Method for preparing and analyzing biopsy sample and biological sample

Abstract

To provide a method for preparing and analyzing a biopsy sample and a biological sample.SOLUTION: A matrix support method and a composition are provided for solidifying and preparing a liquid biopsy and another liquid sample for three-dimensional high-resolution imaging and diagnosis by a microscopic examination.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to matrix-assisted methods and compositions for analyzing biological specimens, particularly liquid biopsies, and other liquid samples by microscopy methods including fluorescence microscopy.

Background Art

[0002] Liquid biopsies are typically obtained from body fluids such as peripheral blood, bone marrow, cerebrospinal fluid, urine, saliva, sputum, tears, semen, or other tissue sources. Biomarkers or components in liquid samples can be evaluated or measured for various diagnostic applications such as disease screening, detection, staging, and surveillance.

[0003] Biomarkers in liquid biopsy can include cell components and extracellular components, and their selection can depend on multiple factors such as medical or treatment status. For example, biomarkers can correspond to antigens or other attributes that distinguish rare circulating cells such as circulating tumor cells (CTCs) and CTC clusters derived from solid tumors or metastases, and circulating endothelial cells (CECs) associated with cardiovascular and other conditions. See, for example, Lim et al. 2019, NPJ Prec. Oncol. 3, 23; Rostami et al. 2019, J. Sci: Adv. Mat. Dev. 4, 1-18; Schmidt et al. 2015, Trends Cardiovasc. Med. 25, 578-587. Such cells can be further processed for genetic abnormalities and other molecular characteristics. Biomarkers can also identify extracellular components such as circulating tumor-derived factors, secreted proteins, released vesicles and exosomes, and cell-free nucleic acids. Cell-free nucleic acids include cell-free tumor DNA (ctDNA) applied to cancer monitoring, and cell-free fetal DNA (cffDNA) found in maternal blood and applied to non-invasive prenatal testing. See Campos et al. 2018, Cancer J. 24, 93-103; Sifakis et al. 2014, Mol. Med. Rep. 11, 30 2367-2372. Subsequent genomics and protein processing can enable further analysis of extracellular biomarkers.

[0004] Typical biopsy methods can encompass several approaches. See, e.g., Harouaka et al., 2014, Pharmacol. Ther. 141, 209-221. One common approach is based on immunoaffinity, such as methods based on microfluidics and microchips that enable the detection of antibodies bound to cellular and extracellular targets. These methods rely on antibody-antigen binding between floating cells and an antibody-coated surface and are thus limited to antigens present on the surface of target cells such as membrane proteins. In addition, the effectiveness of these methods depends on a level of cell surface antigen sufficient to enable efficient and specific recognition by the antibody (e.g., low EpCAM expression on the cell surface can result in insufficient cell-to-chip surface binding). These methods generally have low cell flow rates that preclude effective analysis of complex samples. Thus, the scope, sensitivity, specificity, and throughput of such methods, as well as other immunoaffinity-based methods (such as those based on magnetic beads), are limited.

[0005] Another common approach is cytology, which directly examines cells under a microscope. However, this approach is limited to examining only a small number of cells at a time (e.g., as a single layer on a slide glass) due to the light scattering properties of cells, and thus it is not practical or economically feasible to use this approach to detect rare targets among the millions of cells that can be present in 2 cc of blood.

[0006] In another approach, flow cytometry, thousands of cells per second pass one by one through one or more laser beams, where they can produce different patterns of light scattering (depending, for example, on cell size and granularity) and fluorescence emission (depending on which fluorescent probes are bound to the cell). See, for example, Flow cytometry: retrospective, fundamentals and recent instrumentation, Cytotechnology, 2012 Mar; 64(2): 109-130. However, the flow cytometry method does not provide high resolution and reliability when the cells of interest are rare. For example, they do not provide a direct visual inspection of the cells to confirm potential morphological or functional characteristics of the cells. Further, gating problems can arise based on fluorescence signal intensity and pixels captured by the detector (which do not provide information about the labeling quality or morphological details of the analyzed sample). For example, even small gating changes or perturbations can result in the exclusion of CTC cells and other rare cells (e.g., CEC) with small or weak fluorescence signals.

Summary of the Invention

Problems to be Solved by the Invention

[0007] Considering these and other limitations, further improvements in liquid biopsy analysis are still needed.

[0008] The present invention addresses these and other needs in the art by providing methods and compositions for labeling, dispersing, and capturing biopsy components in a three-dimensional gel or other non-liquid form. When maintained in this form, discrete and rare biomarkers can be detected with high resolution and high sensitivity by rapid imaging methods such as light sheet fluorescence microscopy and other methods. More generally, these methods are applicable to components in any (biological or non-biological) sample, and their resolution can be improved by dispersion in a liquid and subsequent capture and imaging in a non-liquid state.

Means for Solving the Problem

[0009] The disclosure of the present invention provides a matrix-assisted method for preparing and analyzing components in biological and non-biological samples containing liquid biological samples, such as a matrix-assisted method based on gel formation, as further described herein. The liquid specimen can be derived from any source including humans and animals. In embodiments, it can be derived from peripheral blood, bone marrow, cerebrospinal fluid, and liquid biopsies obtained from other tissue sources, all of which can be further processed. In embodiments, it can be derived from a liquid dispersion of materials obtained from other sources including solid sources such as solid tissue biopsies.

[0010] In embodiments, the matrix-assisted method includes adding a solidifying agent (e.g., a gelling agent) to a biological specimen containing biological material; generating a solidified specimen (e.g., a gelled specimen) containing the dispersed biological material; and imaging the solidified specimen to identify one or more components in the biological material. The biological material can include any biomolecule including nucleic acids, proteins, and small molecules, which can, in embodiments, serve as biomarkers for a medical condition or disease state. For example, the biomolecule can serve as a biomarker for rare circulating cells in the blood, such as circulating tumor cells, circulating endothelial cells, and other cells and cell clusters that may be present in a biological specimen. In embodiments, the biological material in the specimen can be concentrated, for example, by concentrating cells from a large volume of blood or other sample source. However, advantageously, the methods of the present disclosure do not require preselection or prescreening of cells. Instead, the methods enable unbiased analysis of samples of non-selected or non-screened cells by detecting biomarker labels such as antibodies or nucleic acid probes bound to the biological material in the specimen, for example, by detecting a labeled antibody that identifies a specific cell surface marker in the specimen.

[0011] In embodiments, as disclosed herein, in any of the methods, the step of adding a solidifying agent can include adding a liquid gel solution, such as low melting point agarose or a hydrogel precursor, to a sample; directly adding an agent to the sample under conditions that allow for the formation of a solid such as a gel; forming a hydrogel or other methods. Before adding the solidifying agent, the method can include subjecting the components of the biological specimen to a fixation procedure as described herein, and can also include labeling one or more biomolecules, such as proteins or nucleic acids, using a molecular probe. Molecular probes include antibodies, dyes, and nucleic acid probes known in the art, including those described herein. In embodiments, the probe can be used to identify circulating tumor cells and clusters, as well as cell-free tumor or fetal-derived DNA, and genetic and structural changes in the cell nucleus such as DNA and chromosomal abnormalities, amplifications, deletions, and translocations.

[0012] In embodiments, the step of adding a solidifying agent to a liquid specimen includes mixing a sample containing a biological material (e.g., a pellet containing the biological material) with a liquid (molten) gel solution having a temperature raised above its gelling point. Thus, in embodiments, a cell pellet, e.g., one labeled and washed in PBS, is resuspended in the gel solution and cooled to form a gel. The resuspension step also allows any trace liquid associated with the pellet after treatment (e.g., PBST) to be diluted and mixed in the gel solution to make a uniform sample for imaging.

[0013] In an alternative embodiment, the step of adding a solidifying agent to a liquid specimen includes mixing a sample containing a biological material (e.g., a pellet containing the biological material) with a mixture or solution containing one or more hydrogel precursors, and changing the state of the mixture to induce solidification (e.g., gelation). The formation of hydrogels is known in the art and can be achieved by various methods according to the present disclosure. For example, a second agent (e.g., Ca +An ion or crosslinking agent) can be added in an amount sufficient to induce gelation of the hydrogel precursor according to methods known in the art.

[0014] For example, the sample can be processed as described herein, including the steps of centrifuging the sample to obtain a pellet, resuspending it in an alginate hydrogel precursor solution, and mixing it with an appropriate amount of CaCl2 solution (e.g., 0.2 M) to initiate gelation. In this exemplary method, a gel is formed within a short time (e.g., about 15 minutes), after which it can be mounted (if necessary) for refractive index matching and for imaging.

[0015] In any method, the step of creating a solidified sample containing the dispersed biological material can include transferring the sample to a sample holder after adding a solidifying agent to cause solidification. In other embodiments, the sample can be prepared directly within the sample holder to which the solidifying agent is added, thus allowing the pre-gelation and solidification steps to be combined within a single tube. The sample can be agitated, shaken, vibrated, or otherwise agitated within the sample holder prior to solidification to ensure dispersion of the material. In embodiments, the solidified sample has a shape suitable for imaging, such as a block, cylindrical shape, or any other shape, that is compatible with the desired imaging system. In embodiments, the solidified sample containing the dispersed biological material is transferred to a clearing (or equilibration) solution to achieve refractive index matching. In other embodiments, refractive index matching is not necessary, for example, due to the appropriate optical properties of the particular solidifying agent used. For example, if a sample of dispersed cells is prepared in a transparent matrix, then labeled molecules on (or within) the surface of the individually dispersed cells can be appropriately imaged without pre-incubation in a refractive index matching material (e.g., a refractive index matching solution). In certain embodiments, solidification includes mixing the biological sample with a refractive index matching material (e.g., a refractive index matching solution), thereby obviating the need for separate treatment with the refractive index matching material.

[0016] The step of imaging the solidified sample can, in embodiments, include imaging a properly shaped solid sample, such as a block, by various microscopy techniques, including fluorescence microscopy, and more specifically fluorescence light sheet microscopy. In embodiments, imaging can include enabling single cell identification in the solidified sample, and more specifically, can include detecting one or more cancer cells, such as, for example, circulating tumor cells or cancer markers. The size of the solidified sample can vary depending on the application, sensitivity, and biomolecules being assayed. In embodiments, imaging can be used to analyze cell-cell interactions, as well as morphological and structural features of cells, such as size, shape, and nucleus-to-cytoplasm ratio, and features of organelles.

Advantages of the Invention

[0017] The method of the present invention is useful in a number of applications, including, but not limited to, evaluating, diagnosing, or monitoring a disease by, for example, microscopically analyzing a liquid and / or tissue biopsy, screening candidate therapeutic agents for their effects on a sample (e.g., a blood or tissue sample) in a disease state, and evaluating the expression of a panel of biomarkers in a sample.

Brief Description of the Drawings

[0018]

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Mode for Carrying Out the Invention

[0019] Detailed Description of the Invention The present disclosure provides matrix-assisted methods and compositions for analyzing liquid samples, such as liquid biopsies, for the presence of one or more biomarkers. In embodiments, the methods and compositions of the present invention are used to solidify dispersed materials in a sample to capture and immobilize them in a three-dimensional state. Subsequently, biomarkers of interest, such as rare disease markers that may be present in the material, can be detected and determined with high sensitivity and high specificity. The matrix-assisted method of the present invention involves the use of a solidifying agent, such as a molten gel solution or a hydrogel precursor, to convert a liquid sample into a solid sample having dispersed components.

[0020] In embodiments, the present disclosure provides a three-dimensional imaging approach for detecting biomarkers, including rare molecules such as cancer cell markers, using matrix-assisted methods and compositions for analyzing liquid biopsies and other liquid samples by a microscope including a fluorescence microscope.

[0021] In an embodiment, a biomarker indicates a cell component such as a component of rare circulating cells such as circulating tumor cells and circulating endothelial cells. For example, a biomarker can include a cell surface protein, a morphological marker, or a nucleic acid sequence that can identify such tumor cells. In an embodiment, a biomarker indicates an extracellular component such as extracellular DNA, protein, or vesicles that indicate a specific disease or health state.

[0022] As described herein, biomarkers can be labeled by known techniques and they can be detected by a wide range of imaging methods, even in complex samples, and more particularly by fluorescence microscopy including light sheet fluorescence microscopy and other microscopy methods.

[0023] Terms and Definitions Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Any methods, devices, and materials similar or equivalent to those described herein can be used in the practice of the present invention. The following definitions are provided to facilitate understanding of certain terms frequently used herein but are not meant to limit the scope of the present disclosure.

[0024] As used herein, the term “about” or “approximately” means a range of values that includes a particular value and that one of ordinary skill in the art would consider reasonably similar to the particular value. In an embodiment, “about” means within one standard deviation using measurements generally accepted in the art. In an embodiment, “about” means a range that extends from + / −10% of a particular numerical value. In an embodiment, “about” means the specified numerical value.

[0025] Regardless of whether the term "about" is explicitly used, all amounts recited in this specification are to be understood as referring to both an approximate value that is the actual given value and the value that would reasonably be inferred based on one of ordinary skill in the art, including equivalent and approximate values resulting from the particular given value's experimental and / or measurement conditions. Thus, for any embodiment of the disclosure where a numerical value begins with "about" or "approximately", the disclosure includes embodiments where the specific value is recited. Conversely, for any embodiment of the disclosure where a numerical value does not begin with "about" or "approximately", the disclosure includes embodiments where the numerical value begins with "about" or "approximately".

[0026] Unless otherwise specified, concentrations provided as percentages or weight (wt)% refer to weight / volume (w / v) concentrations. For example, 2% or 2 wt% of a component in 100 mL of solution corresponds to 2 grams of that component.

[0027] As used herein, the terms "a", "an", and "the" are to be understood to mean both the singular and the plural unless otherwise specified. Thus, "a", "an", and "the" (and, where appropriate, their grammatical variations) refer to one or more.

[0028] Furthermore, items, elements, or components of an embodiment may be described or claimed in the singular, but the plural is intended to be within the scope thereof unless a limitation to the singular is explicitly stated.

[0029] As used herein, the terms "comprising" and "including" are used in their open and non-limiting sense. Other terms and phrases used herein, as well as their variants, should be construed as open-ended and not limiting, unless otherwise expressly stated. By way of example, the term "example" is used to provide illustrative instances of the item under discussion, rather than an exhaustive or limiting list. Terms such as "conventional," "ordinary," "known," and terms of similar import should not be construed as limiting the items described to those available at a given time or point in time, but rather should be construed to include conventional or ordinary techniques that are or may be available or known at any present or future time. Similarly, when this specification refers to techniques that are apparent or known to one of ordinary skill in the art, such techniques include those that are or may be apparent or known to one of ordinary skill in the art at any present or future time.

[0030] As used herein, the term "solidified sample" refers to a sample in a non-liquid form, such as a solid or gel form, where the solid or gel material provides a support matrix for capturing, i.e., immobilizing, biological material in a dispersed state within the three-dimensional sample. Subsequently, the dispersed material within the sample can be efficiently identified and imaged in three dimensions. As used herein, the term "solidifying agent" includes gelling agents that can form gels, as well as, for example, epoxies and other agents that are desirable for supporting the dispersed biological material at high density.

[0031] As used herein, the terms “biological sample” and “biological specimen” (and depending on the context, “sample” or “specimen”) refer to any biological material that contains or is believed to contain biomolecules such as nucleic acids or proteins. Samples that can be manipulated with the compositions and methods provided herein can be obtained from in vivo or in vitro sources, and thus include specimens such as cells, tissues, viruses, and organs dissected from a subject such as a rodent model, as well as specimens such as cells, tissues, and mini-organs grown in vitro. Exemplary biological specimens include, but are not limited to, solid tissues and organs including tissues and organs of the liver, spleen, kidney, lung, intestine, thymus, colon, tonsil, testis, skin, brain, heart, muscle, and pancreas. In embodiments, the sample is an entire organ obtained from an animal including mice, rats, and other animals. In embodiments, the biological specimen is brain tissue or an entire brain from a rodent, more specifically a mouse, etc. Other biological samples include cells, viruses, and other microorganisms. In embodiments, the biological sample is derived from a human, animal, or plant. In embodiments, the sample is derived from a human, a companion animal such as a dog or cat, an agricultural animal such as a cow, sheep, and pig, a rodent such as a rat or mouse, a zoo animal, a primate such as a monkey, etc.

[0032] Exemplary biological samples include, but are not limited to, materials derived from biopsies, bone marrow samples, organ samples, skin fragments, living organisms, and materials obtained from clinical or forensic situations. In embodiments, the biological sample is a tissue sample, preferably an organ sample. The sample can be obtained from an animal or human subject that has or is suspected of having a disease or other pathological condition (normal or diseased), or is considered normal or healthy. Specimens such as organ and tissue samples can be collected and processed using the methods described herein and can be subjected to microscopic analysis immediately after processing or can be stored and subjected to microscopic analysis at a future time, for example, after long-term storage. In embodiments, the methods described herein can be used to analyze live cells, and in other embodiments, the methods described herein can be used to analyze fixed cells.

[0033] In certain embodiments, the biological sample is a liquid biopsy obtained from a body fluid such as peripheral blood, bone marrow, cerebrospinal fluid, urine, saliva, sputum, tears, semen, or other tissue sources.

[0034] As used herein, the term "biomolecule" can be changed to "molecule" and refers to molecules present in a biological sample or specimen. In one aspect, the biomolecule is an endogenous biomolecule. In another aspect, the biomolecule is an exogenous biomolecule. Non-limiting examples of exogenous biomolecules include biomolecules artificially transplanted, such as biomolecules transferred or expressed by a virus or plasmid. Biomolecules include, but are not limited to, proteins, nucleic acids, lipids, carbohydrates, steroids, metabolites, and other subcellular structures or components within cells, tissues, or organs. Non-limiting examples of proteins include enzymes, membrane proteins, transcription factors, synaptic proteins, and nerve markers. In some non-limiting embodiments, the biomolecule is selected from polymer subunits, receptors, receptor subunits, membrane proteins, intermediate filament proteins, membrane pumps, transcription factors, and combinations thereof. In other non-limiting embodiments, the biomolecule is Olig2 (oligodendrocyte transcription factor), NeuN (neuronal nuclear antigen), NKCC2 (Na+K+Cl Cotransporter 2). In other non-limiting embodiments, the biomolecule includes RNA. In still other non-limiting embodiments, the biomolecule includes DNA molecules. In an embodiment, the biomolecule is structurally present, and examples of the structure include flagella, cilia, synapses, synaptic spines, extracellular matrix (ECM), cell walls, cell envelopes, membranes, cytoplasm, Golgi network, mitochondria, endoplasmic reticulum (ER) (e.g., rough ER or smooth ER), nuclei, centrosomes, ribosomes, polyribosomes, lysosomes, liposomes, cytoskeletal components, vesicles, granules, peroxisomes, vacuoles, protoplasts, tonoplasts, plasmodesmata, plastids, chloroplasts, pseudopods, brain vasculature-related structures, brain high-density astrocyte networks, or combinations thereof. In an embodiment, the biomolecule is a cell marker such as a protein expressed on the surface of cancer cells.

[0035] In an embodiment, the "biomolecule" is in a liquid biopsy sample and is evaluated or measured for diagnostic purposes such as screening, detecting, staging, or surveilling (monitoring) for a disease state such as cancer or a medical condition such as a metabolic disorder. In an embodiment, the biomolecule is evaluated or measured in a non-invasive prenatal screening or diagnostic test.

[0036] As used herein, the term "label" refers to any currently known or later discovered technique and reagent that can provide a signal-based indication of the presence or absence of a particular target moiety within a sample of the present disclosure. Non-limiting examples of labeling agents include small molecules, dyes, antibodies, enzymes, nanoparticles, nucleic acid probes, or combinations thereof. In some non-limiting embodiments, the labeling agent includes a label, such as a chromogenic label, a fluorescent label, a radionuclide-binding label, or combinations thereof.

[0037] One aspect of the present disclosure provides a method for analyzing a liquid sample containing biological material for one or more target components. In one exemplary embodiment, the method of the present invention comprises adding a solidifying agent to an analyte obtained from a liquid sample containing biological material, creating a solidified sample containing the dispersed biological material, and imaging the solidified sample to identify one or more target components in the dispersed biological material.

[0038] In embodiments, the method for analyzing a liquid sample according to the present disclosure further comprises labeling an analyte obtained from the liquid sample with one or more probes for one or more target components and / or labeling the solidified sample with one or more probes for one or more target components before adding the solidifying agent. In one exemplary embodiment, the method of the present invention comprises labeling an analyte obtained from the liquid sample with one or more probes for one or more target components before adding the solidifying agent. In embodiments, the method of the present invention further comprises introducing a refractive index matching material into the solidified sample.

[0039] In an embodiment, the liquid sample is a liquid blood sample. For example, a specimen obtained from a liquid blood sample can be processed to remove red blood cells and platelets from the liquid blood sample, and / or it can be a specimen containing peripheral blood mononuclear cell (PBMC) cells isolated from the liquid blood sample. Alternatively, in other applications, red blood cells or other components of the liquid sample can be isolated. Alternatively, the specimen can be obtained from other biological fluids and liquids obtained from mammals other than blood (e.g., humans or rats).

[0040] In certain embodiments, the one or more target components include nucleic acids, proteins, viruses, or vesicles. In certain embodiments, the one or more target components include extracellular targets. In certain embodiments, the one or more target components include cells or intracellular targets.

[0041] In an embodiment, as described in any of the above embodiments, labeling includes contacting a specimen obtained from the liquid sample with a molecular probe and / or contacting the solidified sample from step (b) with a molecular probe. The molecular probe can be, for example, an antibody, a fluorescent dye, or a nucleic acid probe, separately.

[0042] In an embodiment, the method of analyzing a liquid sample according to the present disclosure further includes transferring the specimen to a sample holder. The method of the present invention can further include, in an exemplary embodiment, shaking or vibrating the sample within the sample holder. In an exemplary embodiment, the solidified sample is a solid or gel block suitable for imaging.

[0043] In an embodiment, the method of analyzing a liquid sample according to the present disclosure further includes performing a fixation procedure on the specimen, such as by incubating the specimen or the solidified sample in a fixative (when performed before coagulation). In an exemplary embodiment, the fixative includes glutaraldehyde, formaldehyde, epoxy, or any mixture of two or more thereof.

[0044] As described in any of the above embodiments, imaging can be performed using, for example, microscopy and camera techniques known to those skilled in the art. For example, in an embodiment, imaging is performed by a fluorescence microscopy method such as light sheet fluorescence microscopy. In an exemplary embodiment, imaging identifies the presence or absence of a specific cell type in a solidified sample.

[0045] One exemplary embodiment of the present disclosure provides, but is not limited to, a method for analyzing a liquid blood sample for the presence of rare circulating cells such as, for example, circulating tumor cells. In one embodiment, the method of the present invention comprises labeling a sample containing isolated peripheral blood mononuclear cells (PBMCs) obtained from a liquid blood sample with one or more probes for rare circulating cells, adding a solidifying agent to the labeled sample containing peripheral blood mononuclear cells (PBMCs), creating a solidified sample containing dispersed peripheral blood mononuclear cells (PBMCs), optionally introducing a refractive index matching material into the solidified sample to provide an optically transparent solidified sample having a refractive index suitable for imaging, and imaging the solidified sample or the optically cleared solidified sample to determine the presence of one or more probes, thereby determining the presence of rare circulating cells in the liquid blood sample. The labeling can further comprise adding a probe for white blood cells that can serve as a control, for example.

[0046] In embodiments, the one or more probes recognize cancer-specific antigens or tumor-specific DNA or RNA sequences. For example, the one or more probes can be selected from antibodies or nucleic acid probes. In one aspect, the one or more probes provide detection of one or more of the EpCAM, HER2, CDX2, CK20, CK19, PD / PDL-1, and EGFR antigens or corresponding nucleic acid sequences.

[0047] In an embodiment, the method of analyzing a liquid sample according to any of the above embodiments further includes introducing a refractive index matching material into the solidified sample. In certain embodiments, the optically clarified gelled sample is placed in a sample holder that is immersed in a solution containing the refractive index matching material.

[0048] In one embodiment, the solidifying agent includes a component selected from low melting point agarose, agarose, and a hydrogel precursor. In an exemplary embodiment, the step of creating the solidified sample includes adding the solidifying agent to the sample at a temperature higher than room temperature and bringing the sample to a low temperature at which it becomes a solid gel. In other embodiments, the step of creating the solidified sample includes adding a chemical to the hydrogel precursor to induce gelation.

[0049] In one embodiment, the present disclosure provides a solidified sample suitable for imaging, including a dispersed biological material immobilized within the solidified sample obtained from a liquid biopsy (e.g., a blood sample). The solidified sample can further include a probe for one or more target components in the liquid biopsy. The biological material includes rare circulating cells such as peripheral blood mononuclear cells (PBMCs) and circulating tumor cells, circulating epithelial cells, and circulating endothelial cells.

[0050] Low melting point (LM) agarose is commercially available and is the result of a chemical derivatization process known in the art, such as hydroxyethylation, which reduces the number of intramolecular hydrogen bonds present in standard agarose, thereby resulting in a relatively low melting point and gelation temperature. LM agarose generally has several properties including (i) a relatively low melting and gelation temperature compared to standard agarose, and (ii) higher transparency (gel transparency) compared to standard agarose gels.

[0051] In certain exemplary embodiments, the LM agarose has a gelation temperature ≤ 30 °C (e.g., 26 °C to 30 °C), and / or a melting temperature ≤ 65 °C (both at a 1.5 wt% concentration), and / or a gel strength (at a 1 wt% concentration) ≥ 200 g / cm 2It is molecular biology grade LM agarose having , and can create gels with sieving properties larger than normal melting agarose and higher transparency. Examples of commercially available LM agarose suitable for use according to the present disclosure include, but are not limited to, SeaPrep™ agarose (Lonza catalog number 50302), SeaPlaque™ agarose (Lonza catalog number 50104), and UltraPure™ low melting point agarose (ThermoFisher Scientific catalog number 16500500).

[0052] In embodiments, the labeling agent includes small molecules that can bind to specific target moieties within tissues. Examples of small molecule dyes include DAPI, propidium iodide, lectin, phalloidin, and any other small molecules that can bind to target moieties within tissues. In embodiments, the small molecules essentially generate signals such as fluorescence signals generated by DAPI, propidium iodide, or acridine orange. In embodiments, the small molecules are conjugated to an indicator to generate a signal, for example, in the case of a lectin dye, a fluorescence signal generating indicator, or a non-fluorescent signal generating indicator, for example, a colorimetric indicator (e.g., horseradish peroxidase (HRP) or 3,3'-diaminobenzidine tetrahydrochloride (DAB)). In embodiments, the staining agent includes antibodies further described herein. In embodiments, the staining includes modified nucleic acid strand targeting detection activity. In other embodiments, the staining includes in situ hybridization such as staining that includes nucleotide-based probes that can hybridize to a predetermined sequence of nucleic acids within tissues. In embodiments, the nucleotide-based probes include labels (e.g., one or more of the labels provided above) to enable signal generation and detection of the nucleotide-based probes. In further embodiments, the nucleotide-based probes include fluorescent labels such as fluorescence in situ hybridization (FISH).

[0053] In embodiments, a biological sample, such as a cell, tissue, organ, organism, or organ substructure, provides an endogenous signal, such as an endogenous fluorescent molecule. Examples of endogenous fluorescent molecules include fluorescent protein reporters (e.g., green fluorescent protein (GFP) or red fluorescent protein (RFP)). In other embodiments, the sample is derived from a transgenic model and the fluorescent molecule is expressed by a constitutive or inducible promoter. In still other aspects, the organism is infected with a recombinant virus or transformed with a plasmid encoding a fluorescent protein. Exemplary fluorescent protein reporters include green fluorescent protein (GFP), EGFP (enhanced GFP), BFP (blue fluorescent protein), CFP (cyan), red fluorescent protein (RFP), wtGFP (white GFP), YFP (yellow fluorescent protein), dsRed, mCherry, mVenus, mCitrine, tdTomato, luciferase, mTurquoise2, and the like.

[0054] As described above, a liquid specimen can be labeled with a molecular probe such as an antibody. In embodiments, the antibody is a primary antibody that includes a label that directly or indirectly generates a signal, such as a biotin label, a fluorescent label (fluorophore), an enzyme label (e.g., HRP or DAB), a coenzyme label, a chemiluminescent label, or a radioisotope label. In other aspects, the primary antibody is applied as a single stain (e.g., with or without the use of additional reagents such as labeled streptavidin or an enzyme / coenzyme substrate to provide a signal). In embodiments, the primary antibody does not include a label and is instead detected by a secondary antibody conjugated to a label.

[0055] Examples of fluorophores that can be conjugated to primary or secondary antibodies include Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 680, or Alexa Fluor 750. Other exemplary fluorophores include BODIPY FL, coumarin, Cy3, Cy5, fluorescein (FITC), Oregon Green, Pacific Blue, Pacific Green, Pacific Orange, tetramethylrhodamine (TRITC), Texas Red, APC-eFluor 780, eFluor 450, eFluor 506, eFluor 660, PE-eFluor 610, PerCP-eFluor 710, Super Bright 436, Super Bright 645, Super Bright 702, Super Bright 780, Super Bright 600, Qdot 525, Qdot 565, Qdot 605, Qdot 655, Qdot 705, Qdot 800, R-phycoerythrin (R-PE), and allophycocyanin (APC).

[0056] The fixed sample can be imaged by any microscope-based application, and thus the disclosed subject matter is, although specific embodiments, not limited to the particular imaging techniques used therein. Examples of microscope-based applications include, but are not limited to, immunofluorescence, confocal microscopy, two-photon microscopy, super-resolution microscopy, optical sheet microscopy, and x-ray microscopy. The term "detectable reagent" or "detectable label" refers to a molecule that can be used for the direct or indirect detection of a biomarker. A variety of detectable agents are known in the art and can be readily identified and used by those skilled in the art. Suitable detectable agents include, but are not limited to, fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), Oregon Green (trademark), rhodamine, Texas Red, tetrarhodamine isothiocyanate (TRITC), Cy3, Cy5, Alexa Fluor (registered trademark) 647, Alexa Fluor (registered trademark) 555, Alexa Fluor (registered trademark) 488), fluorescent protein markers (e.g., green fluorescent protein (GFP), phycoerythrin, etc.), enzymes (e.g., luciferase, horseradish peroxidase, alkaline phosphatase, etc.), nanoparticles, biotin, digoxigenin, metals, etc.

[0057] The term "immunofluorescent marker" refers to a detectable reagent that is an antibody or a functional fragment thereof that directs a fluorescent dye to a specific molecule within or on a cell. Immunofluorescent markers can be used in methods for generating immunostaining of a desired sample using a fluorescence microscope. Immunofluorescent markers can also be used in the immunocytochemistry (ICC) method or immunohistochemistry (IHC) method described herein. For example, in the context of the present disclosure, immunofluorescent markers can be used to detect rare circulating cells (e.g., CTCs or CTC mimics) as described herein.

[0058] The term "antibody" refers to any immunoglobulin or its derivatives, whether natural or produced wholly or partially synthetically. All antibody derivatives that maintain specific binding ability can also be used in the methods disclosed herein. The antibodies of the present disclosure can specifically bind to biomarkers. For example, an antibody can specifically bind to a single biomarker (e.g., chondroitin sulfate proteoglycan 4 (CSPG4)). Further, an antibody can be pan-specific. For example, the pan-specific antibodies of the present disclosure can specifically bind to one or more members of a biomarker family (e.g., one or more members of the chondroitin sulfate proteoglycan family including chondroitin sulfate proteoglycan 1, 2, 3, 4, 5, 6, 7, and 8). An antibody can have a binding domain that is homologous or mostly homologous to an immunoglobulin binding domain and can be derived from a natural source or produced partially or wholly synthetically. An antibody can be a monoclonal or polyclonal antibody. In some embodiments, the antibody is a single-chain antibody. In some embodiments, the antibody comprises a single-chain antibody fragment. In some embodiments, the antibody can be an antibody fragment including, but not limited to, Fab, Fab, F(ab)2, scFv, Fv, dsFv diabody, and Fd fragment. Due to their smaller size, antibody fragments can offer advantages over full antibodies in certain applications. Alternatively or additionally, an antibody can include, for example, multiple chains linked to each other by disulfide bonds, and any functional fragment obtained from such a molecule, such fragments retaining the specific binding properties of the parent antibody molecule. Those skilled in the art will understand that the antibody can be provided in any of various forms including, for example, humanized, partially humanized, chimeric, chimeric humanized, etc. Antibodies can be prepared using any suitable methods known in the art. For example, an antibody can be produced enzymatically or chemically by fragmentation of an intact antibody or recombinantly from a gene encoding a partial antibody sequence.

[0059] The term "biomarker" refers to a biological molecule or a fragment of a biological molecule, the changes and / or detection of which can correlate with specific physical conditions or states of rare circulating cells (e.g., CTCs, CTC mimics, or CECs) or other target components. The terms "marker" and "biomarker" are used interchangeably throughout the present disclosure. Such biomarkers include, but are not limited to, nucleotides, nucleic acids, nucleosides, amino acids, sugars, fatty acids, steroids, metabolites, peptides, polypeptides, proteins, carbohydrates, lipids, hormones, antibodies, regions of interest that serve as alternatives to biological macromolecules, and combinations thereof (e.g., glycoproteins, ribonucleoproteins, lipoproteins). The term also encompasses portions or fragments of biological molecules, e.g., peptide fragments of proteins or polypeptides. In the context of the present disclosure, exemplary biomarkers of CTCs, such as circulating melanoma cells (CMCs), include chondroitin sulfate proteoglycan 4 (CSPG4), premelanosome protein (Pmel17), and S100 calcium-binding protein A1 (S100A1).

[0060] Method In embodiments, the present disclosure provides for analyzing substances in a liquid sample that may contain biological or non-biological components. Accordingly, the method can be used to analyze liquid biopsies and other samples containing biological components. Biological components can include cellular material as well as extracellular material such as vesicles and cell-free DNA, secreted proteins, and other cell-free biomolecules.

[0061] In an embodiment, the method includes the step of solidifying a liquid sample, thereby capturing (immobilizing) a dispersed material in a form that can then be imaged by microscopy (or other imaging methods). The resulting scanned image enables the detection of components immobilized in three dimensions and spatially separated in a solid sample, enabling high resolution and high sensitivity. Subsequently, selectively labeled components, such as those labeled with fluorescently labeled antibodies, can be rapidly and sensitively identified by fluorescence microscopy or the like to detect the labeled target of interest.

[0062] Accordingly, the compositions and methods described herein effectively provide a 3D scan or image of a liquid biopsy. Rare biomarkers can include, but are not limited to, rare circulating cells, such as circulating tumor cells (and intact circulating tumor cell clusters), or cell-free nucleic acids, which can appear as discrete signals in the resulting scanned sample, for example. This is in contrast to other methods, such as microfluidics-based applications where such biomarkers are detected indirectly, or conventional cytological methods based on examining small samples of overlapping cells on a standard slide.

[0063] Embodiments of the present disclosure provide the additional advantage that the methods of the present invention do not impose any morphological or cell size cut-off on the components to be analyzed. For example, existing CTC detection techniques based on specific enrichment methods may inadvertently miss rare biomarkers. In addition, multiple processing steps underlying existing CTC detection methods can induce changes in cell biomarkers that can further reduce the sensitivity and accuracy of the detection method. In contrast, embodiments of the methods described herein do not require any specific selection criteria regarding the biological material to be analyzed. Instead, they enable the capture of complex arrays of cells and extracellular materials, regardless of shape and morphology, and enable capture, preservation, and spatial separation in a three-dimensional solidified sample.

[0064] In an exemplary embodiment, the method of the present disclosure enables the discrete separation and identification of individual cells in a solidified sample for spatial dispersion (separation) in the matrix of the solidified sample. Further, the method of the present disclosure enables the analysis of specific details of the identified cells themselves (e.g., morphological details of the cells) and the spatial relationship of the identified cells to other cells in the sample. For example, in an exemplary embodiment, the method of the present disclosure enables the analysis of clusters of cells, and one cell within the cluster can be made the specific cell of interest. The morphology of the specific cell of interest can also be compared, for example, to its unbound state versus the morphology of the cells in the cluster to confirm the clinical progression of the disease state in the subject. In other exemplary embodiments, cell fragments (e.g., cell fragments in white blood cells), or biomolecules secreted by cells, can be analyzed according to the methods disclosed herein.

[0065] Matrix-assisted method The present disclosure provides, in part, a matrix-assisted method for processing and analyzing liquid samples, such as biopsy samples, that may contain rare biomarkers such as circulating tumor cells or cell-free tumor DNA.

[0066] In an exemplary embodiment, the steps in the method are used to create an image of the dispersed components of the sample in a 3D spatial configuration. In an embodiment, the method includes solidifying a liquid sample containing biological material dispersed in a form that enables rapid imaging, such as by light sheet microscopy or other microscopy techniques. In an embodiment, the method includes (a) adding a solidifying agent to a liquid specimen containing biological material, (b) creating a solidified sample containing the biological material, and (c) imaging the solidified sample to identify one or more components in the dispersed biological material.

[0067] In an embodiment, the liquid sample contains biological material and undergoes a plurality of processing steps, which may include, but are not limited to, those shown in the steps shown in FIG. 1, which are described in detail below.

[0068] Step 1: Cell preparation In an embodiment, the method includes preparing or obtaining a liquid sample using a biological sample, where the biological sample can be dispersed and subsequently captured in solid form, thereby enabling high-resolution detection.

[0069] Although shown in FIG. 1 as a blood sample, the liquid sample can be derived from multiple sources, such as bone marrow, cerebrospinal fluid, urine, saliva, sputum, tears, semen, or other fluid sources. It should be further noted that the exemplary disclosure refers to CTC cells, but is equally applicable to other biomarkers that may be present in a liquid biopsy sample.

[0070] In an embodiment, the materials in the liquid sample are obtained from a processed blood sample such as obtained in a liquid biopsy. The processing can include one or more steps. For example, the processing can include red blood cell removal and PBMC cell isolation (collection). More specifically, the blood sample can be subjected to centrifugation to remove red blood cells (erythrocytes) and platelets. The processing may also include fractionating the blood sample into different components by well-known separation techniques such as density gradient centrifugation. For example, such separation techniques can include red blood cell (RBC) removal and peripheral blood mononuclear (PBMC) collection or isolation.

[0071] As shown in FIG. 1, in an exemplary embodiment, a blood sample can be provided in a tube or container containing an anticoagulant, such as a vacuum blood collection tube containing EDTA or heparin. The blood sample can be processed with a cell separation medium such as Lymphoprep™ or FicollPaque™, centrifuged, and the supernatant removed. Alternatively, a PBMC isolation tube can be used (e.g., SepMate™ tube available from Stemcell™ Technologies). The blood sample can, if desired, be subjected to red blood cell lysis according to known techniques such as the application of a commercially available or synthesized ammonium chloride solution (such as the ammonium chloride solution available from Stemcell™ Technologies), which can be performed before or after centrifugation. In other embodiments, red blood cell lysis is not used as a small amount of red blood cells does not affect the imaging and analysis of the sample.

[0072] In an exemplary embodiment, the pellet (e.g., a pellet containing isolated PBMCs) is obtained, for example, from centrifugation and further processed as described below. In one exemplary embodiment, the amount of the pellet can be at least 5 μl, or at least 10 μl, or at least 15 μl. In other exemplary embodiments, a smaller amount of the pellet is processed. In any case, the entire pellet is encapsulated in a gel, which has a size orders of magnitude larger than the typical throughput used in microfluidic processes known in the art.

[0073] In embodiments, the materials dispersed in the liquid sample can be derived from other tissue sources. For example, they can be derived from body fluids other than blood, such as bone marrow, cerebrospinal fluid, urine, saliva, sputum, tears, semen, or biopsies obtained from other body fluid sources. Alternatively, the materials dispersed in the liquid sample can reflect a liquid dispersion of materials from a solid biopsy, such as a tissue sample obtained from a tumor or other solid sample derived from a structure or organ of interest. In embodiments, the dispersed materials can be derived from non-tissue sources. In embodiments, the biological materials in the specimen can be concentrated by concentrating a large amount of the sample, which can be, for example, collecting and pelleting cells from a larger amount of blood, such as 2 ml, 4 ml, 8 ml, or more, or other sample sources.

[0074] In embodiments, the biological materials in the specimen can be concentrated by centrifuging and collecting cell pellets from a relatively large amount of sample, such as a large amount of blood (e.g., 0.5 cc or 1 cc or more) or other tissue sources.

[0075] Before being dispersed in the liquid sample, the materials can undergo additional processing steps. In embodiments, the biological materials can undergo a fixation step before being dispersed in the liquid sample, as further discussed herein. Alternatively, fixation may be performed after the biological materials are collected in the liquid sample. Fixation may also be performed after the cells are labeled. The specific fixatives that can be found for use according to the disclosure herein are not particularly limited and include those known to those skilled in the art. In an exemplary embodiment, the fixative is a solution containing glutaraldehyde, formaldehyde, epoxy, or one or more of the crosslinking products of one or more of the foregoing.

[0076] In the case of rare targets such as CTCs, a large amount of liquid biopsy samples can be continuously collected and collected in a single tube. For example, 2 ml or more of blood can be collected, processed, the cell pellets pooled, and resuspended in a liquid sample buffer, such as PBS.

[0077] In an embodiment, the biological material includes circulating tumor cells (CTCs), circulating tumor cell fragments, circulating tumor cell mimics, circulating epithelial cells (CECs), and similar rare circulating cells.

[0078] In an embodiment, the method of the present invention further includes labeling one or more targets of interest and adding a solidifying agent that enables capturing the dispersed labeling substance in a solid form including a three-dimensional cross-linked network structure. In other words, the solidifying agent provides a solid matrix for supporting 3D visualization of sample components.

[0079] The labeling can include any method known in the art for identifying biomolecules, including immunological and molecular means. For example, a protein, whether on the cell surface, intracellular, or extracellular, the target protein of interest can be labeled with an antibody (or related immunological reagent) that is detected directly, for example, with a fluorescent-conjugated antibody, or indirectly, for example, with immunohistochemistry or a primary antibody and a conjugated secondary antibody. The labeling (e.g., chemical and immunolabeling) can be performed in one step, or in an alternative embodiment, the labeling is a multi-step process.

[0080] For example, multiple antibodies from different host species can be introduced simultaneously, or almost simultaneously, or at different times. In an exemplary embodiment, the primary antibody can be conjugated (or pre-labeled) with a tag such as a fluorescent dye or an enzyme, e.g., a fluorophore or a corresponding secondary antibody, and introduced into the mixture in one step. In certain embodiments, the labeling step generally requires a post-wash, and thus additional centrifugation and supernatant removal steps can result in cell loss or cell damage, potentially reducing signal sensitivity, so one-step labeling is preferred over multi-step labeling. Similarly, biomolecular targets such as DNA or RNA can be labeled with nucleic acid probes that are detected directly or indirectly, such as for fluorescence in situ hybridization (FISH). Optionally, the signal can be further amplified by available techniques such as biotin-streptavidin binding and polymerase chain reaction-based systems. In embodiments, the probes can identify other disease biomarkers such as cell-free tumor or fetal-derived DNA, or can visualize genetic and structural changes in the cell nucleus such as DNA and chromosomal abnormalities, amplifications, deletions, and translocations.

[0081] The sample can also be labeled by other methods known in the art using various dyes targeting cell components, such as DAPI and PI (which bind to nuclear components) and DiD or DiL (which bind to membrane components).

[0082] The labeling may also include other steps such as cell permeabilization or fixation, as appropriate and known in the art, to enable efficient and specific binding of immunological or molecular reagents to the target (biomolecule) of interest. In certain embodiments, the labeling is applied prior to gelation and clearing of the sample. In certain embodiments, the labeling is preferably applied before obtaining a pellet from the sample or separating components, e.g., when the biological material is dispersed in a liquid (e.g., blood) sample.

[0083] Labeling can also be performed after fixing the cells in a gel state. For example, cells collected from a liquid sample (e.g., PBMC obtained from blood) can be introduced into a PFA solution, and a gel can be formed directly before labeling. The gel sample can then be labeled with a probe passively or by a method involving other active immunolabeling approaches, such as electrophoresis or pressure-based approaches. Thus, in embodiments, the processed gel sample can be immunolabeled after solidification (i.e., either labeled for the first time or further labeling is applied). This approach of labeling after gelation may require a longer processing time, but can also enhance the preservation of the number of target cells. In embodiments where labeling occurs after solidification, the fixing means can be performed on the solidified sample after gel formation and then labeled.

[0084] In other embodiments, defatting can be performed on the solidified (e.g., gelled) cell sample, where there is a need or desire to enhance the transparency of the sample for imaging. In embodiments, the solidified sample can be further cross-linked with a hydrogel precursor or epoxy and defatted according to the CLARITY approach. See, for example, “Advances in CLARITY-based tissue clearing and imaging,” Exp Ther. Med. 2008;16(3):1567-1576. However, it should be noted that according to most embodiments, the defatting step is generally not required due to the low layering of cells in the sample dispersed in the solidified sample. As will be described below, in some embodiments, even the refractive index matching step is not required.

[0085] Step 2: Solidification and transparency of the sample In certain embodiments, solidifying the sample involves introducing a solidifying agent (e.g., a gelling agent) that enables the biological material to be captured in a solid form such as a gel form, the solidifying agent being compatible with subsequent imaging and detection of the target labeled biomolecule. Examples of solidifying agents include, but are not limited to, reagents known in the art such as agarose (including low melting point agarose) solutions, polyacrylamide precursors, natural rubber, starch, pectin, agar, and gelatin. In embodiments, such reagents are polysaccharide- or protein-based. See, for example, Kar et al. 2019, Current developments in excipient science: in Fundamentals of Drug Delivery, 29-83. Alternatively, in certain embodiments, the solidifying agent can consist of, or essentially consist of, a refractive index matching solution modified to have an appropriate viscosity as needed to provide a rigid physical gel.

[0086] In embodiments, the solidifying agent is, or includes, a viscosity modifier such as one made from natural or synthetic polymers (e.g., xanthan gum, Pemulen™, Carbopol™, Velvesil™ plus, or other polyacrylic acid derivatives).

[0087] In embodiments, the solidifying agent includes a mixture of polysaccharides (e.g., agar / agarose, gellan gum) that results in a physical gel having a solid three-dimensional matrix or network structure.

[0088] In embodiments, the solidifying agent includes a mixture of a chemical monomer and a crosslinking agent that produces a synthetic chemical gel, i.e., having a chemically crosslinked polymer network.

[0089] Solidification (e.g., gelation) can, in an exemplary embodiment, include dispersing or resuspending the biological material in a gelling material in liquid form, such as a low melting point agarose solution added at a temperature above its gelation point, e.g., 37°C.

[0090] During the pre-gelation stage, according to this exemplary embodiment, the sample remains in a fluid or molten state, and the material can be maintained in a dispersed state until it is transferred to the imaging holder in the third stage. For example, a solidifying agent can be introduced into a labeled specimen (e.g., a labeled PBMC pellet as shown in FIG. 1), and the mixture can be mixed, for example, with a pipette or a vortex mixer. Thus, the gelling agent in certain embodiments is a reversible gelling agent in that, if desired, the gelling agent (and the sample) can be heated to a fluid or molten state.

[0091] In certain embodiments, pre-gelation includes dispersing or resuspending a biological material in a solution containing low melting point agarose. In embodiments, the final concentration of low melting point agarose is greater than 0.3% or greater than 0.5%. In embodiments, the final concentration of low melting point agarose is less than 1.0%, or less than 1.6%, or less than 2%, or less than 10%. In embodiments, the final concentration of low melting point agarose is 0.1% - 10%, or 0.3% - 1.6%, or 0.5% - 1.5% (e.g., about 1%).

[0092] Accordingly, in certain embodiments, pre-gelation includes dispersing or resuspending a biological material in a solution containing agarose (i.e., normal melting point agarose). In embodiments, the final concentration of agarose is greater than 0.3% or greater than 0.5%. In embodiments, the final concentration of agarose is less than 1.0%, or less than 1.6%, or less than 2%, or less than 10%. In embodiments, the final concentration of agarose is 0.1% - 10%, or 0.3% - 1.6%, or 0.5% - 1.5% (e.g., about 1%).

[0093] In certain exemplary embodiments, pre-gelation involves dispersing or resuspending biological material in a solution containing a mixture of low melting point agarose and normal agarose. In certain exemplary embodiments, the gelling agent solution (such as, but not limited to, an agarose or low melting point agarose solution) is combined with a sufficient amount of RI matching material, described below, to provide the desired refractive index of the final solidified gel. In certain exemplary embodiments, the pre-gelation material is dissolved directly in the RI matching solution to constitute the gelling solution.

[0094] In embodiments, by dispersing or resuspending a biological sample in a mixture and forming a gel, generally, the components in the sample are stabilized in a spatial distribution having sufficient properties such as transparency, enabling subsequent imaging. In certain embodiments, a refractive index matching material having a certain refractive index (for example, having a refractive index between 1.3 and 1.6 or between 1.33 and 1.5, or having an RI approximately equivalent to water) can be introduced into the gelling or pre-gelling mixture, while in other embodiments, RI matching materials having other refractive indices can be used. As is known in the art, refractive index matching materials (also called RI compatible or RIM) can penetrate into tissues / cells to achieve tissue / cell transparency, including, but not limited to, CUBIC-R+, RapidClear, RIMS, or ScaleView. See Neuropathol Appl Neurobiol, 2016 Oct;42(6):573-87.

[0095] In certain embodiments, a refractive index matching material is not required. For example, in small samples, the laser light can pass through the solidified sample and excite labeled cells or other target components. That is, the refractive index difference of the solidified sample is not a problem when the laser does not need to penetrate deeply enough to cause refraction of the light. For example, using a two-photon laser with greater power, the laser can penetrate deeper without being bent. Such a system is only limited by the potential photodamage to fluorophores, and this problem does not necessarily require the addition of a refractive index matching material, such as a refractive index matching solution, to avoid such damage.

[0096] In embodiments, pre-gelation involves dispersing or resuspending biological materials in a gelling material, such as a polymerizable material, monomer, or oligomer, that includes one or more hydrogel precursors, such as monomers selected from the group consisting of water-soluble groups containing polymerizable ethylenically unsaturated groups. Monomers or oligomers can include one or more substituted or unsubstituted methacrylates, acrylates, acrylamides, methacrylamides, vinyl alcohols, vinyl amines, allyl amines, and allyl alcohols. The precursors can also include polymerization initiators, crosslinking agents, and other components known in the art, such as those described in, for example, WO2019023214 and WO / 2020 / 013833.

[0097] In embodiments, the method of the invention includes transferring the pre-gelation mixture into a sample well within a holder while in a liquid or molten state. In embodiments, the wells within the holder enable the formation of a solid sample suitable for imaging. For example, the solid sample can be in the form of a block or other shape customized for imaging by fluorescence microscopy. Alternatively, in certain embodiments, the pre-gelation step can be performed in a tube-sample holder combination, thus enabling solidification (e.g., gelation) to occur in the same tube and eliminating the need to subsequently transfer the liquid solution to a separate sample holder.

[0098] In the case of an agarose mixture, solidification or gelation can be achieved by transferring the pre-gelled mixture to a temperature below the gelation point, for example, 4°C. In other embodiments, for example, for hydrogel formation, the transferred sample is subjected to further processing such as the addition of a polymerizing agent.

[0099] After solidification, the sample can be processed by additional steps prior to imaging. For example, the sample can be equilibrated in a refractive index material (e.g., made transparent) to be appropriately adapted for imaging in Step 3 (RI adaptation). Alternatively, in certain embodiments, the RI adaptation material is added simultaneously with the solidifying agent. In yet another embodiment, the RI adaptation material is added with the solidifying agent and then a second round of RI adaptation material is added to the solidified sample to provide the final desired transparency of the sample.

[0100] RI adaptation materials are known in the art and can be used in an amount suitable to provide the desired transparency of the solidified gel sample. In certain embodiments, the RI adaptation material has an RI of from about 1.39 to about 1.65, or from about 1.49 to about 1.55 (e.g., about 1.52). For example, but not limited to, the RI adaptation material can be as described in Table 3 of Neuropathology and Applied Neurobiology, November 2015, ”Bring CLARITY to the human brain: Visualization of Lewy pathology in three dimensions,” Liu et al., available at https: / / www.researchgate.net / figure / Comparison-of-different-refractive-index-matching-solutions- Abbreviations-BABB_tbl3_283493188>.

[0101] Step 3 - Mounting and Imaging of the Sample This step, in an exemplary embodiment, involves mounting the sample onto an image holder and imaging the solid sample by means of suitable imaging means such as fluorescence microscopy.

[0102] In an embodiment, the method of the present invention can include mounting the sample onto an image holder, such as a 3D printed sample holder as shown in FIG. 1, prior to imaging, and imaging the sample, for example, in a water chamber or an RI compatible solution. The sample holder can be customized according to a particular sample preparation. In an embodiment, it may comprise a base and two side walls. In an embodiment, a holder for use with a sample prepared in a viscous medium can have four walls that surround and preserve the spatial configuration of the dispersed components in the sample. In an embodiment, imaging can be performed in a sample cuvette using a standard epifluorescence microscope. The 3D gel sample can be mounted onto the sample holder with the aid of a mounting gel (e.g., agarose, poly-L-lysine, super glue). See, for example, Asano et al., Expansion Microscopy: Protocols for Imaging Proteins and RNA in Cells and Tissue, Current Protocols in cell biology (2018), particularly "Sample mounting" on pages 34 - 36.

[0103] The approach of fluorescence microscopy includes, but is not limited to, conventional confocal microscopy, resonant scanning confocal microscopy, spinning disk microscopy, and light sheet microscopy, as shown in FIG. 1. In certain embodiments, the gel sample is rapidly imaged by a light sheet microscopy method, such as light sheet fluorescence microscopy (LSFM) using a detection lens and an illumination lens, as shown in FIG. 1. In some embodiments, as shown in FIG. 1, a Gaussian beam can be used, or a special beam profile such as a non-diffracting Bessel beam can be used. Such imaging can scan substances in a 3D sample block and evaluate the individual presence of labeled biomarkers. As is known in the art, a processor can communicate with the microscope, receive outputs therefrom, and combine adjacent object regions that have been continuously imaged (i.e., "stitching"). See, for example, the techniques and devices disclosed in U.S. Pat. Nos. 10,746,981, 10,876,870 and U.S. Patent Application Publication Nos. 2016 / 0041099 and International Patent Application Publication No. 2017 / 031249. These can be used in accordance with the subject matter of the present disclosure.

[0104] Other detection light sources and corresponding microscope applications can also be used. For example, an optical microscope can be used to evaluate visible dye-stained cells, including cells stained by, for example, H&E (hematoxylin and eosin) or immunohistochemistry (IHC). Further, X-ray microscopy can be used to detect liquid components labeled with appropriate metal tags. Other imaging techniques can also be used, including images obtained by a high-resolution camera or related light detection system included in a commercially available smartphone (e.g., an iPhone® or Android®-based smartphone).

[0105] Step 4 - Visualization and Analysis The method of the present invention can also be used to evaluate, diagnose, or monitor a disease. For example, a liquid biopsy (e.g., whole blood processed as described above) can be microscopically analyzed to detect the presence of rare cells therein. For example, the methods of the present disclosure can detect, for example, colorectal adenocarcinoma cells, leukemia cells, types of cancer, the extent to which those cancers have developed, and whether the cancers respond to therapeutic intervention.

[0106] In embodiments, the methods disclosed herein can be used to detect cell biomarkers associated with other diseases and disorders such as inflammatory, metabolic, gastrointestinal, endocrine, immune, musculoskeletal, cardiovascular, cardiopulmonary, genitourinary, liver, respiratory, viral, and neurological diseases and disorders, according to embodiments of the present disclosure.

[0107] In addition to detecting the presence of biomarkers such as specific cells or extracellular components (e.g., circulating tumor-derived factors, secreted proteins, released vesicles and exosomes, and cell-free nucleic acids and other biomarkers), the methods disclosed herein can be used to confirm cell-cell interactions, cell volume, nucleus-cytoplasm ratio, and other phenomena. In embodiments, imaging can be used to analyze cell morphology and structure, which includes analysis of changes in cell morphology and structure compared to their native or healthy state. For example, imaging methods can be used to analyze the appearance of cells, e.g., their size, shape, or other external characteristics. Additionally, imaging methods can be used to analyze the morphology and structure of internal cell components such as the nucleus, endoplasmic reticulum, Golgi apparatus, mitochondria, or other organelles.

[0108] As another example, a biopsy can be prepared by liquid dispersion of a sample of diseased tissue from the kidney, heart, stomach, liver, pancreas, intestine, brain, etc., and can determine the state of the tissue, the extent to which the disease has developed, the likelihood of tissue success, etc. The methods herein can be used to evaluate morphological changes in cell populations that may indicate a disease state, for example, through the use of dyes that target membrane or cell components.

[0109] This method can also be used for other applications. In one application, liquid biological samples can be used to screen candidate therapeutic agents for their effects on tissues or diseases. For example, liquid samples obtained from a subject, such as a mouse, rat, dog, primate, human, etc., that have been contacted with a candidate agent can be prepared by the methods disclosed herein and microscopically analyzed for one or more cellular or tissue parameters, i.e., attributes or characteristics of intracellular components that can be measured.

[0110] In another application, the method of the present invention can also be used to visualize the distribution of genetically encoded markers in liquid samples prepared by dispersing materials from tissues. Such markers can include, for example, chromosomal abnormalities (inversions, duplications, translocations), loss of genetic heterozygosity, and the presence of genetic markers indicative of a predisposition to a disease state or a healthy state. Such detection can be useful, for example, in diagnosing and monitoring diseases in personalized medicine, paternity studies, or other applications.

[0111] CTC Detection In one exemplary embodiment, the methods disclosed herein are used to detect CTCs by immunological or molecular means for diagnosis and to address their clinical significance. CTCs are rare cells that circulate in the blood or other fluids along with millions of other circulating cells. The method of the present invention captures such rare cells in a solidified 3D form of a complex liquid sample or liquid biopsy. The sample can then be imaged, for example, by light sheet fluorescence microscopy, and any CTCs can be detected as individual signals within the sample block. Advantageously, the method of the present invention enables such CTC detection without reducing their biological heterogeneity. They can also involve less interference than that required for microfluidic or conventional cytological processing, which can disrupt the morphology or interfere with the sensitivity.

[0112] Examples The disclosure of this specification is further illustrated by the following non-limiting examples. These examples are to be understood as merely illustrative and are not to be construed as limiting the scope of one or more embodiments and the scope defined by the appended claims.

[0113] Example 1 - Matrix-Assisted Detection of CTCs in Patient Blood Samples Background CTCs are rare cells that circulate in blood or other fluids along with millions of other circulating cells belonging to, for example, the hematopoietic compartment and do not adhere spontaneously. These poor adhesion properties make it difficult the existing methods in the art for detecting CTCs, such as the use of solid supports for isolating and immobilizing CTCs. The methods disclosed herein enable the detection of complex 3D representations of liquid samples without limiting the biological heterogeneity of such rare cells when they are present and can reduce interventions that may disrupt the morphology. Furthermore, methods based on modified supports and matrices coated with anti-adhesion molecules (or other binding proteins) can induce biological responses that change the CTC morphology, resulting in inaccurate analysis. Immunoisolation relies on the strong affinity between the coated antibody and cell membrane proteins. Low expression of the target surface protein or low antibody affinity can result in a low capture rate of the target cells. Similarly, cytocentrifugation of cells onto a support such as a microscope slide, subsequent fixation, and subsequent labeling can disrupt the cells or their morphology and interfere with diagnostic evaluation. In contrast, the microscope slide approach requires coating the cells in a monolayer fashion to enable imaging, which significantly reduces the throughput or number of cells that can be imaged and analyzed.

[0114] Microfluidics, nanostructures, and channel-dependent detection methods can be subject to similar limitations. See, e.g., WO2012016136; WO2013049636. Such methods can induce flow stress on cell components in a liquid sample and may compromise their morphology and other characteristics. More generally, such methods typically select a homogeneous population of cells based on the size or expression of surface membrane proteins, which will limit the biological heterogeneity suitable for diagnostic analysis of clinical or biological samples.

[0115] Therefore, it is desirable to identify biopsy methods that can minimize long operations, interaction with external stimuli, and long-term processing. Such improvements can help improve both the natural state and heterogeneity of cells and other components in liquid biopsies, and thereby provide more accurate tools for diagnostic applications, such as early diagnosis of possible metastatic processes.

[0116] CTCs in peripheral blood can be regarded as an expansion of tumors. Tumors are typically heterogeneous, which means that through mutations, tumors can develop several different cell types. Each cell type can have its own characteristics, which can range from mild to progressive. CTCs are rare cancer cells released from tumors into the bloodstream and are thought to play an important role in cancer metastasis. See, e.g., Harouaka et al., 2014, Pharmacol. Ther. 141, 209-221.

[0117] U.S. Patent Application Publication No. 2019 / 0113423, which is incorporated herein by reference, describes methods in which molecular characteristics such as membrane proteins or DNA / RNA information in tumors can be indicators of treatment outcomes. This reference involves fixing or embedding a sample such as solid tissue or a solid cell pellet in a solution of hydrogel monomers, crosslinking the monomers, clearing the crosslinked sample, staining the cleared sample with one or more detectable markers, and imaging the stained sample using COLM or a similar imaging process.

[0118] In contrast, the disclosure herein provides, in certain embodiments, a method for analyzing individual cells (e.g., individual cells from a liquid biological sample) dispersed and captured in a three-dimensional solidified sample. Cell concentration or selection is not required, and all cells can be visually screened by labeling and imaging the sample.

[0119] In certain embodiments, without the general need for defatting (e.g., SDS-based defatting) or other sample clearing methods, certain embodiments of the methods disclosed herein are defined by not including such defatting or other sample clearing steps. See, for example, Jensen and Berg, 2017, J. Chem. Neuroanat. 86, 19-34. Thus, more cell information is preserved compared to methods including, for example, U.S. Patent Application Publication No. 2019 / 0113423 and methods involving CLARITY or other clearing protocols. According to the disclosure herein, each individual cell or cell cluster can be visualized and analyzed separately in a three-dimensional array, thereby providing comprehensive cell information such as cell size, morphology, biomarker distribution, and nuclear-to-cytoplasmic ratio.

[0120] Multiple types of tumors, such as lung cancer, liver cancer, and colon cancer, as well as other cancers that can be detected as CTCs in liquid biopsies, can be identified according to the disclosure herein. For example, when detecting CTCs from a liquid biopsy (e.g., a blood sample) using the EpCAM marker, the information obtained can indicate whether a patient has cancer (or other characteristics related to risk, prognosis, and treatment response) by providing information such as the number (or type) of cancer cells in 1 cc, 2 cc, 3 cc, 4 cc, or more of blood. EpCAM is an epithelial marker that indicates invasive cancer cells that have undergone epithelial-mesenchymal transition (EMT), which is a major cause of cancer invasion. Cancer invasion usually begins with EMT from a small population of tumor cells that stimulate angiogenesis, providing a route for cells to invade the bloodstream as CTCs.

[0121] There can be several different cancer cells derived from different tissues present in the bloodstream, and different biomarkers can be used to identify them. For example, but not limited to, multiple cancer markers such as HER2 (breast), CDX2 (colon), CK20 (colorectal, transitional cell carcinoma, and Merkel cell carcinoma), CK19 (breast), PD / PDL1 (several cancers including NSCLC, melanoma, and renal cell), and EGFR (lung) can be used to identify CTCs. Identification of different CTC cell subtypes in a blood sample can provide information about the origin of the cancer. This information can then lead to more detailed follow-up studies, such as high-resolution analysis by MRI, to identify the location of the lesion for pathological examination and tissue biopsy.

[0122] Cell heterogeneity in the blood can also apply to healthy cells such as white blood cells. According to the disclosure herein, the differences between CTCs and white blood cells can be known by applying parameters such as cell morphology (e.g., the nucleus of CTCs can be larger than that of white blood cells) and molecular markers (e.g., EpCAM CTC+ / WBC & CD45 CTC- / WBC+).

[0123] Furthermore, since CTCs escape from the primary tumor into the bloodstream, they are generally highly invasive. Such invasive cells can make it difficult to treat and cure cancer because of their ability to metastasize and their high potential for mutation, which can increase the opportunity for resistance to chemotherapy and other therapeutic interventions. This underscores the importance and value of identifying the molecular characteristics of such invasive cells in order to determine an appropriate treatment plan. For example, if CTCs present in the blood show high levels of PDL-1, immunotherapy is likely to be a more effective approach. PDL-1 levels can be determined, for example, using a PDL-1 antibody probe to quantify the number of PDL-1+ CTCs across all CTCs in the sample. Tumorigenesis is almost always heterogeneous, meaning that one tumor site does not represent all tumor populations and CTCs can be derived from all tumor sites, so this measurement can help, for example, to predict the effectiveness of PD-1 / PDL-1 inhibitory treatments (https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC6627043 / ). As provided by the methods of the disclosure herein, since CTCs are rare in the blood, such predictive measurements are most accurate when CTCs are efficiently captured and analyzed.

[0124] Another example according to the disclosure of this specification involves determining the CTC count and cell type, and administering treatment based on this determination (e.g., determining whether to continue the current treatment course or initiate a different or additional treatment course). In the case of cancer patients undergoing chemotherapy or cell therapy, the CTC count can indicate treatment efficacy. In stage III colon cancer patients, the CTC count in 1 cc of blood can range from tens of thousands in advanced cases to hundreds in less advanced cases. In some advanced stage III cases, after 3 months of chemotherapy or other therapeutic interventions, the CTC count may drop to 5000, and may even approach zero after 12 months. However, in some cases, the CTC count may only decrease to about 2000 after 6 months of chemotherapy and then rise back to tens of thousands 6 months later. This indicates drug resistance of cancer cells to therapeutic interventions. For example, chemotherapy may have killed all drug-sensitive CTCs, but other subtypes that were resistant would not be affected.

[0125] In accordance with the disclosure of this subject matter, by periodically (e.g., weekly or monthly) performing a blood test for CTC detection and identification, physicians and medical professionals can quickly verify tumor resistance to chemotherapy and accordingly modify the treatment plan. Thus, the tests here address the above limitations by describing formulations and methods for visualizing biopsy samples in three dimensions by capturing dispersed components in the sample in a solidified state. This approach has a number of advantages, such as increasing the sensitivity of the analysis by separating individual components, enabling the use of rapid imaging techniques such as light sheet fluorescence microscopy, and increasing the specificity of the analysis by reducing the number of processing steps that can interfere with the morphology and integrity of sample components such as cell markers.

[0126] Materials and Methods Sample Collection and Fixation: A blood sample (e.g., 2 cc, 8 cc, or 10 cc) is collected from a subject, and red blood cells are removed by density gradient centrifugation using standard methods, such as centrifugation at 1000 rpm for 5 minutes at room temperature. See, for example, Farahinia et al. 2020, Circulating Tumor Cell Separation of Blood Cells and Sorting in novel Microfluidic approach: a review. 10.20944 / preprints202010.0622.v1; and Lowes et al., 2014, Circulating tumor cells as a 25 real-time liquid biopsy: isolation and detection systems, molecular characterization, and clinical applications; in Pathobiology of human disease: a dynamic encyclopedia of disease mechanisms (eds, McManus and Mitchell).

[0127] The supernatant is removed, transferred to another tube, and centrifuged again to collect the remaining cells, including CTCs and other biological components. The pellet is optionally resuspended in a fixative solution, such as a 4% paraformaldehyde solution, shaken for several minutes, centrifuged, washed in phosphate-buffered saline (PBS), and centrifuged again. The pellet is resuspended in blocking buffer, shaken for several minutes, and centrifuged again.

[0128] Biomarker labeling: The fixed and washed pellet is resuspended in blocking and permeabilization buffer, placed on a shaker for several minutes, centrifuged, and resuspended in a pre-mixed labeling solution for 30 - 60 minutes. Depending on the binding affinity of the antibody, which can directly affect the final imaging quality, the pellet can be resuspended and incubated in the labeling mixture for a longer time (e.g., up to 10 hours or 20 hours) if necessary.

[0129] The target protein biomarker can be labeled directly, e.g., with a fluorescent-conjugated antibody, or indirectly, e.g., with an antibody (or related fragment or derivative) detected by immunohistochemistry or a primary antibody and a conjugated secondary antibody. Similarly, the nucleic acid of interest can be labeled with a molecular probe detected directly or indirectly. Optionally, the signal can be further amplified by available techniques such as biotin-streptavidin binding and polymerase chain reaction-based systems. Optionally, the sample can be centrifuged and the pellet can be washed one or more times.

[0130] In this example, 200 μL of a labeling solution of PBST blocking buffer is mixed with propidium iodide (PI) (1:2000) for nuclear staining, an EpCAM antibody (1:250 at an antibody concentration exceeding 0.1 mg / ml) for cancer cell detection, a CD45 antibody (1:250 antibody concentration) for immune cell detection, and a secondary antibody compatible with the primary antibody host (a two-fold amount relative to the primary antibody). Fab from Jackson ImmunoResearch Laboratories, Inc. (https: / / www.jacksonimmuno.com / catalog / 31) was used.

[0131] Alternatively, sequential labeling can be performed as follows: starting with incubation with the primary antibody for 60 minutes or more and washing, then the secondary antibody and washing, then staining with PI in PBS solution for 5 minutes, and then washing. For other targets, surface proteins can be labeled with an antibody or DNA / RNA targets can be labeled with a probe. Consecutive labeling with a secondary antibody or a direct conjugate of a fluorescent molecule can also be used. Biotinylated antibodies can also be used with boosted signals, e.g., based on the binding affinity for streptavidin, as known in the art. After addition of the labeled antibody or any probe or chemical dye, optionally, the pellet can be resuspended in 4% PFA at room temperature for 10 minutes to fix all labeling substances on the cells and prevent them from being washed away during the fixation process.

[0132] Solidification: Following the labeling reaction, the sample is optionally washed one or more times and then resuspended in a solution containing a solidifying agent (e.g., a gelling agent). The solidifying agent can be, for example, a low melting point agarose (LMP) solution capable of solidifying (e.g., gelling), or a hydrogel precursor that can be polymerized by adding a sufficient amount of a cross-linking agent and an ion-containing component (e.g., Ca2 + ions-containing component) to induce gelation. For example, the amount of the cross-linking agent introduced can be adjusted according to whether a gel-type solid is desired or increased to provide a higher density of solidified sample.

[0133] In an embodiment, the solution containing the solidifying agent contains liquid agarose, e.g., 1.2% or 2.4% or 10%, in an RI-compatible solution to facilitate subsequent imaging. Alternatively, the solidifying agent (e.g., gelling agent) is present in the solution with a solvent that cross-links and / or solidifies (e.g., gels) at a lower temperature and / or a higher ion concentration, or melts at a higher temperature and / or a lower ion concentration.

[0134] In this example, the gel solution containing the dispersed material is transferred to a desired imaging holder, such as a holder containing sample wells having a block shape (or other desired shape), at 25 - 37 °C, and the holder can be shaken or vibrated to ensure dispersion of the material in each well. The holder containing the sample solution is transferred to a lower temperature so that gelation occurs in the wells. For example, incubate the sample dispersed in LMP agarose at 4 °C for about 15 - 30 minutes.

[0135] It should be noted that the initial liquid blood sample has millions of non-cancerous cells that dilute and obscure the CTCs present at very low levels. Gelation enables the cells to be dispersed and separated within a 3D space for imaging (described below). One important point is that millions of cells are condensed from a large volume (e.g., in 10 cc of blood) into a 20 μl gel block (2.71 mm 3 ), and then it becomes possible to be imaged within an acceptable imaging period. In alternative embodiments, the volume can be further reduced to enable rapid high-resolution imaging (e.g., up to 20x or 40x). In certain embodiments, the cell pellet is reduced to give the smallest possible volume. In such situations where a relatively small volume is used, due to the high density of lipid bilayers (cell membranes), attention may be required for gel clearing due to the proximity distance between cells, but it can be solved, for example, using an optimized RI matching solution or a higher power laser such as a two-photon microscope.

[0136] Imaging: Once the gel block (with the dispersed contents) is formed, the gel block is cleared by immersion in a refractive index (RI) matching solution for about 30 minutes and becomes transparent for imaging. The RI-matched gel sample can then be imaged to detect the presence of many biomarkers that are not discretely separated individually within the gel block.

[0137] Such imaging can be performed by microscopy methods including efficient fluorescence imaging methods such as light sheet microscopy. In the case of fluorescently labeled biomarkers, for example, samples can be rapidly imaged by light sheet fluorescence microscopy (LSFM) using a non-diffracting Bessel beam. For example, by using a light sheet microscope, it becomes possible to image hundreds or thousands of cells from a single field of view (FOV). For example, by setting the imaging z-step to 1 micron, each cell with an average cell diameter of 15 microns is imaged approximately 10 - 15 times from top to bottom. Different from indirect gating on summary plots by flow cytometry, it is clear that LFSM data obtained from the imaged samples can visually inspect the validity, intensity, distribution, etc. of antibody labeling for individual cells.

[0138] The application of the method herein can rapidly detect rare biomarkers such as CTCs in a solid array and enable spatial discrimination of labeled biomarkers. By reducing the need for processing steps that can reduce sample heterogeneity or change their properties, the method of the present invention also provides a more sensitive and accurate diagnostic approach.

[0139] Furthermore, the method described herein allows PBMCs to be fixed immediately after RBC removal, enabling capture of their distribution and association in their original state. Advantageously, this enables direct visualization of sample characteristics such as cell-cell interactions, cell clusters (e.g., circulating tumor microemboli (CTM)), and other characteristics that may indicate disease states or provide other prognostic or diagnostic uses.

[0140] Example 2 - 3D Matrix-Based Imaging of Blood Samples Cell Preparation: Anticoagulated blood collected from humans in an EDTA-coated tube was transferred to a 15-ml test tube, and a cell separation solution (e.g., Ficoll (registered trademark)) was added. Density gradient centrifugation (1200 RPM) was performed on the mixture for 5 minutes. After centrifugation, the buffy coat (the fraction containing white blood cells and platelets) was transferred to a 2-ml test tube without collecting the separation medium. The resulting mixture was centrifuged again at 500 g for 5 minutes to form a cell pellet at the bottom of the tube. The supernatant was carefully removed without disturbing the cell pellet.

[0141] Labeling: Next, the pellet was resuspended in 200 μl of a labeling solution (1:1000 PI in PBS - 4% PFA) to label the cell nuclei. The labeling solution was added and incubated at 4°C for 20 minutes. Then, the mixture was centrifuged at 500 g for 5 minutes, and the supernatant was carefully collected and discarded. Next, 1 ml of a 4% PFA solution in PBS was added to fix the chemical dye at 4°C for 20 minutes, and the mixture was centrifuged again at 500 g for 5 minutes, and the supernatant was discarded again.

[0142] Solidification: On the other hand, a solidification solution mixture containing about 0.5 wt% of LM agarose was prepared. The solidification solution was heated in a microwave until it melted beyond its gelling point and then cooled to room temperature, where it was provided in a liquid state until it reached the gelling point again. Using a pipette, 20 μl of the cooled solidification solution was slowly added to the pellet to resuspend the pellet. The solidification solution was added slowly to avoid the formation of air bubbles. The mixture was brought to 4°C and maintained for a period (less than about 30 minutes) that allowed gel formation.

[0143] Mounting and RI adaptation: Once the sample had gelled, the gel was carefully removed from the tube with a pipette by packing the sides of the gel, and a 20 μl volume of gel was obtained. The gel was then mounted at room temperature on a sample holder (in this case, a 3D gel rod with a diameter of 2 mm, pre-made in a holding shape mounted on a 3D printed plastic surface, with the same composition as the solidification solution) and cooled at 4 °C for 10 minutes to gel. The sample gel on the holder was immersed in the RI compatible solution for 5 - 30 minutes to prepare for imaging.

[0144] Imaging: The sample was transferred to a light sheet imaging chamber and imaged (10X objective lens). For a 20 μl gel sample, it took approximately 3 minutes to image a single channel at a resolution of 10X and a z-step of 4 μm, although the imaging time can be decreased by decreasing the gel volume (increasing the cell density), decreasing the imaging resolution (for lower magnification purposes or higher z-step), or optimizing microscope settings such as the z-step, and by other means known to those skilled in the art.

[0145] Figures 2A - 2C show images obtained from the light sheet imaging chamber.

[0146] The nuclei of the stained white blood cells are shown in cyan. In Figure 2A, the image represents the entire 20 μl gel sample viewed from above. Figure 2A is a reduced image of a 100 μm stack of 25×4 μm images. In Figure 2B, the image represents the entire 20 μl gel sample from a side view. In Figure 2C, the image is magnified to capture the individual white blood cells in more detail, and the small horizontal line in the lower left corner is equal to a scale of 20 μm.

[0147] Example 3 - Cell Compatibility Cells from the CACO2 cell line obtained from ATCC and, similarly, cells from the HL60 cell line obtained from ATCC were recovered and processed as described below. The CACO2 cells were cultured and recovered by trypsinization. HL60 is a suspension cell line and does not require trypsinization. The CACO2 and HL60 cells were each recovered, washed with PBS, and centrifuged to recover the cell pellet.

[0148] Immunolabeling - Cancer cell line CACO2 The CACO2 cells were stained with trypan blue and counted using a cell counter. An appropriate number of cells were resuspended in 100 μl of PBS. The EpCAM (Dako) antibody was added to the cell solution at a ratio of 1:100, and the secondary antibody was added at a molar ratio of primary antibody to secondary antibody of 1:2.

[0149] The mixture was shaken at room temperature for 30 minutes. The mixture was centrifuged at 500 g for 5 minutes, and the supernatant was carefully removed. The cell pellet was resuspended and washed in PBS, centrifuged again at 500 g for 5 minutes, and the supernatant was discarded. The cells were then resuspended and incubated in 1 ml of PI solution:4% PFA (1:1000) at 4 °C for 20 minutes to fix the bound antibody and label the cell nuclei. The mixture was centrifuged again at 500 g for 5 minutes, and the supernatant was discarded.

[0150] Immunolabeling - Leukocyte cell line HL60 The HL60 cells were stained with trypan blue and counted using a cell counter. An appropriate number of cells were collected in 100 μl of PBS. The CD45 (Dako) antibody was added to the cell solution at a ratio of 1:100, and the secondary antibody was added at a molar ratio of primary antibody to secondary antibody of 1:2.

[0151] The mixture was shaken at room temperature for 30 minutes. The mixture was centrifuged at 500 g for 5 minutes, and the supernatant was carefully removed. 1 ml of PBS was added for washing, and the mixture was centrifuged again at 500 g for 5 minutes. The supernatant was carefully collected again and discarded. Then, the cells were resuspended and incubated in 1 ml of PI solution: 4% PFA (1:1000) at 4 °C for 20 minutes to fix the conjugated antibody and label the cell nuclei at 4 °C for 20 minutes. The mixture was centrifuged again at 500 g for 5 minutes, and the supernatant was carefully collected again and discarded.

[0152] Solidification: On the other hand, a solidification solution containing about 1 wt% of LM agarose was prepared. The solidification solution was heated with a microwave to melt it and then cooled to room temperature. There, it was finally provided in a gel state. Using a pipette, 20 μl of the cooled solidification solution was slowly added to the pellet to resuspend the pellet. The solidification solution was added slowly to avoid bubble formation. The mixture was brought to 4 °C and maintained for a period (less than about 20 minutes) to allow gel formation.

[0153] Imaging: Both gels with immunolabeled CACO2 and HL60 cells were imaged with a light sheet microscope equipped with a 10x objective lens as described above.

[0154] Figure 3A shows CD45 / PI-stained HL60 cells captured by light sheet microscopy. Figure 3A is a digitally enlarged view as indicated by the scale bar in the lower left corner. The white bar corresponds to 50 μm. Yellow indicates PI staining that labels the nucleus, and green indicates CD45 immunolabeling. Figures 3B - 3C show EpCAM / PI-stained CACO2 cells captured by light sheet microscopy. The images were taken with a 10x objective lens. Figure 3B shows the entire gel volume. Figure 3C is a digitally enlarged view as indicated by the scale bar. That is, in Figure 3B, the scale bar represents 300 μm, and in Figure 3C, the scale bar represents 50 μm.

[0155] Example 4 - Multiplex Labeling of CACO2 Cells in Blood Samples Spiking of cancer cells in blood samples Cancer cell count A study was conducted to simulate CTC detection in cancer patients. The study was designed to evaluate the rare cell detection efficiency of the 3D liquid biopsy method and the cell loss rate during the cell preparation process.

[0156] Cell preparation: Samples of CACO2 cells were trypsinized, counted, and seeded in freshly collected peripheral blood samples in appropriate amounts to prepare blood samples with added cancer cells. Peripheral blood mononuclear cells (PBMCs) were collected through a red blood cell lysis approach. Anticoagulated blood collected from humans in EDTA-coated tubes was transferred to 15 ml tubes. 8 c.c. of ammonium chloride solution was added to 1 c.c. of blood to lyse red blood cells. The lysis process was carried out on ice for 15 minutes, centrifuged, the supernatant was removed, and the collected PBMC cell pellet containing the added cancer cells was resuspended in 100 μL of PBS and prepared for the next process.

[0157] EpCAM (Dako) and CD45 (Dako) antibodies were added to the cell solution at 1:100, and the appropriate secondary antibody was added at a molar ratio of primary antibody to secondary antibody of 1:2 to perform the labeling process in Step 1. The cell samples were incubated at room temperature for 1 hour to bind the antibodies. Next, the samples were centrifuged at 500 g for 5 minutes to recover the pellet. The pellet was washed with 1 ml of PBS, centrifuged again, and the supernatant was removed. Next, PI:4% PFA (1:1000) was added to the pellet to fix the labeled antibodies and stain the nuclei for 30 minutes. Next, the cells were centrifuged again to recover the pellet.

[0158] The cell pellet was mixed in the solidification solution as described above. Next, the mixed PBMC and CACO2 cells were identified in the sample, and the ability to detect multiple cell types using multiple antibody target labels in the sample was confirmed. Figure 4A shows the full 3D gel data of the multiplex labeling of CACO2 in PBMC, where the EpCAM marker is shown in magenta and PI is shown in blue. Figure 4B shows an enlarged stack image of CACO2-EpCAM (magenta), leukocyte-CD45 (green), and PI (blue). Figures 4C and 4D show images of the identified CTCs with and without EpCAM (magenta signal) for confirmation.

[0159] In these stacked or separated figures, in the 3D image, the EpCAM label for cancer cell detection is a feasible approach. CACO2 shows high EpCAM expression but no CD45 expression, and PBMC (leukocytes) show variable CD45 expression but no EpCAM expression.

[0160] Cell count: Trypsinized CACO2 was counted, seeded into 2 cc of blood, stained, labeled as described in the following table, and three sets of gels (a, b, and c) were prepared.

[0161]

Table 1

[0162] As described in Examples 1, 2, and 3, a three-dimensional gel was formed and imaged by light sheet microscopy. Each image was denoised and features were enhanced for all immunolabeled signals for cell detection. Cells with positive EpCAM and PI signals and negative CD45 were counted as positive, cells with negative EpCAM signals and positive PI signals were counted as negative, and signals with negative PI signals were counted as null. Other parameters such as cell or nuclear circularity, cell / nuclear volume, and nuclear cytoplasmic ratio (N:C ratio) can be quantified for positive cancer cells using MATLAB-based or Python-based cell detection algorithms.

[0163] Figure 4A shows the output from 3D images of three replicate sets of gel samples with 2000 added CACO2 cells.

[0164] To better visualize the detected results, the 3D imaging data was shown in a maximum projection manner where all 2D images were stacked on top of each other to generate single-plane imaging data. EpCAM (color)-labeled CACO2 cells can be easily spotted visually by digitally zooming in on the dataset. EpCAM-positive cells are screened for negative CD45 signals and positive PI signals. The cell detection algorithm screens all cells using these parameters and outputs the identified CTC images using the separated and combined signals as shown in Figure 4B. These images are stored in a single file for each sample, and all detected cell images are further confirmed by the human eye or a signal detection algorithm.

[0165] The following table shows the final cell counts from three sets of gels, a, b, and c. The detected results were within a reasonable approximation of the seeded (added) cell numbers.

[0166]

Table 2

[0167] The total number of cells in the whole gel can be quantified using the total PI-positive signals. Dividing the total number of positive cancer cells by the total number of PI signals gives a ratio. Multiplying this ratio by one million yields the number of cancer cells per one million PBMCs.

[0168] Example 5 - Detection of CTCs in Cancer Patients An IRB review was conducted to detect the presence of CTCs in the blood of colorectal cancer patients. This review was designed to evaluate the rare cell detection efficiency of the 3D liquid biopsy method. The enrolled patients included healthy subjects and disease subjects. The following examples show patients with stage II - stage IV colorectal cancer with liver or lung metastases.

[0169] Preparation of blood samples: Anticoagulated blood collected from humans in EDTA-coated tubes was transferred to 15 ml tubes. Peripheral blood mononuclear cells (PBMCs) were collected through a red blood cell lysis approach. For 2 c.c. of blood, 16 c.c. of ammonium chloride solution was added to lyse the red blood cells. The lysis process was carried out on ice for 15 minutes, followed by centrifugation and removal of the supernatant. The collected PBMC cell pellet containing the added cancer cells was resuspended in 100 μL of PBS and prepared for the next process.

[0170] Patient A EpCAM (Dako) and CD45 (Dako) antibodies were added to the cell solution at a ratio of 1:100, and the appropriate secondary antibody was added at a molar ratio of 1:2 of primary antibody to secondary antibody to perform the step 1 labeling process. The cell sample was incubated at room temperature for 1 hour to bind the antibodies. Next, the sample was centrifuged at 500 g for 5 minutes to recover the pellet. The pellet was washed with 1 ml of PBS, centrifuged again, and the supernatant was removed. Next, 1:1000 PI:4% PFA was added to the pellet to fix the labeled antibodies and stain the nuclei for 30 minutes. Next, the cells were centrifuged again to recover the pellet.

[0171] The cell pellet was mixed in a solidification solution (1% LM agarose) and imaged as described above. Figure 5A shows 3D gel data (single stack block) with a resolution of 0.42 μm × 0.42 μm × 1 μm, where the EpCAM marker is shown in magenta, the CD45 marker is shown in green, and PI is shown in blue. Figure 5B shows an enlarged image of EpCAM-positive, CD45-negative, and PI-positive cell targets. When screening for CTC positivity using the above parameters, 195 CTCs were detected in approximately 160,000 PBMCs. This corresponds to 1,218 CTCs per 5 million PBMCs.

[0172] Patient B CK20 (Ventana) and EpCAM (Dako) antibodies were added to the cell solution at 1:100, and a compatible secondary antibody was added at a molar ratio of primary antibody to secondary antibody of 1:2 to perform the step 1 labeling process. Collection, gelation, and imaging of the labeled cell pellet were performed as described above for Patient A. Figure 6A shows two consecutive layers of 3D gel data (600 μm stack from a 1200 μm stack) with a resolution of 0.42 μm × 0.42 μm × 1 μm, where the CK20 marker is shown in yellow, the EpCAM marker is shown in magenta, and PI is shown in blue. Figure 6B shows a zoomed-in single cell image of CK20-positive, EpCAM-positive, and PI-positive cell targets. When screening for positive CTCs using the above parameters, 12 CTCs were detected in approximately 46,200 PBMCs. This corresponds to 259 CTCs per 1 million PBMCs.

[0173] In contrast to other methods of viewing cells in complex samples such as tissue samples, the method of the present invention does not require a clearing step (a clearing step) that removes lipids or destroys other cell information in order to achieve tissue transparency for 3D imaging. Instead, the method herein achieves complete cell sorting and 3D visualization of complete cell information in a liquid sample by combining gelation of a cell sample dispersed in three-dimensional space with refractive index matching. It is a novel approach for screening rare cells or any cells in a liquid biopsy sample using simple antibody tagging.

[0174] It will be understood by those skilled in the art that the examples and embodiments described herein do not limit the scope of the present invention. It will be apparent to those skilled in the art that this specification, including the examples, is intended merely as an illustration and that various modifications and changes can be made in the present invention without departing from the scope or purpose of the invention as defined by the appended claims. Furthermore, while specific details in the disclosure herein are provided to give a sufficient understanding of the present invention as defined by the appended claims, it will be apparent to those skilled in the art that certain embodiments can be practiced without these details.

[0175] Furthermore, in certain instances, well-known methods, procedures, or other specific details are not described in order to avoid unnecessarily obscuring aspects of the invention as defined by the appended claims.

[0176] Numbered embodiments The present disclosure further targets the following embodiments. 1. (a) A method for analyzing a biological sample, comprising the steps of: adding a solidifying agent to a specimen containing biological material; (b) creating a solidified sample containing the dispersed biological material; and (c) imaging the solidified sample to identify one or more components in the dispersed biological material. 2. The method according to embodiment 1, wherein the specimen is obtained from a liquid blood sample. 3. The method according to embodiment 2, wherein the sample is obtained from a liquid blood sample by a process comprising removing red blood cells and platelets from the liquid blood sample. 4. The method according to any one of the preceding embodiments, further comprising labeling a biomolecular target in the sample before adding the solidifying agent. 5. The method according to embodiment 4, wherein the biomolecular target is a nucleic acid, a protein, or a vesicle. 6. The method according to embodiment 4 or 5, wherein the labeling comprises contacting the sample with a molecular probe. 7. The method according to embodiment 6, wherein the molecular probe is an antibody. 8. The method according to embodiment 6, wherein the molecular probe is a fluorescent dye. 9. The method according to embodiment 6, wherein the molecular probe is a nucleic acid probe. 10. The method according to any one of embodiments 1 to 9, wherein the biomolecular target is an extracellular molecular target. 11. The method according to any one of embodiments 1 to 9, wherein the biomolecular target is an intracellular molecular target. 12. The method according to any one of the preceding embodiments, further comprising transferring the sample to a sample holder after adding the solidifying agent. 13. The method according to embodiment 12, further comprising shaking the sample in the sample holder. 14. The method according to embodiment 12, further comprising vibrating the sample in the sample holder. 15. The method according to any one of the preceding embodiments, wherein the solidified sample is a solid block suitable for imaging. 16. The method according to any one of the preceding embodiments, wherein before imaging, the solidified sample containing the dispersed biological material is transferred to a solution to ensure the desired transparency of the sample for imaging. 17. The method according to embodiment 16, wherein the solution is a refractive index matching solution. 18. The method according to any one of the preceding embodiments, wherein step (c) further comprises detecting one or more cancer cells or cancer markers in the solidified sample. 19. The method according to any one of the preceding embodiments, wherein the imaging is performed with a fluorescence microscope. 20. The method according to embodiment 19, wherein the imaging is performed by light sheet fluorescence microscopy. 21. The method according to any one of the preceding embodiments, wherein the curing agent is a gelling agent and the cured sample is a gel sample. 22. The method according to any one of the preceding embodiments, wherein, before step (a), the biological specimen is subjected to a fixation procedure. 23. The method according to embodiment 22, wherein the fixation procedure includes incubating the biological specimen in a fixative. 24. The method according to embodiment 23, wherein the fixative contains glutaraldehyde or formaldehyde. 25. The method according to any one of the preceding embodiments, wherein step (c) enables the identification of single cells in the cured sample.

Claims

1. A matrix-assisted method for analyzing a liquid sample containing biological material for one or more target components, comprising: (a) adding a solidifying agent to a specimen obtained from the liquid sample containing the biological material; (b) creating a solidified sample containing the dispersed biological material for capturing and immobilizing the dispersed biological material in a three-dimensional state; (c) introducing a refractive index matching material into the solidified sample; and (d) imaging the solidified sample to identify one or more target components in the dispersed biological material. The method comprises: The solidifying agent comprises a mixture of a chemical monomer and a cross-linking agent and forms a synthetic chemical gel having a chemically cross-linked polymer network.

2. Furthermore, (i) before adding the solidifying agent, labeling the specimen obtained from the liquid sample in step (a) with one or more probes for the one or more target components; and / or (ii) labeling the solidified sample from step (b) with one or more probes for the one or more target components. The method according to claim 1, further comprising the steps of:

3. The method according to claim 2, wherein the labeling step comprises contacting the specimen obtained from the liquid sample in step (a) with a molecular probe and / or contacting the solidified sample obtained from step (b) with a molecular probe, and the molecular probe is an antibody, a fluorescent dye, or a nucleic acid probe.

4. The method according to claim 1, wherein the one or more target components include a cell target or an intracellular target.

5. The method according to claim 1, further comprising introducing the optically transparent gelled solid sample obtained from step (c) into a sample holder immersed in a solution containing a refractive index matching material.

6. The method according to claim 1, wherein the liquid sample is a liquid blood sample.

7. The specimen is obtained from the liquid blood sample by a process comprising removing red blood cells and platelets from the liquid blood sample and comprises peripheral blood mononuclear cell (PBMC) cells isolated from the liquid blood sample. The method according to claim 6. **Claim 8** (i) labeling a sample containing isolated peripheral blood mononuclear cells (PBMCs) obtained from a liquid blood sample with one or more probes for one or more rare circulating cells before adding a solidifying agent and / or labeling the matrix-supported sample obtained from step (b) with one or more probes for one or more rare circulating cells, and (ii) imaging the optically cleared solidified sample obtained from step (c) to determine the presence of one or more probes and thereby determine the presence of rare circulating cells in the liquid blood sample. The method according to claim 7, further comprising the above steps. **Claim 9** The method according to claim 8, wherein the labeling further comprises adding a probe for white blood cells. **Claim 10** The method according to claim 8 or 9, wherein the one or more probes recognize a cancer-specific antigen or a tumor-specific DNA sequence or a tumor-specific RNA sequence. **Claim 11** The method according to claim 8 or 9, wherein the one or more probes are selected from antibody or nucleic acid probes. **Claim 12** The method according to claim 11, wherein the one or more probes provide detection of one or more of EpCAM, HER2, CDX2, CK20, CK19, PD / PDL-1 and EGFR antigens or corresponding nucleic acid sequences. **Claim 13** The method according to claim 8 or 9, wherein the one or more rare circulating cells include circulating tumor cells. **Claim 14** The method according to claim 8 or 9, wherein the one or more rare circulating cells include circulating epithelial cells or circulating endothelial cells. **Claim 15** The method according to any one of claims 1 to 9, wherein before step (a), the biological sample is subjected to a fixation treatment, and the fixation treatment includes incubating the biological sample in a fixing solution. **Claim 16** The method according to any one of claims 1 to 9, wherein imaging is performed by fluorescence microscopy. **Claim 17** The method according to claim 16, wherein imaging is performed by light-sheet fluorescence microscopy (LSFM). **Claim 18** The method according to claim 1, further comprising a step of performing a degreasing treatment on the solidified sample.

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