, the marker perturbation score is 0.5; (iii) when -2.5 ≤ MAD Z-score ≤ 2.5, the marker perturbation score is 0. The method according to Embodiment 35 or 36.

[0174] Embodiment 38. The method according to any one of the preceding embodiments, further comprising evaluating a reference stimulus, wherein the method includes determining a reference marker perturbation score.

[0175] Embodiment 39. The method according to Embodiment 38, wherein the reference marker perturbation score is 0, and a marker perturbation score greater than 0 indicates that the stimulus has condensate regulation characteristics.

[0176] Embodiment 40. The method of any one of embodiments 2 to 39, wherein the global condensate perturbation score indicates that the stimulus selectively modulates the one or more condensate types of a cellular composition subjected to the stimulus.

[0177] Embodiment 41. The method of any one of embodiments 2 to 39, wherein the global condensate perturbation score indicates that the stimulus non-selectively modulates the one or more condensate types of the cellular composition treated with the stimulus.

[0178] Embodiment 42. The method of any one of embodiments 2 to 39, wherein the global condensate perturbation score indicates that the stimulus does not substantially modulate one or more target condensate types of a cellular composition treated with the stimulus.

[0179] Embodiment 43. The method of any one of embodiments 27 to 42, wherein the global condensate perturbation score is based on the marker perturbation score and the second marker perturbation score.

[0180] Embodiment 44. The method of any one of embodiments 2 to 43, wherein the global condensate perturbation score is calculated by dividing the sum of all marker perturbation scores by the number of marker perturbation scores.

[0181] Embodiment 45. The method of any one of embodiments 2 to 44, wherein the method comprises determining a reference global condensate perturbation score.

[0182] Embodiment 46. The method of embodiment 45, wherein the reference global condensate perturbation score is 1, and (i) a global condensate perturbation score greater than 0 and less than 1 indicates that the stimulus selectively modulates the one or more condensate types in the cellular composition, and (ii) a global condensate perturbation score of 0 indicates that the stimulus does not substantially modulate the one or more condensate types in the cellular composition.

[0183] Embodiment 47. The method described in embodiment 46, wherein, for a global condensate perturbation score greater than 0 and less than 1, the smaller the global condensate perturbation score of the stimulus, the more selective the stimulus. [Example]

[0184] Example 1 This example describes a methodology for identifying stimuli with condensate modulation properties for multiple condensate types. Specifically, stimuli with selective condensate modulation properties are identified using the methodology described herein.

[0185] HeLa cells were grown in a T175 flask at 37°C and 5% CO2 in DMEM + GlutaMAX containing 10% FBS and 1% PS to 100% confluence. After washing excess cell culture medium with 5 mL of PBS, HeLa cells were removed from the T175 flask using 5 mL of TrypLe. A solution of 100,000 cells per mL was used for cell plating. Using a MultiFlo™ with a 5 μL cassette, 6 μL of medium from the 100,000 cell / mL solution was plated into each well of a 1536-well plate, resulting in 600 cells / well. The plate was then centrifuged at 1000 rpm for 1 minute to ensure that the medium and cells reached the bottom of the well. The plate was then stored in a humidified chamber in a cell culture incubator.

[0186] Approximately 48 hours after plating, candidate compounds were dispensed onto the cells using a Beckman Echo 650 / 550 acoustic liquid handler. The cells were incubated for 6 hours in a humidified chamber in a cell culture incubator at 37°C and 5% CO2.

[0187] Following the treatment period, cells were fixed within the wells of the plate. Using a MultiFlo equipped with a 1 μL cassette, 3 μL of 12% PFA was added directly to the cell culture medium in the 1536-well plate for 10 minutes to fix the cells. Using an EL-406 liquid handler, the fixation medium was aspirated and the plate was washed with PBS. Three washes with PBS were completed. After fixation, the cells were permeabilized. Using an EL-406 equipped with a 5 μL cassette, the medium in the 1536-well plate was aspirated and 6 μL of a PBS + 0.5% BSA solution containing 0.5% Triton® X-100 was added to the cells for 10 minutes. Using an EL-406 liquid handler, the medium was aspirated and the plate was washed with PBS containing 0.5% BSA. Three washes with PBS were completed.

[0188] After fixation and permeabilization, cells were stained using a two-antibody system (unlabeled primary antibody and labeled secondary antibody) and dye to visualize cellular features. Specifically, the primary antibody was diluted to a working concentration in PBS + 0.5% BSA, and 6 μL of this solution was added to each well of a 1536-well plate using a MultiFlo™ with a 1 μL cassette equipped with a metal tip. Incubation with the primary antibody was carried out overnight at 4°C. The primary antibody was then washed away using EL-406, and the cells were washed three times with PBS containing 0.5% BSA. Next, the secondary antibody was diluted 1:500 in PBS + 0.5% BSA, and 6 μL of this solution was added to each well of the plate using a MultiFlo™ with a 5 μL cassette. The secondary antibody was incubated for 1 hour at room temperature. The secondary antibody was then washed away using EL-406, and the cells were washed three times with PBS + 0.5% BSA. After antibody staining, cells were stained with Hoechst (nuclear stain) and CMB (cell mask blue). Specifically, 3 μL of PBS + 0.5% BSA containing 6 μg / mL Hoechst and 12 μg / mL CMB was added to 6 μL of PBS + 0.5% BSA in a 1536-well plate using a Multiflo equipped with a 3 μL cassette. The Hoechst and CMB solutions were not washed from the plate. The plate was sealed and stored at 4°C until imaging.

[0189] Images of stained cells were acquired with a Phenix Plus confocal microscope.

[0190] Using the acquired images, marker perturbation scores and global condensate perturbation scores were calculated using the coefficient of variation (CV) and the dispersion index (D). Specifically, CV and D were calculated separately for both the nuclear and cytoplasmic compartments. CV was calculated as the STD^2 / mean of pixel intensities within a given compartment. D was calculated as the STD^2 / mean of pixel intensities within a given compartment.

[0191] A corrected Z-score for each marker stained in each treatment condition tested was then calculated using the CV and D. The result was a Z-score for each treatment condition that represented how different the CV or D was compared to the negative control-treated wells. The Z-score was calculated as:

number

number

[0192] Using the marker perturbation scores, a global condensate perturbation score was calculated for each treatment condition. This score reflected how extensively the treatment condition perturbed all condensates. The global condensate perturbation score ranged from 0 to 1 and was calculated as the sum of all marker perturbation scores divided by the total number of markers stained.

[0193] Marker perturbation scores and global condensate perturbation scores were visualized using Spotfire. As shown in Figure 2, the marker perturbation scores and global condensate perturbation scores for 60 compounds at a concentration of 4 μM are evaluated across 12 condensate types using two markers for each condensate type. As shown in Figure 2, the different compounds tested have very different condensate specificities, as demonstrated by the wide range of global condensate perturbation scores, and the methods provided herein enable the identification of, for example, selective condensate modulators.

[0194] Example 2 This example describes a methodology for identifying stimuli with condensate modulation properties for multiple condensate types. Specifically, stimuli with selective condensate modulation properties are identified using the methodology described herein.

[0195] Experimental Method U2OS cells were grown in a T175 flask to 90% confluence in DMEM containing 10% FBS and 1% PS at 37°C and 5% CO2. After washing excess cell culture medium with 5 mL of PBS, U2OS cells were removed from the T175 flask using 5 mL of TrypLe. A solution of 62,500 cells per mL was used for cell plating. Using a Multidrop Combi with a small-tube metal tip dispense cassette, 8 μL of medium from the 62,500 cell / mL solution was plated into each well of a 1536-well plate, resulting in 500 cells / well. The plate was left at room temperature for 15 minutes to allow the cells to settle to the bottom of the plate before being placed in a humidified chamber in a cell culture incubator.

[0196] Approximately 48 hours after plating, candidate compounds were dispensed onto the cells using a Beckman Echo 650 acoustic liquid handler. The cells were incubated for 6 hours in a humidified chamber in a cell culture incubator at 37°C and 5% CO2.

[0197] Following the treatment period, the medium was aspirated using EL-406, and then 4% PFA solution was dispensed using the EL-406 syringe dispenser. After 10 minutes, the fixation medium was aspirated using EL-406, and the cells were washed three times with PBS. After fixation, the cells were permeabilized. Using EL-406, the PBS in the 1536-well plate was aspirated, and 8 μL of a PBS + 0.5% BSA solution containing 0.5% Triton X-100 was added to the cells for 10 minutes. The medium was aspirated using EL-406, and the plate was washed three times with PBS containing 0.5% BSA.

[0198] After fixation and permeabilization, cells were stained using a two-antibody system (unlabeled primary antibody and labeled secondary antibody) and dye to visualize cellular features. The two-antibody system used a first antibody specific for a condensate-type first marker and a second antibody specific for a second condensate-type second marker, with the first and second antibodies derived from different organisms, e.g., mouse and rabbit. Specifically, the primary antibody was diluted to a working concentration in PBS + 0.5% BSA, and 6 μL of this solution was added to each well of a 1536-well plate using a Multidrop Combi with a small-tube metal tip dispense cassette. The primary antibody incubation was carried out overnight at 4°C. The primary antibody was then washed off using EL-406, and the cells were washed three times with PBS containing 0.5% BSA. Next, the secondary antibody was diluted 1:1000 in PBS + 0.5% BSA, and 6 μL of this solution was added to each well of the plate using a Multidrop Combi with a small tube metal tip dispenser cassette. The secondary antibody was incubated for at least 1 hour at room temperature. The secondary antibody was then washed off using EL-406, and the cells were washed three times with PBS + 0.5% BSA. After antibody staining, the cells were stained with Hoechst (nuclear stain) and CMO (cell mask orange). Specifically, after aspiration using EL-406, 6 μL of PBS + 0.5% BSA containing 6 μg / mL Hoechst and 12 μg / mL CMO was added to the cells. The Hoechst and CMB solutions were not washed from the plate. The plate was sealed and stored at 4°C until imaging.

[0199] Images of stained cells were acquired with a Phenix Plus confocal microscope.

[0200] Image analysis protocol where global condensate perturbation scores are based on the average of marker perturbation scores Using the acquired images, marker perturbation scores and global condensate perturbation scores were calculated using multiple image features extracted by Harmony image analysis software. The conceptual goal of the marker perturbation score was to combine all relevant information captured in the multiple extracted features to produce a single numerical value that represents the extent to which a stimulus alters the staining pattern of a marker compared to the control. The conceptual goal of the global condensate perturbation score was to combine all of the individual marker perturbation scores to produce a single numerical value that represents the extent to which a stimulus alters the staining pattern of all measured condensates compared to the control. One quantitative method for achieving these goals is described below. All features were calculated separately for both the nuclear and cytoplasmic compartments, in two channels containing condensate marker staining. The features can be subdivided into three categories including texture (Gabor maximum, Gabor minimum, Haralick contrast, Haralick correlation, Haralick homogeneity, Haralick sum variance, SER bright, SER dark, SER edge, SER hole, SER ridge, SER saddle, SER spot, SER valley), intensity (coefficient of variation, mean intensity, contrast, standard deviation), and morphology (area, circularity, perimeter, width, length, width-to-length ratio).

[0201] A corrected Z-score was then calculated for each feature assessed for each marker stained in each treatment condition tested. The result was an individual Z-score for each feature for each marker in each treatment condition that represents how different the feature was compared to the negative control-treated wells. The Z-score was calculated as:

number

number

[0202] Using the marker perturbation scores, a global condensate perturbation score was calculated for each treatment condition. This score reflected how extensively the treatment condition perturbed all condensates. The global condensate perturbation score ranged from 0 to 1 and was calculated as the average of all marker perturbation scores.

[0203] Marker perturbation scores and global condensate perturbation scores were visualized using Spotfire. As shown in Figures 3A and 3B, marker perturbation scores and global condensate perturbation scores for 40 compounds at a concentration of 3.33 μM were evaluated across 22 condensate markers (11 condensate types). As shown in Figures 3A and 3B, the different candidate compounds tested had very different condensate specificities, as demonstrated by the wide range of global condensate perturbation scores, demonstrating that the methods provided herein enable the identification of, for example, selective condensate modulators. Image analysis protocol in which a global condensate perturbation score is based on the sum of marker perturbation scores

[0204] A corrected Z-score was then calculated for each feature assessed for each marker stained in each treatment condition tested. The result was an individual Z-score for each feature for each marker in each treatment condition that represents how different the feature was compared to the negative control-treated wells. The Z-score was calculated as:

number

number

[0205] Using the marker perturbation scores, a global condensate perturbation score was calculated for each treatment condition, which reflected how extensively the treatment condition perturbed all condensates, and was calculated as the sum of all marker perturbation scores.

[0206] Marker perturbation scores and global condensate perturbation scores were visualized using Spotfire. As shown in Figures 4A and 4B, the marker perturbation scores and global condensate perturbation scores for 40 compounds at a concentration of 3.33 μM were evaluated across 22 condensate markers (11 condensate types). As shown in Figures 4A and 4B, the different candidate compounds tested had very different condensate specificities, as demonstrated by the wide range of global condensate perturbation scores, demonstrating that the methods provided herein enable the identification of, for example, selective condensate modulators.

Claims

1. 1. A method for identifying stimulation of condensate adjustment characteristics for one or more condensate types, comprising: (a) subjecting a cell composition comprising a cell type to said stimulus; (b) measuring at least one marker characteristic in at least a portion of the cell composition subjected to said stimulation, the measuring, wherein the marker associates with at least one of the one or more condensate types before and / or after the cell composition is subjected to the stimulus; (c) determining a marker perturbation score for each marker in the cell composition subjected to the stimulus based on the measured characteristic of each marker; and (d) identifying the stimulation of the condensate regulatory property from the marker perturbation score.

2. determining a global condensate perturbation score, wherein the global condensate perturbation score is based on at least one marker perturbation score; 2. The method of claim 1, further comprising the determining step, wherein the identifying step of the stimulation of the condensate modulation characteristic can be identified from at least one marker perturbation score and / or a global condensate perturbation score.

3. 10. The method of any one of the preceding claims, wherein the step of measuring the characteristic measures two markers for each of the at least one or more condensate types in at least a portion of the cell composition subjected to the stimulation.

4. 10. The method of any one of the preceding claims, wherein said measuring said characteristic measures at least one marker for at least two or more condensate types.

5. The method of any one of claims 1 to 4, wherein each marker is independently a lipid, a polypeptide, or a nucleic acid.

6. The method of any one of claims 1 to 4, wherein each marker is independently a polypeptide.

7. Each marker is (i) is within the condensate type; (ii) the cell composition is partitioned into the condensate type after being subjected to the stimulus; or (iii) the cell composition is excluded from the condensate type after being subjected to the stimulation.

8. The method of any one of claims 1 to 7, wherein each marker is independently a condensate scaffold polypeptide or a nucleic acid.

9. The method of claim 8 , wherein at least one marker is a condensate scaffold polypeptide.

10. The method of any one of claims 1 to 7, wherein each marker is independently a condensate client polypeptide or nucleic acid.

11. 2. The method of any one of the preceding claims, wherein measuring the characteristic of the at least one marker in at least a portion of the cellular composition subjected to the stimulation comprises staining at least a portion of the cellular composition for the marker.

12. The method of claim 11, wherein the staining is immunofluorescence (IF) staining.

13. 2. The method of claim 1, wherein measuring the characteristic of the at least one marker for at least the portion of the cellular composition comprises imaging at least the portion of the cellular composition.

14. The method of claim 13 , wherein the imaging comprises fluorescent imaging.

15. 10. The method of any one of the preceding claims, wherein at least one of one or more different condensate types is formed in the cell composition after being subjected to the stimulus.

16. 10. The method of any one of the preceding claims, wherein the stimulus is selected from the group consisting of an exogenous compound, an exogenous peptidic substance, an exogenous genetic material, a stressor, an environmental stimulus, and combinations thereof.

17. 10. The method of any one of the preceding claims, wherein the stimulus is an exogenous compound.

18. 18. The method of claim 17, wherein the compound is a small molecule therapeutic candidate or a precursor thereof.

19. 19. The method of claim 17 or 18, wherein the cell composition is subjected to the compound at a known concentration.

20. 10. The method of any one of the preceding claims, further comprising subjecting the cellular composition to a second stimulus; measuring a characteristic of the at least one marker in at least a portion of the cellular composition that has been subjected to the second stimulus; and independently determining a marker perturbation score for each marker in the cellular composition that has been subjected to the second stimulus based on the measured characteristic of each marker in the cellular composition that has been subjected to the second stimulus.

21. 21. The method of claim 20, further comprising comparing a marker perturbation score for a marker in a cell composition subjected to the stimulus with a marker perturbation score for a marker in a cell composition subjected to the second stimulus.

22. 22. The method of claim 20 or 21, further comprising determining a second global condensate perturbation score to evaluate the second stimulation of the condensate modulatory property, wherein the second global condensate perturbation score is based on at least one marker perturbation score of a marker in a cellular composition subjected to the second stimulation.

23. 23. The method of claim 22, further comprising comparing the global condensate perturbation score to the second global condensate perturbation score.

24. 10. The method of any one of the preceding claims, further comprising determining at least one property associated with at least one of the one or more condensate types and / or markers.

25. The at least one characteristic associated with the at least one of the one or more condensate types and / or the markers is: (i) the location of the condensate type; (ii) the distribution of the condensate types and / or the markers; (iii) the number of condensate types; (iv) the size of the condensate type; (v) the ratio of the amount of the condensate type to the amount of a control condensate; (vi) functional activity associated with said condensate type; (vii) the condensate-type composition; (viii) co-localization of the condensate type with a biomolecule; (ix) the diffusion coefficient of the condensate-type component; (x) the stability of the condensate type; (xi) dissolving or reducing the size of said condensate type; (xii) the surface area of ​​the condensate type; (xiii) the sphericity of the condensate type; (xiv) the fluidity of the condensate type; (xv) solidification of said condensate type; (xvi) the location of the marker; (xvii) the amount of said marker or a precursor thereof; (xviii) condensate partitioning of the marker into the condensate types; (xix) a functional activity associated with the marker; (xx) aggregation of the marker; (xxi) the post-translational modification status of the marker; and (xxii) the amount of a degradation product of the marker.

26. 10. The method of any one of the preceding claims, wherein at least one of the one or more condensate types is selected from the group consisting of cleavage bodies, p-granules, histone locus bodies, multivesicular bodies, neuronal RNA granules, nuclear gems, nuclear pore complexes, nuclear speckles, nuclear stress bodies, nucleoli, Oct1 / PTF / transcription (OPT) domains, paraspeckles, juxtanucleolar compartments, PML nucleoli, PML oncogenic domains, Polycomb bodies, processing bodies, Sam68 nucleoli, stress granules, splicing speckles, P62 bodies, Cajal bodies, heterochromatin, transcription condensates, and centrosomes.

27. The method further comprises measuring a characteristic of a second marker in at least a portion of the cell composition that has been subjected to the stimulation, 2. The method of any one of the preceding claims, comprising measuring whether the second marker is associated with at least one of the one or more condensate types before and / or after the cell composition is subjected to the stimulus.

28. 28. The method of claim 27, further comprising determining a second marker perturbation score for the second marker in the cell composition subjected to the stimulus based on the measured characteristic of the second marker.

29. 29. The method of claim 27 or 28, wherein the marker perturbation score indicates whether the stimulus modulates association between the marker and the at least one of the one or more condensate types in the cellular composition, and the second marker perturbation score indicates whether the stimulus modulates association between the second marker and the at least one of the one or more condensate types in the cellular composition.

30. 30. The method of any one of claims 27 to 29, wherein the marker and the second marker are associated with the same condensate type.

31. 30. The method of any one of claims 27 to 29, wherein the marker and the second marker are associated with different condensate types.

32. 10. The method of any one of the preceding claims, wherein each marker perturbation score is independently based on at least one characteristic associated with the one or more condensate types and / or at least one of the markers.

33. 10. The method of any one of the preceding claims, wherein each marker perturbation score is independently determined based on the coefficient of variation (CV) of the marker, wherein the CV is determined based on the standard deviation (STD) of the distribution intensity of the marker divided by the mean distribution intensity of the marker.

34. 34. The method of claim 33, wherein the distribution intensity of each marker is based on pixel analysis of one or more images of the one or more cellular compositions or portions thereof.

35. 35. The method of claim 33 or 34, wherein each marker perturbation score is based on the median absolute deviation (MAD) Z-score of the CV of the marker.

36. 36. The method of claim 35, further comprising obtaining the MAD Z-score for the marker.

37. (i) if the MAD Z-score is greater than 5 or less than −5, then the marker perturbation score is 1; (ii) if 2.5<MAD Z-score≦5, or −5≦MAD Z-score<−2.5, then the marker perturbation score is 0.5; (iii) if -2.5≦MAD Z-score≦2.5, then the marker perturbation score is 0.

38. 32. The method of any one of claims 1-31, wherein said determining said marker perturbation score for a marker comprises extracting one or more features from an image of a cellular composition subjected to said stimulus.

39. 39. The method of claim 38, wherein the one or more features are based on one or more of one or more texture features, one or more intensity features, or one or more morphological features.

40. 40. The method of claim 38 or 39, wherein said determining the marker perturbation scores for markers comprises measuring a plurality of features associated with the markers; calculating a modified Z-score for each feature of each marker based on the measured plurality of features; calculating a feature change score for each feature of each marker based on the associated modified Z-score and proportional scale; and then aggregating the feature change scores of a top percentage of the feature change scores.

41. 41. The method of claim 40, wherein the aggregating the modified Z-scores comprises summing or averaging.

42. 10. The method of any one of the preceding claims, further comprising evaluating a reference stimulus, said evaluating comprising determining a reference marker perturbation score.

43. 43. The method of claim 42, wherein the reference marker perturbation score is 0 and wherein the marker perturbation score greater than 0 indicates that the stimulus has condensate modulatory properties.

44. 44. The method of any one of claims 2-43, wherein the global condensate perturbation score indicates that the stimulus selectively modulates the one or more condensate types of a cellular composition subjected to the stimulus.

45. 44. The method of any one of claims 2-43, wherein the global condensate perturbation score indicates that the stimulus non-selectively modulates the one or more condensate types of a cellular composition treated with the stimulus.

46. 44. The method of any one of claims 2-43, wherein the global condensate perturbation score indicates that the stimulus does not substantially modulate one or more target condensate types of a cellular composition treated with the stimulus.

47. 47. The method of any one of claims 28 to 46, wherein the global condensate perturbation score is based on the marker perturbation score and the second marker perturbation score.

48. 48. The method of any one of claims 2 to 47, wherein the global condensate perturbation score is calculated by dividing the sum of all marker perturbation scores by the number of marker perturbation scores.

49. 48. The method of any one of claims 2 to 47, wherein the global condensate perturbation score is calculated by summing all marker perturbation scores.

50. 50. The method of any one of claims 2 to 49, wherein the method comprises determining a reference global condensate perturbation score.

51. the reference global condensate perturbation score is 1; (i) the global condensate perturbation score is greater than 0 and less than 1, indicating that the stimulus selectively modulates the one or more condensate types in the cellular composition; 51. The method of claim 50, wherein (ii) the global condensate perturbation score of 0 indicates that the stimulus does not substantially modulate the one or more condensate types in the cellular composition.

52. 52. The method of claim 51 , wherein, for the global condensate perturbation score greater than 0 and less than 1, the smaller the global condensate perturbation score of the stimulus, the more selective the stimulus.

Images