JP2024000599A - Method for preparing and analyzing biopsy sample and biological sample - Google Patents

Abstract

PROBLEM TO BE SOLVED: 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

COPYRIGHT: (C)2024,JPO&INPIT

Description

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

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.

Biomarkers in liquid biopsies can include cellular and extracellular components, and their selection can depend on multiple factors, such as the medical or treatment condition. For example, biomarkers are antigens 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. or can correspond to other attributes. 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), which is applied in cancer monitoring, as well as cell-free fetal DNA (cffDNA), which is found in maternal blood and is applied in non-invasive prenatal testing. 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.

A typical biopsy method can include several approaches. For example, Harouaka et al. , 2014, Pharmacol. Ther. See 141, 209-221. One common approach is based on immunoaffinity, such as microfluidics and microchip-based methods, which allow detection of antibodies bound to cellular and extracellular targets. These methods rely on antibody-antigen binding between floating cells and antibody-coated surfaces and are therefore limited to antigens present on the target cell surface, such as membrane proteins. In addition, the effectiveness of these methods depends on sufficient levels of cell surface antigen to allow efficient and specific recognition by antibodies (e.g., low EpCAM expression on the cell surface (can provide sufficient cell-to-chip surface binding). These methods generally have low cell flow rates that preclude effective analysis of complex samples. Therefore, 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.

Another common approach is cytology, which examines cells directly under a microscope. However, this approach is limited to examining only a small number of cells at a time (e.g., as a monolayer on a glass slide) due to the light scattering nature of the cells, and therefore using this approach, 2 cc. Detecting rare targets in the millions of cells that may be present even in blood is not practical or economically feasible.

In another approach, flow cytometry, thousands of cells per second are passed one by one through one or more laser beams, where they are detected with different light scattering (depending on cell size and granularity, for example). Patterns and (depending on which fluorescent probes are bound to the cells) fluorescent emissions can result. See, eg, Flow cytometry: retrospective, fundamentals and recent instrumentation, Cytotechnology, 2012 Mar; 64(2): 109-130. However, flow cytometry methods do not provide high resolution and reliability if the cells of interest are rare. For example, they do not provide direct visual inspection of cells to confirm potential morphological or functional properties of the cells. Furthermore, gating problems can arise based on the fluorescence signal intensity and the 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 (eg, CEC) that are small in size or have weak fluorescent signals.

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

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

The present disclosure provides matrix-assisted methods for preparing and analyzing components in biological and non-biological samples, including liquid biological samples, such as gel formation, as further described herein. A matrix support method based on the present invention is presented. Liquid samples can come from any source, including humans and animals. In embodiments, it can be derived from liquid biopsies obtained from peripheral blood, bone marrow, cerebrospinal fluid, and other tissue sources, all of which can be further processed. In embodiments, it may be derived from liquid dispersions of materials obtained from other sources, including solid sources such as solid tissue biopsies.

In embodiments, the matrix-assisted method includes adding a solidifying agent (e.g., a gelling agent) to a biological sample containing biological material; and imaging the solidified sample to identify one or more components in the biological material. Biological materials can include any biological molecule, including nucleic acids, proteins, and small molecules, which, in embodiments, can serve as biomarkers of medical or disease conditions. For example, biomolecules can serve as biomarkers 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 sample. In embodiments, biological material in a sample 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 pre-selection or pre-screening of cells; instead, the methods detect labeled antibodies that identify specific cell surface markers in the sample, e.g. allows unbiased analysis of samples of unselected or unscreened cells by detecting biomarker labels, such as antibodies or nucleic acid probes, bound to biological material in the sample.

In embodiments, in any of the methods as disclosed herein, adding a solidifying agent comprises adding a liquid gel solution, such as a low melting point agarose or a hydrogel precursor, to the sample; a gel, etc. adding the drug directly to the sample under conditions that allow the formation of a solid; forming a hydrogel, or other methods. Prior to adding the solidifying agent, the method can include subjecting the components of the biological sample to a fixation procedure, as described herein, and using molecular probes, such as proteins or 20 nucleic acids. may also include labeling one or more biomolecules. Molecular probes include antibodies, dyes, and nucleic acid probes known in the art, including those described herein. In embodiments, probes are 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 aberrations, amplifications, deletions, and translocations. It can be used for.

In embodiments, adding the solidifying agent to the liquid sample comprises adding the solidifying agent to the liquid sample (e.g., a pellet containing the biological material) to the liquid sample having an elevated temperature above its gel point. melting) including mixing with the gel solution. Thus, in embodiments, a cell pellet, eg, labeled and washed in PBS, is resuspended in a gel solution and cooled into a gel. The resuspension step also allows any trace liquid associated with the pellet after processing (eg, PBST) to be diluted and mixed in the gel solution to create a homogeneous sample for imaging.

In an alternative embodiment, adding a solidification agent to a liquid sample comprises adding a sample containing biological material (e.g., a pellet containing biological material) to a mixture or solution containing one or more hydrogel precursors. and altering the state of the mixture to induce solidification (eg, gelation). Formation of hydrogels is known in the art and can be accomplished by a variety of methods according to the present disclosure. For example, a second agent (eg, Ca ⁺ ions or a crosslinking agent) can be added in an amount sufficient to induce gelation of the hydrogel precursor according to methods known in the art.

For example, centrifuging the sample to obtain a pellet, resuspending it in an alginate hydrogel precursor solution, and mixing with an appropriate amount of _CaCl2 (e.g., 0.2 M) to initiate gelation. A sample can be processed as described herein, including the steps of: In this exemplary method, the gel is formed within a short period of time (eg, about 15 minutes) and can then be mounted for index matching (if needed) and for imaging.

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

Imaging the solidified sample, in embodiments, comprises imaging a suitably shaped solid sample, such as a block, by various microscopy techniques, including fluorescence microscopy, more specifically fluorescent light sheet microscopy. can include. In embodiments, the imaging enables single cell identification in the solidified sample, and more specifically comprises detecting one or more cancer cells, e.g. circulating tumor cells or cancer markers. I can do it. The size of the solidified sample can vary depending on the application, sensitivity, and biomolecule being assayed. In embodiments, imaging can be used to analyze cell-cell interactions and morphological and structural characteristics of cells, such as size, shape, and nuclear-to-cytoplasmic ratio, as well as organelle characteristics. can.

The methods of the invention are useful for evaluating, diagnosing, or monitoring a disease, e.g., by microscopically analyzing liquid and/or tissue biopsies, their use on samples (e.g., blood or tissue samples) in a disease state. It is useful in a number of applications including, but not limited to, screening candidate therapeutic agents for efficacy and assessing the expression of a panel of biomarkers in a sample.

FIG. 1 is a schematic diagram of a matrix-assisted protocol for preparing and analyzing liquid biological samples, according to one embodiment of the present disclosure. Steps in the protocol can include (1) cell preparation, (2) sample gelation and clearing, (3) sample mounting and imaging, and (4) sample visualization and analysis. 2A-2C show images obtained from a light sheet microscopic imaging chamber as described in Example 2. Scale bar (20 μm) in Figure 2C. 3A-3C show images obtained from a light sheet microscopic imaging chamber as described in Example 3. Scale bars: Figure 3A (50 μm); Figure 3B (300 μm); Figure 3C (50 μm). 4A-4D show images obtained from a light sheet microscopic imaging chamber as described in Example 4. Scale bar in Figure 4B (30 μm). 5A and 5B show 3D gel data for Patient A as described in Example 5. Scale bar in Figure 5A (150 μm). 6A and 6B show 3D gel data for Patient B, as described in Example 5. Scale bars: Figure 6A (200 μm); Figure 6B (2 μm).

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 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 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 with dispersed components.

In embodiments, the present disclosure provides methods 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 microscopy, including fluorescence microscopy. We provide a three-dimensional imaging approach for

In embodiments, the biomarker is indicative of a cellular component, such as a component of rare circulating cells, such as circulating tumor cells and circulating endothelial cells. For example, biomarkers can include cell surface proteins, morphological markers, or nucleic acid sequences that can identify such tumor cells. In embodiments, a biomarker is indicative of an extracellular component such as extracellular DNA, protein, or vesicles that is indicative of a particular disease or health condition.

Biomarkers can be labeled by known techniques, as described herein, and they can be labeled even in complex samples by a wide range of imaging methods, more specifically by light sheet fluorescence microscopy. Detected by fluorescence microscopy, including microscopy and other microscopy methods.

Terms and Definitions Unless otherwise defined, technical and scientific terms used herein have the same meanings 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 invention. The following definitions are provided to facilitate understanding of certain terms frequently used herein and are not meant to limit the scope of the disclosure.

As used herein, the words "about" or "approximately" mean a range of values that includes a particular value, and which one of ordinary skill in the art would consider to be reasonably similar to the particular value. In embodiments, "about" means within a standard deviation using measurements generally accepted in the art. In embodiments, "about" means a range extending +/-10% of the specified value. In embodiments, "about" means the specified numerical value.

All quantities set forth herein, whether or not the term "about" is explicitly used, may be reasonably inferred based on the actual values given and by one of ordinary skill in the art. It is understood that it refers to both values of approximation (including equivalents and approximations resulting from experimental and/or measurement conditions of such a given value). Therefore, for any embodiment of this disclosure in which a numerical value begins with "about" or "approximately," this disclosure includes embodiments in which specific values are recited. Conversely, for any embodiment of this disclosure where the number does not begin with "about" or "approximately," this disclosure includes embodiments where the number begins with "about" or "approximately."

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

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

Furthermore, although an item, element, or component of an embodiment may be described or claimed in the singular, the plural is intended to be included therein unless limitation to the singular is explicitly stated. be done.

The terms "comprising" and "comprising" are used herein in their open, non-limiting sense. Other terms and phrases used herein, and variations thereof, unless expressly stated otherwise, should be construed as open-ended rather than limiting. As an example of the foregoing, the term "example" is used to provide an illustrative instance of the item under discussion, rather than an exhaustive or restrictive list thereof. Terms such as "conventional," "usual," "known" and words of similar meaning shall be construed to limit the items described to those available at a given time or as of a given point in time. but should instead be construed to include conventional or conventional techniques that may be available or known at any time now or in the future. Similarly, when this specification refers to techniques that are obvious or known to those skilled in the art, such techniques may not be obvious or known to those skilled in the art now or at any time in the future. It encompasses the technologies that are currently in use.

As used herein, the term "solidified sample" refers to a sample in non-liquid form, e.g. solid or gel form, where the solid or gel material captures biological material in a dispersed state in a three-dimensional sample. , i.e. provides a support matrix for immobilization. Subsequently, dispersed materials in the sample can be efficiently identified and imaged in three dimensions. As used herein, the term "solidifying agent" refers to gelling agents capable of forming gels, as well as epoxies and others desirable for supporting dense, dispersed biological materials. Contains agents.

As used herein, the terms "biological sample" and "biological specimen" (and, depending on the context, "sample" or "specimen") include biological molecules such as nucleic acids or proteins. Refers to any biological material that is or is believed to contain. Samples that can be manipulated with the compositions and methods provided herein can be obtained from in vivo or in vitro sources, such as cells, tissues, viruses dissected from subjects such as rodent models, and organs, and samples such as cells, tissues, and mini-organs grown in vitro. Exemplary biological samples include, but are not limited to, liver, spleen, kidney, lung, intestine, thymus, colon, tonsils, testes, skin, brain, heart, muscle, and pancreatic tissue. and solid tissues and organs, including organs. In embodiments, the sample is a whole organ obtained from an animal, including mice, rats, and other animals. In embodiments, the biological sample is brain tissue or a whole brain, such as from a rodent, more specifically a mouse. 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 a pig, a rodent such as a rat or mouse, a zoo animal, e.g. a primate such as a monkey.

Exemplary biological samples include, but are not limited to, material derived from biopsies, bone marrow samples, organ samples, skin fragments, living organisms, and material 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 suffering from or suspected of having a disease or other condition (normal or diseased), or considered normal or healthy. Samples 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 stored and stored at a future time, e.g. , can be subjected to microscopic analysis 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. be able to.

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.

As used herein, the term "biomolecule" is interchangeable with "molecule" and refers to a molecule present in a biological sample or specimen. In one aspect, the biomolecule is an endogenous biomolecule. In another embodiment, the biomolecule is an exogenous biomolecule. Non-limiting examples of exogenous biomolecules include artificially implanted biomolecules, such as biomolecules transferred or expressed by viruses or plasmids. 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 neural markers.
In some non-limiting embodiments, the biomolecule is selected from macromolecular 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 yet other non-limiting embodiments, biomolecules include DNA molecules. In embodiments, the biomolecule is present on a structure, examples of structures 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), nucleus, centriole, ribosome, polyribosome, lysosome, liposome, cytoskeletal component, vesicle, granule, peroxisome, vacuole, protoplast, tonoplast , plasmodesmata, chloroplasts, pseudopodia, blood vessel-related structures of the brain, the brain's dense astrocyte network, or combinations thereof. In embodiments, the biomolecule is a cell marker, such as a protein expressed on the surface of cancer cells.

In embodiments, the "biomolecule" is in a liquid biopsy sample and is used for screening, detection, staging, or surveillance (monitoring), etc., for a disease condition, such as cancer, or a medical condition, such as a metabolic disorder. Evaluated or measured for diagnostic use. In embodiments, the biomolecule is evaluated or measured in a non-invasive prenatal screening or diagnostic test.

As used herein, the term "label" refers to any currently known or future label that can provide a signal-based indication of the presence or absence of a particular target moiety within a sample of the present disclosure. Refers to any techniques and reagents discovered. 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 comprises a label, such as a chromogenic label, a fluorescent label, a radionuclide-binding label, or a combination thereof.

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

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

In embodiments, the liquid sample is a liquid blood sample. For example, a sample 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 processed to remove peripheral blood mononuclear cells isolated from the liquid blood sample. (PBMC) cells. Alternatively, in other applications, red blood cells or other components of a fluid sample can be isolated. Alternatively, samples can be obtained from other biological fluids and fluids obtained from mammals (eg, humans or rats) other than blood.

In certain embodiments, one or more target components include a nucleic acid, a protein, a virus, or a vesicle. In certain embodiments, one or more targeting components include extracellular targets. In certain embodiments, the one or more targeting components include cellular or intracellular targets.

In embodiments, labeling comprises contacting the sample obtained from the liquid sample with a molecular probe and/or contacting the solidified sample from step (b) with a molecular probe, as described in any of the embodiments above. including contact with. Molecular probes may separately be, for example, antibodies, fluorescent dyes or nucleic acid probes.

In embodiments, the method of analyzing a liquid sample according to the present disclosure further includes transferring the sample to a sample holder. The method of the invention, in exemplary embodiments, can further include 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.

In embodiments, methods of analyzing liquid samples according to the present disclosure include performing a fixation procedure on the sample, such as by incubating the sample (if performed prior to solidification) or the solidified sample in a fixative solution. further including. In an exemplary embodiment, the fixative includes glutaraldehyde, formaldehyde, epoxy, or a mixture of any two or more thereof.

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

One exemplary embodiment of the present disclosure provides a method of analyzing a liquid blood sample for the presence of rare circulating cells, such as, but not limited to, circulating tumor cells. In one embodiment, the method of the invention includes labeling a sample containing isolated peripheral blood mononuclear cells (PBMC) obtained from a liquid blood sample with one or more probes for rare circulating cells. adding a solidifying agent to a labeled sample containing peripheral blood mononuclear cells (PBMC); creating a solidified sample containing dispersed peripheral blood mononuclear cells (PBMC); optionally, a refractive index matching material; introducing the solidified sample into the solidified sample to provide an optically clear solidified sample having a refractive index suitable for imaging, and imaging the solidified sample or the optically cleared solidified sample to provide one or more solidified samples. and thereby determining the presence of rare circulating cells in a liquid blood sample. Labeling can further include adding a probe for leukocytes, which can serve as a control, for example.

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

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

In one embodiment, the solidifying agent comprises a component selected from low melting point agarose, agarose, and hydrogel precursors. In an exemplary embodiment, creating a solidified sample includes adding a solidifying agent to the sample at a temperature above room temperature and bringing the sample to a low temperature where it becomes a solid gel. In other embodiments, creating the solidified sample includes adding an agent to the hydrogel precursor to induce gelation.

In one embodiment, the present disclosure provides a solidified sample suitable for imaging that includes dispersed biological material obtained from a liquid biopsy (eg, a blood sample) and immobilized within a solidified test sample. The solidified sample can further include probes for one or more target components in the liquid biopsy. Biological materials include peripheral blood mononuclear cells (PBMCs) and rare circulating cells such as circulating tumor cells, circulating epithelial cells, and circulating endothelial cells.

Low melting point (LM) agarose is commercially available and has undergone modifications such as hydroxyethylation, which reduce the number of intrachain hydrogen bonds present in standard agarose, thereby resulting in relatively low melting and gelation temperatures. It is the result of chemical derivatization processes known in the art. LM agarose generally has several properties including (i) relatively lower melting and gelling temperatures compared to standard agarose, and (ii) higher clarity (gel clarity) compared to standard agarose gels. have characteristics.

In certain exemplary embodiments, the LM agarose has a gelling 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 or molecular biology grade LM agarose with gel strength (at 1 wt% concentration) ≧200 g/cm ² , which can make gels with greater sieving properties and higher transparency than regular molten agarose. Examples of commercially available LM agaroses suitable for use with the present disclosure include SeaPrep™ Agarose (Lonza Cat. No. 50302), SeaPlaque™ Agarose (Lonza Cat. No. 50104), and UltraPure™ Low Melting Point Agarose (ThermoFisher Scien. tific catalog number 16500500), but are not limited to these.

In embodiments, the labeling agent comprises a small molecule that can bind to a specific target moiety within the tissue. Examples of small molecule dyes include DAPI, propidium iodide, lectins, phalloidin, and any other small molecule that can bind to a target moiety within a tissue. In embodiments, the small molecule inherently produces a signal, such as a fluorescent signal produced by DAPI, propidium iodide, or acridine orange. In embodiments, the small molecule is conjugated to an indicator to generate a signal, e.g., in the case of a lectin dye, a fluorescent signal-generating indicator, or a non-fluorescent signal-generating indicator, e.g., a colorimetric indicator (e.g., horseradish peroxidase (HRP)). or 3,3'-diaminobenzidine tetrahydrochloride (DAB)). In embodiments, the stain comprises an antibody as further described herein. In embodiments, the staining comprises a modified nucleic acid strand targeted detection activity. In other embodiments, the staining includes in situ hybridization, such as staining that includes a nucleotide-based probe that can hybridize to a predetermined sequence of nucleic acids within the tissue. In embodiments, the nucleotide-based probe includes a label (eg, one or more of the labels provided above) to enable signal generation and detection of the nucleotide-based probe. In further embodiments, the nucleotide-based probe includes a fluorescent label, such as fluorescence in situ hybridization (FISH).

In embodiments, a biological sample, eg, a cell, tissue, organ, organism, or organ substructure, provides an endogenous signal, eg, an endogenous fluorescent molecule. Examples of endogenous fluorescent molecules include fluorescent protein reporters such as 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 yet 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, etc.

As mentioned above, liquid samples can be labeled with molecular probes such as antibodies. In embodiments, the antibody is directly or indirectly conjugated with 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. This is the primary antibody containing the generated label. In other aspects, the primary antibody is applied as a single stain (with or without additional reagents, e.g., labeled streptavidin or enzyme/coenzyme substrates to provide a signal). Ru. In embodiments, the primary antibody does not include a label and is instead detected by a secondary antibody conjugated to a label.

Examples of fluorophores that can be conjugated to a primary or secondary antibody include Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Flu or 568, Alexa Fluor 594, Examples include 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 B right 780, Super Bright 600, Qdot 525, Qdot 565, Qdot 605, Qdot 655, Qdot 705, Qdot 800, R-phycoerythrin (R-PE), and allophycocyanin (APC).

Solidified samples can be imaged by any microscopy-based application, and thus the disclosed subject matter is a particular embodiment, but not limited to the particular imaging technique used therein. do not have. 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 direct or indirect detection of a biomarker. A wide 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 fluorescent dyes such as fluorescein isothiocyanate (FITC), Oregon Green™, rhodamine, Texas Red, tetrarhodamine isothiocynate (TRITC), Cy3, Cy5, Alexa Fluor® 647, Alexa Fluor® 555, Alexa Fluor® 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.

The term "immunofluorescent marker" refers to a detectable reagent that is an antibody or functional fragment thereof that directs a fluorescent dye to a specific molecule in or on a cell. Immunofluorescent markers can be used in methods to generate immunostaining of desired samples using fluorescence microscopy. Immunofluorescent markers can also be used in the immunocytochemistry (ICC) or immunohistochemistry (IHC) methods described herein. For example, in the context of this disclosure, immunofluorescent markers can be used to detect rare circulating cells (eg, CTCs or CTC mimetics), as described herein.

The term "antibody" refers to any immunoglobulin or derivative thereof, whether natural or wholly or partially synthetically produced. Any antibody derivative that maintains specific binding ability can also be used in the methods disclosed herein. Antibodies of the present disclosure are capable of specifically binding a biomarker. For example, an antibody may specifically bind a single biomarker, such as chondroitin sulfate proteoglycan 4 (CSPG4). Additionally, antibodies can be pan-specific. For example, the pan-specific antibodies of the present disclosure may include one or more members of the biomarker family (e.g., one of the chondroitin sulfate proteoglycan families, including chondroitin sulfate proteoglycans 1, 2, 3, 4, 5, 6, 7, and 8). (one or more members). Antibodies can have binding domains that are homologous or largely homologous to immunoglobulin binding domains, and can be derived from natural sources or produced partially or wholly synthetically. Antibodies can be monoclonal or polyclonal antibodies. 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. Because of their smaller size, antibody fragments can offer advantages over whole antibodies in certain applications. Alternatively or in addition, antibodies can include multiple chains linked to each other by, for example, disulfide bonds, and any functional fragments obtained from such molecules, such fragments having specific characteristics of the parent antibody molecule. retains its binding properties. Those skilled in the art will appreciate that antibodies can be provided in any of a variety of forms, including, for example, humanized, partially humanized, chimeric, chimeric humanized, and the like. Antibodies can be prepared using any suitable art-known method. For example, antibodies can be produced enzymatically or chemically by fragmentation of intact antibodies, or recombinantly from genes encoding partial antibody sequencing.

The term "biomarker" refers to a biological molecule or a fragment of a biological molecule, the alteration and/or detection of which is associated with rare circulating cells (e.g., CTCs, CTC mimetics, or CECs) or other target components. may be correlated with certain physical conditions or conditions. The terms "marker" and "biomarker" are used interchangeably throughout this disclosure. Such biomarkers include nucleotides, nucleic acids, nucleosides, amino acids, sugars, fatty acids, steroids, metabolites, peptides, polypeptides, proteins, carbohydrates, lipids, hormones, antibodies, and their role as surrogates for biological macromolecules. including, but not limited to, biological molecules containing regions of interest that perform the following functions, and combinations thereof (e.g., glycoproteins, ribonucleoproteins, lipoproteins). The term also encompasses parts or fragments of biological molecules, such as peptide fragments of proteins or polypeptides. In the context of this disclosure, exemplary biomarkers of CTCs, such as circulating melanoma cells (CMCs), include chondroitin sulfate proteoglycan 4 (CSPG4), premelanosomal protein (Pmel17) and S100 calcium-binding protein A1 (S100A1). is included.

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

In embodiments, the method includes solidifying the liquid sample, thereby capturing (immobilizing) the dispersed material in a form that can be subsequently imaged by microscopy applications (or other imaging methods). The resulting scanned image allows detection of three-dimensionally immobilized and spatially separated components in solid samples, allowing high resolution and sensitivity. Selectively labeled components, such as those labeled with fluorescently labeled antibodies, can then be sensitively and rapidly identified, such as by fluorescence microscopy, to detect labeled targets of interest.

Thus, the compositions and methods described herein effectively provide 3D scans or images of liquid biopsies. Rare biomarkers include, for example, rare circulating cells, such as circulating tumor cells (and intact circulating tumor cell clusters), or cell-free nucleic acids, which may appear as discrete signals in the resulting scanned sample; Not limited to these. This is in contrast to other methods such as microfluidic-based applications where such biomarkers are detected indirectly, or traditional cytological methods based on examining small samples of cells in duplicate on standard slides. are in contrast.

In embodiments of the present disclosure, the methods of the invention provide the additional advantage of not imposing any morphological or cellular size cutoffs on the components analyzed. For example, existing CTC detection techniques based on certain enrichment methods may inadvertently miss rare biomarkers. In addition, the multiple processing steps underlying existing CTC detection methods can induce changes in cellular biomarkers that can further reduce the sensitivity and accuracy of the detection method. In contrast, the method embodiments described herein do not require any specific selection criteria regarding the biological material to be analyzed. Instead, they allow the capture of complex arrays of cells and extracellular material, regardless of shape and morphology, allowing them to be captured, preserved, and spatially separated in three-dimensional coagulated samples. .

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

Matrix-Assisted Methods The present disclosure provides, in part, matrix-assisted methods for processing and analyzing liquid samples, such as biopsy samples, which may contain rare biomarkers such as circulating tumor cells or cell-free tumor DNA.

In an exemplary embodiment, the steps in the method are used to create an image of the distributed components of the sample in a 3D spatial configuration. In embodiments, the method includes solidifying a liquid sample containing dispersed biological material in a form that allows for rapid imaging, such as by light sheet microscopy or other microscopy techniques. In embodiments, the method comprises: (a) adding a solidifying agent to a liquid sample comprising biological material; (b) creating a solidified sample comprising biological material; (c) adding the solidified sample to a liquid sample comprising biological material; imaging to identify one or more components in the dispersed biological material.

In embodiments, the liquid sample includes biological material and undergoes multiple processing steps, which may include those illustrated in the process illustrated in FIG. 1, described in detail below. Not limited.

Step 1: Cell Preparation In embodiments, the method includes preparing or procuring a liquid sample with a biological sample, which may be dispersed and subsequently captured in solid form; This allows high resolution detection.

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

In embodiments, the material in the liquid sample is obtained from a processed blood sample, such as from a liquid biopsy. Processing can include one or more steps. For example, processing can include removing red blood cells and isolating (harvesting) PBMC cells. More specifically, a blood sample can be subjected to centrifugation to remove red blood cells (red blood cells) and platelets. 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.

As shown in FIG. 1, in an exemplary embodiment, a blood sample may be provided in a tube or container containing an anticoagulant, such as a vacuum blood collection tube containing EDTA or heparin. Blood samples can be treated with a cell separation medium such as Lymphoprep™ or FicollPaque™, centrifuged, and the supernatant removed. Alternatively, PBMC isolation tubes can be used (eg, SepMate™ tubes available from Stemcell™ Technologies). Blood samples can be collected, if desired, according to protocols known to those skilled in the art, such as by application of commercially available or synthetic ammonium chloride solutions (such as the ammonium chloride solution available from Stemcell™ Technologies). Red blood cells can also be subjected to erythrocyte lysis according to known techniques, which can be carried out before or after centrifugation. In other embodiments, red blood cell lysis is not used because trace amounts of red blood cells do not affect sample imaging and analysis.

In an exemplary embodiment, a pellet (eg, a pellet containing isolated PBMC) is obtained, eg, from centrifugation, and is further processed as described below. In certain exemplary embodiments, the amount of pellet can be at least 5 μl, or at least 10 μl, or at least 15 μl. In other exemplary embodiments, smaller amounts of pellets are processed. In any case, the entire pellet is encapsulated in the gel, which has a size orders of magnitude larger than the typical throughputs used in microfluidic processes known in the art.

In embodiments, the dispersed material in the liquid sample can be derived from other tissue sources. For example, they may be derived from biopsies obtained from body fluids other than blood, such as bone marrow, cerebrospinal fluid, urine, saliva, sputum, tears, semen, or other sources of body fluids. Alternatively, the dispersed material in the liquid sample may reflect liquid dispersion of material 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 material can be derived from non-tissue sources. In embodiments, biological material in a sample may be concentrated by concentrating a large amount of sample, such as a larger amount of blood, e.g., 2 ml, 4 ml, 8 ml, or more, or other samples. The cells can be collected and pelleted from the source.

In embodiments, the biological material in the sample is present in relatively large amounts, such as by centrifuging and collecting cell pellets from large volumes of blood (e.g., 0.5 cc or 1 cc or more) or other tissue sources. It can be concentrated by concentrating it from the sample.

Prior to being dispersed into a liquid sample, the material may undergo additional processing steps. In embodiments, the biomaterial can undergo a fixation step before being dispersed into a liquid sample, as discussed further herein. Alternatively, fixation may be performed after the biological material has been collected into a liquid sample. Fixation may also be performed after the cells have been labeled. The particular fixatives that may find 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 one or more of glutaraldehyde, formaldehyde, epoxy, or one or more crosslinking products of the foregoing.

For rare targets such as CTCs, large volumes of liquid biopsy samples can be collected serially and collected into a single tube. For example, 2 ml or more of blood can be collected, processed, and cell pellets pooled and resuspended in liquid sample buffer, such as PBS.

In embodiments, 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.

In embodiments, the methods of the invention include adding a solidifying agent that allows labeling one or more targets of interest and capturing the dispersed labeled substance in a solid form comprising a three-dimensional cross-linked network. further including. In other words, the solidifying agent provides a solid matrix to support 3D visualization of sample components.

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

For example, multiple antibodies from different host species can be introduced at or about the same time, or at different times. In an exemplary embodiment, the primary antibody can be conjugated (or prelabeled) with a tag, such as a fluorescent dye or enzyme, e.g., a fluorophore or a corresponding secondary antibody, and introduced into the mixture in one step. be able to. In certain embodiments, one-step labeling is preferred, as the labeling step typically requires post-washing, and thus further centrifugation and supernatant removal steps can result in cell loss or damage, potentially reducing signal sensitivity. 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). If desired, the signal can be further amplified by available techniques such as biotin-streptavidin binding and polymerase chain reaction-based systems. In embodiments, probes can identify other disease biomarkers, such as cell-free tumor or fetal-derived DNA, or genetic and structural DNA and chromosomal abnormalities in the cell nucleus, such as amplifications, deletions, and translocations. changes can be visualized.

Samples can also be prepared using a variety of dyes that target cellular components, including, for example, fluorescent dyes such as DAPI and PI (which bind nuclear components) and DiD or DiL (which bind membrane components). Labeling can also be done by other methods known in the art.

Labels can also be used for cell permeabilization, as appropriate and known in the art, to enable efficient and specific binding of immunological or molecular reagents to targets of interest (biomolecules). Alternatively, other steps such as fixation may be included. In some embodiments, the label is applied before gelling and clearing the sample. In certain embodiments, the label is preferably applied before pelleting or separating components from the sample, eg, if the biological material is dispersed in a liquid (eg, blood) sample.

Labeling can also be performed after fixing the cells in a gel state. For example, cells collected from a liquid sample (eg, PBMCs obtained from blood) can be introduced into a PFA solution and directly form a gel before labeling. The gel sample can then be labeled with the probe passively or by other active immunolabeling approaches, such as those involving electrophoresis or pressure-based approaches. Thus, in embodiments, after solidification, the processed gel sample can be immunolabeled (ie, labeled for the first time or with additional labeling applied). Although this approach of post-gelation labeling may require longer processing times, it may also enhance preservation of target cell numbers. In embodiments where labeling occurs after solidification, the fixation means can be performed on the solidified sample after gel formation, which can then be labeled.

In other embodiments, delipidation can be performed on solidified (eg, gelled) cell samples, where there is a need or desire to increase sample transparency for imaging. In embodiments, the solidified sample can be further crosslinked with a hydrogel precursor or epoxy and degreased according to the CLARITY approach. For example, "Advances in CLARITY-based tissue clearing and imaging," Exp Ther. Med. See 2008;16(3):1567-1576. However, it is noted that, according to most embodiments, a delipidation step is generally not required due to the low stacking of cells in the sample that are dispersed in the solidified sample. As explained below, in some embodiments, even an index matching step is not required.

Step 2: Solidifying and Clearing the Sample In some embodiments, solidifying the sample includes a solidifying agent (e.g., a gelling agent) that allows the biological material to be captured in a solid form, such as a gel form. and the introduction of a solidifying agent compatible with subsequent imaging and detection of labeled biomolecules of interest. Solidifying agents include reagents known in the art such as, but not limited to, agarose (including low melting point agarose) solutions, polyacrylamide precursors, natural gum, starch, pectin, agar, and gelatin. can be mentioned. In embodiments, such reagents are polysaccharide- or protein-based. For example, Kar et al. 2019, Current developments in experimental science: in Fundamentals of Drug Delivery, 29-83. Alternatively, in some embodiments, the solidification agent consists of, or consists essentially of, an index-matching solution itself, optionally modified to provide the appropriate viscosity to provide a rigid physical gel. can become.

In embodiments, the solidifying agent is a viscosity-adjusting agent, such as one made from natural or synthetic polymers (e.g., xanthan gum, Pemulen™, Carbopol™, Velvesil™ plus, or other polyacrylic acid derivatives). is or contains an agent.

In embodiments, the solidifying agent comprises a mixture of polysaccharides (eg, agar/agarose, gellan gum) that produces a physical gel with a solid three-dimensional matrix or network structure.

In embodiments, the solidifying agent includes a mixture of chemical monomers and crosslinking agents that produce a synthetic chemical gel, ie, has a chemically crosslinked polymer network.

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

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

In certain embodiments, pregelling comprises dispersing or resuspending the 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 between 0.1% and 10%, or between 0.3% and 1.6%, or between 0.5% and 1.5% (e.g., about 1%). .

Accordingly, in certain embodiments, pregelling comprises dispersing or resuspending the biological material in a solution containing agarose (ie, 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% to 10%, or 0.3% to 1.6%, or 0.5% to 1.5% (eg, about 1%).

In certain exemplary embodiments, pregelling involves dispersing or resuspending the biological material in a solution comprising a mixture of low melting point agarose and regular agarose. In certain exemplary embodiments, the gelling agent solution (such as, but not limited to, agarose or a low melting point agarose solution) is sufficient as described below to provide the desired refractive index of the final solidified gel. combined with a quantity of RI compatible material. In certain exemplary embodiments, the pre-gelled material is dissolved directly into the RI compatible solution to constitute the gelled solution.

In embodiments, dispersing or resuspending a biological sample in a mixture to form a gel generally stabilizes the components in the sample in a spatial distribution with sufficient properties, such as transparency, and Allows for subsequent imaging. In some embodiments, an index matching material having a refractive index (e.g., having a refractive index between 1.3 and 1.6 or 1.33 and 1.5, or having an RI approximately equal to water) can be introduced into the gelling or pregelling mixture, although RI matching materials with other refractive indices can be used in other embodiments. As known in the art, refractive index matching materials (also referred to as RI compatible or RIM) can penetrate into tissues/cells to achieve tissue/cell transparency, and CUBIC-R+, Including, but not limited to, RapidClear, RIMS, or ScaleView. See Neuropathol Appl Neurobiol, 2016 Oct; 42(6):573-87.

In some embodiments, index matching material is not required. For example, in small samples, laser light can pass through the solidified sample and excite labeled cells or other target components. That is, refractive index differences in the solidified sample are not a problem when the laser does not need to penetrate deeply to cause light refraction. For example, using a two-photon laser with more power allows the laser to penetrate deeper without being bent. Such systems are limited only by possible photodamage of the fluorophore, and this problem does not necessarily require the addition of an index-matching material, e.g. an index-matching solution, to avoid such damage. .

In embodiments, pregelling comprises one or more hydrogel precursors, e.g., polymerizable materials, monomers or oligomers comprising monomers selected from the group consisting of water-soluble groups containing polymerizable ethylenically unsaturated groups. dispersing or resuspending the biological material in the gelling material. The monomer or oligomer can include one or more substituted or unsubstituted methacrylates, acrylates, acrylamides, methacrylamides, vinyl alcohols, vinyl amines, allyl amines, allyl alcohols. The precursor also includes polymerization initiators, crosslinkers, and other components known in the art, as described, for example, in WO2019023214 and WO/2020/013833.

In embodiments, the methods of the invention include transferring the pre-gelled mixture to a sample well in a holder while in a liquid or molten state. In embodiments, wells within the holder allow for the formation of solid samples suitable for imaging. For example, the solid sample can be in the shape of a block or other form customized for imaging by fluorescence microscopy. Alternatively, in some embodiments, the pre-gelation step can be performed in a tube-sample holder combination, thus allowing solidification (e.g., gelation) to occur in the same tube and keeping the liquid solution The need for subsequent transfer to a separate sample holder can be eliminated.

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

After solidification, the sample can be processed by additional steps before imaging. For example, the sample can be equilibrated in a refractive index material to be properly adapted (eg, cleared) for imaging in step 3 (RI adaptation). Alternatively, in some embodiments, the RI compatible material is added at the same time as the solidifying agent. In yet another embodiment, the RI compatible material is added with a solidifying agent and then a second round of RI compatible material is added to the solidified sample to provide the final desired clarity of the sample.

RI compatible materials are known in the art and can be used in amounts appropriate to provide the desired clarity of the solidified gel sample. In certain embodiments, the RI compatible material has an RI of about 1.39 to about 1.65, or about 1.49 to about 1.55 (eg, about 1.52). For example, but not limited to, RI-compatible materials can be 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>.

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

In embodiments, the method of the invention includes, prior to imaging, mounting the sample on an image holder, such as a 3D printed sample holder as shown in FIG. imaging the sample within the sample. Sample holders can be customized according to specific sample preparation. In embodiments, it may include a base and two side walls. In embodiments, holders such as those used for samples prepared in viscous media may have four walls that enclose and preserve the spatial organization of dispersed components in the sample. In embodiments, imaging can be performed in a sample cuvette using a standard epifluorescence microscope. 3D gel samples can be mounted on sample holders with the help of mounting gels (eg agarose, poly-L-lysine, superglue). For example, Asano et al., Expansion Microscopy: Protocols for Imaging Proteins and RNA in Cells and Tissues, Current Protocols in cell biology (2018), especially “Sample mounting” on pages 34-36.

Fluorescence microscopy approaches include, but are 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, gel samples are rapidly imaged by light sheet microscopy, such as light sheet fluorescence microscopy (LSFM) using a detection lens and an illumination lens, as shown in FIG. In some embodiments, a Gaussian beam may be used, as shown in FIG. 1, or a special beam profile such as a undiffracted Bessel beam may be used. Such imaging allows the material in the 3D sample block to be scanned and evaluated for the individual presence of labeled biomarkers. As is known in the art, the processor can communicate with and receive output from the microscope to combine (i.e., "stitch") successively imaged adjacent object regions. See, for example, the techniques and devices disclosed in US Pat. These can be used according to the subject matter of the present disclosure.

Other detection light sources and corresponding microscopy applications can also be used. For example, light microscopy can be used to evaluate visible dye-stained cells, including cells stained by, for example, H&E (hematoxylin and eosin) or immunohistochemistry (IHC). Additionally, X-ray microscopy can be used to detect liquid components labeled with appropriate metal tags. Other imaging techniques can also be used, including images captured by high-resolution cameras or associated light detection systems included in commercially available smartphones (e.g., iPhone® or Android®-based smartphones).

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

In embodiments, the methods disclosed herein can be used to treat inflammatory, metabolic, gastrointestinal, endocrine, immunological, musculoskeletal, cardiovascular, cardiopulmonary, genitourinary, hepatic, respiratory, It can be used to detect cellular biomarkers associated with viruses and other diseases and disorders, such as neurological diseases and disorders.

In addition to detecting the presence of biomarkers such as specific cellular 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, nucleocytoplasmic ratio, and other phenomena. In embodiments, imaging can be used to analyze cell morphology and structure, including analysis of changes in cell morphology and structure compared to their natural or healthy state. For example, imaging methods can be used to analyze the appearance of cells, such as their size, shape, or other external characteristics. Additionally, imaging methods can be used to analyze the morphology and structure of internal cellular components such as the nucleus, endoplasmic reticulum, Golgi apparatus, mitochondria, or other organelles.

As another example, a biopsy is prepared by liquid dispersion of a sample of affected tissue from the kidney, heart, stomach, liver, pancreas, intestine, brain, etc., and determines the condition of the tissue, the degree to which the disease has developed, and the success of the tissue. Possibilities etc. can be determined. The methods herein can be used to assess morphological changes in a cell population that may be indicative of a disease state, eg, through the use of dyes that target membranes or cellular components.

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, a liquid sample obtained from a subject, such as a mouse, rat, dog, primate, human, etc., that has been contacted with a candidate agent can be prepared by the methods disclosed herein, and one or more cells or microscopically analyzed for tissue parameters, i.e. attributes or characteristics of intracellular components that can be measured.

In another application, the method of the invention can also be used to visualize the distribution of genetically encoded markers in liquid samples prepared by dispersing material from tissue. Such markers can include, for example, chromosomal abnormalities (inversions, duplications, translocations), loss of genetic heterozygosity, the presence of genetic markers indicative of a predisposition to a disease or health condition. Such detection may be useful, for example, in diagnosing and monitoring diseases, such as in personalized medicine, paternity studies, or other applications.

CTC Detection In certain exemplary embodiments, the methods disclosed herein are used to detect CTCs by immunological or molecular means for diagnosis and to address their clinical significance. used. CTCs are rare cells that circulate in blood or other fluids along with millions of other circulating cells. The method of the invention captures such rare cells in solidified 3D form in complex liquid samples or liquid biopsies. When the sample is subsequently imaged, for example by light sheet fluorescence microscopy, any CTCs can be detected as individual signals in the sample block. Advantageously, the method of the invention allows such CTC detection without reducing their biological heterogeneity. They may also contain less interference than is required for processing by microfluidics or conventional cytology, which may disrupt morphology or interfere with sensitivity.

EXAMPLES The disclosure herein is further illustrated by the following non-limiting examples.
It is understood that these examples are illustrative only; they are to be construed as limiting the scope of one or more embodiments, and as defined by the appended claims. Shouldn't.

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 the hematopoietic compartment, for example, and do not spontaneously adhere. It is. These poor adhesive properties complicate existing methods in the art for detecting CTCs, such as the use of solid supports to isolate and immobilize CTCs. The method disclosed herein allows to detect complex 3D representations of liquid samples without limiting their biological heterogeneity when such rare cells are present, and , reducing interventions that can destroy morphology. Furthermore, methods based on modified supports and matrices coated with anti-adhesion molecules (or other binding proteins) can induce biological responses that change CTC morphology, resulting in inaccurate analysis. Immunofixation relies on strong affinity between coated antibodies and cell membrane proteins. Low expression of target surface proteins or low antibody affinity may result in low capture rates of target cells. Similarly, cytocentrifugation, subsequent fixation and subsequent labeling of cells onto a support such as a microscope slide may destroy the cells or disrupt their morphology, interfering with diagnostic evaluation. In contrast, the microscope slide approach requires coating cells in a monolayer fashion to enable imaging, which greatly reduces throughput or the number of cells imaged and analyzed.

Detection methods that rely on microfluidics, nanostructures, and channels can suffer from similar limitations. For example, see WO2012016136; WO2013049636. Such methods can induce flow stress on cellular components in liquid samples, impairing their morphology and other characteristics. More generally, such methods typically select homogeneous populations of cells based on the size or expression of surface membrane proteins, making them suitable for diagnostic analysis of clinical or biological samples. This will limit scientific heterogeneity.

Therefore, it is desirable to uncover a biopsy method that can minimize lengthy operations, interacting external stimuli, and long-term processing. Such improvements could help improve both the native state and heterogeneity of cells and other components in liquid biopsies, and thereby improve diagnostic applications, such as early diagnosis of possible metastatic processes. Provide more accurate tools for

CTCs in peripheral blood can be considered as tumor extension. Tumors are typically heterogeneous, meaning that through mutations, a tumor can develop several different cell types within it. 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 have an important role in cancer metastasis. For example, Harouaka et al. , 2014, Pharmacol. Ther. See 141, 209-221.

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

In contrast, the disclosure herein provides, in certain embodiments, methods for analyzing individual cells dispersed and captured in a three-dimensional solidified sample (e.g., individual cells from a liquid biological sample). do. No enrichment or selection of cells is required and all cells can be visually screened by labeling and imaging the sample.

In some embodiments, there is no general need for delipidation (e.g., SDS-based delipidation) or other sample clearing methods, and certain embodiments of the methods disclosed herein do not require such delipidation or other sample clearing methods. Defined by not including a transparency step. For example, Jensen and Berg, 2017, J. Chem. Neuroanat. 86, 19-34. reference. Thus, more cellular information is preserved compared to, for example, methods involving US Patent Application Publication No. 2019/0113423 and CLARITY or other clearing protocols. According to the disclosure herein, each individual cell or cell cluster can be visualized and analyzed separately from each other in a three-dimensional array, which allows for cell size, morphology, biomarker distribution, nucleocytoplasmic ratio, etc. can provide comprehensive cellular information.

Multiple types of tumors, such as lung, liver, and colon cancers, as well as other cancers that can be detected as CTCs in liquid biopsies, can be identified according to the disclosure herein. For example, upon detection of CTCs from a liquid biopsy (e.g., blood sample) using the EpCAM marker, the information obtained may include, for example, the number of cancer cells in the blood of 1 cc, 2 cc, 3 cc, 4 cc, or more. Providing information such as (or type) can indicate whether a patient has cancer (or other characteristics related to risk, prognosis, and treatment response). EpCAM is an epithelial marker that indicates infiltrating cancer cells that have undergone epithelial-mesenchymal transition (EMT), which is the main cause of cancer invasion. Cancer invasion typically begins with EMT from a small population of tumor cells that stimulates blood vessel proliferation, providing a pathway for cells to enter the bloodstream as CTCs.

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

Cellular heterogeneity in blood can also apply to healthy cells such as white blood cells. According to the disclosure herein, by applying parameters such as cell morphology (e.g., CTC nuclei can be larger than leukocyte nuclei) and molecular markers (e.g., EpCAMCTC+/WBC&CD45 CTC-/WBC+), You can know the difference between CTC and white blood cells.

Additionally, CTCs are generally highly invasive because they escape from the primary tumor into the bloodstream. Such invasive cells can make cancer difficult to treat and cure due to their ability to metastasize, as well as their high likelihood of mutation, which increases the chance of resistance to chemotherapy and other therapeutic interventions. can be done. This confirms the importance and value in identifying the molecular characteristics of such invasive cells in order to determine appropriate treatment regimens. For example, if the CTCs present in the blood exhibit high levels of PDL-1, immunotherapy is likely to be a more effective approach. PDL-1 levels can be determined using, for example, a PDL-1 antibody probe to quantify the number of PDL-1+CTCs across all CTCs in a sample. Since tumor development is almost always heterogeneous, i.e. one tumor site does not represent the entire tumor population and CTCs can originate from all tumor sites, this measurement is useful for example in PD-1/PDL- 1 may be useful in predicting the effectiveness of inhibitory treatment. (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6627043/). Because CTCs are rare in blood, such predictive measurements are most accurate when CTCs are efficiently captured and analyzed, as provided by the methods disclosed herein.

Another example according to the disclosure herein involves determining CTC numbers and cell types and administering therapy based on this determination (e.g., continuing the current course of treatment or starting a different or additional course of treatment). (including deciding whether to do so). For cancer patients undergoing chemotherapy or cell therapy, CTC counts can indicate treatment effectiveness. In stage III colon cancer patients, the number of CTCs 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 intervention, the CTC count can drop to 5000 and even approach zero after 12 months. However, in some cases, the CTC count decreases to only about 2000 after 6 months of chemotherapy, and then rises again to tens of thousands 6 months later. This indicates drug resistance of cancer cells to therapeutic intervention. For example, chemotherapy may have killed all drug-sensitive CTCs, but other subtypes that were resistant would be unaffected.

In accordance with the present subject disclosure, by performing blood tests for CTC detection and identification on a regular basis (e.g., weekly or monthly), physicians and health care professionals can quickly verify tumor resistance to chemotherapy and The treatment plan can be modified accordingly. Therefore, the present study addresses the above limitations by describing a formulation and method for visualizing biopsy samples in three dimensions by capturing dispersed components in the sample in a solidified state. This approach increases the sensitivity of analysis by separating individual components, allows the use of rapid imaging techniques such as light-sheet fluorescence microscopy, and improves the morphology and integrity of sample components such as cellular markers. It has a number of advantages, including increasing the specificity of the assay by reducing the number of processing steps that can interfere with the sensitivity of the assay;

Materials and Methods Sample collection and fixation:
A blood sample (eg, 2 cc, 8 cc, or 10 cc) is taken from the subject and red blood cells are removed by standard methods, eg, density gradient centrifugation using centrifugation at 1000 rpm for 5 minutes at room temperature. 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 char activation, and clinical applications; in Pathobiology of human disease: a dynamic encyclopedia of disease mechanisms (eds , McManus and Mitchell).

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

Biomarker label:
The fixed and washed pellet is resuspended in blocking and permeabilization buffer, placed on a shaker for several minutes, centrifuged, and resuspended in premixed labeling solution for 30-60 minutes. Depending on the binding affinity of the antibody, which can directly affect the final imaging quality, resuspend the pellet and incubate in the labeling mixture for a longer period of time (e.g. up to 10 or 20 hours). can do.

The protein biomarker of interest is an antibody (or related fragment or derivative) detected directly, e.g., with a fluorescently conjugated antibody, or indirectly, e.g., by immunohistochemistry or with a primary antibody and a conjugated secondary antibody. can be marked with. Similarly, nucleic acids of interest can be labeled with molecular probes that are detected directly or indirectly. If desired, 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 washed one or more times.

In this example, 200 μL of labeling solution in PBST blocking buffer was combined with propidium iodide (PI) (1:2000) for nuclear staining and EpCAM antibody (antibody concentration >0.1 mg/ml) for cancer cell detection. (at 1:250), a CD45 antibody (1:250 antibody concentration) for immune cell detection, and a secondary antibody compatible with the primary antibody host (2x volume relative to the primary antibody). Jackson ImmunoResearch Laboratories, Inc. Fab from (https://www.jacksonimmuno.com/catalog/31) was used.

Alternatively, sequential labeling can be performed as follows: starting with incubation with primary antibody for 60 minutes or more and washing, then secondary antibody and washing, then staining with PI in PBS solution for 5 minutes, Then wash. For other targets, surface proteins can be labeled with antibodies or DNA/RNA targets can be labeled with probes. Sequential labeling with secondary antibodies or direct conjugates of fluorescent molecules can also be used. Biotinylated antibodies can also be used with boosted signals, eg, based on binding affinity for streptavidin, as known in the art. After addition of the labeled antibody or any probe or chemical dye, the pellet can optionally be resuspended in 4% PFA for 10 minutes at room temperature to immobilize all the labeled material onto the cells and remove them during the solidification step. Prevent them from being washed away.

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

In embodiments, the solution containing the solidifying agent contains liquid agarose, such as 1.2% or 2.4% or 10%, in an RI compatible solution to facilitate subsequent imaging. Alternatively, the solidifying agent (e.g., gelling agent) crosslinks and/or becomes solid (e.g., gel-like) at lower temperatures and/or higher ionic concentrations, or present in solution with a solvent that melts at

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

Note that the original liquid blood sample has millions of non-cancerous cells obscuring the CTCs, which are rare and present at very low levels. Gelation allows cells to be dispersed and separated in 3D space for imaging (described below). One important point is that millions of cells can be condensed from a large volume (e.g., 10 cc of blood) into a 20 μl gel block (2.71 mm ³ ) and then imaged within an acceptable imaging period. This means that it will be possible to In alternative embodiments, the capacity may be further reduced to allow rapid high-resolution imaging (eg, up to 20x or 40x). In certain embodiments, the cell pellet is reduced to give the smallest possible volume. In such situations where relatively small volumes are used, care may be required in gel clearing due to the close distance between cells due to the dense lipid bilayer (cell membrane), e.g. This can be solved using optimized RI matched solutions or higher power lasers such as two-photon microscopy.

Imaging:
Once the gel block (with dispersed contents) is formed, it is cleared by soaking in a refractive index (RI) matching solution for about 30 minutes to make it clear for imaging. The RI-compatible gel sample can then be imaged to detect the presence of many biomarkers that are not individually discretely separated in the gel block.

Such imaging can be performed by microscopy, 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 undiffracted Bessel beam. For example, using a light sheet microscope allows hundreds or thousands of cells to be imaged 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 from top to bottom at approximately 10-15x magnification. Unlike indirect gating on summary plots by flow cytometry, LFSM data obtained from imaged samples allows for visual inspection of antibody labeling, such as adequacy, intensity, and distribution, for individual cells. It is clear that this can be done.

Application of the method here allows rare biomarkers such as CTCs to be rapidly detected on solid-state arrays, allowing spatial discrimination of labeled biomarkers. By reducing the need for processing steps that may reduce sample heterogeneity or alter their properties, the methods of the invention also provide a more sensitive and accurate diagnostic approach.

Furthermore, the methods described herein allow PBMCs to be fixed immediately after RBC removal, allowing capture of their distribution and association in their original state. Advantageously, this may indicate cell-cell interactions, cell clusters (e.g., Circulating Tumor Microemboli (CTM)), and others that may indicate disease status or provide other prognostic or diagnostic uses. allows direct visualization of sample properties, such as the properties of

Example 2 - 3D matrix-based imaging of blood samples Cell preparation:
Transfer anticoagulated blood from a human in an EDTA-coated tube to a 15 ml test tube, add cell separation solution (e.g. Ficoll®), and subject the mixture to density gradient centrifugation (1200 RPM) for 5 minutes. went. After centrifugation, the buffy coat (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.

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

Solidification:
On the other hand, a solidification solution mixture containing about 0.5 wt% LM agarose was prepared. The solidified solution was heated in the microwave until it melted above its gel point, then cooled to room temperature, where it was again provided in a liquid state until the gel point was reached. With a pipette, 20 μl of chilled solidification solution was slowly added to the pellet to resuspend it. The solidification solution was added slowly to avoid bubble formation. The mixture is brought to 4° C. and maintained for a period of time (less than about 30 minutes) to allow gel formation.

Mounting and RI Compatibility:
Once the sample gelled, the gel was carefully removed from the tube with a pipette by popping the side of the gel to obtain a 20 μl volume of gel. The gel was then mounted on a sample holder (in this case a 2 mm diameter 3D gel rod (same composition as the solidifying solution, prefabricated into a holding shape mounted on a 3D printed plastic surface) at room temperature and incubated for 10 min at 4 °C. Cool for minutes to gel. Sample gel on holder was immersed in RI compatible solution for 5-30 minutes to prepare for imaging.

Imaging:
Samples were transferred to a light sheet imaging chamber and imaged (10X objective). For a 20 μl gel sample, it took approximately 3 minutes to image a single channel at 10X resolution and 4 μm z-step, but the imaging time decreases the gel volume. (increasing cell density), decreasing the imaging resolution (lower magnification objective or higher z-step), or optimizing microscope settings such as z-step, and as known to those skilled in the art. It can be reduced by other means.

2A-2C show images obtained from the light sheet imaging chamber.

Stained leukocyte nuclei are shown in blue-green. In Figure 2A, the image represents the entire 20 μl gel sample viewed from above. FIG. 2A is a 100 μm stack reduced image of a 25×4 μm image. In Figure 2B, the image represents the entire 20 μl gel sample from the side view. In Figure 2C, the image has been magnified to capture individual leukocytes in more detail, and the small horizontal line in the lower left corner is equal to the 20 μm scale.

Example 3 - Cell Suitability Cells from the CACO2 cell line obtained from ATCC and from the HL60 cell line also obtained from ATCC were collected and processed as briefly described below. CACO2 cells were cultured and harvested by trypsinization. HL60 is a suspension cell line and does not require trypsinization. CACO2 and HL60 cells were each harvested, washed with PBS, and centrifuged to collect cell pellets.

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

The mixture was shaken for 30 minutes at room temperature. The mixture was centrifuged at 500g for 5 minutes and the supernatant was carefully removed. Cell pellets were washed by resuspending in PBS, centrifuged again at 500 g for 5 min, and the supernatant was discarded. Cells were then resuspended and incubated in 1 ml PI solution:4% PFA (1:1000) for 20 min at 4°C to fix bound antibodies and label cell nuclei. The mixture was centrifuged again at 500g for 5 minutes and the supernatant was discarded.

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

The mixture was shaken for 30 minutes at room temperature. The mixture was centrifuged at 500 g for 5 min, the supernatant was carefully removed, 1 ml of PBS was added to wash, the mixture was centrifuged again at 500 g for 5 min, and the supernatant was again carefully collected and discarded. Cells were then resuspended and incubated in 1 ml PI solution:4% PFA (1:1000) for 20 min at 4°C to fix bound antibodies and label cell nuclei for 20 min at 4°C. The mixture was centrifuged again at 500g for 5 minutes and the supernatant was again carefully collected and discarded.

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

Imaging:
Both gels with immunolabeled CACO2 and HL60 cells were imaged on a light sheet microscope with a 10x objective as previously described.

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

Example 4 - Multiplexed labeling of CACO2 cells in blood samples Spiking of cancer cells in blood samples Cancer cell counting Studies were 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 rate of cell loss during the cell preparation process.

Cell preparation:
Samples of CACO2 cells were trypsinized, counted, and seeded in appropriate amounts into freshly drawn peripheral blood samples to generate cancer cell-loaded blood samples. Peripheral blood mononuclear cells (PBMC) were collected through a red blood cell lysis approach. Anticoagulated blood collected from humans in EDTA-coated tubes was transferred to 15 ml tubes. Blood 1c. c. For ammonia chloride solution 8c. c. was added to lyse the red blood cells. The lysis process was performed on ice for 15 min, centrifuged, the supernatant was removed, and the collected PBMC cell pellet containing spiked cancer cells was resuspended in 100 μL of PBS and prepared for the next process.

EpCAM (Dako) and CD45 (Dako) antibodies were added to the cell solution at 1:100 and compatible secondary antibodies were added at a 1:2 primary to secondary antibody molar ratio for the labeling process in step 1. I did it. Cell samples were incubated for 1 hour at room temperature to allow antibody binding. The samples were then centrifuged at 500g for 5 minutes to collect 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 antibody and stain the nuclei for 30 minutes. The cells were then centrifuged again to collect the pellet.

Cell pellets were mixed into solidification solution as described above. Mixed PBMC and CACO2 cells were then identified in the sample, confirming the ability to detect multiple cell types using multiple antibody target labels in the sample. Figure 4A shows the complete 3D gel data of multiple labeling of CACO2 in PBMCs, where the EpCAM marker is shown in magenta and PI is shown in blue. Figure 4B shows enlarged stack images of CACO2-EpCAM (magenta), leukocytes-CD45 (green) and PI (blue). Figures 4C and 4D show images of identified CTCs with and without EpCAM (magenta signal) for confirmation.

In these figures, stacked or separated, in 3D images, EpCAM labeling for cancer cell detection is a viable approach. CACO2 shows high EpCAM expression but no CD45 expression, and PBMCs (white blood cells) show variable CD45 expression but no EpCAM expression.

Cell counting:
Trypsinized CACO2 was counted, plated in 2 cc of blood, stained and labeled as described in the table below, and three sets of gels (a, b, and c) were prepared.

Three-dimensional gels were formed and imaged by light sheet microscopy as described in Examples 1, 2, and 3. Each image was denoised and feature 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 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. can.

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

To better visualize the detected results, the 3D imaging data were shown in maximum projection mode, where all 2D images were stacked on top of each other to produce a 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 signal and positive PI signal. The cell detection algorithm uses these parameters to screen all cells and uses the separated and combined signals to output an identified CTC image, as shown in FIG. 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 signal detection algorithm.

The table below shows the final cell counts from three sets of gels, a, b, and c. The results detected were within a reasonable approximation of the number of cells seeded (added).

The total number of cells in the whole gel can be quantified using the total PI positive signal. Dividing the total number of positive cancer cells by the total number of PI signals outputs a ratio. Multiplying this ratio by 1 million gives the number of cancer cells in 1 million PBMCs.

Example 5 - CTC Detection in Cancer Patients An IRB study was conducted to detect the presence of CTCs in the blood of colon cancer patients. This study was designed to evaluate the efficiency of rare cell detection of the 3D liquid biopsy method. Enrolled patients included healthy and diseased subjects. In the examples below, patients with stage II to stage IV colon cancer with metastases to the liver or lungs are presented.

Blood sample preparation:
Anticoagulated blood collected from humans in EDTA-coated tubes was transferred to 15 ml tubes. Peripheral blood mononuclear cells (PBMC) were collected through a red blood cell lysis approach. Blood 2c. c. For ammonia chloride solution 16c. c. was added to lyse the red blood cells. The lysis process was performed on ice for 15 minutes, centrifuged and the supernatant removed. The collected PBMC cell pellet containing spiked cancer cells was resuspended in 100 μL of PBS and prepared for the next process.

Patient A
Step 1 labeling process was performed by adding EpCAM (Dako) and CD45 (Dako) antibodies to the cell solution at 1:100 and matching secondary antibodies at a 1:2 primary to secondary antibody molar ratio. went. Cell samples were incubated for 1 hour at room temperature to allow antibody binding. The samples were then centrifuged at 500g for 5 minutes to collect the pellet. The pellet was washed with 1 ml of PBS, centrifuged again and the supernatant was removed. Next, 1:1000 PI:4% in PFA was added to the pellet to fix the labeled antibody and stain the nuclei for 30 minutes. The cells were then centrifuged again to collect the pellet.

Cell pellets were mixed in 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 x 0.42 μm x 1 μm, with EpCAM markers shown in magenta, CD45 markers in green, and PI in blue. shown. Figure 5B shows a magnified image of EpCAM-positive, CD45-negative, and PI-positive cell targets. When screening for CTC positivity using the above parameters, we were able to detect 195 CTCs in approximately 160,000 PBMCs. This corresponds to 1,218 1CTC per 5 million PBMCs.

Patient B
Step 1 labeling process was performed by adding CK20 (Ventana) and EpCAM (Dako) antibodies to the cell solution at 1:100 and matching secondary antibodies at a 1:2 primary to secondary antibody molar ratio. went. Collection, gelation, and imaging of labeled cell pellets were performed as described above for Patient A. Figure 6A shows two consecutive stacks of 3D gel data (600 μm stack from 1200 μm stack) with a resolution of 0.42 μm x 0.42 μm x 1 μm, where the CK20 marker is shown in yellow and the EpCAM marker is Shown in magenta and PI in blue. Figure 6B shows zoomed-in single cell images of CK20 positive, EpCAM positive, and PI positive cell targets. When positive CTCs were screened using the above parameters, 12 CTCs could be detected in approximately 46,200 PBMCs. This corresponds to 259 CTCs per million PBMCs.

In contrast to other methods of viewing cells in complex samples, such as tissue samples, our method removes lipids or removes other cells to achieve tissue transparency for 3D imaging. There is no need for a transparency process (clearing step) that destroys information. Instead, the method here achieves complete cell sorting and 3D visualization of complete cellular information within a liquid sample by combining gelation and refractive index matching of a cell sample dispersed in three-dimensional space. . It is a novel approach to screen rare or arbitrary cells in liquid biopsy samples using simple antibody tagging.

It will be understood by those skilled in the art that the examples and embodiments described herein do not limit the scope of the invention. This specification, including the examples, is intended to be illustrative only, and various modifications and changes may be made in the invention without departing from the scope or purpose of the invention as defined by the appended claims. It will be obvious to those skilled in the art that this can be done.
Furthermore, although certain details in this disclosure are provided to provide a thorough understanding of the invention as defined by the appended claims, certain embodiments may be practiced without these details. Those skilled in the art will understand this.

Moreover, in certain instances, well-known methods, procedures, or other specific details are set forth to avoid unnecessarily obscuring aspects of the invention as defined by the appended claims. It has not been.

Numbered Embodiments The present disclosure is further directed to the following embodiments.
1. A method for analyzing a biological sample, the method comprising: adding a solidifying agent to a sample containing biological material; creating a solidified sample containing dispersed biological material; A method comprising imaging a solidified sample to identify one or more components of the solidified sample.
2. 2. The method of embodiment 1, wherein the sample is obtained from a liquid blood sample.
3. 3. The method of embodiment 2, wherein the sample is obtained from a liquid blood sample by a process that includes removing red blood cells and platelets from the liquid blood sample.
4. 12. The method as in any one of the preceding embodiments, further comprising labeling a biomolecular target in the sample prior to adding the solidifying agent.

5. 5. The method of embodiment 4, wherein the biomolecular target is a nucleic acid, protein, or vesicle.
6. 6. The method of embodiment 4 or 5, wherein the labeling comprises contacting the sample with a molecular probe.
7. 7. The method of embodiment 6, wherein the molecular probe is an antibody.

8. 7. The method of embodiment 6, wherein the molecular probe is a fluorescent dye.
9. 7. The method of embodiment 6, wherein the molecular probe is a nucleic acid probe.
10. The method according to any one of embodiments 1-9, wherein the biomolecular target is an extracellular molecular target.
11. The method according to any one of embodiments 1-9, wherein the biomolecular target is an intracellular molecular target.
12. The method of any one of the preceding embodiments, further comprising transferring the sample to a sample holder after adding the solidifying agent.
13. 13. The method of embodiment 12, further comprising shaking the sample in a sample holder.
14. 13. The method of embodiment 12, further comprising vibrating the sample within a sample holder.
15. A method as in any one of the preceding embodiments, wherein the solidified sample is a solid block suitable for imaging.
16. as in any one of the preceding embodiments, wherein, prior to imaging, the solidified sample containing the dispersed biological material is transferred into a solution to ensure the desired transparency of the sample for imaging; the method of.
17. 17. The method of embodiment 16, wherein the solution is an index matching solution.
18. A method as in 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. A method according to any one of the preceding embodiments, wherein the imaging is performed with a fluorescence microscope.
20. 20. The method of embodiment 19, wherein the imaging is performed by light sheet fluorescence microscopy.
21. A method as in any one of the preceding embodiments, wherein the solidifying agent is a gelling agent and the solidified sample is a gel sample.
22. A method according to any one of the preceding embodiments, wherein, before step (a), the biological sample is subjected to a fixation procedure.
23. 23. The method of embodiment 22, wherein the fixation step comprises incubating the biological sample in a fixative.
24. 24. The method of embodiment 23, wherein the fixative comprises glutaraldehyde or formaldehyde.
25. A method according to any one of the preceding embodiments, wherein step (c) allows identification of single cells in the solidified sample.

Claims (40)

A method of analyzing a liquid sample containing biological material for one or more target components, the method comprising:
(a) adding a solidifying agent to a sample obtained from a liquid sample containing said biological material;
(b) creating a solidified sample containing dispersed biological material;
(c) imaging the solidified sample to identify one or more target components in the dispersed biological material;
method including. Further, (i) labeling the sample obtained from the liquid sample in step (a) with one or more probes for the one or more target components before adding the solidifying agent: 2. The method of claim 1, further comprising: and/or (ii) labeling the solidified sample from step (b) with one or more probes for one or more target components. 1 . The method of claim 1 , further comprising the step of labeling the sample obtained from the liquid sample in step (a) with one or more probes for the one or more target components before adding the solidifying agent. The method described in. 2. The method of claim 1, further comprising introducing an index matching material into the solidified sample. 2. The method of claim 1, wherein the liquid sample is a liquid blood sample. 6. The method of claim 5, wherein the sample is obtained from the liquid blood sample by a process that includes removing red blood cells and platelets from the liquid blood sample. 7. The method of claim 6, wherein the sample comprises peripheral blood mononuclear cells (PBMC) cells isolated from the liquid blood sample. 2. The method of claim 1, wherein the one or more target components include a nucleic acid, a protein, a virus, or a vesicle. The labeling step comprises contacting the sample obtained from the liquid sample in step (a) with a molecular probe; and/or contacting the solidified sample obtained in step (b) with a molecular probe. , the method according to claim 2. 10. The method of claim 9, wherein the molecular probe is an antibody. 10. The method of claim 9, wherein the molecular probe is a fluorescent dye. 10. The method of claim 9, wherein the molecular probe is a nucleic acid probe. 2. The method of claim 1, wherein the one or more targeting components include an extracellular target. 2. The method of claim 1, wherein the one or more target components include cellular or intracellular targets. 2. The method of claim 1, further comprising transferring the sample to a sample holder. 16. The method of claim 15, further comprising shaking or vibrating the sample in the sample holder. 2. The method of claim 1, wherein the solidified sample is a solid or gel block suitable for imaging. 2. The method of claim 1, wherein the imaging is performed by fluorescence microscopy. 2. The method of claim 1, wherein the imaging is performed by light sheet fluorescence microscopy. 2. The method of claim 1, further comprising a fixation step on the sample. 21. The method of claim 20, wherein the fixation step comprises incubating the sample or the solidified sample in a fixative. 22. The method of claim 21, wherein the fixative comprises glutaraldehyde, formaldehyde, epoxy, or a mixture of any two or more thereof. 2. The method of claim 1, wherein the imaging identifies the presence or absence of particular cell types in the solidified sample. A method of analyzing a liquid blood sample for the presence of rare circulating cells, the method comprising:
(a) labeling a sample containing isolated peripheral blood mononuclear cells (PBMC) obtained from a liquid blood sample with one or more probes for rare circulating cells;
(b) adding a solidifying agent to a labeled sample containing peripheral blood mononuclear cells (PBMC);
(c) creating a solidified sample containing dispersed peripheral blood mononuclear cells (PBMC);
(d) optionally introducing a refractive index matching material into the solidified sample to provide an optically cleared solidified sample with a refractive index suitable for imaging; and (e) one or more of the solidified sample from step (c) or the optionally cleared solidified sample from step (d) in order to determine the presence of the probe and thereby the presence of rare circulating cells in the liquid blood sample. The process of imaging;
method including. 25. The method of claim 24, wherein the labeling further comprises adding a probe for leukocytes. 25. The method of claim 24, wherein the one or more probes recognize a cancer-specific antigen or a tumor-specific DNA or RNA sequence. 27. The method of claim 26, wherein the one or more probes are selected from antibodies or nucleic acid probes. 28. The method of claim 27, wherein the one or more probes provide for detection of one or more of EpCAM, HER2, CDX2, CK20, CK19, PD/PDL-1 and EGFR antigens or corresponding nucleic acid sequences. . 25. The method of claim 24, wherein the solidification agent comprises a component selected from low melting agarose, agarose, and a hydrogel precursor. 25. The method of claim 24, further comprising introducing an index matching material into the solidified sample. 25. Producing a solidified sample comprises adding a solidifying agent above room temperature to the sample and allowing the sample to achieve a reduced temperature such that it becomes a solid gel. the method of. 25. The method of claim 24, wherein creating the solidified sample includes adding a reagent to the hydrogel precursor to induce gelation. 25. The method of claim 24, wherein the optically cleared gelled sample is introduced into a sample holder immersed in a solution containing an index matching material. 25. The method of claim 24, wherein imaging is performed using light sheet microscopy. 25. The method of claim 24, wherein the rare circulating cells are circulating tumor cells. A solidified sample suitable for imaging, comprising dispersed biological material obtained from a liquid biopsy and immobilized within the solidified sample. 37. The solidified sample of claim 36, further comprising a probe for one or more target components in the liquid biopsy. 37. The solidified sample of claim 36, wherein the liquid biopsy is a blood sample and the biological material comprises peripheral blood mononuclear cells (PBMC). 39. The clotted sample of claim 38, further comprising rare circulating cells. 40. The solidified sample of claim 39, wherein the rare circulating cells are selected from circulating tumor cells, circulating epithelial cells, and circulating endothelial cells.