Methods and devices for the isolation of tumor cells

Abstract

This specification describes methods and compositions for isolating cancer cells using positively charged surfaces such as beads, microparticles, and capillaries. These isolation methods and compositions can be used in the diagnosis, treatment, and therapeutic preparations of cancer.

Description

[Technical Field]

[0001] This disclosure generally relates to methods for binding to cancer cells, as well as to functionalized microspheres and surfaces (e.g., capillaries). More specifically, this disclosure relates to isolation matrices of functionalized microspheres or functionalized capillaries for isolating cancer cells from biological samples, and to columns containing such isolation matrices. The methods and compositions disclosed herein can, among other things, be used to diagnose or treat cancer. [Background technology]

[0002] Tumors are abnormal growths of body tissue and can be cancerous (malignant) or non-cancerous (benign). Tumors, especially cancerous tumors, pose a serious threat to human health, and their early detection is crucial for effective treatment or cure. However, conventional tumor detection methods struggle to detect cancer before symptoms appear or at an early stage of metastasis. For example, conventional methods fail to identify approximately 40% of cancer patients who require more or intensified treatment. It is also important to detect any early signs of cancer spread after cancer treatment in order to assess the effectiveness of treatment and whether and what kind of follow-up treatment is needed. Conventional cancer detection techniques, such as X-ray imaging and nuclear magnetic resonance (NMR) imaging, cannot provide reliable information for the above critical applications.

[0003] Recent studies and clinical trials have shown that cancer invasion into the human body can occur very early in tumor development. Early detection and early systemic therapy would lead to a reduction in cancer mortality. Metastasis initiated by tumor cells transported from the primary tumor to vital distant organs via the circulation is known to be a major cause of cancer-related death. Early diffusion of tumor cells into lymph nodes or bone marrow in the peripheral blood is called circulating tumor cells (CTCs). CTCs can still be present in the peripheral blood of a patient even after the removal of the primary tumor.

[0004] CTCs are essential in establishing metastasis, and their detection and isolation are important tools for assessing the aggressiveness of a given tumor and its potential for subsequent growth in distant organs. CTC detection may also represent the early and initial detection of cancer in a patient. Specific and highly sensitive detection and isolation of CTCs can be used to identify the initial presence of cancer, as well as to assess the overall state of cancer development or metastasis, survivability, and treatment response. CTC removal from a patient's blood may also be useful for treating certain cancers.

[0005] Current CTC detection and isolation methods based on molecular biomarkers are hampered by limitations in method specificity and sensitivity. Therefore, novel targeting strategies are needed to explore other biophysical properties of cancer cells. Unlike current CTC detection methods based on molecular biomarkers, this disclosure can be applied independently to the detection of any type of cancer cell. [Overview of the project] [Problems that the invention aims to solve]

[0006] This disclosure provides functionalized microspheres and surfaces (e.g., capillaries) that can bind to cancer cells based on the negative charge of the cancer cells and the opposite positive charge of the functional groups. Microspheres and capillaries, which may be made from glass or other materials as described herein, can be functionalized. [Means for solving the problem]

[0007] In a first embodiment, the present disclosure provides a method for isolating cancer cells from a biological sample, comprising: a) passing a biological sample containing cancer cells through an isolation matrix containing functionalized microspheres; and b) collecting the biological sample flowing through the isolation matrix into a first aliquot, wherein the cancer cells are bound to the isolation matrix.

[0008] In some embodiments disclosed herein, the functionalized microspheres are functionalized with positively charged functional groups. In some embodiments, the functionalized microspheres are functionalized with amines, polyethyleneimine (PEI), and / or guanidine groups. In some embodiments, the microspheres comprise glass, polymers, or resins. In some embodiments, the microspheres have a diameter of 500 μm to 600 μm.

[0009] In embodiments disclosed herein, the biological sample includes blood. In some embodiments, the biological sample is obtained from a subject having or suspected of having cancer. In some embodiments, the cancer includes hematological cancer or solid tumors. Some embodiments disclosed herein include eluting cancer cells bound to an isolation matrix into a second aliquot. Some embodiments include administering a first aliquot to a subject and returning it. Some embodiments include detecting the presence or absence of cancer cells in the biological sample. Some embodiments include lysing the eluted cancer cells to obtain a lysate. Some embodiments include incorporating the lysate into a cancer vaccine. Some embodiments include determining the mRNA copy number from the cancer cell lysate. In some embodiments, the mRNA copy number is determined by quantitative RT-PCR. Some embodiments include calculating the number of cancer cells bound to functionalized microspheres.

[0010] In one embodiment, the disclosure includes a cancer vaccine prepared by a process, which includes a) contacting a biological sample containing cancer cells with a positively charged surface, the cancer cells binding to the positively charged surface; b) collecting the biological sample flowing through an isolation matrix into a first aliquot; c) lysing the cancer cells to obtain a cancer cell lysate in a second aliquot; and d) incorporating the cancer cell lysate into yeast cell wall particles (YCWP).

[0011] In one aspect, the present disclosure provides a method of treating cancer in a patient, the method comprising extracting cancer cells from the patient's blood by passing blood through an isolated matrix comprising functionalized microspheres that bind to cancer cells.

[0012] In some embodiments disclosed herein, the functionalized microspheres are functionalized with a positively charged functional group. In some embodiments, the functionalized microspheres are functionalized with an amine, polyethyleneimine (PEI), and / or a guanidine group. In some embodiments, the microspheres comprise glass, a polymer, or a resin. In some embodiments, the microspheres have a diameter of 500 μm to 600 μm.

[0013] In some embodiments disclosed herein, the patient has a blood cancer or a malignant cancer.

[0014] Some embodiments disclosed herein include eluting the bound cancer cells from the isolated matrix. Some embodiments disclosed herein include lysing the eluted cells to obtain a lysate and incorporating the lysate into a cancer vaccine for treating the patient.

[0015] In one aspect, the present disclosure provides a method of detecting cancer cells in a patient, the method comprising: a) passing a biological sample from the patient through an isolated matrix of functionalized microspheres, wherein the functionalized microspheres bind to cancer cells; b) eluting the cancer cells from the matrix; and c) detecting the presence or absence of cancer cells in the biological sample.

[0016] In some embodiments disclosed herein, the patient has a blood cancer or a malignant cancer.

[0017] In some embodiments disclosed herein, the functionalized microspheres are functionalized with positively charged functional groups. In some embodiments, the functionalized microspheres are functionalized with amines, polyethyleneimine (PEI), and / or guanidine groups. In some embodiments, the microspheres comprise glass, polymer, or resin. In some embodiments, the microspheres have a diameter of 500 μm to 600 μm.

[0018] In one aspect, the present disclosure provides a cancer cell isolation matrix comprising functionalized microspheres comprising positively charged functional groups, wherein the functionalized microspheres bind to cancer cells. In some embodiments disclosed herein, the functionalized microspheres are functionalized with positively charged functional groups. In some embodiments, the functionalized microspheres are functionalized with amines, polyethyleneimine (PEI), and / or guanidine groups. In some embodiments, the microspheres comprise glass, polymer, or resin. In some embodiments, the microspheres have a diameter of 500 μm to 600 μm.

[0019] In one aspect, the present disclosure provides a kit for purifying cancer cells from a biological sample, the kit comprising functionalized microspheres that bind to cancer cells derived from the biological sample.

[0020] In one embodiment, the present disclosure provides a method for preparing a column for isolating cancer cells, comprising: a) preparing an isolation matrix comprising functionalized microspheres; and b) depositing the isolation matrix into a container having an inlet and an outlet. In some embodiments of the herein disclosure, the functionalized microspheres are functionalized with positively charged functional groups. In some embodiments, the functionalized microspheres are functionalized with amines, polyethyleneimine (PEI), and / or guanidine groups. In some embodiments, the microspheres comprise glass, polymer, or resin. In some embodiments, the microspheres have a diameter of 500 μm to 600 μm.

[0021] In some embodiments, preparing functionalized microspheres involves coating the microspheres with a 5% 3-aminopropyltriethoxysilane solution. In some embodiments, preparing functionalized microspheres involves coating the microspheres with a 5% silane coupling agent. In some embodiments, preparing functionalized microspheres involves coating the microspheres with polyethyleneimine (PEI). In some embodiments, preparing functionalized microspheres involves coating the microspheres with aminoguanidine. In some embodiments, depositing an isolation matrix involves depositing 0.1 to 1 ml of functionalized microspheres into a container.

[0022] In one embodiment, the present disclosure provides a method for isolating cancer cells from a biological sample, comprising: a) passing the biological sample containing cancer cells through a functionalized capillary; and b) collecting the biological sample flowing through the capillary into a first aliquot, wherein the cancer cells are bound to the capillary. In some embodiments, the capillary is functionalized with an amine, polyethyleneimine (PEI), and / or guanidine group.

[0023] In one embodiment, the disclosure provides a method for isolating cancer cells from a biological sample, comprising contacting the biological sample containing cancer cells with a positively charged surface, wherein the cancer cells bind to the positively charged surface. In some embodiments, the positively charged surface is functionalized with amines, polyethyleneimine (PEI), and / or guanidine groups. In some embodiments, the positively charged surface is made from glass, polymer, or resin. In some embodiments, the positively charged surface is selected from beads, microparticles, capillaries, blood collection tubes, microscope slides, and microscope slide coverslips. In some embodiments, the biological sample includes blood. In some embodiments, the cancer includes hematological cancer or solid tumors. In some embodiments, the biological sample is obtained from a subject having or suspected of having cancer.

[0024] In some embodiments, the method further includes detecting the presence or absence of cancer cells in a biological sample. In some embodiments, the method further includes lysing cancer cells to obtain a cancer cell lysate. In some embodiments, the method further includes incorporating the cancer cell lysate into a cancer vaccine. In some embodiments, the method further includes calculating the number of cancer cells bound to a positively charged surface.

[0025] In one embodiment, the present disclosure provides a cancer vaccine comprising yeast cell wall particles (YCWP) and a cancer cell lysate prepared as described herein.

[0026] In some embodiments, YCWP is modified by capping with a silicate. In some embodiments, the silicate is selected from the group comprising tetraethyl orthosilicate, tetramethyl orthosilicate, tetrapropyl orthosilicate, and tetrabutyl orthosilicate. Some embodiments include one or more adjuvants, excipients, and preservatives.

[0027] In one embodiment, the present disclosure provides a method for delivering a vaccine to a subject, which includes administering a vaccine as disclosed herein to the subject.

[0028] In one embodiment, the Disclosure provides a method for treating or preventing cancer, which includes administering a vaccine, such as those disclosed herein, to a subject in need thereof.

[0029] Some embodiments involve administering the vaccine subcutaneously, orally, or intravenously. Some embodiments involve administering the vaccine into the dermis of the target area.

[0030] In one embodiment, the disclosure provides a method for treating cancer in a patient, comprising: extracting cancer cells from the patient's blood by contacting the patient's blood with a positively charged surface, wherein the cancer cells bind to the positively charged surface; and returning the blood to the patient after contact with the positively charged surface. In some embodiments, the positively charged surface is functionalized with amines, polyethyleneimine (PEI), and / or guanidine groups. In some embodiments, the positively charged surface is made from glass, polymer, or resin. In some embodiments, the positively charged surface is selected from beads, microparticles, capillaries, blood collection tubes, microscope slides, and microscope slide coverslips. In some embodiments, the biological sample includes blood. In some embodiments, the cancer is hematological cancer or includes solid tumors. In some embodiments, the biological sample is obtained from a subject having or suspected of having cancer.

[0031] In one embodiment, the Disclosure provides a method for detecting cancer cells in a subject, comprising: isolating cancer cells from a biological sample according to the method disclosed herein; and detecting the presence or absence of cancer cells in the biological sample. In some embodiments, cancer is a blood cancer, a solid tumor, or a malignant cancer.

[0032] In one embodiment, the disclosure provides a device for isolating cancer cells, comprising a positively charged surface containing an amine group, polyethyleneimine (PEI), a guanidine group, or any combination thereof. In some embodiments, the positively charged surface is made from glass, a polymer, or a resin. In some embodiments, the positively charged surface is selected from beads, microparticles, capillaries, blood collection tubes, microscope slides, and microscope slide covers.

[0033] The following drawings and detailed descriptions are illustrative and descriptive, but are not intended to be limiting. [Brief explanation of the drawing]

[0034] [Figure 1] Bright-field and GFP images of the SW620 colon cancer cell line before and after passing approximately 1.0 × 10⁵ cells through a column prepared with 0.5 ml of 5% 3-aminopropyltriethoxysilane bead matrix are shown (A-D). Colon cancer cells are visible under green fluorescence (E-H). All cancer cells were captured by the bead matrix, as indicated by the absence of stained cells in F-G. [Figure 2] Bright-field and GFP images of the SW620 colon cancer cell line before and after passing approximately 1.0 × 10⁵ cells through a column prepared with 0.5 ml of 10% 3-aminopropyltrimethoxysilane bead matrix are shown (A-D). Colon cancer cells are visible under green fluorescence (E-H). Most cancer cells were captured by the bead matrix, as shown by the very few stained cells in F-G. [Figure 3]Bright-field and GFP images of the T47D breast cancer cell line before and after passing approximately 1.0 × 10⁵ cells through a column prepared with 0.5 ml of 5% 3-aminopropyltriethoxysilane bead matrix are shown (A-D). Breast cancer cells are visible under green fluorescence (E-H). Most cancer cells were captured by the bead matrix, as shown by the very few stained cells in F-G. [Figure 4] Bright-field and GFP images of the T47D breast cancer cell line before and after passing approximately 1.0 × 10⁵ cells through a column prepared with 0.5 ml of 10% 3-aminopropyltrimethoxysilane bead matrix are shown (A-D). Breast cancer cells are visible under green fluorescence (E-H). Most cancer cells were captured by the bead matrix, as shown by the very few stained cells in F-G. [Figure 5] Bright-field and GFP images of the A549 lung cancer cell line before and after passing approximately 1.0 × 10⁵ cells through a column prepared with 0.5 ml of 5% 3-aminopropyltriethoxysilane bead matrix are shown (A-D). Lung cancer cells are visible under green fluorescence (E-H). Most cancer cells were captured by the bead matrix, as shown by the very few stained cells in F-G. [Figure 6] Bright-field and GFP images of the A549 lung cancer cell line before and after passing approximately 1.0 × 10⁵ cells through a column prepared with 0.5 ml of 10% 3-aminopropyltrimethoxysilane bead matrix are shown (A-D). Lung cancer cells are visible under green fluorescence (E-H). Most cancer cells were captured by the bead matrix, as shown by the very few stained cells in F-G. [Figure 7] Bright-field and GFP images of the A549 lung cancer cell line before and after passing approximately 1.0 × 10⁵ cells through a column prepared with 0.8 ml of 5% 3-aminopropyltriethoxysilane bead matrix are shown (A-D). Lung cancer cells are visible under green fluorescence (E-H). Most cancer cells were captured by the bead matrix, as shown by the very few stained cells in F-G. [Figure 8] Bright-field and GFP images of the A549 lung cancer cell line before and after passing approximately 3.0 × 10⁴ cells through a column prepared with 0.5 ml of 5% 3-aminopropyltriethoxysilane bead matrix are shown (A-D). Lung cancer cells are visible under green fluorescence (E-H). Most cancer cells were captured by the bead matrix, as shown by the very few stained cells in F-G. [Figure 9] Bright-field and GFP images of CCRF-SB acute lymphoblastic leukemia (ALL) cells before and after passing approximately 1.0 × 10⁵ cells through a column prepared with 0.5 ml of 5% 3-aminopropyltriethoxysilane bead matrix are shown (A-D). ALL cells are visible under green fluorescence (E-H). All cancer cells were captured by the bead matrix, as indicated by the absence of stained cells in F-G. [Figure 10] Bright-field and GFP images of CCRF-SB acute lymphoblastic leukemia (ALL) cells before and after passing approximately 1.0 × 10⁵ cells through a column prepared with 0.5 ml of 10% 3-aminopropyltrimethoxysilane bead matrix are shown (A-D). ALL cells are visible under green fluorescence (E-H). Most cancer cells were captured by the bead matrix, as shown by the very few stained cells in F-G. [Figure 11] Microscopic images (A-D) of a mixture of 1.0 × 10⁵ colon cancer cells and 400 μl of non-cancerous WBCs before and after passing through a 5% 3-aminopropyltriethoxysilane bead matrix are shown. Colon cancer cells are visible under green fluorescence (E-H). All tumor cells were captured by the bead matrix, as indicated by the absence of stained cells in F-G. The presence of cells in B indicates that non-cancerous WBCs were not captured by the bead matrix. [Figure 12]Microscopic images (A-D) of a mixture of 1.0 × 10⁶ colon cancer cells and 200 μl of non-cancerous WBCs before and after passing through a 3-aminopropyltriethoxysilane bead matrix are shown. Colon cancer cells are visible under green fluorescence (E-H). All tumor cells were captured by the bead matrix, as indicated by the absence of stained cells in F-G. The presence of cells in B indicates that non-cancerous WBCs were not captured by the bead matrix. [Figure 13] This image shows breast cancer cells trapped on positively charged glass beads within a 1.5 mm mini-column made of clear glass. The cells can be clearly visualized, analyzed, and evaluated using fluorescently labeled antibody markers. [Figure 14] Examples of columns used in the embodiments described herein are shown. [Figure 15] This shows a standard curve of cell number versus protein concentration established using the CCRF-SB cell line, which is used to determine bead capacity. [Figure 16] The number of PCR cycles required to detect (A) beta-actin and (B) Her2 in unbound PBMCs (PBMCs), bead-bound SKOV3 ovarian cancer cells (beads), and unbound SKOV3 ovarian cancer cells (SKOV3) is shown. Error bars indicate the standard deviation for n=3. [Figure 17] This shows the standard cycle count versus cell count curve created using SK-BR-3 cells. "Cells Only" is the standard curve showing SKOV3 cells versus CQ value. "Beads with Cells" shows the number of cells bound to the beads versus the CQ value. [Figure 18] One embodiment of a method used to quantify the number of cells in a sample (white box), as well as a method for detecting and amplifying circulating cancer cells in a patient (white and gray boxes), is shown. [Figure 19]The graph shows the number of PCR cycles required to detect (A) beta-actin, (B) Her2, and (C) CD45 in the sample before incubation with beads (total PBMCs), bead-conjugated breast cancer cells from patient samples (beads with cells / beads), and remaining cells from patient samples that did not adhere to beads (after isolation). Error bars indicate the standard deviation for n=3. [Modes for carrying out the invention]

[0035] This disclosure generally relates to the fields of cancer diagnosis and treatment, each of which utilizes the isolation of cancer cells from biological samples (e.g., blood, plasma, or serum of a subject who has or is suspected of having cancer). More specifically, this disclosure provides methods, compositions, and kits suitable for isolating cancer cells from biological samples. The disclosed methods, compositions, and kits are based on the ability of positively charged surfaces, such as microparticles (e.g., glass microbeads) or capillaries, to capture negatively charged cancer cells.

[0036] definition Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this technology belongs.

[0037] As used herein, the term “about” refers to a value within 10% above or below the stated numerical expression and the stated value. For example, the term “about 5 nM” refers to both the specified value of 5 nM and the disclosure of a range of 4.5 nM to 5.5 nM.

[0038] As used herein, the singular forms "a," "an," and "the" refer to multiple objects unless otherwise explicitly stated. For example, "microparticle" refers to two or more microparticles, "polynucleotide" refers to two or more polynucleotides, and "cell" refers to two or more cells.

[0039] As used herein, the terms “particle,” “bead,” or “sphere” are interchangeable and may include any shape or composition. Particles, beads, or spheres may have any diameter greater than 1 mm. In some embodiments, particles, beads, or spheres may have diameters greater than 1 mm, 1.5 mm, 2 mm, 2.5 mm, or 3 mm. In some embodiments, particles, beads, or spheres may have diameters between 1 mm and 10 mm. In some embodiments, particles, beads, or spheres may have diameters between 1.5 mm and 5 mm. In some embodiments, particles, beads, or spheres may have diameters between 1.7 mm and 2.5 mm. In some embodiments, particles, beads, or spheres may have average diameters of about 1 mm, about 1.5 mm, about 1.7 mm, about 2 mm, about 2.5 mm, about 3 mm, about 5 mm, about 7.5 mm, about 10 mm, about 15 mm, or about 20 mm. In some embodiments, particles, beads, or spheres are solid. In some embodiments, the particles, beads, or spheres are hollow. In some embodiments, the particles, beads, or spheres are porous or non-porous. In some embodiments, the particles, beads, or spheres comprise one or more different materials. In some embodiments, the particles, beads, or spheres comprise or consist of glass, nylon, hydrogel, ceramic, metal, and / or any other suitable material. In some embodiments, the particles, beads, or spheres are magnetic. In some embodiments, the particles, beads, or spheres are coated with a metallic surface such as nickel. In some embodiments, the particles, beads, or spheres are functionalized.

[0040] As used herein, the term “microparticles” refers to particles of any shape or composition having a diameter of 1 μm to 1,000 μm. In some embodiments, microparticles have a diameter of 200 μm to 800 μm. In some embodiments, microparticles have a diameter of 400 μm to 600 μm. In some embodiments, microparticles have an average diameter of about 1 μm, about 100 μm, about 200 μm, about 300 μm, about 400 μm, about 500 μm, about 600 μm, about 700 μm, about 800 μm, about 900 μm, or about 1,000 μm. In some embodiments, microparticles are solid. In some embodiments, microparticles are hollow. In some embodiments, microparticles are porous or non-porous. In some embodiments, microparticles comprise one or more different materials. In some embodiments, the microparticles include or consist of glass, nylon, hydrogel, ceramic, metal, and / or any other suitable material. In some embodiments, the microparticles are magnetic. In some embodiments, the microparticles are coated with a metal surface such as nickel. In some embodiments, the microparticles are functionalized. In some embodiments, the microparticles may be microspheres or nanoparticles.

[0041] As used herein, the term “matrix” refers to a collection of microparticles having an average diameter of 1 μm to 1,000 μm. In some embodiments, the average diameter of the microparticles is 200 μm to 800 μm. In some embodiments, the average diameter of the microparticles is 400 μm to 600 μm. In some embodiments, the microparticles have an average diameter of about 1 μm, about 100 μm, about 200 μm, about 300 μm, about 400 μm, about 500 μm, about 600 μm, about 700 μm, about 800 μm, about 900 μm, or about 1,000 μm. In some embodiments, the matrix comprises a mixture of two or more microparticles of different sizes. For example, in some embodiments, the matrix may include microparticles having an average diameter of about 1 μm and microparticles having an average diameter of 500 μm to 600 μm. In some embodiments, spaces exist between the microparticles of the matrix. In some embodiments, the spaces have a maximum diameter of up to 500 μm. In some embodiments, the space has a maximum diameter of up to 250 μm. In some embodiments, the space has a maximum diameter of up to 125 μm. In some embodiments, the space has a maximum diameter of up to 50 μm. In some embodiments, the matrix may contain particles, beads, or spheres having an average diameter greater than 1 mm.

[0042] As used herein, the term “column” refers to a structure or device that contains a matrix. The column can be any shape or size suitable for holding the matrix. In some embodiments, the column is a syringe. In some embodiments, the column includes an inlet and an outlet. The inlet is the point of entry of a sample, solution, buffer, or reagent into the column. The inlet can be an opening into the column or an opening in a conduit leading directly or indirectly to the column. The outlet is an opening through which the sample, sample component, or reagent exits the column. The sample component and reagent exiting the chamber can be waste, i.e., sample component that will not be used further, or sample component or reagent to be recovered, such as a reusable reagent or target cells to be further analyzed or manipulated. The outlet can be an opening in the column adjacent to a collection container or an opening in a conduit leading directly or indirectly from the column to a collection container. In some embodiments, the column may be connected to an automated system that controls the flow of cells and other reagents through the column and out of the column into one or more collection containers. The column may include additional components such as a mesh or filter to prevent matrix loss from the outlet or outlet clogging.

[0043] As used herein, the term “functionalization” refers to any process of modifying a material by introducing physical, chemical, or biological properties that are different from those originally found on the material. Generally, functionalization involves introducing functional groups into / on a material. As used herein, a functional group is a specific group of atoms within a molecule responsible for characteristic chemical reactions of those molecules. As used herein, a functional group may be positively or negatively charged. In some embodiments, positively charged functional groups may include amines, aldehydes, polyethyleneimines (PEIs), and / or guanidine groups. In some embodiments, functionalization may include coupling agents for attaching the groups to microparticles.

[0044] As used herein, the term “sample” refers to a biological sample. In some embodiments, the term “sample” refers to a clinical sample obtained from a patient. In embodiments, a sample is obtained from a biological source (i.e., a “biological sample”) such as tissue, body fluid, or microorganisms collected from a subject. Sample sources include, but are not limited to, mucus, sputum (processed or unprocessed), bronchoalveolar lavage (BAL), bronchial lavage (BW), blood, body fluid, cerebrospinal fluid (CSF), urine, plasma, serum, or tissue (e.g., biopsy material). In some embodiments, a sample may be a mixture of several different cell types or may consist of a single cell type. In some embodiments, a solid sample, such as a tissue biopsy, may be prepared into a fluid sample by, for example, chemical, enzymatic, or physical dissociation of cells. In some embodiments, a sample may be cultured cells or cell lines. In some embodiments disclosed herein, the cell line may be a colon cancer cell line such as SW620 cells, a breast cancer cell line such as T47D cells, a lung cancer cell line such as A549 cells, or an acute lymphoblastic leukemia cell line such as CCRF-SB. In some embodiments, the cells may be leukocytes (WBCs) purified from a blood sample.

[0045] As used herein, the “cell surface charge” or “static charge” of a cell refers to the net positive, negative, or neutral charge on the cell surface. It has recently been discovered that cancer cells are negatively charged on their cell surface. The disclosures herein utilize the net negative charge on the surface of cancer cells to isolate them from biological samples.

[0046] As used herein, the terms “cancer” and “tumor” are interchangeable and refer to the presence of cells that have characteristics typical of cancer-causing cells, such as uncontrolled growth, immortality, metastatic ability, rapid growth and proliferation rates, and certain characteristic morphological features. Cancer cells are often in the form of tumors, but such cells can exist alone in an animal or can be non-tumor-forming cancer cells. As used herein, the term “cancer” includes precancerous conditions and malignant cancers. Examples of cancers may include cancers of the blood, prostate, breast, colon, brain, lung, head and neck, ovaries, bladder, kidneys and testes, melanoma, liver, pancreas, and other gastrointestinal tract. In some embodiments, cancer may be circulating tumor cells (CTCs), i.e., tumor or cancer cells that circulate in the vascular system, lymphatic vessels, or other fluids. CTCs include, but are not limited to, leukemia cells or cells detached from a primary tumor.

[0047] As used herein, “control” is an alternative sample used in an experiment for comparative purposes. A control may be “positive” or “negative.” As used herein, “control cell sample” or “reference cell sample” refers to cells derived from a control sample or reference sample. In certain embodiments, a reference cell sample or control cell sample is wild-type or non-cancerous cells. In certain embodiments, a reference cell sample is purified or isolated (e.g., it is removed from its natural state). In other embodiments, a reference cell sample is from a non-tumor sample, e.g., a blood control, tumor-adjacent normal tissue (NAT), or any other non-cancerous sample derived from the same or different subject.

[0048] As used herein, the term “concentrate” means to increase the relative concentration of one sample component in one sample relative to other sample components (which may result in a decrease in the concentration of other sample components), or to increase the absolute concentration of one sample component. For example, as used herein, “concentrating” cancer cells from a sample includes increasing the proportion of cancer cells to all cells or other components in the sample; concentrating cancer cells in a blood sample may mean increasing the concentration of cancer cells in the sample (e.g., by reducing the volume of the sample), or increasing the proportion of cells that are cancerous among the cells present by decreasing the concentration or number of other cellular components in the blood sample; and “concentrating” cancer cells in a sample may mean increasing the concentration of cancer cells in the sample, such as by reducing the volume of the sample or by reducing the number of “non-cancerous” cells in the sample.

[0049] As used herein, the term “isolation” refers to the process by which one or more components of a sample are spatially separated from one or more other components of the sample. Isolation can be carried out such that one or more desired sample components are moved or held in one or more areas of an isolation apparatus, and at least a portion of the remaining components are moved away from the one or more areas to which the one or more desired sample components are moved and / or held, or one or more sample components are held in one or more areas, and at least a portion of the remaining components are removed from said one or more areas. Alternatively, one or more components of a sample can be moved and / or held in one or more areas, and one or more sample components can be removed from said one or more areas. It is also possible to move one or more sample components in one or more areas and move one or more desired sample components or one or more components of a sample in one or more other areas. Isolation can be achieved, for example, by filtration, or by the use of physical, chemical, electrical, or magnetic forces. Non-limiting examples of forces that can be used for separation are electrostatic force, gravity, mass flow, dielectrophoretic force, traveling wave dielectrophoretic force, and electromagnetic force.

[0050] As used herein, the term “capture” refers to a type of isolation in which one or more parts or components of a sample are retained within or on one or more areas of a surface, chamber, tip, bead particles, tube, or any container containing the sample, and the remainder of the sample can be removed from that area. For example, the isolation matrix described herein may be used to capture cancer cells from a sample.

[0051] As used herein, the terms “subject” and “patient” refer to an organism receiving diagnosis or treatment for a particular disease or condition. Examples of subjects and patients include mammals such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, guinea pigs, members of the Bovidae family (such as cattle, bison, buffalo, and yaks), sheep, and horses, among others. Patients who may be diagnosed using the methods described herein may or may not be presenting symptoms of the disease. Patients who may be diagnosed using the methods described herein may have a genetic predisposition or lifestyle risk to the disease. For example, in some embodiments, the methods described herein may be used for early detection. Patients who may be diagnosed using the methods described herein may have previously recovered from the disease or be in remission. Patients who may be treated using the compositions and methods described herein may have an established disease. In this case, the patient has been diagnosed with the disease and has been presenting symptoms of the disease over a long period of time (e.g., over a course of days, weeks, months, or years). Alternatively, a patient may be presenting symptoms for a particular disease but has not yet been diagnosed with that disease by a physician. Other patients who may be treated with the compositions and methods described herein include patients who have been diagnosed with a disease or disorder, who may or may not be exhibiting symptoms of the disease. Patients who may be treated with the compositions and methods described herein also include patients who have not been diagnosed with a disease or disorder, who may or may not be exhibiting symptoms of the disease, but who have a genetic predisposition or lifestyle risk to the disease.

[0052] As used herein, the terms “to treat” or “treatment” refer to therapeutic actions aimed at inhibiting or slowing (reducing) an undesirable physiological change or impairment. Beneficial or desirable clinical outcomes of treatment include, but are not limited to, symptom relief, reduction of disease severity, stable (i.e., non-worsening) state of disease, delayed or slowed disease progression, improvement or mitigation of symptoms, and (partial or complete) remission, whether detectable or undetectable. Persons requiring treatment include those who already have a condition or impairment, as well as those who are prone to developing a condition or impairment, or whose condition or impairment should be inhibited.

[0053] This disclosure recognizes that the screening, diagnosis, prognosis, and treatment of many conditions, including cancer, may depend on the detection, isolation, and concentration of rare cells from complex samples. Often, concentration can be achieved by one or more separation steps. In particular, this disclosure recognizes that the concentration or isolation of rare cells, including malignant cells, from patient samples, e.g., the isolation of cancerous cells from patient fluid samples, can aid in the detection and typing of such malignant cells, and thus in diagnostic decisions, as well as the development of therapeutic modalities for patients.

[0054] Positively charged surface and isolated matrix This disclosure relies on recent findings that cancer cells are negatively charged on their cell surface. Accordingly, this disclosure provides several positively charged surfaces, including but not limited to microparticles and capillaries, that have been generated and tested for their ability to bind and capture cancer cells.

[0055] The microparticles described herein may be of any shape or size and may be made of any suitable material. For example, the microparticles may include one or more of glass, nylon, hydrogel, ceramic, metal, and / or any other suitable material. These microparticles may be made of a homogeneous material or may be coated with another material such as nickel. For example, in some embodiments, the microparticles are glass microspheres. In embodiments, the microparticles are nickel-coated glass microspheres.

[0056] The capillaries described herein may have any suitable volume, length, and diameter, and may be made of any suitable material. For example, the capillaries may include one or more of glass, nylon, hydrogel, ceramic, metal, and / or any other suitable material. These capillaries may be made of a homogeneous material or may be coated with another material such as nickel. In some embodiments, the capillaries are made of glass. In some embodiments, the capillaries are made of nickel-coated glass.

[0057] In addition to capillaries and microparticles (e.g., microspheres), other surfaces and substrates may also be used for the purposes of this disclosure. For example, beads of a suitable size made from glass, nylon, hydrogel, ceramic, metal, and / or any other suitable material may be functionalized as disclosed herein to provide a positively charged surface on which cancer cells can be bound. Other useful surfaces and substrates include, but are not limited to, beads, meshes, films, membranes, dishes, wells, epitubes, blood collection tubes, bags (e.g., blood bags), beakers, microscope slides, microscope coverslips, microfluidic chambers, pipette tips, stirrers (e.g., magnetic stirrers), or any other surface or substrate on which fluid samples containing cancer cells can pass or be collected.

[0058] Glass microspheres, capillaries, and other surfaces or substrates can be functionalized by the methods described herein to generate a positive charge on the surface of the microsphere or capillary. For example, chemical or electrostatic reactions may be used to functionalize the surface with positively charged moieties such as amines, polyethyleneimine (PEI), and / or guanidine groups. In some embodiments, the surface of microparticles, the surface of a capillary, or other surface may be positively charged without functionalization.

[0059] In some embodiments, amine-coated surfaces such as amine-coated glass beads or capillaries can be prepared. For example, one or more glass beads may be used. In some embodiments, the glass beads may have a diameter of less than 1 mm, e.g., 500-600 μm. In some embodiments, the glass beads may have a diameter greater than 1 mm, e.g., 1.7-2.5 mm. In some embodiments, the glass beads are prepared by etching in 30% NaOH. The etched glass beads are washed five times with ddH2O to ensure the removal of NaOH, and then washed again three times with ethanol to ensure the removal of ddH2O. The beads are then treated with a 5% 3-aminopropyltriethoxysilane solution by gently rotating them at room temperature for 2 hours. The beads are washed again three times with ethanol to ensure the removal of the 3-aminopropyltriethoxysilane solution, and then washed again three times with ddH2O to ensure the removal of ethanol. Next, the beads are frozen at -84°C, then freeze-dried to remove water and form siloxane bonds. The same protocol can be used to coat the inside of capillaries or the surface of other devices such as coverslips, but instead of gently rotating them, the capillaries of the device are placed in a 5% 3-aminopropyltriethoxysilane solution for 2 hours, during which time the capillaries are frequently moved up and down to mix the solution.

[0060] In some embodiments, the amine-coated surface may be prepared with 10% 3-aminopropyltriethoxysilane to produce a 10% 3-aminopropyltriethoxysilane-coated surface. In some embodiments, the amine-coated surface may be prepared with 5% 3-aminopropyltrimethoxysilane to produce a 5% 3-aminopropyltrimethoxysilane-coated surface. In some embodiments, the amine-coated surface may be prepared with 10% 3-aminopropyltrimethoxysilane to produce a 10% 3-aminopropyltrimethoxysilane-coated surface.

[0061] In some embodiments, aldehyde-coated surfaces, such as aldehyde-coated glass beads or capillaries, can be prepared. For example, one or more glass beads may be used. In some embodiments, the glass beads may have a diameter of less than 1 mm, e.g., 500-600 μm. In some embodiments, the glass beads may have a diameter greater than 1 mm, e.g., 1.7-2.5 mm. In some embodiments, the glass beads are prepared by etching in 30% NaOH. The etched glass beads are washed five times with ddH2O to ensure the removal of NaOH, and then washed three more times with ethanol to ensure the removal of ddH2O. A 5% silane coupling agent solution can be prepared by dissolving 2.5 ml of triethoxysilylbutyraldehyde in 50 ml of water in a 4% ethanol solution, and rotating the solution in a plastic tube at room temperature for 5 minutes to allow hydrolysis and form a reactive silanol. The beads are then treated with the 5% silane coupling agent solution by gently rotating at room temperature for 2 hours. The beads are washed three more times with ethanol to ensure removal of the coupling agent, and then three more times with ddH2O to ensure removal of the ethanol. The beads are then frozen at -84°C and then freeze-dried to remove water and form siloxane bonds. The same protocol can be used to coat the inside of capillaries or the surface of other devices such as coverslips, but instead of gently rotating them, the capillaries of the devices are left in a 5% silane coupling agent solution for 2 hours, during which time the capillaries are frequently moved up and down to mix the solution.

[0062] In some embodiments, PEI-coated surfaces such as polyethyleneimine (PEI)-coated glass beads or capillaries can be prepared. For example, one or more glass beads may be used. In some embodiments, the glass beads may have a diameter of less than 1 mm, e.g., 500-600 μm. In some embodiments, the glass beads may have a diameter greater than 1 mm, e.g., 1.7-2.5 mm. The PEI surface may be prepared by using a high pH, ​​or by using a high-low pH reaction, or by using an electrostatic reaction.

[0063] For example, in embodiments where a high pH reaction is used, the amine-containing protein solution can be prepared at a concentration of 10 mg / ml by dissolving 2.5 g of branched PEI in 50 ml of 0.1 M sodium borate (pH 9.5). The aldehyde-coated beads prepared as described above are washed three times in PBS to neutralize the pH of the beads, and 35 ml of PEI solution and 5 M sodium borohydride in 350 μl of 1 N NaOH are added to the beads and rotated at room temperature for 2 hours. The beads are then washed five times in 30 ml of PBS to ensure the removal of unreacted PEI.

[0064] In embodiments where a high-low pH reaction is used, two solutions of amine-containing protein are prepared at a concentration of 10 mg / ml by dissolving 2.5 g of branched polyethyleneimine (PEI) in 50 ml each of 0.1 M sodium borate (pH 9.5) and 0.1 M sodium phosphate and 0.15 M NaCl (pH 7.2), respectively, to produce high pH and low pH PEI solutions. The aldehyde-coated beads prepared as described above are washed three times in PBS to neutralize the pH of the beads. The beads are first resuspended in 35 ml of high pH PEI solution and gently swirled for 15 minutes. The supernatant is then removed, and the beads are resuspended in 35 ml of low pH PEI solution. Next, 5 M sodium borocyanohydride in 350 μl of 1N NaOH is added to the beads, and they are swirled at room temperature for 2 hours. The beads are then washed five times in 30 ml of PBS to ensure the removal of unreacted PEI.

[0065] In embodiments where an electrostatic reaction is used, glass beads are prepared by etching in 30% NaOH. The etched glass beads are washed five times with ddH2O to ensure the removal of NaOH, and then washed again three times with ethanol to ensure the removal of ddH2O. The aminoguanidine solution is prepared by dissolving 2 g of aminoguanidine hydrochloride in 10 ml of DMSO. Once completely dissolved, 40 ml of MCF, pH 6.0 buffer is added to bring the total volume to 50 ml. Next, 40 ml of the aminoguanidine solution is added to the washed beads and rotated at room temperature for 2 hours. The beads are then washed three times with 30 ml of PBS to ensure the removal of unreacted aminoguanidine.

[0066] In some embodiments, histidine-coated glass beads or histidine-coated surfaces such as capillaries can be prepared. For example, one or more glass beads may be used. In some embodiments, the glass beads may have a diameter of less than 1 mm, e.g., 500-600 μm. In some embodiments, the glass beads may have a diameter greater than 1 mm, e.g., 1.7-2.5 mm. A 5 mg / ml histidine-containing solution is prepared by dissolving 25 mg of N-acetyl-L-histidine in 5 ml of MES buffer (pH 6). A 0.5-0.1 M EDC concentration solution is prepared by dissolving 250 mg of EDC in the 5 ml of MES buffer solution of N-acetyl-L-histidine prepared above. The 10% 3-aminopropylmethoxysilane-coated beads or any amine-coated beads prepared as above are rotated with EDC containing N-acetyl-L-histidine in MES buffer at room temperature for 2 hours to react. Next, the beads are washed three times with 30 ml of ddH2O to ensure the removal of any unreacted solution. The same protocol can be used to coat the inside of capillaries or the surface of other devices such as coverslips, but instead of gently rotating them, the capillaries or devices are left in a solution of EDC with N-acetyl-L-histidine in MES buffer for 2 hours, during which time the capillaries or devices are frequently moved up and down to mix the solution.

[0067] For the purposes of this disclosure, the isolation matrix may include a column or other suitable housing packed with positively charged microspheres or beads as described herein. For example, a column may be packed with multiple microspheres (e.g., glass microspheres) or beads (e.g., glass beads) functionalized such that the surface of the microparticles exhibits one or more positively charged moieties, such as amines, polyethyleneimine (PEI), and / or guanidine groups. Similarly, the isolation matrix may include a single narrow capillary, multiple capillaries, or other suitable tube / chamber, functionalized to have a positively charged inner surface and having a diameter such that cells can easily pass through the capillary or chamber while making significant contact with its inner surface. Negatively charged CTCs may bind to an inner surface functionalized with one or more positively charged moieties, such as amines, polyethyleneimine (PEI), and / or guanidine groups.

[0068] In some embodiments, cells can be treated with positively charged nickel-coated microparticles or nanoparticles before being applied to isolation or capture on a microparticle column or capillary tube. These particles adhere to negatively charged CTCs by simple electrostatic interactions, resulting in the CTCs being decorated on the surface of these microparticles or nanonickel particles. In this embodiment, such decorated CTCs bind to the surface of glass microparticles, such as histidine-coated glass microspheres, or to the inner surface of capillaries, such as histidine-coated capillaries, but this binding occurs not by electrostatic interactions, but by chelation between their nickel surface decoration and the imidazole groups of the histidine moieties. A unique advantage of this embodiment is that such decorated cells can be easily removed from the microbead column or capillary tube by elution with imidazole, which breaks the chelate bonds that previously held them in place. This allows the captured cells to be more easily released from the column and used for additional downstream purposes, such as cancer diagnostics and other applications described herein.

[0069] Columns and capillaries containing isolation matrix Columns and capillaries can be prepared to contain an isolation matrix through which biological samples can pass, and which captures and isolates positively charged microparticles or negatively charged cancer cells from the sample. Columns or capillaries may be of any shape or size suitable for containing the matrix and should include inputs and outputs that allow samples to enter and exit the column.

[0070] Figure 14 shows an exemplary workflow for preparing the column of this disclosure. When microparticles are deposited on the column, they form a matrix with spaces between the microparticles, allowing cells and other components of the sample to be filtered through the matrix.

[0071] Alternatively, cancer cells can be bound to or isolated from a biological sample (e.g., blood) by bringing the biological sample into contact with a positively charged surface as disclosed herein. Such a surface can be prepared by functionalizing it according to the methods disclosed herein.

[0072] Use of isolated cancer cells Cancer cells captured using the methods and compositions described herein can be used for a number of downstream purposes. The cancer cells may be utilized directly in the column while attached to microparticles, the cancer cells may be lysed without eluting from the column, the cancer cells may remain on the column, or the cancer cells may be eluted from the column and used for one or more downstream purposes, including but not limited to those described herein.

[0073] In some embodiments, the disclosed methods may be used to identify the presence of cancer cells in a blood sample from a subject for the purpose of diagnosing cancer, and the presence of cancer cells in a blood sample from said patient indicates the presence of a tumor in the subject's body. The disclosed methods for detecting cancer or circulating tumor cells (CTCs) enable early detection of cancer before signs or symptoms of cancer become otherwise apparent. As shown in the examples provided herein, the disclosed methods and compositions are highly sensitive and can isolate cancer cells / CTCs, which are negligibly small portions of a sample. Thus, for example, a blood sample can be taken from a subject and passed through a positively charged matrix or surface disclosed herein to detect previously unknown cancer in the subject. Any cells that bind to the matrix or surface are expected to be cancer cells and would indicate that the subject has cancer or a tumor. In this way, early-stage detection of previously unidentified cancer in a subject is possible. These methods are particularly useful for detecting or diagnosing certain types of cancer, such as pancreatic cancer and ovarian cancer, which otherwise often evade detection until later stages.

[0074] Additionally, or alternatively, the presence of cancer cells may indicate the possibility of tumor progression or metastasis. In some embodiments, the disclosure may be used to detect the presence of cancer in patients who have already received treatment. For example, the disclosure may be used to monitor cancer remission / recurrence or to detect minimal residual disease. In some embodiments, cancer cells are isolated using the methods described herein, and definitive identification of the cancer cells is obtained by labeling the cells with one or more cancer marker-specific conjugates (e.g., antibodies). In some embodiments, cancer cells are isolated using the methods described herein, and definitive identification of the cancer cells is obtained by detecting the genetic signature of the cancer cells.

[0075] In some embodiments of this disclosure, the type of cancer present in a sample may be unknown. Therefore, in some embodiments, cancer cells are isolated using the methods described herein, and the cells are visualized using fluorescent cell markers or colorimetric markers to enable counting and / or determination of the presence of bound cancer cells. In some embodiments, the type of cancer cells may be identified after their isolation from the sample. Identification may be achieved, for example, using cancer type-specific biomarkers that can be bound by fluorescent labeling. Labeled cancer cells may be further analyzed using spectral imaging, fluorescence microscopy, visible light microscopy, or manual or automated image analysis. In some embodiments, cancer cells may be genotyped to identify the type of cancer. Unlike other current methods of identifying CTCs by specific tumor type biomarkers, captured cells in this disclosure are defined simply by their negative surface charge. Thus, any cells bound to these described matrices are cancer cells, providing a positive diagnosis of cancer in the patient from whom the cells originated. The type of cancer cells and their primary tissue may be determined by tissue-specific biomarkers that are not cancer-specific.

[0076] In some embodiments, the disclosed compositions and methods may be used to monitor disease progression, response to therapy, or relapse / recurrence in patients with cancer. In some embodiments, the number of cells captured using the methods herein is determined during disease progression, during a treatment regimen lasting several weeks, months or longer, or at different time intervals after discontinuation of the treatment regimen. An increase in the number of cancer cells over time indicates a lack of response to therapy, relapse, a higher risk of metastasis, a worse prognosis, a shorter predicted survival time, or progression to a higher stage of cancer or a higher rate of tumor growth, or a combination of the above. A decrease or no change in the number of cancer cells indicates a favorable response to therapy, a stable state of the disease, or tumor reduction or remission, or a combination of the above.

[0077] In some embodiments, cancer cells isolated from cancer patients may be characterized for their nucleic acid content. RNA and / or DNA from isolated cancer cells may be analyzed, and the genetic content and / or gene expression patterns may be characterized in cancer cells using one or more of the following methodologies: single nucleotide polymorphism analysis, quantitative PCR, RT-PCR, quantitative RT-PCR, FISH, DNA sequencing, multiplex PCR, DNA methylation determination, quantification of total DNA content, whole genome amplification (WGA), CGH, laser dissection microscopy (LDM), amplification from RNA, oligonucleotide ligation assay (OLA), chromosomal immunoprecipitation (CHIP), Southern blotting, hybridization, amplification, ligation, and enzyme assays. DNA and / or RNA may be isolated from matrix-bound cancer cells simply by lysing these cells on the matrix and recovering the lysates.

[0078] In some embodiments, the genetic content of cancer cells is analyzed to identify mutations that can confer higher growth rates, or chromosomal deletions spanning tumor suppressor genes or chromosomal amplifications of tumor-promoting genes. In some embodiments, characterization of the genetic content of cancer cells isolated from a subject is used to tailor the course of personalized treatment specific to that subject or a particular cancer phenotype.

[0079] Some embodiments of this disclosure may involve culturing and in vitro proliferation of cancer cells isolated from biological samples obtained from cancer patients. In some embodiments, patient-specific cultured cells may be used to assess the progress of an experimental cancer therapy or drug candidate in a clinical study. While subjects in such studies may be human, they will often include other mammals such as mice, rats, dogs, and monkeys. In this embodiment, the method can be used to provide clinical endpoints for measuring the efficacy of an experimental cancer therapy or drug candidate that are faster and more quantitative than efficacy, metastasis, or recurrence data alone. This provides a faster and more quantitative assessment of the efficacy of the therapeutic agent under investigation and provides additional information on how the therapeutic agent affects the probability of metastasis and recurrence of the treated cancer. Thus, it provides more information about the overall efficacy of the experimental therapy and reduces the time required for clinical trials.

[0080] In some embodiments, cultured cancer cells obtained from blood samples of cancer patients are used to test the efficacy of a candidate anticancer drug or drug combination in vitro before administering the drug(s) to the patient, or before deciding whether to continue administering the drug(s). In other embodiments, such cultured cancer cells are used to test a new drug candidate or other experimental therapy as part of a clinical trial, or even as a primary screening for efficacy.

[0081] In some embodiments, cancer cells isolated from cancer patients using the methods of the present disclosure are immortalized by in vitro culture and selection. This culture and selection may or may not be assisted by transfection of cells with SV40 T antigen or telomerase or other preferred methods. The immortalized cells can then be used to test the efficacy of anticancer drugs, to screen for new anticancer drugs, or for any other investigation requiring immortalized cell lines.

[0082] In some embodiments, cancer cells isolated from cancer patients using the methods or compositions of the present disclosure may be used in invasive assays.

[0083] In some embodiments, cancer cells isolated from cancer patients may be used for the purpose of personalized immunotherapy, and proteins or nucleic acids obtained from cancer cells isolated from cancer patients, or combinations thereof, are incubated with WBCs or WBC subfractions from said patients to stimulate a cancer-specific immune response. The WBCs or WBC subfractions exposed to cancer cell tumor antigens are then reinoculated into the patients.

[0084] In some embodiments, after isolating cancer cells from a blood sample using the methods or compositions described herein, the cancer cells can be characterized using several immunoassays. For example, the cancer cells can be lysed, the lysate centrifuged, and subjected to an ELISA assay. In this case, specific target proteins expressed in the cancer cells can be directly detected. This provides a profile of protein content in the cancer cells and allows for monitoring how the cancer cell phenotype changes during the course of the disease or during therapeutic treatment.

[0085] In some embodiments, isolated cancer cells can be characterized by one or more functional or enzymatic assays. Telomerase activity has been identified in lung cancer cells, as well as in cancer cells from many other cancers. Telomerase activity assays can be used to further characterize circulating tumor cells isolated by the depletion method of this disclosure or by positive selection methods well known to those skilled in the art. In this case, a telomerase repeat amplification protocol (TRAP) can be performed. Once cancer cells are isolated, telomerase is extracted using a CHAPS-based surfactant buffer or any other suitable method. The supernatant of the cell lysate is used as a template for a telomerase extension reaction by PCR. A fluorescent PCR product is generated using fluorescently labeled primers, followed by capillary electrophoresis. A larger amount of fluorescent PCR product or a longer telomerase repeat amplification product indicates higher telomerase activity in the cancer cells in the sample, which can serve as an indicator of tumor aggressiveness or the number or proportion of cancer cells in the enriched sample.

[0086] In recent years, antibody-based therapies have achieved great clinical success and are now part of the standard arsenal used by clinicians to combat cancer. The methods of this disclosure provide a unique approach to monitoring the efficacy and effectiveness of antibody-based therapies. In some embodiments, this disclosure can be used to detect interactions between circulating cancer cells in the blood of cancer patients and immunotherapeutic agents, such as humanized exogenous antibodies used in therapy. Whether isolated cancer cells are bound to therapeutic antibodies can be determined by isolating the cancer cells and examining them for the presence of such antibodies.

[0087] In some embodiments, monitoring the interaction between therapeutic antibodies and cancer cells present in a blood sample can be used to assess a patient's response to therapy. If the percentage of cancer cells bound to an immunotherapy agent such as an antibody exceeds a predetermined value or increases over time in measurements at different time points, a favorable treatment outcome is predicted. If the percentage of cancer cells bound to the antibody falls below a predetermined value or decreases over time in measurements at different time points, an unfavorable treatment outcome is predicted.

[0088] In some embodiments, cancer cells isolated using the disclosed methods or compositions can be used to prepare patient-specific cancer vaccines. For example, cancer cells may be lysed and filled into vaccine particles, such as yeast cell wall particles (YCWPs), which can be administered to a patient to stimulate the patient's immune system to attack cancer. In some embodiments, the YCWPs are capped. In some embodiments, the YCWPs are capped with silicates. In some embodiments, the silicates include tetraethyl orthosilicate, tetramethyl orthosilicate, tetrapropyl orthosilicate, or tetrabutyl orthosilicate. In some embodiments, the YCWPs are not capped. YCWPs suitable for encapsulating cancer cell lysates are known in the art and are described, for example, in PCT / US2013 / 063091 (WO2014 / 040089).

[0089] The disclosed methods for isolating or capturing cancer cells and CTCs can further be used as a treatment by removing cells from the patient. While surgical excision of solid tumors is often the first-line treatment for many forms of cancer, such an option has not been available for hematological cancers until now. The disclosed methods for isolating or capturing cancer cells / CTCs can be used to treat hematological cancers such as leukemia and lymphoma. Similarly, the disclosed methods for isolating or capturing cancer cells / CTCs can treat, prevent, or minimize the risk of metastasis of other forms of cancer (e.g., solid tumors) by removing CTCs and cancer cells from circulation that may potentially invade tissues outside the primary tissue of a given cancer. Therefore, in some embodiments, the methods described herein can be used to remove cancer cells from the blood of a cancer patient and return the blood to the patient. For example, the blood of a hematological cancer patient may be "filtered" and cancer cells may be captured by the isolation matrix described herein. After removal of cancer cells, the blood may be returned to the patient. This process may be repeated until the patient's cancer is gone or until a significant reduction in circulating cancer cells occurs. Cancers suitable for treatment by these methods include, but are not limited to, any blood cancers such as leukemia and lymphoma. Such treatments may utilize existing apheresis or dialysis techniques to pass the blood of the target through a matrix or substrate containing a positively charged surface, as disclosed herein, thereby allowing CTCs to come into contact with and bind to the charged surface or matrix.

[0090] Compositions and kits This specification also includes kits for functionalizing microparticles or surfaces, preparing isolation matrices, and / or preparing columns for isolating cancer cells. In some embodiments, the kit may include unfunctionalized microparticles or surfaces and reagents for functionalizing said microparticles or surfaces. The kit may include one or more already functionalized and / or positively charged microparticles or surfaces. In some embodiments, the kit may include reagents and apparatus for preparing matrices, e.g., buffers, tubing, and other necessary apparatus. In some embodiments, the kit may include components for preparing columns, e.g., syringes, buffers, and tubing.

[0091] In some embodiments, the kits described herein may additionally or alternatively include reagents and apparatus used for downstream processing or analysis of captured cancer cells. For example, the kits may include reagents or apparatus for culturing isolated cells, identifying isolated cells using cancer cell biomarkers, or extracting and analyzing nucleic acids from captured cancer cells.

[0092] The kit may further include one or more of the following: washing buffer and / or washing reagent, hybridization buffer and / or hybridization reagent, labeling buffer and / or labeling reagent, and detection means. The buffers and / or reagents are typically optimized for the amplification / detection technique targeted by the kit. Protocols for using these buffers and reagents to perform different steps of the procedure may also be included in the kit.

[0093] The Disclosure further provides a column for use in the disclosed method. The column may comprise a plurality of beads or microparticles having a positively charged surface as a result of functionalization with one or more positively charged moieties, such as one or more amines, polyethyleneimine (PEI), and / or guanidine groups. Alternatively, the Disclosure provides a capillary having a positively charged surface as a result of functionalization with one or more positively charged moieties, such as one or more amines, polyethyleneimine (PEI), and / or guanidine groups.

[0094] Examples of Embodiments Embodiment 1. A method for isolating cancer cells from a biological sample, comprising: a) passing a biological sample containing cancer cells through an isolation matrix containing functionalized microspheres; and b) collecting the biological sample flowing through the isolation matrix into a first aliquot, wherein the cancer cells are bound to the isolation matrix.

[0095] Embodiment 2. The method according to Embodiment 1, wherein the functionalized microsphere is functionalized with a positively charged functional group.

[0096] Embodiment 3. The method according to Embodiment 1 or 2, wherein the functionalized microsphere is functionalized with an amine, polyethyleneimine (PEI), and / or a guanidine group.

[0097] Embodiment 4. The method according to any one of Embodiments 1 to 3, wherein the microsphere comprises glass, polymer, or resin.

[0098] Embodiment 5. The method according to any one of Embodiments 1 to 4, wherein the microspheres have a diameter of 500 μm to 600 μm.

[0099] Embodiment 6. The method according to any one of Embodiments 1 to 5, wherein the biological sample includes blood.

[0100] Embodiment 7. The method according to any one of Embodiments 1 to 6, wherein the biological sample is obtained from a subject that has cancer or is suspected of having cancer.

[0101] Embodiment 8. The method according to Embodiment 7, wherein the cancer is a blood cancer or includes a solid tumor.

[0102] Embodiment 9. The method according to any one of Embodiments 1 to 8, further comprising eluting the cancer cells bound to the isolation matrix into a second aliquot.

[0103] Embodiment 10. The method according to any one of Embodiments 7 to 9, wherein the first aliquot is administered to a subject and returned.

[0104] Embodiment 11. The method according to any one of Embodiments 1 to 9, further comprising detecting the presence or absence of cancer cells in the biological sample.

[0105] Embodiment 12. The method according to any one of Embodiments 7 to 11, further comprising lysing the cancer cells to obtain a cancer cell lysate.

[0106] Embodiment 13. The method according to Embodiment 12, further comprising incorporating the cancer cell lysate into a cancer vaccine.

[0107] Embodiment 14. The method according to Embodiment 12, further comprising determining the mRNA copy number from the cancer cell lysate.

[0108] Embodiment 15. The method according to Embodiment 14, wherein the mRNA copy number is determined by quantitative RT-PCR.

[0109] Embodiment 16. The method according to any one of Embodiments 1 to 15, further comprising calculating the number of cancer cells bound to the functionalized microspheres.

[0110] Embodiment 17. A cancer vaccine prepared by a process, the process comprising: a) contacting a biological sample containing cancer cells with a positively charged surface, wherein the cancer cells bind to the positively charged surface; b) lysing the cancer cells to obtain a cancer cell lysate; and c) incorporating the cancer cell lysate into yeast cell wall particles (YCWP).

[0111] Embodiment 18. A cancer vaccine comprising a) yeast cell wall particles (YCWP) and b) the cancer cell lysate described in Embodiment 12.

[0112] Embodiment 19. The vaccine according to Embodiment 17 or 18, wherein the YCWP is modified by capping with silicate.

[0113] Embodiment 20. The vaccine according to Embodiment 19, wherein the silicate is selected from the group comprising tetraethyl orthosilicate, tetramethyl orthosilicate, tetrapropyl orthosilicate, and tetrabutyl orthosilicate.

[0114] Embodiment 21. The vaccine according to any one of Embodiments 17 to 20, further comprising one or more adjuvants, excipients, and preservatives.

[0115] Embodiment 22. A method for delivering a vaccine to a subject, comprising administering the vaccine described in any one of Embodiments 17 to 21 to the subject.

[0116] Embodiment 23. A method for treating or preventing cancer, comprising administering a vaccine described in any one of Embodiments 17 to 21 to a subject in need thereof.

[0117] Embodiment 24. The method according to Embodiment 22 or 23, wherein the vaccine is administered subcutaneously, orally, or intravenously.

[0118] Embodiment 25. The method according to Embodiment 22 or 23, wherein the vaccine is administered to the dermis of the subject.

[0119] Embodiment 26. A method for treating cancer in a patient, comprising extracting cancer cells from the patient's blood by passing the blood through an isolation matrix containing functionalized microspheres that bind to cancer cells.

[0120] Embodiment 27. The method according to Embodiment 26, wherein the functionalized microsphere is functionalized with a positively charged functional group.

[0121] Embodiment 28. The method according to Embodiment 26 or 27, wherein the functionalized microsphere is functionalized with an amine, polyethyleneimine (PEI), and / or a guanidine group.

[0122] Embodiment 29. The method according to any one of Embodiments 26 to 28, wherein the microsphere comprises glass, polymer, or resin.

[0123] Embodiment 30. The method according to any one of Embodiments 26 to 29, wherein the microspheres have a diameter of 500 μm to 600 μm.

[0124] Embodiment 31. The method according to any one of Embodiments 26 to 30, wherein the patient has blood cancer or malignant cancer.

[0125] Embodiment 32. The method according to any one of Embodiments 26 to 31, further comprising eluting the bound cancer cells from the isolation matrix.

[0126] Embodiment 33. The method according to Embodiment 32, further comprising lysing the eluted cells to obtain a lysate and incorporating the lysate into a cancer vaccine for treating the patient.

[0127] Embodiment 34. A method for detecting cancer cells in a patient, comprising: a) passing a biological sample from the patient through an isolation matrix of functionalized microspheres, wherein the functionalized microspheres bind to cancer cells; b) eluting the cancer cells from the matrix; and c) detecting the presence or absence of cancer cells in the biological sample.

[0128] Embodiment 35. The method according to Embodiment 34, wherein the patient has blood cancer or malignant cancer.

[0129] Embodiment 36. The method according to Embodiment 34 or 35, wherein the functionalized microsphere is functionalized with an amine, polyethyleneimine (PEI), and / or a guanidine group.

[0130] Embodiment 37. The method according to any one of Embodiments 34 to 36, wherein the microsphere comprises glass, polymer, or resin.

[0131] Embodiment 38. The method according to any one of Embodiments 34 to 37, wherein the microspheres have a diameter of 500 μm to 600 μm.

[0132] Embodiment 39. A cancer cell isolation matrix comprising functionalized microspheres containing positively charged functional groups, wherein the functionalized microspheres bind to cancer cells.

[0133] Embodiment 40. The cancer cell isolation matrix according to Embodiment 39, wherein the functionalized microspheres are functionalized with an amine, polyethyleneimine (PEI), and / or a guanidine group.

[0134] Embodiment 41. The cancer cell isolation matrix according to Embodiment 39 or 40, wherein the microsphere comprises glass, polymer, or resin.

[0135] Embodiment 42. The cancer cell isolation matrix according to any one of Embodiments 39 to 41, wherein the microspheres have a diameter of 500 μm to 600 μm.

[0136] Embodiment 43. A kit for purifying cancer cells from a biological sample, comprising functionalized microspheres, wherein the functionalized microspheres bind to cancer cells derived from the biological sample.

[0137] Embodiment 44. A method for preparing a column for isolating cancer cells, comprising: a) preparing an isolation matrix, the isolation matrix comprising functionalized microspheres; and b) depositing the isolation matrix into a container, the container having an inlet and an outlet.

[0138] Embodiment 45. The method according to Embodiment 44, wherein the functionalized microsphere is functionalized with a positively charged functional group.

[0139] Embodiment 46. The method according to Embodiment 44 or 45, wherein the functionalized microsphere is functionalized with an amine, polyethyleneimine (PEI), and / or a guanidine group.

[0140] Embodiment 47. The method according to any one of Embodiments 44 to 46, wherein the microsphere comprises glass, polymer, or resin.

[0141] Embodiment 48. The method according to any one of Embodiments 44 to 47, wherein the microspheres have a diameter of 500 μm to 600 μm.

[0142] Embodiment 49. The method according to any one of Embodiments 44 to 48, wherein the preparation of the functionalized microspheres comprises coating the microspheres with a 5% 3-aminopropyltriethoxysilane solution.

[0143] Embodiment 50. The method according to any one of Embodiments 44 to 49, wherein the preparation of the functionalized microspheres comprises coating the microspheres with a 5% silane coupling agent.

[0144] Embodiment 51. The method according to any one of Embodiments 44 to 50, wherein the preparation of the functionalized microspheres comprises coating the microspheres with polyethyleneimine (PEI).

[0145] Embodiment 52. The method according to any one of Embodiments 44 to 50, wherein the preparation of the functionalized microspheres comprises coating the microspheres with aminoguanidine.

[0146] Embodiment 53. The method according to any one of Embodiments 44 to 51, wherein the deposition of the isolation matrix comprises depositing 0.1 to 1 ml of functionalized microspheres into a container.

[0147] Embodiment 54. A method for isolating cancer cells from a biological sample, comprising: a) passing a biological sample containing cancer cells through a functionalized capillary; and b) collecting the biological sample flowing through the capillary into a first aliquot, wherein the cancer cells are bound to the capillary.

[0148] Embodiment 55. The method according to Embodiment 54, wherein the capillary is functionalized with an amine, polyethyleneimine (PEI), and / or a guanidine group.

[0149] Embodiment 56. A method for extracting cancer cells from a biological sample, comprising contacting the biological sample containing cancer cells with a positively charged surface, wherein the cancer cells bind to the positively charged surface.

[0150] Embodiment 57. The method according to Embodiment 56, wherein the positively charged surface is functionalized with an amine, polyethyleneimine (PEI), and / or guanidine groups.

[0151] Embodiment 58. The method according to Embodiment 56 or 57, wherein the positively charged surface is made from glass, polymer, or resin.

[0152] Embodiment 59. The method according to any one of Embodiments 56 to 58, wherein the positively charged surface is selected from beads, microparticles, capillaries, blood collection tubes, microscope slides, and microscope slide covers.

[0153] Embodiment 60. The method according to any one of Embodiments 56 to 59, wherein the biological sample includes blood.

[0154] Embodiment 61. The method according to any one of Embodiments 56 to 60, wherein the biological sample is obtained from a subject that has cancer or is suspected of having cancer.

[0155] Embodiment 62. The method according to Embodiment 61, wherein the cancer is a blood cancer or includes a solid tumor.

[0156] Embodiment 63. The method according to any one of Embodiments 56 to 62, further comprising detecting the presence or absence of cancer cells in the biological sample.

[0157] Embodiment 64. The method according to any one of Embodiments 56 to 62, further comprising lysing the cancer cells to obtain a cancer cell lysate.

[0158] Embodiment 65. The method according to Embodiment 64, further comprising incorporating the cancer cell lysate into a cancer vaccine.

[0159] Embodiment 66. The method according to any one of Embodiments 56 to 65, further comprising calculating the number of cancer cells bound to the positively charged surface.

[0160] Embodiment 67. A cancer vaccine comprising a) yeast cell wall particles (YCWP) and b) the cancer cell lysate described in Embodiment 64.

[0161] Embodiment 68. The vaccine according to Embodiment 67, wherein the YCWP is modified by capping with a silicate optionally selected from tetraethyl orthosilicate, tetramethyl orthosilicate, tetrapropyl orthosilicate, and tetrabutyl orthosilicate.

[0162] Embodiment 69. The vaccine according to Embodiment 67 or 68, further comprising one or more adjuvants, excipients, and preservatives.

[0163] Embodiment 70. A method for delivering a vaccine to a subject, comprising administering to the subject the vaccine described in any one of Embodiments 67 to 69.

[0164] Embodiment 71. A method for treating or preventing cancer, comprising administering a vaccine described in any one of Embodiments 67 to 69 to a subject in need thereof.

[0165] Embodiment 72. The method according to Embodiment 70 or 71, wherein the vaccine is administered subcutaneously, orally, or intravenously.

[0166] Embodiment 73. The method according to Embodiment 70 or 71, wherein the vaccine is administered to the dermis of the target.

[0167] Embodiment 74. A method for treating cancer in a patient, comprising: extracting the patient's blood by bringing it into contact with a positively charged surface, wherein the cancer cells bind to the positively charged surface; and returning the blood to the patient after contact with the positively charged surface.

[0168] Embodiment 75. The method according to Embodiment 75, wherein the positively charged surface is functionalized with an amine, polyethyleneimine (PEI), and / or guanidine groups.

[0169] Embodiment 76. The method according to Embodiment 74 or 75, wherein the positively charged surface is made from glass, polymer, or resin.

[0170] Embodiment 77. The method according to any one of Embodiments 74 to 76, wherein the positively charged surface is selected from beads, microparticles, capillaries, blood collection tubes, microscope slides, and microscope slide covers.

[0171] Embodiment 78. The method according to any one of Embodiments 74 to 77, wherein the patient has blood cancer or malignant cancer.

[0172] Embodiment 79. A method for detecting cancer cells in a subject, comprising: isolating cancer cells from a biological sample according to the method described in any one of Embodiments 56 to 60; and detecting the presence or absence of cancer cells in the biological sample.

[0173] Embodiment 80. The method according to Embodiment 79, wherein the patient has a blood cancer, a cancer including a solid tumor, or a malignant cancer.

[0174] Embodiment 81. A device for isolating cancer cells, comprising a positively charged surface containing an amine group, polyethyleneimine (PEI), a guanidine group, or any combination thereof.

[0175] Embodiment 82. The device according to Embodiment 81, wherein the positively charged surface is made from glass, polymer, or resin.

[0176] Embodiment 83. The device according to Embodiment 81 or 82, wherein the positively charged surface is selected from beads, microparticles, capillaries, blood collection tubes, microscope slides, and microscope slide covers. [Examples]

[0177] The following examples are provided to explain how the compositions and methods claimed herein can be carried out, prepared and evaluated, and are intended to be purely illustrative and not to limit the scope of this disclosure.

[0178] Method: The following materials and methods were used in the examples. Microsphere Sensualization Amine-coated glass beads 5% 3-aminopropyltriethoxysilane beads: 10 g of MO-SCI OL-GLO0191B5-2338 500-600 μm glass beads were weighed into a 50 ml tube. To etch the beads, the beads were rotated for 1 hour in 30 ml of 30% NaOH solution prepared by adding 10 ml of 1 M NaOH to 20 ml of ddH2O. A 4% ethanol solution of water was prepared by mixing 4 ml of ddH2O with 96 ml of ethanol and adjusting the pH to 4.5-5.5 with acetic acid. A 50 ml solution of 5% 3-aminopropyltriethoxysilane was prepared by dissolving 2.5 ml of 3-aminopropyltriethoxysilane in 50 ml of 4% ethanol solution of water and rotating the solution in a plastic tube at room temperature for 15 minutes to allow hydrolysis and form reactive silanols. Next, the etched glass beads were washed five times with 30 ml of ddH2O to ensure the removal of NaOH. Then, the beads were washed three times with 30 ml of ethanol to ensure the removal of ddH2O. Next, the beads were reacted with 35 ml of 5% 3-aminopropyltriethoxysilane solution by gently rotating them at room temperature for 2 hours. Next, the beads were washed three times with 30 ml of ethanol to ensure the removal of the 3-aminopropyltriethoxysilane solution, and then washed three times with 30 ml of ddH2O to ensure the removal of the ethanol. Next, the beads were frozen at -84°C and then lyophilized to remove water and form siloxane bonds. The same protocol can be used to coat the inside of capillaries or the surface of other devices such as coverslips, but instead of gently rotating, the capillaries or other devices are left in 5% 3-aminopropyltriethoxysilane solution for 2 hours, during which time the capillaries or devices are frequently moved up and down to mix the solution.

[0179] 10% 3-aminopropyltriethoxysilane beads: 10% 3-aminopropyltriethoxysilane beads were prepared using the same protocol as used to prepare the 5% 3-aminopropyltriethoxysilane beads described above, except that 5 ml of 3-aminopropyltriethoxysilane was dissolved in a 4% ethanol solution of water. All other steps were the same. The same protocol can be used to coat the inside of capillaries or the surface of other devices such as coverslips, but instead of gently rotating, the capillaries or other devices should be left in the 10% 3-aminopropyltriethoxysilane solution for 2 hours, during which time the capillaries or devices should be frequently moved up and down to mix the solution.

[0180] 5% 3-aminopropyltrimethoxysilane beads: 10 g of MO-SCI OL-GLO0191B5-2338 500-600 μm glass beads were weighed into a 50 ml tube. To etch the beads, the beads were rotated for 1 hour in 30 ml of a 30% NaOH solution prepared by adding 10 ml of 1 M NaOH to 20 ml of ddH2O. A 50 ml solution of 5% 3-aminopropyltrimethoxysilane was prepared by dissolving 2.5 ml of 3-aminopropyltrimethoxysilane in 50 ml of methanol and rotating the solution in a plastic tube at room temperature for 15 minutes to allow hydrolysis and form a reactive silanol. Next, the etched glass beads were washed 5 times with 30 ml of ddH2O to ensure removal of NaOH. Next, the beads were washed 3 times with 30 ml of methanol to ensure removal of ddH2O. Next, the beads were reacted by gently rotating them in 35 ml of a 5% 3-aminopropyltrimethoxysilane solution at room temperature for 2 hours. The beads were then washed three times with 30 ml of methanol to ensure removal of the 3-aminopropyltrimethoxysilane solution, and then three times with 30 ml of ddH2O to ensure removal of the methanol. The beads were then frozen at -84°C and lyophilized to remove water and form siloxane bonds. The same protocol can be used to coat the inside of capillaries or the surface of other devices such as coverslips, but instead of gently rotating, the capillaries or devices are left in a 5% 3-aminopropyltrimethoxysilane solution for 2 hours, during which time the capillaries or devices are frequently moved up and down to mix the solution.

[0181] 10% 3-aminopropyltrimethoxysilane beads: 10% 3-aminopropyltriethoxysilane beads were prepared using the same protocol as used to prepare the 5% 3-aminopropyltrimethoxysilane beads described above, except that 5 ml of 3-aminopropyltrimethoxysilane was dissolved in methanol. All other steps were the same. The same protocol can be used to coat the inside of capillaries or the surface of other devices such as coverslips, but instead of gently rotating, the capillaries or other devices should be left in the 10% 3-aminopropyltrimethoxysilane solution for 2 hours, during which time the capillaries or devices should be frequently moved up and down to mix the solution.

[0182] Aldehyde-coated glass beads Beads were coated with triethoxysilylbutyraldehyde, a silane coupling agent. First, 10 g of MO-SCI OL-GLO0191B5-2338 500-600 μm glass beads were weighed into a 50 ml tube. To etch the beads, the beads were rotated for 1 hour in 30 ml of 30% NaOH solution, which was prepared by adding 10 ml of 1 M NaOH to 20 ml of ddH2O. A 4% ethanol-water solution was prepared by mixing 4 ml of ddH2O with 96 ml of ethanol and adjusting the pH to 4.5-5.5 with acetic acid. A 50 ml solution of 5% silane coupling agent was prepared by dissolving 2.5 ml of triethoxysilylbutyraldehyde in 50 ml of the 4% ethanol-water solution and rotating the solution in a plastic tube at room temperature for 5 minutes to allow hydrolysis and form a reactive silanol. Next, the etched glass beads were washed five times with 30 ml of ddH2O to ensure the removal of NaOH. Then, the beads were washed three times with 30 ml of ethanol to ensure the removal of ddH2O. Next, the beads were reacted with 35 ml of 5% silane coupling agent solution by gently rotating them at room temperature for 2 hours. Next, the beads were washed three times with 30 ml of ethanol to ensure the removal of the silane coupling agent solution, and then three times with 30 ml of ddH2O to ensure the removal of the ethanol. Next, the beads were frozen at -84°C and then freeze-dried to remove water and form siloxane bonds. The same protocol can be used to coat the inside of capillaries or the surface of other devices such as coverslips, but instead of gently rotating, the capillaries or other devices are left in the 5% silane coupling agent solution for 2 hours, during which time the capillaries or devices are frequently moved up and down to mix the solution.

[0183] Polyethyleneimine (PEI) coated glass beads Preparation by chemical reaction (high pH): A solution of amine-containing protein was prepared at a concentration of 10 mg / ml by dissolving 2.5 g of branched PEI in 50 ml of 0.1 M sodium borate (pH 9.5). 10 g of the aldehyde-coated beads prepared as described above were weighed into a 50 ml tube and washed three times with PBS to neutralize the pH of the beads. 35 ml of PEI solution and 5 M sodium borohydride in 350 μl of 1 N NaOH were added to the beads, and the mixture was rotated at room temperature for 2 hours. The beads were then washed five times with 30 ml of PBS to ensure the removal of unreacted PEI.

[0184] Preparation by chemical reaction (high-low pH): Two solutions of amine-containing protein at a concentration of 10 mg / ml were prepared by dissolving 2.5 g of branched polyethyleneimine (PEI) in 50 ml each of 0.1 M sodium borate (pH 9.5) and 0.1 M sodium phosphate and 0.15 M NaCl (pH 7.2), respectively, to produce high pH and low pH PEI solutions. 10 g of the aldehyde-coated beads prepared as described above were weighed into a 50 ml tube and washed three times with PBS to neutralize the pH of the beads. The beads were first resuspended in 35 ml of high pH PEI solution and gently rotated for 15 minutes. The supernatant was then removed and the beads were resuspended in 35 ml of low pH PEI solution. Next, 5 M sodium borohydride in 350 μl of 1 N NaOH was added to the beads, and they were rotated at room temperature for 2 hours. The beads were then washed five times with 30 ml of PBS to ensure the removal of unreacted PEI.

[0185] Preparation by electrostatic reaction: 10 g of MO-SCI OL-GLO0191B5-2338 500-600 μm glass beads were weighed into a 50 ml tube. To etch the beads, the beads were rotated for 1 hour in 30 ml of 30% NaOH solution, which was prepared by adding 10 ml of 1 M NaOH to 20 ml of ddH2O. Next, the beads were washed five times with 30 ml of ddH2O to ensure the removal of NaOH, and resuspended in 35 ml of PEI solution, which was prepared by dissolving 5 g of branched polyethyleneimine (PEI) in 35 ml of PBS, and rotated at room temperature for 2 hours. Next, the beads were washed five times with 30 ml of PBS to ensure the removal of unreacted PEI.

[0186] Guanidine-coated glass beads Preparation by electrostatic reaction: 10 g of MO-SCI OL-GLO0191B5-2338 500-600 μm glass beads were weighed into a 50 ml tube. To etch the beads, the beads were rotated for 1 hour in 30 ml of 30% NaOH solution, which was prepared by adding 10 ml of 1 M NaOH to 20 ml of ddH2O. Next, the beads were washed five times with 30 ml of ddH2O to ensure removal of NaOH. Next, an aminoguanidine solution was prepared by dissolving 2 g of aminoguanidine hydrochloride in 10 ml of DMSO. Once completely dissolved, 40 ml of MCF, pH 6.0 buffer was added to bring the total volume to 50 ml. Next, 40 ml of the aminoguanidine solution was added to the washed beads and rotated at room temperature for 2 hours. Next, the beads were washed three times with 30 ml of PBS to ensure removal of unreacted aminoguanidine.

[0187] Histidine-coated glass beads Preparation by chemical reaction: A histidine-containing solution was prepared at a concentration of 5 mg / ml by dissolving 25 mg of N-acetyl-L-histidine in 5 ml of MES buffer (pH 6). A 0.5-0.1 M EDC solution was prepared by dissolving 250 mg of EDC in the 5 ml of MES buffer solution of N-acetyl-L-histidine prepared above. 10 g of 10% 3-aminopropylmethoxysilane-coated beads prepared as described above were weighed into a 50 ml tube. Alternatively, any amine-coated beads may be used. The EDC containing N-acetyl-L-histidine in the MES buffer was added to the tube containing the amino beads and reacted by rotation at room temperature for 2 hours. After the reaction time, the beads were washed three times with 30 ml of ddH2O to ensure the removal of unreacted solution. The same protocol can be used to coat the inside of capillaries or the surface of other devices such as coverslips, but instead of gently rotating them, the capillaries or other devices are left in a solution of EDC containing N-acetyl-L-histidine in MES buffer for 2 hours, during which time the capillaries or devices are frequently moved up and down to mix the solution.

[0188] cell culture Cells were cultured in T75 or T25 flasks using DMEM cell culture medium containing 10% fetal bovine serum. Cells were incubated at 37°C in a 5% CO2 incubator. The culture medium was changed every 2-3 days, and cells were reseeded into new T75 or T25 flasks if the population became too large or if cells were needed for the experiment. All cell lines except CCRF-SB were adherent cell lines; to collect these cells, the culture medium was removed, the cell layer was briefly rinsed with PBS, and then 1-2 ml of trypsin-EDTA was added to the flask to disperse the cells. The cells were then collected, the flask was washed again with PBS, and all excess cells were collected. The cells were then centrifuged at 500xg for 5 minutes. The supernatant was discarded, and the cells were resuspended in a volume sufficient to perform cell counting on a hemocytometer. Based on cell counting, 1.0 × 10⁶ cells were obtained. 5 ~1.0×10 6The cells were retained for the further procedures described below, and the remaining cells were centrifuged at 500xg for 5 minutes, then resuspended in culture medium and re-seeded in a new flask. The CCRF-SB cell line is a suspended cell line, and to collect these cells, a sufficient volume of culture medium was removed and the cells were centrifuged at 500xg for 5 minutes. The supernatant was then discarded and the cells were resuspended in a volume sufficient to perform cell counting on a hemocytometer. Based on the cell count, 1.0 × 10⁶ cells were obtained. 5 ~1.0×10 6 The cells were retained for the further steps described below, and the remaining cells were centrifuged at 500xg for 5 minutes, then resuspended in culture medium and placed in a new flask.

[0189] Red blood cell lysis and whole blood wash 5 ml of human whole blood was collected in a 15 ml centrifuge tube and washed with 5 ml of PBS to remove any serum. The blood was then centrifuged at 500 x g for 5 minutes, and the supernatant was removed. 5 ml of ACK lysis buffer was added to the washed blood and incubated at room temperature for 7-10 minutes. Next, 5 ml of PBS was added, and the sample was centrifuged at 500 x g for 5 minutes. The supernatant was removed, and the pellet was resuspended in 5 ml of ACK lysis buffer and incubated for a further 7-10 minutes. Then, 5 ml of PBS was added, and the sample was again centrifuged at 500 x g for 5 minutes. The lysis, incubation, and centrifugation steps were repeated until the pellet turned white. Next, the pellet was resuspended in 5 ml of PBS. In some embodiments, human whole blood was simply washed to remove plasma and serum that have a negative charge and may interfere with charged microparticles or capillaries. 5 ml of human whole blood was collected in a 15 ml centrifuge tube and washed with 5 ml of PBS to remove any serum and / or plasma. Next, the blood was centrifuged at 500xg for 5 minutes, and the supernatant was removed. This washing step was repeated two more times. Finally, the washed blood was brought to a volume of 5 ml in PBS and was ready for adding cells for use on a microparticle column or capillary tube as described herein.

[0190] cell staining 1.0 × 105 ~1.0×10 6 Cells were collected from the culture at ~1.0×10 6 and resuspended in 1 ml of PBS. The cells were stained with 12 μl of a pre-made solution of calcein AM dye in 100 μl of DMSO and incubated in the dark on ice for 1 - 3 hours. Next, the cells were centrifuged at 500×g for 5 minutes. The supernatant was discarded, the cells were washed with 1 ml of PBS, and centrifuged again at 500×g for 5 minutes. The supernatant was discarded, and the cells were resuspended in 1 ml of PBS.

[0191] Cells mixed with nickel-coated particles prior to isolation In some instances, prior to applying the cells to isolation or capture on a microparticle column or a single capillary, the cells are treated with positively charged nickel-coated microparticles or nanoparticles. 1.0×10 5 ~1.0×10 6 Cells were collected from the culture at ~1.0×10 6 and resuspended in 1 ml of PBS. The cells were stained using the method described above. 100 μl (≈1.0×10 5 ) of the stained cells were added to a tube, and 1 - 100 μl of nickel-coated nanoparticles or microparticles were added to the cells such that there were approximately 100 - 1,000 nickel particles per cell. The mixture was made up to 500 ml with PBS and rotated for 15 minutes to react the nickel particles with the cells. After incubation, the cells with the nickel particle solution were ready for use on a microparticle column or capillary as described in this disclosure.

[0192] Column preparation ​​The column was prepared by placing a 40 μm cell strainer mesh between a 1 ml syringe and a 23 gauge needle. The mesh was used to prevent beads from clogging the needle tip. Using a funnel, an appropriate amount (e.g., 0.5 ml) of dry functionalized glass microspheres was added to an Eppendorf tube and the volume was increased to 1.5 ml with PBS. The tip of a plastic transfer pipette was cut off and the microparticles in PBS were carefully pipetted into the column until they reached the 0.5 ml mark on the syringe. Air bubbles and gaps between the beads were removed as much as possible. The column was washed five times with 500 μl of PBS to ensure it was completely washed. The column was run until the dripping stopped.

[0193] Capillary tube preparation and procedure Capillaries were prepared by treating them with different functional groups as described above. A single capillary was used for each run. Cells were collected and stained as described above. 100 μl (approximately 1.0 × 10⁻⁶) 5 Add (1) stained cells to a tube ("initial cell solution") and increase the volume to 500 μl with 400 μl of PBS. Mix the cells thoroughly and add them to a capillary tube using a micropipette with a long tip, carefully pipetting the cells into the inside of the capillary tube to ensure that the cell solution is in contact with the sides of the capillary tube. Add the cells 100 μl at a time, taking care to avoid air bubbles, and collect the flow-through into a second tube ("pass-through"). Once the capillary tube has stopped moving, wash the capillary tube with 500 μl of PBS, adding 100 μl at a time, taking care to avoid air bubbles, and collect the flow-through into a third tube ("first wash"). Once the capillary tube has stopped moving, perform a second wash on the capillary tube with 500 μl of PBS, adding 100 μl at a time, taking care to avoid air bubbles, and collect the flow-through into a fourth tube ("second wash"). Once the capillary tubes stopped functioning, they were analyzed under a microscope to observe the adhesion of green fluorescent cells to the inner surface of the capillaries.

[0194] Single bead diagnostic In some embodiments, a single functionalized bead was used in the assay described herein. For example, a single bead functionalized as described above and having a diameter large enough to be handled with tweezers was used. The bead had a diameter greater than 1 mm, or more specifically, 1.7 mm to 2.5 mm. The bead was placed in a tube with the patient blood sample and rotated at room temperature for 1 hour. The bead was then removed from the tube, washed with PBS, and the cells were lysed and used for downstream analysis as described below.

[0195] Cancer cell lysis The binding of tumor cells to the positively charged beads or matrix disclosed herein is extremely strong, making it difficult to remove the cells intact. Therefore, tumor cell lysates were produced by exposing the matrix containing the bound circulating tumor cells (CTCs) to lysis conditions such as rapid freeze-thaw cycles, or by chemical lysis in all diagnostic and therapeutic steps after capturing tumor cells from patient blood.

[0196] Example 1. Isolation of colon cancer cells Approximately 1.0×10 6 Individual SW620 colon cancer cells were stained using the method described above. 100 μl (approximately 1.0 × 10⁶) 5Add (1) stained cells to a tube ("initial cell solution") and increase the volume to 500 μl with 400 μl of PBS. Mix the cells thoroughly and add them to a column prepared as described above, either with 0.5 ml of 5% 3-aminopropyltriethoxysilane or 10% 3-aminopropyltrimethoxysilane beads. Add the cells 100 μl at a time, taking care to avoid bubbles, and collect the flow-through in a second tube ("pass-through"). Once the column has stopped dropping, wash the column with 500 μl of PBS, adding 100 μl at a time, taking care to avoid bubbles, and collect the flow-through in a third tube ("first wash"). Once the column has stopped dropping, perform a second wash on the column with 500 μl of PBS, adding 100 μl at a time, taking care to avoid bubbles, and collect the flow-through in a fourth tube ("second wash"). Once the column dripping stopped, the needle was capped, and cells from each of the four tubes were analyzed under a microscope.

[0197] Figure 1 shows bright-field and green fluorescence images of 10 μl of colon cancer cells passed through a 5% 3-aminopropyltriethoxysilane bead column, including initial cell solution (A and E), pass-through samples (B and F), first wash samples (C and G), and second wash samples (D and H). Viable colon cancer cells are visualized as green fluorescence under a microscope using calcein AM staining and green light. While multiple cells are visible in the initial cell solution, no cells are observed in the pass-through samples, as well as in the first and second wash samples, indicating that viable colon cancer cells remained in the column and were captured by the positively charged 5% 3-aminopropyltriethoxysilane beads. Similar results were obtained when the experiment was repeated.

[0198] Figure 2 shows bright-field and green fluorescence images of 10 μl each of initial cell solution (A and E), pass-through samples (B and F), first wash samples (C and G), and second wash samples (D and H) of colon cancer cells that passed through a 10% 3-aminopropyltrimethoxysilane bead column. Viable colon cancer cells are visualized as green fluorescence when observed under a microscope with green light by calcein AM staining. Multiple cells can be seen in the initial cell solution, but no cells are seen in the pass-through samples, as well as in the first and second wash samples, indicating that viable colon cancer cells remained in the column and were captured by the positively charged 10% 3-aminopropyltriethoxysilane beads.

[0199] Each of the 5% 3-aminopropyltriethoxysilane or 10% 3-aminopropyltrimethoxysilane bead columns showed similar results in capturing colon cancer cells.

[0200] Example 2. Isolation of breast cancer cells Approximately 5.0×10 5 T47D cancer cells were stained using the method described above. 100 μl (approximately 1.0 × 10⁴) 5Add (1) stained cells to a tube ("initial cell solution") and increase the volume to 500 μl with 400 μl of PBS. Mix the cells thoroughly and add them to a column prepared as described above, either with 0.5 ml of 5% 3-aminopropyltriethoxysilane or 10% 3-aminopropyltrimethoxysilane beads. Add the cells 100 μl at a time, taking care to avoid bubbles, and collect the flow-through in a second tube ("pass-through"). Once the column has stopped dropping, wash the column with 500 μl of PBS, adding 100 μl at a time, taking care to avoid bubbles, and collect the flow-through in a third tube ("first wash"). Once the column has stopped dropping, perform a second wash on the column with 500 μl of PBS, adding 100 μl at a time, taking care to avoid bubbles, and collect the flow-through in a fourth tube ("second wash"). Once the column dripping stopped, the needle was capped, and cells from each of the four tubes were analyzed under a microscope.

[0201] Figure 3 shows bright-field and green fluorescence images of 10 μl each of initial cell solution (A and E), pass-through samples (B and F), first wash samples (C and G), and second wash samples (D and H) of breast cancer cells that passed through a 5% 3-aminopropyltriethoxysilane bead column. Viable breast cancer cells are visualized as green fluorescence when observed under a microscope with green light by calcein AM staining. Multiple cells are visible in the initial cell solution, but only a few cells are visible in the pass-through samples, and no cells are visible in either the first or second wash samples. This indicates that viable breast cancer cells remained in the column and were captured by the positively charged 5% 3-aminopropyltriethoxysilane beads.

[0202] Figure 4 shows bright-field and green fluorescence images of 10 μl each of initial cell solution (A and E), pass-through samples (B and F), first wash samples (C and G), and second wash samples (D and H) of breast cancer cells that passed through a 10% 3-aminopropyltrimethoxysilane bead column. Viable breast cancer cells are visualized as green fluorescence when observed under a microscope with green light by calcein AM staining. Multiple cells can be seen in the initial cell solution, but only a small number of cells are seen in the pass-through samples, as well as in the first and second wash samples, indicating that viable breast cancer cells remained in the column and were captured by the positively charged 10% 3-aminopropyltrimethoxysilane beads.

[0203] Each of the 5% 3-aminopropyltriethoxysilane or 10% 3-aminopropyltrimethoxysilane bead columns showed similar results in capturing breast cancer cells.

[0204] Example 3. Isolation of lung cancer cells Approximately 1.0×10 5 A549 lung cancer cells were stained using the method described above. 100 μl (approximately 1.0 × 10⁶) 5Add (1) stained cells to a tube ("initial cell solution") and increase the volume to 500 μl with 400 μl of PBS. Mix the cells thoroughly and add them to a column prepared as described above, either with 0.5 ml of 5% 3-aminopropyltriethoxysilane or 10% 3-aminopropyltrimethoxysilane beads. Add the cells 100 μl at a time, taking care to avoid bubbles, and collect the flow-through in a second tube ("pass-through"). Once the column has stopped dropping, wash the column with 500 μl of PBS, adding 100 μl at a time, taking care to avoid bubbles, and collect the flow-through in a third tube ("first wash"). Once the column has stopped dropping, perform a second wash on the column with 500 μl of PBS, adding 100 μl at a time, taking care to avoid bubbles, and collect the flow-through in a fourth tube ("second wash"). Once the column dripping stopped, the needle was capped, and cells from each of the four tubes were analyzed under a microscope.

[0205] Figure 5 shows bright-field and green fluorescence images of 10 μl of lung cancer cells passed through a 5% 3-aminopropyltriethoxysilane bead column, including initial cell solution (A and E), pass-through samples (B and F), first wash samples (C and G), and second wash samples (D and H). Viable lung cancer cells are visualized as green fluorescence under a microscope using calcein AM staining. Multiple cells are visible in the initial cell solution, but only a small number of cells are visible in the pass-through samples, first wash samples, and second wash samples. This indicates that the majority of viable lung cancer cells remained in the column and were captured by the positively charged 5% 3-aminopropyltriethoxysilane beads, but some cells were not captured. Similar results were obtained when the experiment was repeated.

[0206] Figure 6 shows bright-field and green fluorescence images of 10 μl each of initial cell solution (A and E), pass-through samples (B and F), first wash samples (C and G), and second wash samples (D and H) of lung cancer cells that passed through a 10% 3-aminopropyltrimethoxysilane bead column. Viable lung cancer cells are visualized as green fluorescence when observed under a microscope with green light by calcein AM staining. Multiple cells can be seen in the initial cell solution, but only a small number of cells can be seen in the pass-through samples, first wash samples, and second wash samples. This indicates that the majority of viable lung cancer cells remained in the column and were captured by the positively charged 10% 3-aminopropyltrimethoxysilane beads, but some cells were not captured.

[0207] Each of the 5% 3-aminopropyltriethoxysilane or 10% 3-aminopropyltrimethoxysilane bead columns showed similar results in capturing lung cancer cells. However, because the number of uncaptured cells was significantly higher than in any of the other cell lines examined, the 5% 3-aminopropyltriethoxysilane column was repeated once with a higher number of beads and the same number of cells, and again with the same number of beads and fewer cells. These experiments were set up to determine whether increasing the number of beads would require a higher volume column for this cell line.

[0208] For experiments conducted with a larger number of beads and the same number of cells, the experiment was carried out in the same manner as above, except that the column was prepared with 0.8 ml of 5% 3-aminopropyltriethoxysilane beads. The results can be seen in Figure 7. Multiple cells can be seen in the initial cell solution, but fewer cells are seen in the pass-through sample, first wash sample, and second wash sample compared to the 0.5 ml column. This indicates that this cell line may require a higher volume to improve cell binding.

[0209] For experiments conducted with the same number of beads and fewer cells, use 30 μl (approximately 3.0 × 10⁻⁶). 4 The experiment was conducted in the same manner as above, except that (1) cells were used and the volume was increased to 500 μl with 470 μl of PBS. The results can be seen in Figure 8. Multiple cells can be seen in the initial cell solution, but fewer cells are seen in the pass-through sample, first wash sample, and second wash sample compared to experiments using more cells. This again indicates that this cell line may require a higher volume to improve cell binding.

[0210] Example 4. Isolation of acute lymphoblastic leukemia cells Approximately 1.0×10 6 CCRF-SB acute lymphoblastic leukemia cells were stained using the method described above. 100 μl (approximately 1.0 × 10⁶) 5 Add (1) stained cells to a tube ("initial cell solution") and increase the volume to 500 μl with 400 μl of PBS. Mix the cells thoroughly and add them to a column prepared as described above, either with 0.5 ml of 5% 3-aminopropyltriethoxysilane or 10% 3-aminopropyltrimethoxysilane beads. Add the cells 100 μl at a time, taking care to avoid bubbles, and collect the flow-through in a second tube ("pass-through"). Once the column has stopped dropping, wash the column with 500 μl of PBS, adding 100 μl at a time, taking care to avoid bubbles, and collect the flow-through in a third tube ("first wash"). Once the column has stopped dropping, perform a second wash on the column with 500 μl of PBS, adding 100 μl at a time, taking care to avoid bubbles, and collect the flow-through in a fourth tube ("second wash"). Once the column dripping stopped, the needle was capped, and cells from each of the four tubes were analyzed under a microscope.

[0211] Figure 9 shows bright-field and green fluorescence images of 10 μl of acute lymphoblastic leukemia cancer cells that passed through a 5% 3-aminopropyltriethoxysilane bead column, for initial cell solution (A and E), pass-through samples (B and F), first wash samples (C and G), and second wash samples (D and H). Viable acute lymphoblastic leukemia cancer cells are visualized as green fluorescence when observed under a microscope with green light by calcein AM staining. Multiple cells are visible in the initial cell solution, but only a few cells are visible in the pass-through samples, and no cells are visible in either the first or second wash samples. This indicates that viable acute lymphoblastic leukemia cancer cells remained in the column and were captured by the positively charged 5% 3-aminopropyltriethoxysilane beads.

[0212] Figure 10 shows bright-field and green fluorescence images of 10 μl each of the initial cell solution (A and E), pass-through samples (B and F), first wash samples (C and G), and second wash samples (D and H) of acute lymphoblastic leukemia cancer cells that passed through a 10% 3-aminopropyltrimethoxysilane bead column. Viable acute lymphoblastic leukemia cancer cells are visualized as green fluorescence when observed under a microscope with green light by calcein AM staining. Multiple cells can be seen in the initial cell solution, but only a small number of cells are seen in the pass-through samples, as well as in the first and second wash samples, indicating that viable acute lymphoblastic leukemia cancer cells remained in the column and were captured by the positively charged 10% 3-aminopropyltrimethoxysilane beads.

[0213] Each of the 5% 3-aminopropyltriethoxysilane or 10% 3-aminopropyltrimethoxysilane bead columns showed similar results in capturing acute lymphoblastic leukemia cancer cells.

[0214] Example 5. Isolation of cancer cells in the presence of non-cancerous WBCs. To determine whether a positively charged bead matrix can isolate cancer cells from a mixture of cancer and non-cancerous cells, SW620 colon cancer cells were mixed with purified non-cancerous leukocytes, and the mixture was passed through a column.

[0215] Approximately 1.0×10 6 Individual SW620 colon cancer cells were stained using the method described above, and WBCs were purified using the hemolysis protocol described above. 100 μl (approximately 1.0 × 10⁶) 5 Stained tumor cells were mixed with 200 μl of WBCs in PBS in a tube ("initial cell solution") and the volume was increased to 500 μl with 200 μl of PBS. The cells were thoroughly mixed and added to a column of either 0.5 ml of 5% 3-aminopropyltriethoxysilane or 10% 3-aminopropyltrimethoxysilane beads, prepared as described above. Cells were added 100 μl at a time, taking care to avoid air bubbles, and the flow-through was collected in a second tube ("pass-through"). Once the dripping of the column stopped, the column was washed with 500 μl of PBS, adding 100 μl at a time, taking care to avoid air bubbles, and the flow-through was collected in a third tube ("first wash"). Once the column dripping stopped, the column was washed a second time with 500 μl of PBS, adding 100 μl at a time to ensure air bubbles were avoided, and the flow-through was collected in the fourth tube ("Second Wash"). Once the column dripping stopped, the needle was capped, and cells from each of the four tubes were analyzed under a microscope.

[0216] Figure 11 shows bright-field and green fluorescence images of 10 μl each of the initial cell solution (A and E), pass-through samples (B and F), first wash samples (C and G), and second wash samples (D and H) of a colon cancer cell / WBC mixture passed through a 5% 3-aminopropyltriethoxysilane bead column. Both colon cancer cells and WBCs are visible in bright-field images. Viable colon cancer cells are visualized as green fluorescence when observed under a microscope with green light after calcein AM staining. Multiple viable colon cancer cells are visible in the initial cell solution, but no viable colon cancer cells are visible in the pass-through samples, first wash samples, and second wash samples. On the other hand, WBCs are visible in bright-field images of each of the pass-through samples, first wash samples, and second wash samples. This indicates that viable colon cancer cells remained in the column and were captured by positively charged 5% 3-aminopropyltriethoxysilane beads, while non-cancerous WBCs were not captured.

[0217] The experiment was repeated by increasing the amount of WBCs in the mixture. This experiment was conducted in the same manner as above, except that 400 μl of WBCs in PBS was used. The results can be seen in Figure 12. Again, no viable colon cancer cells were observed in the pass-through sample, the first wash sample, and the second wash sample, and WBCs were visible in bright-field imaging for each of the pass-through sample, the first wash sample, and the second wash sample. This confirms that viable colon cancer cells remained in the column and were captured by the positively charged 5% 3-aminopropyltriethoxysilane beads, but non-cancerous WBCs were not captured.

[0218] Example 7. Isolation of cancer cells using a mini-column. A single capillary containing 10% positively charged 3-aminopropyltrimethoxysilane beads, prepared as described above, was used as a "mini-column" for each run. Cells were collected and stained as described above. 100 μl (approximately 1.0 × 10⁶) 5100 μl of stained cells were added to a tube ("initial cell solution") and the volume was increased to 500 μl with 400 μl of PBS. The cells were thoroughly mixed and added to a mini-column using a micropipette with a long tip. Cells were added 100 μl at a time, taking care to avoid air bubbles, and the flow-through was collected in a second tube ("pass-through"). Once the mini-column stopped working, it was washed with 500 μl of PBS, adding 100 μl at a time, taking care to avoid air bubbles, and the flow-through was collected in a third tube ("first wash"). Once the mini-column stopped working, it was washed a second time with 500 μl of PBS, adding 100 μl at a time, taking care to avoid air bubbles, and the flow-through was collected in a fourth tube ("second wash"). Once the mini-column stopped working, it was analyzed under a microscope to confirm the adhesion of green fluorescent cells to positively charged glass beads.

[0219] Figure 13 shows a green fluorescence image of breast cancer cells trapped on positively charged glass beads in a 1.5 mm transparent glass mini-column. The narrow diameter of the capillaries allows for clear visualization, analysis, and evaluation of the cells using fluorescently labeled antibody markers, enabling determination of the primary tissue.

[0220] Example 8. Measurement of bead volume For example, the bead capacity was measured to determine the number of beads required for downstream applications such as vaccine preparation. One amine-coated glass bead, prepared as described above, with a diameter of 1.7–2.5 mm, was measured in 1 × 10⁻⁶ units. 6 The individual cells were incubated in 1 ml of PBS for 1 hour. After incubation, a single bead was washed three times with PBS. 40 μl of 1% SDS was added to the bead to lyse the cells, and the beads were incubated for 30 minutes. After incubation, the beads and lysed cells were centrifuged at 10,000 × g for 5 minutes. The absorbance of the supernatant was read at 280 nm on a nanodrop and compared to a standard curve prepared as described below. This procedure was then repeated using five beads.

[0221] A standard curve was established using the CCRF-SB cell line (human acute lymphoblastic leukemia). The set cell count in 20 μl of PBS was 1 × 10⁶. 3 , 2.5×10 3 , 5×10 3 , 1 x 10 4 , and 1.5 × 10 4 The protein was dissolved in 20 μl of 1% SDS. After incubation for 30 minutes, the tube was centrifuged at 10,000 × g for 5 minutes. The absorbance of the supernatant was read at 280 nm on a nanodrop, and the protein μg / μl per cell count was graphed (Figure 15).

[0222] result The cell lysate produced from a single bead had an absorbance of 0.02725 at 280 nm. The number of cells was determined using the equation derived from the standard curve: Y = 1.165e-005*X + 0.009881. Therefore, one bead contained a volume of 1490.90 cells.

[0223] The five beads had an absorbance of 0.0876 at 280 nm. The number of cells was determined using an equation generated from the curve. Accordingly, the five beads had a volume of 6676.88 cells, and therefore, one bead had a volume of 1335.38 cells.

[0224] conclusion From this data, it was concluded that one amine-coated charged bead binds to approximately 1400 cells. This information can be used for downstream applications, for example, to prepare 1 × 10⁶ lysates sufficient for the oncolytic vaccine described herein. 6 A certain number of cells are needed. Therefore, 1 × 10 6 Approximately 750 beads are needed to capture a single cell, and a column containing 750 or more beads is sufficient to capture enough leukemia cells or other blood cancer cells from a patient's blood to produce an effective therapeutic cancer vaccine.

[0225] Example 9. Quantification of circulating cancer cells For diagnostic use, the nucleic acid content of the lysate, i.e., the mRNA copy number determined by quantitative RT-PCR, can be used to determine the number of tumor cells bound to each bead.

[0226] Amine-coated glass beads were prepared as described herein and functionalized with 10% 3-aminopropyltriethoxysilane. 10 million human PBMC cells, 10 million human SKOV3 breast cancer cells, and 160 human SKOV3 cells were spiked and prepared in 50 μl final volume PBS. The cells were transferred to U-bottom wells in a 96-well plate with a single glass bead having a diameter of 1.7–2.5 mm in each well. The plate was shaken at 100 rpm for 1 hour at room temperature. The beads were picked up with tweezers and washed three times with PBS. The beads were then placed in PCR tubes and lysed in 50 μl lysis buffer. 10 μl of the cell lysate was used for the reverse transcription reaction in a final volume of 20 μl. 1 microliter of the reverse transcription reaction was used for quantitative PCR analysis.

[0227] The number of amplification cycles required to observe the PCR product was measured using beta-actin PCR primers (housekeeping genes) (Figure 16A). Similarly, the number of amplification cycles required to observe the PCR product was measured using Her2 PCR primers (Figure 16B).

[0228] Using the number of cycles, we created a standard curve demonstrating that a single human breast cancer cell can be determined by quantitative PCR analysis in approximately 37 cycles (Figure 17). This standard curve can be used to determine the number of breast cancer cells in a sample. Similar curves can be prepared for different cancer samples according to the protocol outlined in the flowchart (white box) in Figure 18 to quantify the number of cancer cells in the sample.

[0229] Example 10. Detection and identification of circulating cancer cells in patient samples Using the flowchart (white and gray boxes) protocol in Figure 18, cancer cells in patients can be quantified, detected, and identified using the method described herein. PBMCs from 7.5 ml of patient blood were resuspended in 200 μl of PBS. 10 μl of cells were used for circulating cancer cell isolation with two glass beads in 100 μl of PBS. After incubation at room temperature for 1 hour with shaking at 100 rpm, the glass beads were isolated and washed three times with PBS. 10 μl of the original PBMCs, PBMCs from the circulating cancer cell isolation, and the glass beads were all lysed in 50 μl of lysis buffer. 10 μl of the lysate was used for first-strand cDNA synthesis in a 20 μl reaction. The first-strand cDNA was diluted 10-fold, and 1 μl was used for quantitative PCR amplification in a 20 μl reaction.

[0230] result As shown in Figure 19, these results clearly indicate that the circulating cancer cells captured from the patient's blood sample by the beads were breast cancer cells expressing Her2 and beta-actin, and that only breast cancer cells were captured. The captured circulating cancer cells did not show contamination by lymphocytes, which constitute the majority of cells in the patient's blood sample, as the high-sensitivity CD45 PCR assay was completely negative (Figure 19C).

Claims

1. A method for extracting cancer cells from a biological sample, a) Passing a biological sample containing cancer cells through an isolation matrix containing functionalized microspheres, b) Collecting the biological sample flowing through the isolation matrix into a first aliquot, Includes, The cancer cells bind to the isolation matrix. method.

2. The method according to claim 1, wherein the functionalized microsphere is functionalized with a positively charged functional group.

3. The method according to claim 1 or 2, wherein the functionalized microsphere is functionalized with an amine, polyethyleneimine (PEI), and / or a guanidine group.

4. The method according to any one of claims 1 to 3, wherein the microsphere comprises glass, polymer, or resin.

5. The method according to any one of claims 1 to 4, wherein the microsphere has a diameter of 500 μm to 600 μm.

6. The method according to any one of claims 1 to 5, wherein the biological sample includes blood.

7. The method according to any one of claims 1 to 6, wherein the biological sample is obtained from a subject that has cancer or is suspected of having cancer.

8. The method according to claim 7, wherein the cancer is a blood cancer or includes a solid tumor.

9. The method according to any one of claims 1 to 8, further comprising eluting the cancer cells bound to the isolation matrix into a second aliquot.

10. The method according to any one of claims 7 to 9, wherein the first aliquot is administered to and returned to the subject.

11. The method according to any one of claims 1 to 9, further comprising detecting the presence or absence of cancer cells in the biological sample.

12. The method according to any one of claims 7 to 11, further comprising lysing the cancer cells to obtain a cancer cell lysate.

13. The method according to claim 12, further comprising incorporating the cancer cell lysate into a cancer vaccine.

14. The method according to claim 12, further comprising determining the mRNA copy number from the cancer cell lysate.

15. The method according to claim 14, wherein the mRNA copy number is determined by quantitative RT-PCR.

16. The method according to any one of claims 1 to 15, further comprising calculating the number of cancer cells bound to the functionalized microspheres.

17. A cancer vaccine prepared by a process, wherein the process is a) Contacting a biological sample containing cancer cells with a positively charged surface, wherein the cancer cells bind to the positively charged surface. b) Lysing the cancer cells to obtain a cancer cell lysate, c) Incorporating the cancer cell lysate into yeast cell wall particles (YCWP), Cancer vaccines, including...

18. a) Yeast cell wall particles (YCWP), b) The cancer cell lysate according to claim 12, Cancer vaccines, including...

19. The vaccine according to claim 17 or 18, wherein the YCWP is modified by capping with silicate.

20. The vaccine according to claim 19, wherein the silicate is selected from the group comprising tetraethyl orthosilicate, tetramethyl orthosilicate, tetrapropyl orthosilicate, and tetrabutyl orthosilicate.

21. The vaccine according to any one of claims 17 to 20, further comprising one or more adjuvants, excipients, and preservatives.

22. A method for delivering a vaccine to a target, comprising administering the vaccine described in any one of claims 17 to 21 to the target.

23. A method for treating or preventing cancer, comprising administering a vaccine according to any one of claims 17 to 21 to a subject in need thereof.

24. The method according to claim 22 or 23, wherein the vaccine is administered subcutaneously, orally, or intravenously.

25. The method according to claim 22 or 23, wherein the vaccine is administered to the dermis of the subject.

26. A method for treating cancer in a patient, comprising extracting cancer cells from the patient's blood by passing the blood through an isolation matrix containing functionalized microspheres that bind to cancer cells.

27. The method according to claim 26, wherein the functionalized microsphere is functionalized with a positively charged functional group.

28. The method according to claim 26 or 27, wherein the functionalized microsphere is functionalized with an amine, polyethyleneimine (PEI), and / or a guanidine group.

29. The method according to any one of claims 26 to 28, wherein the microsphere comprises glass, polymer, or resin.

30. The method according to any one of claims 26 to 29, wherein the microspheres have a diameter of 500 μm to 600 μm.

31. The method according to any one of claims 26 to 30, wherein the patient has blood cancer or malignant cancer.

32. The method according to any one of claims 26 to 31, further comprising eluting the bound cancer cells from the isolation matrix.

33. The method according to claim 32, further comprising lysing the eluted cells to obtain a lysate, and incorporating the lysate into a cancer vaccine for treating the patient.

34. A method for detecting cancer cells in a patient, a) Passing a biological sample from the patient through an isolation matrix of functionalized microspheres, wherein the functionalized microspheres bind to and pass cancer cells. b) Eluting the cancer cells from the matrix, c) To detect the presence or absence of cancer cells in the biological sample, Methods that include...

35. The method according to claim 34, wherein the patient has blood cancer or malignant cancer.

36. The method according to claim 34 or 35, wherein the functionalized microsphere is functionalized with an amine, polyethyleneimine (PEI), and / or a guanidine group.

37. The method according to any one of claims 34 to 36, wherein the microsphere comprises glass, polymer, or resin.

38. The method according to any one of claims 34 to 37, wherein the microsphere has a diameter of 500 μm to 600 μm.

39. A cancer cell isolation matrix comprising functionalized microspheres containing positively charged functional groups, wherein the functionalized microspheres bind to cancer cells.

40. The cancer cell isolation matrix according to claim 39, wherein the functionalized microspheres are functionalized with an amine, polyethyleneimine (PEI), and / or a guanidine group.

41. The cancer cell isolation matrix according to claim 39 or 40, wherein the microsphere comprises glass, polymer, or resin.

42. The cancer cell isolation matrix according to any one of claims 39 to 41, wherein the microspheres have a diameter of 500 μm to 600 μm.

43. A kit for purifying cancer cells from a biological sample, comprising functionalized microspheres, wherein the functionalized microspheres bind to cancer cells derived from the biological sample.

44. A method for preparing a column for isolating cancer cells, a) Preparing an isolation matrix, wherein the isolation matrix contains functionalized microspheres, b) Depositing the isolation matrix into a container, wherein the container has an inlet and an outlet, Methods that include...

45. The method according to claim 44, wherein the functionalized microsphere is functionalized with a positively charged functional group.

46. The method according to claim 44 or 45, wherein the functionalized microsphere is functionalized with an amine, polyethyleneimine (PEI), and / or a guanidine group.

47. The method according to any one of claims 44 to 46, wherein the microsphere comprises glass, polymer, or resin.

48. The method according to any one of claims 44 to 47, wherein the microsphere has a diameter of 500 μm to 600 μm.

49. The method according to any one of claims 44 to 48, wherein preparing the functionalized microspheres comprises coating the microspheres with a 5% 3-aminopropyltriethoxysilane solution.

50. The method according to any one of claims 44 to 49, wherein preparing the functionalized microspheres comprises coating the microspheres with a 5% silane coupling agent.

51. The method according to any one of claims 44 to 50, wherein preparing the functionalized microspheres comprises coating the microspheres with polyethyleneimine (PEI).

52. The method according to any one of claims 44 to 50, wherein preparing the functionalized microspheres comprises coating the microspheres with aminoguanidine.

53. The method according to any one of claims 44 to 51, wherein depositing the isolation matrix includes depositing 0.1 to 1 ml of functionalized microspheres into the container.

54. A method for extracting cancer cells from a biological sample, a) Passing a biological sample containing cancer cells through a functionalized capillary, b) Collecting the biological sample flowing through the capillary into a first aliquot, Includes, The aforementioned cancer cells bind to the capillaries. method.

55. The method according to claim 54, wherein the capillary is functionalized with an amine, polyethyleneimine (PEI), and / or a guanidine group.

56. A method for extracting cancer cells from a biological sample, comprising contacting the biological sample containing cancer cells with a positively charged surface, wherein the cancer cells bind to the positively charged surface.

57. The method according to claim 56, wherein the positively charged surface is functionalized with an amine, polyethyleneimine (PEI), and / or a guanidine group.

58. The method according to claim 56 or 57, wherein the positively charged surface is made of glass, polymer, or resin.

59. The method according to any one of claims 56 to 58, wherein the positively charged surface is selected from beads, microparticles, capillaries, blood collection tubes, microscope slides, and microscope slide coverslips.

60. The method according to any one of claims 56 to 59, wherein the biological sample includes blood.

61. The method according to any one of claims 56 to 60, wherein the biological sample is obtained from a subject that has cancer or is suspected of having cancer.

62. The method according to claim 61, wherein the cancer is a blood cancer or includes a solid tumor.

63. The method according to any one of claims 56 to 62, further comprising detecting the presence or absence of cancer cells in the biological sample.

64. The method according to any one of claims 56 to 62, further comprising lysing the aforementioned cancer cells to obtain a cancer cell lysate.

65. The method according to claim 64, further comprising incorporating the cancer cell lysate into a cancer vaccine.

66. The method according to any one of claims 56 to 65, further comprising calculating the number of cancer cells bound to the positively charged surface.

67. a) Yeast cell wall particles (YCWP), b) The cancer cell lysate according to claim 64, Cancer vaccines, including...

68. The vaccine according to claim 67, wherein the YCWP is modified by capping with a silicate optionally selected from tetraethyl orthosilicate, tetramethyl orthosilicate, tetrapropyl orthosilicate, and tetrabutyl orthosilicate.

69. The vaccine according to claim 67 or 68, further comprising one or more adjuvants, excipients, and preservatives.

70. A method for delivering a vaccine to a subject, comprising administering the vaccine according to any one of claims 67 to 69 to the subject.

71. A method for treating or preventing cancer, comprising administering a vaccine according to any one of claims 67 to 69 to a subject in need thereof.

72. The method according to claim 70 or 71, wherein the vaccine is administered subcutaneously, orally, or intravenously.

73. The method according to claim 70 or 71, wherein the vaccine is administered to the dermis of the subject.

74. A method for treating cancer in a patient, comprising: extracting cancer cells from the patient's blood by bringing the patient's blood into contact with a positively charged surface, wherein the cancer cells bind to the positively charged surface; and returning the blood to the patient after contact with the positively charged surface.

75. The method according to claim 75, wherein the positively charged surface is functionalized with an amine, polyethyleneimine (PEI), and / or a guanidine group.

76. The method according to claim 74 or 75, wherein the positively charged surface is made of glass, polymer, or resin.

77. The method according to any one of claims 74 to 76, wherein the positively charged surface is selected from beads, microparticles, capillaries, blood collection tubes, microscope slides, and microscope slide covers.

78. The method according to any one of claims 74 to 77, wherein the patient has blood cancer or malignant cancer.

79. A method for detecting cancer cells in a subject, comprising: isolating cancer cells from a biological sample according to the method described in any one of claims 56 to 60; and detecting the presence or absence of cancer cells in the biological sample.

80. The method according to claim 79, wherein the patient has a blood cancer, a cancer including a solid tumor, or a malignant cancer.

81. A device for isolating cancer cells, comprising a positively charged surface containing an amine group, polyethyleneimine (PEI), a guanidine group, or any combination thereof.

82. The device according to claim 81, wherein the positively charged surface is made of glass, polymer, or resin.

83. The device according to claim 81 or 82, wherein the positively charged surface is selected from beads, microparticles, capillaries, blood collection tubes, microscope slides, and microscope slide covers.

Images