Methods and devices for separation of tumor cells

Abstract

Described herein are methods and compositions for isolating cancer cells using positively charged surfaces, such as beads, microparticles, and capillaries. The methods and compositions for isolation can be used for cancer diagnosis, treatment and therapeutic agent preparation.

Description

Technical Field

[0001] This disclosure generally relates to methods and functionalized microspheres and surfaces (e.g., capillaries) for binding cancer cells. More specifically, this disclosure relates to separation matrices of functionalized microspheres or capillaries and columns comprising said separation matrices for removing cancer cells from biological samples. The methods and compositions disclosed herein can be used for diagnosing or treating cancer, etc. 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 obtaining effective treatment or a cure. However, detecting cancer before symptoms appear or in the early stages of tumor metastasis presents a challenge for traditional tumor detection methods. For example, conventional methods fail to identify approximately 40% of cancer patients who require additional or enhanced therapy. Equally important is detecting any early signs of cancer spread after treatment to assess the effectiveness of the treatment and whether and what follow-up treatment is needed. Traditional cancer detection techniques, such as X-ray imaging and magnetic resonance (NMR) imaging, cannot provide reliable information for these critical applications.

[0003] Recent studies and clinical research have shown that cancer can invade the body very early in tumor development. Early detection and early systemic treatment will reduce cancer mortality. Metastasis, caused by tumor cells traveling from the primary tumor to vital distant organs via circulation, is known to be a major cause of cancer-related death. Tumor cells that spread early in the peripheral blood to lymph nodes or bone marrow are called circulating tumor cells (CTCs or CTCs). Even after the removal of the primary tumor, CTCs may still be present in the patient's peripheral blood.

[0004] Central cytokines (CTCs) are crucial for establishing metastasis, and the detection and isolation of CTCs are important tools for assessing the invasiveness of a given tumor and its potential for subsequent growth in distant organs. CTC detection can also represent early and initial detection of cancer in a patient. Specific and sensitive detection and isolation of CTCs can be used to identify the initial presence of cancer and overall cancer development or metastatic status, survival probability, and assessment of treatment response. Removal of CTCs from a patient's blood may also be used to treat certain cancers.

[0005] Current methods for detecting and isolating cancer cells (CTCs) based on molecular biomarkers are limited by method specificity and sensitivity. Therefore, novel targeting strategies are needed to explore other biophysical properties of cancer cells. Unlike current molecular biomarker-based CTC detection methods, this disclosure is universal for detecting any type of cancer cell. Summary of the Invention

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

[0007] In a first aspect, this disclosure provides a method for removing cancer cells from a biological sample, the method comprising: a) passing a biological sample containing cancer cells through a separation matrix comprising functionalized microspheres; and b) collecting the biological sample flowing through the separation matrix in a first aliquot, wherein the cancer cells are bound to the separation 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 diameter of the microspheres is between 500 µm and 600 µm.

[0009] In the embodiments disclosed herein, the biological sample includes blood. In some embodiments, the biological sample is obtained from a subject who has or is suspected of having cancer. In some embodiments, the cancer is a hematologic malignancy or includes a solid tumor. Some embodiments disclosed herein include eluting cancer cells bound to a separation matrix into a second aliquot. Some embodiments include administering a first aliquot back to the subject. 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 lysates. Some embodiments include incorporating the lysates into a cancer vaccine. Some embodiments include determining the mRNA copy number from the cancer cell lysates. In some embodiments, the mRNA copy number is determined by quantitative RT-PCR. Some embodiments include calculating the number of cancer cells bound to the functionalized microspheres.

[0010] In one aspect, this disclosure includes a cancer vaccine prepared by a 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) collecting the biological sample flowing through a separation matrix in a first aliquot; c) lysing the cancer cells to obtain cancer cell lysates in a second aliquot; and d) incorporating the cancer cell lysates into yeast cell wall particles (YCWPs).

[0011] In one aspect, this disclosure provides a method for treating a patient’s cancer, the method comprising extracting cancer cells from the patient’s blood by passing blood through a separation matrix containing functionalized microspheres that bind to cancer cells.

[0012] 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 diameter of the microspheres is between 500 µm and 600 µm.

[0013] In some of the embodiments disclosed herein, the patient has hematologic malignancies or other cancers.

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

[0015] In one aspect, this disclosure provides a method for detecting cancer cells in a patient, the method comprising: a) passing a biological sample from the patient through a separation matrix of functionalized microspheres, wherein the functionalized microspheres bind 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 of the embodiments disclosed herein, the patient has hematologic malignancies or other cancers.

[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, polymers, or resins. In some embodiments, the diameter of the microspheres is between 500 µm and 600 µm.

[0018] In one aspect, this disclosure provides a cancer cell separation matrix comprising functionalized microspheres containing positively charged functional groups, wherein the functionalized microspheres bind 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, polymers, or resins. In some embodiments, the diameter of the microspheres is between 500 µm and 600 µm.

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

[0020] In one aspect, this disclosure provides a method for preparing a column for cancer cell isolation, the method comprising: a) preparing an isolation matrix, wherein the isolation matrix comprises functionalized microspheres; and b) depositing the isolation matrix into a container having an inlet and an outlet. 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 diameter of the microspheres is between 500 µm and 600 µm.

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

[0022] In one aspect, this disclosure provides a method for removing cancer cells from a biological sample, the method comprising: a) passing a biological sample containing cancer cells through a functionalized capillary; and b) collecting the biological sample flowing through the capillary in 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 a guanidine group.

[0023] In one aspect, this disclosure provides a method for removing cancer cells from a biological sample, the method 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 an amine, polyethyleneimine (PEI), and / or a guanidine group. In some embodiments, the positively charged surface is made of 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 coverslips. In some embodiments, the biological sample comprises blood. In some embodiments, the cancer is a hematologic malignancy or includes a solid tumor. In some embodiments, the biological sample is obtained from a subject who has or is 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 cancer cell lysates. In some embodiments, the method further includes incorporating the cancer cell lysates 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 aspect, this disclosure provides a cancer vaccine comprising yeast cell wall particles (YCWP) and cancer cell lysates prepared as described herein.

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

[0027] In one aspect, this disclosure provides a method for delivering a vaccine to a subject, the method comprising administering to the subject the vaccine as disclosed herein.

[0028] In one aspect, this disclosure provides a method for treating or preventing cancer, which includes administering a vaccine, as disclosed herein, to a subject in need of such treatment.

[0029] Some embodiments include subcutaneous, oral, or intravenous administration of the vaccine. Some embodiments include administration of the vaccine to the dermis of the subject.

[0030] In one aspect, this disclosure provides a method of treating a patient's cancer, the method comprising: extracting cancer cells from the patient's blood by contacting the patient's blood with a positively charged surface, wherein the cancer cells are bound to the positively charged surface; and returning the blood to the patient after contacting the positively charged surface. In some embodiments, the positively charged surface is functionalized with an amine, polyethyleneimine (PEI), and / or a guanidine group. In some embodiments, the positively charged surface is made of 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 coverslips. In some embodiments, the biological sample comprises blood. In some embodiments, the cancer is a hematologic cancer or includes solid tumors. In some embodiments, the biological sample is obtained from a subject who has or is suspected of having cancer.

[0031] In one aspect, this disclosure provides a method for detecting cancer cells in a subject, the method comprising: removing 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, the cancer is a hematologic cancer, a cancer comprising a solid tumor, or a malignant cancer.

[0032] In one aspect, this disclosure provides an apparatus for isolating cancer cells, the apparatus comprising a positively charged surface containing amine groups, polyethyleneimine (PEI), guanidine groups, or any combination thereof. In some embodiments, the positively charged surface is made of 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 figures and detailed descriptions are exemplary and illustrative, but not intended to be limiting. Attached Figure Description

[0034] Figure 1 The SW620 colon cancer cell line was shown to be approximately 1.0 x 10⁻⁶ cells / mL. 5 Bright-field and GFP images (A to D) of cells before and after column preparation using 0.5 ml of 5% 3-aminopropyltriethoxysilane bead matrix. Colon cancer cells are visible under green fluorescence views (E to H). All cancer cells are captured by the bead matrix, as indicated by the lack of stained cells in F to G.

[0035] Figure 2 The SW620 colon cancer cell line was shown to be approximately 1.0 x 10⁻⁶ cells / mL. 5 Bright-field and GFP images (A to D) of cells prepared using 0.5 ml of 10% 3-aminopropyltrimethoxysilane bead matrix before and after column preparation. Colon cancer cells are visible under green fluorescence views (E to H). Most cancer cells are captured by the bead matrix, as indicated by a very small number of stained cells in F to G.

[0036] Figure 3 The T47D breast cancer cell line was shown to be approximately 1.0 x 10⁻⁶ cells. 5 Bright-field and GFP images (A to D) of cells before and after column preparation using 0.5 ml of 5% 3-aminopropyltriethoxysilane bead matrix. Breast cancer cells are visible under green fluorescence views (E to H). Most cancer cells are captured by the bead matrix, as indicated by a very small number of stained cells in F to G.

[0037] Figure 4 The T47D breast cancer cell line was shown to be approximately 1.0 x 10⁻⁶ cells. 5 Bright-field and GFP images (A to D) of cells prepared using 0.5 ml of 10% 3-aminopropyltrimethoxysilane bead matrix before and after column preparation. Breast cancer cells are visible under green fluorescence views (E to H). Most cancer cells are captured by the bead matrix, as indicated by a very small number of stained cells in F to G.

[0038] Figure 5The A549 lung cancer cell line was shown to be approximately 1.0 x 10⁻⁶ cells. 5 Cells were analyzed using bright-field and GFP images (A to D) before and after column preparation with 0.5 ml of 5% 3-aminopropyltriethoxysilane bead matrix. Lung cancer cells are visible under green fluorescence views (E to H). Most cancer cells are trapped by the bead matrix, as indicated by a very small number of stained cells in F to G.

[0039] Figure 6 The A549 lung cancer cell line was shown to be approximately 1.0 x 10⁻⁶ cells. 5 Bright-field and GFP images (A to D) of cells prepared using 0.5 ml of 10% 3-aminopropyltrimethoxysilane bead matrix before and after column preparation. Lung cancer cells are visible under green fluorescence views (E to H). Most cancer cells are captured by the bead matrix, as indicated by a very small number of stained cells in F to G.

[0040] Figure 7 The A549 lung cancer cell line was shown to be approximately 1.0 x 10⁻⁶ cells. 5 Cells were analyzed using bright-field and GFP images (A to D) before and after column preparation with 0.8 ml of 5% 3-aminopropyltriethoxysilane bead matrix. Lung cancer cells are visible under green fluorescence views (E to H). Most cancer cells were captured by the bead matrix, as indicated by a very small number of stained cells in F to G.

[0041] Figure 8 The A549 lung cancer cell line was shown to be approximately 3.0 x 10⁻⁶ cells. 4 Cells were analyzed using bright-field and GFP images (A to D) before and after column preparation with 0.5 ml of 5% 3-aminopropyltriethoxysilane bead matrix. Lung cancer cells are visible under green fluorescence views (E to H). Most cancer cells are trapped by the bead matrix, as indicated by a very small number of stained cells in F to G.

[0042] Figure 9 The image shows CCRF-SB acute lymphoblastic leukemia (ALL) cells at approximately 1.0 x 10⁻⁶. 5 Bright-field and GFP images (A to D) of cells prepared using 0.5 ml of 5% 3-aminopropyltriethoxysilane bead matrix before and after column chromatography. ALL cells are visible under green fluorescence views (E to H). All cancer cells are captured by the bead matrix, as indicated by the lack of stained cells in F to G.

[0043] Figure 10 The image shows CCRF-SB acute lymphoblastic leukemia (ALL) cells at approximately 1.0 x 10⁻⁶. 5Bright-field and GFP images (A to D) of cells prepared using 0.5 ml of 10% 3-aminopropyltrimethoxysilane bead matrix before and after column preparation. ALL cells are visible under green fluorescence views (E to H). Most cancer cells are captured by the bead matrix, as indicated by a very small number of stained cells in F to G.

[0044] Figure 11 It shows 1.0 x 10 5 A mixture of colon cancer cells and 400 µl of non-cancerous white blood cells (WBCs) is viewed under a microscope before and after passing through a 5% 3-aminopropyltriethoxysilane bead matrix (A to D). Colon cancer cells are visible under green fluorescence views (E to H). All tumor cells are captured by the bead matrix, as indicated by the lack of stained cells in F to G. The presence of cells in B indicates that the bead matrix did not capture non-cancerous WBCs.

[0045] Figure 12 It shows 1.0 x 10 6 A mixture of colon cancer cells and 200 µl of non-cancerous white blood cells (WBCs) is viewed in the microscopic fields (A to D) before and after passing through a 3-aminopropyltriethoxysilane bead matrix. Colon cancer cells are visible under green fluorescence views (E to H). All tumor cells are captured by the bead matrix, as indicated by the lack of stained cells in F to G. The presence of cells in B indicates that the bead matrix did not capture non-cancerous WBCs.

[0046] Figure 13 Breast cancer cells captured on positively charged glass beads within a 1.5 mm micropillar of clear glass are shown. These cells are clearly visualized, analyzed, and evaluated using fluorescently labeled antibody markers.

[0047] Figure 14 An example of a column used in the embodiments described herein is shown.

[0048] Figure 15 A standard curve of cell number versus protein concentration established using the CCRF-SB cell line is shown for determining bead capacity.

[0049] Figure 16 The PCR cycle numbers for detecting (A) β-actin and (B) Her2 in unbound PBMCs (PBMCs), bead-bound SKOV3 ovarian cancer cells (beads), and unbound SKOV3 ovarian cancer cells (SKOV3) are shown. The error bars show the standard deviation for n=3.

[0050] Figure 17The standard curves of cycle number versus cell number generated using SK-BR-3 cells are shown. "Cells Only" shows the curve of SKOV3 cells versus CQ value as a standard curve. "Beads with Cells" shows the number of cells bound to the beads and the CQ value.

[0051] Figure 18 An example of a method for quantifying the number of cells in a sample (white box) and a method for detecting and amplifying circulating cancer cells in a patient (white box and gray box) are shown.

[0052] Figure 19 The PCR cycle number for detecting (A) β-actin, (B) Her2, and (C) CD45 in a sample is shown before incubation with beads (whole PBMCs), breast cancer cells bound to beads from a patient sample (beads / beads with cells), and remaining cells from a patient sample that were not attached to beads (after separation). The error bars show the standard deviation for n=3. Detailed Implementation

[0053] This disclosure generally relates to the fields of cancer diagnosis and treatment, each of which utilizes the isolation of cancer cells from biological samples, such as 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.

[0054] definition

[0055] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which they pertain.

[0056] As used herein, the term “about” refers to both the numerical term and a value not exceeding or less than 10% of the stated value. For example, the term “about 5 nM” simply discloses both the stated value of 5 nM and the range of 4.5 nM to 5.5 nM.

[0057] As used herein, unless the content explicitly states otherwise, the singular forms “a / an” and “described” include plural indicators. Thus, for example, “particle” includes two or more particles, “polynucleotide” includes two or more polynucleotides, “cell” refers to two or more cells, etc.

[0058] As used herein, the terms “particle,” “bead,” or “sphere” are used interchangeably and can include any shape or composition. Particles, beads, or spheres can have any diameter greater than 1 mm. In some embodiments, particles, beads, or spheres can have a diameter greater than 1 mm, greater than 1.5 mm, greater than 2 mm, greater than 2.5 mm, or greater than 3 mm. In some embodiments, particles, beads, or spheres can have a diameter between 1 mm and 10 mm. In some embodiments, particles, beads, or spheres can have a diameter between 1.5 mm and 5 mm. In some embodiments, particles, beads, or spheres can have a diameter between 1.7 mm and 2.5 mm. In some embodiments, particles, beads, or spheres can have an average diameter 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, 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 are composed 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.

[0059] As used herein, the term "particle" refers to a particle of any shape or composition having a diameter between 1 µm and 1000 µm. In some embodiments, the particle has a diameter between 200 µm and 800 µm. In some embodiments, the particle has a diameter between 400 µm and 600 µm. In some embodiments, the particle has a 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 1000 µm. In some embodiments, the particle is solid. In some embodiments, the particle is hollow. In some embodiments, the particle is porous or non-porous. In some embodiments, the particle comprises one or more different materials. In some embodiments, the particle includes or is composed of glass, nylon, hydrogel, ceramic, metal, and / or any other suitable material. In some embodiments, the particle is magnetic. In some embodiments, the particle is coated with a metallic surface, such as nickel. In some embodiments, the particle is functionalized. In some embodiments, the particle may be a microsphere or a nanoparticle.

[0060] As used herein, the term "matrix" refers to a collection of microparticles having an average diameter between 1 µm and 1000 µm. In some embodiments, the average diameter of the microparticles is between 200 µm and 800 µm. In some embodiments, the average diameter of the microparticles is between 400 µm and 600 µm. In some embodiments, the microparticles have a 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 1000 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 comprise microparticles having an average diameter of about 1 µm and microparticles having an average diameter between 500 µm and 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 spaces have 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 comprise particles, beads, or spheres having an average diameter greater than 1 mm.

[0061] As used herein, the term "column" refers to a structure or device that contains a matrix. A column can be of any shape or size suitable for containing a matrix. In some embodiments, a column is a syringe. In some embodiments, a column includes an inlet and an outlet. An inlet is the point at which a sample, solution, buffer, or reagent enters the column. The inlet can be an opening in the column or an opening in a conduit leading directly or indirectly to the column. An outlet is the opening through which a sample, sample component, or reagent leaves the column. Sample components and reagents leaving the chamber can be waste, i.e., sample components no longer to be used, or sample components or reagents to be recovered, such as reusable reagents or target cells to be further analyzed or manipulated. An outlet can be an opening in the column near a collection container or an opening in a conduit leading directly or indirectly from the column to the collection container. In some embodiments, the column can be connected to an automated system that controls the flow of cells and other reagents through 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 blockage.

[0062] As used herein, the term "functionalization" refers to any process that modifies a material by introducing physical, chemical, or biological properties that differ 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 atomic group within a molecule that is responsible for the characteristic chemical reactions of those molecules. As used herein, functional groups can be positively or negatively charged. In some embodiments, positively charged functional groups may include amine, aldehyde, polyethyleneimine (PEI), and / or guanidine groups. In some embodiments, functionalization may include coupling agents to attach groups to particles.

[0063] 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, the 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 fluid (BAL), bronchial lavage fluid (BW), blood, body fluid, cerebrospinal fluid (CSF), urine, plasma, serum, or tissue (e.g., biopsy material). In some embodiments, the sample may be a mixture of multiple different cell types or contain a single cell type. In some embodiments, solid samples, such as tissue biopsies, may be prepared into fluid samples, for example, by chemical, enzymatic, or physical dissociation of cells. In some embodiments, the 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 white blood cells (WBCs) purified from a blood sample.

[0064] As used herein, the term "cell surface charge" or "electrostatic charge" refers to the net positive, negative, or neutral charge on the cell surface. Cancer cells have recently been found to have a negatively charged cell surface. The disclosures described herein utilize the net negative charge on the surface of cancer cells to isolate cancer cells from biological samples.

[0065] As used herein, the terms “cancer” and “tumor” are used interchangeably and refer to cells exhibiting typical characteristics of cancerous cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rates, and certain distinctive morphological features. Cancer cells typically take the form of tumors, but such cells can exist alone in an animal or can be non-tumorigenic cancer cells. As used herein, the term “cancer” includes both precancerous and malignant cancers. Examples of cancers can include blood cancers, prostate cancer, breast cancer, colon cancer, brain cancer, lung cancer, head and neck cancer, ovarian cancer, bladder cancer, kidney cancer and testicular cancer, melanoma, liver cancer, pancreatic cancer, and other gastrointestinal cancers. In some embodiments, cancer can be in the form of circulating tumor cells (CTCs), i.e., tumors or cancer cells circulating in the vascular system, lymphatic vessels, or other fluids. CTCs include, but are not limited to, leukemia cells or cells shed from a primary tumor.

[0066] As used herein, a “control” is an alternative sample used in an experiment for comparative purposes. A control can be “positive” or “negative.” As used herein, a “control cell sample” or “reference cell sample” refers to cells derived from a control or reference sample. In some embodiments, the reference or control cell sample is wild-type or non-cancerous. In some embodiments, the reference cell sample is purified or isolated (e.g., removed from its natural state). In other embodiments, the reference cell sample is derived from a non-tumor sample, such as a blood control, normal adjacent tumor (NAT), or any other non-cancerous sample from the same or different subjects.

[0067] As used herein, the term “enrichment” refers to increasing the relative concentration of a sample component with respect to other sample components (which may be a result of decreasing the concentration of other sample components), or increasing the absolute concentration of a sample component. For example, as used herein, “enriching” cancer cells from a sample includes increasing the proportion of cancer cells in the sample relative to all cells or other components. Enriching cancer cells in a blood sample can refer to increasing the concentration of cancer cells in the sample (e.g., by reducing the sample volume) or reducing the concentration or number of other cellular components in the blood sample to increase the percentage of cells present as cancer cells. Furthermore, “enriching” cancer cells in a sample can refer to increasing their concentration in the sample, such as by reducing the sample volume or reducing the number of “non-cancerous” cells in the sample.

[0068] As used herein, the term "separation" refers to the process in which one or more components of a sample are spatially separated from one or more other components of the sample. Separation can be performed such that one or more sample components of interest are transferred to or retained in one or more regions of a separation device, and at least some of the remaining components are transferred away from one or more sample components of interest being transferred to and / or retained in one or more regions, or where one or more sample components are retained in one or more regions and at least some or the remaining components are removed from said one or more regions. Alternatively, one or more components of a sample can be transferred to and / or retained in one or more regions, and one or more sample components can be removed from said one or more regions. It is also possible to transfer one or more sample components to one or more regions and to transfer one or more sample components of interest or one or more components of a sample to one or more other regions. Separation can be achieved, for example, by filtration or by using physical, chemical, electrical, or magnetic forces. Non-limiting examples of forces that can be used for separation are electrostatic forces, gravity, mass flow, dielectric force, traveling wave dielectric force, and electromagnetic force.

[0069] As used herein, the term "capture" refers to a type of separation in which one or more portions or sample components are retained in or on one or more areas of a surface, chamber, chip, bead, tube, or any container containing the sample, from which any remaining portion of the sample may be removed. For example, the separation matrix described herein can be used to capture cancer cells from a sample.

[0070] As used herein, the terms “subject” and “patient” refer to an organism receiving a diagnosis or treatment for a specific 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. Patients who can be diagnosed using the methods described herein may or may not exhibit symptoms of the disease. Patients who can be diagnosed using the methods described herein may have a genetic predisposition or lifestyle risk for the disease. For example, in some embodiments, the methods described herein can be used for early detection. Patients who can be diagnosed using the methods described herein may have previously recovered from or be in remission of the disease. Patients who can be treated using the compositions and methods described herein may have a known disease in which case the patient has been diagnosed with the disease and has exhibited symptoms of the disease over a longer period of time (e.g., over days, weeks, months, or years). Alternatively, a patient may have symptoms of a specific disease but has not yet been diagnosed with the disease by a physician. Other patients who can be treated using the compositions and methods described herein include those who have been diagnosed with a disease or condition and may or may not yet exhibit symptoms of the disease. Patients who can be treated with the compositions and methods described herein include those who have not yet been diagnosed with a disease or disorder and may have or may not yet exhibit symptoms of the disease, but have a genetic predisposition or lifestyle risk to the disease.

[0071] As used herein, the term "treatment" refers to therapeutic treatment in which the aim is to suppress or slow down (alleviate) undesirable physiological changes or conditions. Beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms, reduction of disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or reduction of the disease state, and parity (whether partial or complete), whether detectable or undetectable. Subjects requiring treatment include those who already have the condition or condition, those who are susceptible to the condition or condition, or those who wish to prevent the condition or condition.

[0072] This disclosure recognizes that the screening, diagnosis, prognosis, and treatment of many conditions, including cancer, may depend on the detection, isolation, and enrichment of rare cells from complex samples. Enrichment can typically be accomplished through one or more isolation steps. In particular, this disclosure recognizes that enriching or isolating rare cells, including malignant cells, from patient samples, such as isolating cancer cells from patient bodily fluid samples, can aid in the detection and typing of such malignant cells and thus contribute to diagnostic decisions and the development of treatments for patients.

[0073] Positively charged surfaces and separation matrices

[0074] This disclosure relies on recent findings that cancer cells carry a negative charge on their cell surface. Therefore, this disclosure provides several positively charged surfaces, including but not limited to microparticles and capillaries, for generating these microparticles and capillaries and testing their ability to bind and capture cancer cells.

[0075] The microparticles described herein can have any shape or size and be made of any suitable material. For example, the microparticles can be composed of one or more of glass, nylon, hydrogel, ceramic, metal, and / or any other suitable material. These microparticles can be homogeneous materials or coated with another material, such as nickel. For example, in some embodiments, the microparticles are glass microspheres. In an embodiment, the microparticles are nickel-coated glass microspheres.

[0076] The capillaries described herein can have any suitable volume, length, and diameter, and are made of any suitable material. For example, capillaries can be composed of one or more of glass, nylon, hydrogel, ceramic, metal, and / or any other suitable material. These capillaries can be made of a homogeneous material or coated with another material, such as nickel. In some embodiments, the capillary is glass. In some embodiments, the capillary is nickel-coated glass.

[0077] Other surfaces and substrates besides capillaries and microparticles (e.g., microspheres) may also be used for the purposes of this disclosure. For example, beads made of glass, nylon, hydrogel, ceramic, metal, and / or any other suitable material and having suitable dimensions can be functionalized as disclosed herein to provide a positively charged surface capable of binding cancer cells. Other useful surfaces and substrates include, but are not limited to, beads, meshes, membranes, films, culture dishes, pores, epitaxial tubes, 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 through which fluids containing cancer cells can pass or be collected.

[0078] Glass microspheres, capillaries, and other surfaces or substrates can be functionalized using the methods described herein to generate a positive charge on the surface of the microspheres or capillaries. For example, surfaces can be functionalized with positively charged portions, such as amines, polyethyleneimine (PEI), and / or guanidine groups, using chemical or electrostatic reactions. In some embodiments, the surface of the microparticles, capillaries, or other surfaces can be positively charged without functionalization.

[0079] For example, in some embodiments, an amine-coated surface, such as amine-coated glass beads or capillaries, can be prepared. For example, one or more glass beads can be used. In some embodiments, the glass beads may have a diameter of less than 1 mm (e.g., between 500 µm and 600 µm). In some embodiments, the glass beads may have a diameter of greater than 1 mm (e.g., between 1.7 mm and 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 removal of NaOH, and then washed three times again in ethanol to ensure removal of ddH₂O. The beads are then treated with a 5% 3-aminopropyltriethoxysilane solution by gently rotating for 2 h at room temperature. The beads are washed three times again with ethanol to ensure removal of the 3-aminopropyltriethoxysilane solution, and then washed three times again in ddH₂O to ensure removal of ethanol. The beads are then frozen at -84°C and then freeze-dried to remove water and form siloxane bonds. The same procedure can be used to coat the inside of capillaries or the surface of other devices (such as coverslips), but instead of gently rotating, the capillary of the device is placed in a 5% solution of 3-aminopropyltriethoxysilane for 2 hours while frequently moving the capillary up and down to mix the solution.

[0080] In some embodiments, an amine-coated surface can be prepared using 10% 3-aminopropyltriethoxysilane to produce a 10% 3-aminopropyltriethoxysilane-coated surface. In some embodiments, an amine-coated surface can be prepared using 5% 3-aminopropyltrimethoxysilane to produce a 5% 3-aminopropyltrimethoxysilane-coated surface. In some embodiments, an amine-coated surface can be prepared using 10% 3-aminopropyltrimethoxysilane to produce a 10% 3-aminopropyltrimethoxysilane-coated surface.

[0081] In some embodiments, an aldehyde-coated surface, such as aldehyde-coated glass beads or capillaries, can be prepared. For example, one or more glass beads can be used. In some embodiments, the glass beads may have a diameter of less than 1 mm (e.g., between 500 µm and 600 µm). In some embodiments, the glass beads may have a diameter of greater than 1 mm (e.g., between 1.7 mm and 2.5 mm). In some embodiments, the glass beads are prepared by etching in 30% NaOH. The etched glass beads are washed 5 times with ddH2O to ensure removal of NaOH, and then washed again 3 times in ethanol to ensure removal of ddH2O. A 5% silane coupling agent solution can be prepared by dissolving 2.5 ml of triethoxysilylbutyraldehyde in 50 ml of 4% water in an ethanol solution, and rotating the solution in a plastic tube at room temperature for 5 min to allow hydrolysis and formation of reactive silanols. The beads are then treated with the 5% silane coupling agent solution by gently rotating at room temperature for 2 h. The beads were washed three times with ethanol to ensure removal of the coupling agent, and then washed three more times in ddH2O to ensure removal of ethanol. The beads were then frozen at -84°C and freeze-dried to remove water and form siloxane bonds. The same procedure can be used to coat the inside of capillaries or the surface of other devices (such as coverslips), but instead of gently rotating, the capillary of the device is placed in a 5% silane coupling agent solution for 2 hours while frequently moving the capillary up and down to mix the solution.

[0082] In some embodiments, a polyethyleneimine (PEI) coated surface can be prepared, such as PEI-coated glass beads or capillaries. For example, one or more glass beads can be used. In some embodiments, the glass beads may have a diameter of less than 1 mm (e.g., between 500 µm and 600 µm). In some embodiments, the glass beads may have a diameter of greater than 1 mm (e.g., between 1.7 mm and 2.5 mm). The PEI surface can be prepared using a high pH or a high-low pH reaction, or by using an electrostatic reaction.

[0083] For example, in an embodiment using a high pH reaction, an amine-containing protein solution with a concentration of 10 mg / ml can be prepared by dissolving 2.5 g of branched PEI in 50 ml of 0.1 M sodium borate (pH 9.5). The acetaldehyde-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 350 µl of 5 M sodium cyanoborohydride in 1 N NaOH solution are added to the beads, and the mixture is rotated for 2 hours at room temperature. The beads are then washed five times in 30 ml of PBS to ensure removal of unreacted PEI.

[0084] In the example using the high-low pH reaction, two amine-containing protein solutions with 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, 0.15 M NaCl (pH 7.2) to produce a high-pH PEI solution and a low-pH PEI solution, respectively. The acetaldehyde-coated beads prepared as described above were washed three times in PBS to neutralize the pH of the beads. First, the beads were resuspended in 35 ml of the high-pH PEI solution and gently swirled for 15 min. Then, the supernatant was removed, and the beads were resuspended in 35 ml of the low-pH PEI solution. Then, 350 µl of 5 M sodium cyanoborohydride in 1 N NaOH solution was added to the beads, and swirled for 2 hr at room temperature. The beads were then washed five times in 30 ml of PBS to ensure removal of unreacted PEI.

[0085] In the example using an electrostatic reaction, glass beads were prepared by etching in 30% NaOH. The etched glass beads were washed five times with ddH₂O to ensure removal of NaOH, and then washed three more times in ethanol to ensure removal of ddH₂O. An aminoguanidine solution was prepared by dissolving 2 g of aminoguanidine hydrochloride in 10 ml of DMSO. Once completely dissolved in 40 ml of MCF, a pH 6.0 buffer was added to bring the total volume to 50 ml. The 40 ml aminoguanidine solution was then added to the washed beads and rotated at room temperature for 2 h. The beads were then washed three times with 30 ml of PBS to ensure removal of unreacted aminoguanidine.

[0086] In some embodiments, a histidine-coated surface, such as histidine-coated glass beads or capillaries, can be prepared. For example, one or more glass beads can be used. In some embodiments, the glass beads may have a diameter of less than 1 mm (e.g., between 500 µm and 600 µm). In some embodiments, the glass beads may have a diameter of greater than 1 mm (e.g., between 1.7 mm and 2.5 mm). A 5 mg / ml solution containing histidine 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 prepared above is prepared by dissolving 250 mg of EDC in 5 ml of MES buffer solution containing N-acetyl-L-histidine. Beads coated with 10% 3-aminopropylmethoxysilane or any amine-coated beads prepared as described above are rotated together with the MES buffer solution containing N-acetyl-L-histidine EDC for 2 hours at room temperature to react. Then wash the beads three times with 30 ml of ddH2O to ensure the removal of unreacted solution. The same procedure can be used to coat the inside of capillaries or the surface of other devices (such as coverslips), but instead of gently rotating, place the capillary or device in a MES buffer solution containing N-acetyl-L-histidine EDC for 2 hours while frequently moving the capillary or device up and down to mix the solution.

[0087] For the purposes of this disclosure, the separation matrix may comprise a column or other suitable shell filled with positively charged microspheres or beads as described herein. For example, a column may be filled with a plurality of functionalized microspheres (e.g., glass microspheres) or beads (e.g., glass beads) such that the surface of the particles exhibits positively charged portions, such as one or more amines, polyethyleneimine (PEI), and / or guanidine groups. Similarly, the separation matrix may comprise a single capillary, multiple capillaries, or other suitable tubes / chambers functionalized such that their inner surfaces are positively charged and have a diameter that allows cells to easily pass through the capillary or chamber and make significant contact with its inner surface. Negatively charged CTCs can bind to the functionalized inner surface through positively charged portions, such as one or more amines, polyethyleneimine (PEI), and / or guanidine groups.

[0088] In some embodiments, cells can be treated with positively charged nickel-coated microparticles or nanoparticles before being applied to microbead columns or capillaries for separation or capture. These particles adhere to negatively charged CTCs via simple electrostatic interactions, resulting in CTC decoration on the surface of these nickel microparticles or nickel nanoparticles. In this embodiment, such decorated CTCs bind to the surface of glass microparticles (such as histidine-coated glass microspheres) or the inner surface of capillaries (such as histidine-coated capillaries) not through electrostatic interactions but through chelation between their nickel surface decoration and the imidazole groups of the histidine moiety. A specific advantage of this embodiment is that such modified cells can be easily removed from microbead columns or capillaries by elution with imidazole, which disrupts the chelate bonds that previously held them in place. This allows for easier release of captured cells from the column and for use in additional downstream purposes, such as cancer diagnosis and other uses described herein.

[0089] Columns and capillaries containing separation matrix

[0090] Columns and capillaries can be prepared to contain a separation matrix, allowing biological samples to pass through the matrix, and positively charged particles or capillaries to capture and separate negatively charged cancer cells from the sample. The column or capillary can be of any shape or size suitable for containing the matrix and should include inlets and outlets to allow samples to enter and exit the column.

[0091] Figure 14 An exemplary workflow for preparing the columns of this disclosure is described. Once the microparticles have been deposited into the column, they form a matrix such that there are spaces between the microparticles that allow cells and other components of the sample to be filtered through the matrix.

[0092] Alternatively, cancer cells can be bound to or separated from biological samples (e.g., blood) by contacting the biological sample with a positively charged surface as disclosed herein. Such surfaces can be prepared by functionalization according to the methods disclosed herein.

[0093] Uses of isolating cancer cells

[0094] Cancer cells captured using the methods and compositions described herein can be used for a variety of downstream purposes. Cancer cells can be used directly on the column while still attached to the microparticles; cancer cells can be lysed without eluting from the column; cancer cells can be retained on the column; or cancer cells can be eluted from the column and used for one or more downstream purposes, including but not limited to those described herein.

[0095] In some embodiments, the disclosed methods can be used to identify the presence of cancer cells in a blood sample from a subject for cancer diagnosis purposes, wherein the presence of cancer cells in the blood sample from the patient indicates the presence of a tumor in the subject's body. The disclosed methods for detecting cancer or circulating tumor cells (CTCs) allow for early detection of cancer before signs or symptoms of cancer become apparent. As illustrated in the examples provided herein, the disclosed methods and compositions are highly sensitive and capable of separating cancer cells / CTCs as a very small fraction of a sample. Therefore, previously unknown cancers in a subject can be detected, for example, by obtaining a blood sample from the subject and passing that blood sample through a positively charged matrix or surface disclosed herein. Any cells that bind to the matrix or surface are expected to be cancer cells and will indicate that the subject has cancer or a tumor. In this way, previously unidentified cancers in a subject can be detected early. These methods are particularly useful for detecting or diagnosing certain types of cancer, such as pancreatic and ovarian cancer, which are often detected at a late stage.

[0096] Alternatively or concurrently, the presence of cancer cells can indicate the likelihood of tumor progression or metastasis. In some embodiments, this disclosure can be used to detect the presence of cancer in patients who have already received treatment. For example, this disclosure can be used to monitor cancer remission / relapse or detect minimal residual disease. In some embodiments, cancer cells are isolated using the methods described herein, and a positive 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 a positive identification of the cancer cells is obtained by detecting the genetic characteristics of the cancer cells.

[0097] In some embodiments of this disclosure, the type of cancer present in the sample may be unknown. Therefore, in some embodiments, cancer cells are isolated using the methods described herein, and the cells are visualized using fluorescent or colorimetric markers to allow counting and / or determination of the presence of bound cancer cells. In some embodiments, the type of cancer cells can be identified after isolation from the sample. For example, identification can be achieved using cancer type-specific biomarkers that can bind via fluorescent labeling, for example. The labeled cancer cells can be further analyzed using spectral imaging, fluorescence microscopy, visible light microscopy, or manual or automated image analysis. In some embodiments, cancer cells can be genotyped to identify the cancer type. Unlike other current methods for identifying CTCs using specific tumor type biomarkers, cells captured in this disclosure are simply defined as cancer cells by their negative surface charge. Therefore, any cell bound to these described matrixes is a cancer cell and provides a positive diagnosis of cancer for patients of cell origin. The type of cancer cells and their tissue of origin can be determined using non-cancer-specific tissue-specific biomarkers.

[0098] In some embodiments, the disclosed compositions and methods can be used to monitor disease progression, response to treatment, or relapse / recurrence in patients with cancer. In some embodiments, the number of cells captured using the methods described herein is determined at different time intervals during disease progression, during a treatment regimen lasting weeks, months, or longer, or after the treatment regimen has been interrupted. An increase in the number of cancer cells over time indicates a lack of response to treatment, relapse, a higher risk of metastasis, a poorer prognosis, a shorter expected survival, progression to a higher stage of cancer, or a faster 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 treatment, a stable disease state, or tumor shrinkage or remission, or a combination of the above.

[0099] In some embodiments, nucleic acid content can be used to characterize cancer cells isolated from cancer patients. One or more of the following methods can be used to analyze RNA and / or DNA from isolated cancer cells and characterize the genetic content and / or gene expression patterns in cancer cells: single nucleotide polymorphism analysis, quantitative PCR, RT-PCR, quantitative RT-PCR, FISH, DNA sequencing, multiplex PCR, determination of DNA methylation, quantification of total DNA content, whole genome amplification (WGA), CGH, laser dissecting microscopy (LDM), RNA amplification, oligonucleotide ligation assay (OLA), chromosome immunoprecipitation (CHIP), Southern blotting, hybridization, amplification, ligation, and enzyme assays. DNA and / or RNA can be isolated from matrix-bound cancer cells simply by lysing these cells on the matrix and recovering the lysates.

[0100] In some embodiments, the purpose of analyzing the genetic content of cancer cells is to identify the presence of mutations that may confer a higher proliferation rate or chromosomal deletions across 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 a personalized treatment process specific to the subject or a particular cancer phenotype.

[0101] Some embodiments of this disclosure may include the culture 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 clinical studies of experimental cancer therapies or drug candidates. Subjects in such trials may be humans, but typically they will include other mammals such as mice, rats, dogs, monkeys, etc. In this respect, the method can be used to provide clinical endpoints to measure the efficacy of experimental cancer therapies or drug candidates, which are faster and more quantitative than individual efficacy, metastasis, or recurrence data. This provides a faster and more quantitative assessment of the efficacy of the treatment being tested and provides additional information on how the therapeutic agent affects the probability of metastasis and recurrence of the cancer being treated. Therefore, it provides more information on the overall effectiveness of the experimental therapy and reduces the time required for clinical trials.

[0102] In some embodiments, cultured cancer cells obtained from blood samples of cancer patients are used to test the efficacy of candidate anticancer drugs or drug combinations in vitro before administering the drug to the patient or before deciding whether to continue administering the drug. In other embodiments, such cultured cancer cells are used to test new candidate drugs or other experimental therapies as part of clinical trials or even preliminary screening for efficacy.

[0103] In some embodiments, cancer cells isolated from cancer patients using the methods of this disclosure are immortalized through in vitro culture and selection, which may or may not be assisted by transfection of cells with SV40 T antigen or telomerase or other suitable methods. The immortalized cells can then be used to test the efficacy of anticancer agents, screen for new anticancer agents, or any other research requiring immortalized cell lines.

[0104] In some embodiments, cancer cells isolated from cancer patients using the methods or compositions of this disclosure can be used for invasiveness assays.

[0105] In some embodiments, cancer cells isolated from a cancer patient can be used for personalized immunotherapy purposes, wherein proteins or nucleic acids, or combinations thereof, obtained from cancer cells isolated from the cancer patient are incubated together with white blood cells (WBCs) or WBC subfractions from the patient to stimulate a cancer-specific immune response. The WBCs or WBC subfractions exposed to cancer cell tumor antigens are then re-inoculated into the patient.

[0106] In some embodiments, after isolating cancer cells from a blood sample using the methods or compositions described herein, various immunoassays can be used to characterize the cancer cells. For example, cancer cells can be lysed and the lysate centrifuged, and an ELISA assay performed. In this case, specific proteins of interest expressed in the cancer cells can be detected directly. This can provide an overview of the protein content in the cancer cells and allow monitoring of how the cancer cell phenotype changes during the disease process or during therapeutic treatment.

[0107] 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 cancer cells from many other cancers. Telomerase activity assays can be used to further characterize circulating tumor cells isolated using the elimination methods of this disclosure or 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 cleansing buffer or any other suitable method. The supernatant of the cell lysate is used as a template for telomerase extension reactions in PCR. Fluorescent PCR products are generated using fluorescently labeled primers and then measured by capillary electrophoresis. The greater the amount of fluorescent PCR product generated or the greater the length of the telomerase repeat amplification product, the higher the telomerase activity of cancer cells in the sample, which can serve as an indicator of tumor invasiveness or the number or fraction of cancer cells in the enriched sample.

[0108] In recent years, antibody-based therapies have achieved significant clinical success and are now part of the standard arsenal of clinicians in the fight against cancer. The methods disclosed herein provide a unique approach to monitoring the effects and efficacy of antibody-based therapies. In some embodiments, this disclosure can be used to detect the interaction between circulating cancer cells in the blood of a cancer patient and immunotherapeutic agents used for treatment, such as humanized exogenous antibodies. Whether isolated cancer cells bind to a therapeutic antibody can be determined by isolating the cancer cells and examining them for the presence of such antibodies.

[0109] In some embodiments, monitoring the interaction between a therapeutic antibody and cancer cells present in a blood sample can be used to assess a patient’s response to treatment, wherein a favorable outcome is predicted when the fraction of cancer cells binding to an immunotherapeutic agent (such as an antibody) is above a predetermined value or increases over time for measurements at different time points, and an unfavorable outcome is predicted when the fraction of cancer cells binding to the antibody is below a predetermined value or decreases over time for measurements at different time points.

[0110] 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 can be lysed and packaged 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 the cancer. In some embodiments, the YCWPs are capped. In some embodiments, the YCWPs are capped with a silicate ester. In some embodiments, the silicate ester includes 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 described, for example, in PCT / US2013 / 063091 (WO2014 / 040089).

[0111] The disclosed methods for isolating or capturing cancer cells and CTCs can be further used for treatment by removing cells from a patient. Surgical resection of solid tumors is often the first-line treatment for many forms of cancer, but such an option has not yet been available for hematologic malignancies. The disclosed methods for isolating or capturing cancer cells / CTCs can be used to treat hematologic malignancies such as leukemia and lymphoma. Similarly, the disclosed methods for isolating or capturing cancer cells / CTCs can be used to treat, prevent, or minimize the risk of metastasis to other forms of cancer (e.g., solid tumors) by removing CTCs and cancer cells from circulation that may invade tissues other than the primary tissue of a given cancer. Thus, 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 hematologic malignancies patient may be “filtered” and the cancer cells may be captured by the isolation matrix described herein. After the cancer cells are removed, the blood may be returned to the patient. This process may be repeated until the patient no longer has cancer or the circulating cancer cells are significantly reduced. Cancers suitable for treatment according to these methods include, but are not limited to, any hematologic malignancies such as leukemia and lymphoma. Such treatments can utilize existing apheresis or dialysis techniques to pass a subject's blood through a matrix or substrate that includes a positively charged surface as disclosed herein, thereby allowing CTCs to contact and bind to the charged surface or matrix.

[0112] Compositions and kits

[0113] This document also includes kits for functionalizing microparticles or surfaces, preparing separation 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 functionalized and / or positively charged microparticles or surfaces. In some embodiments, the kit may include reagents and apparatus for preparing matrices, such as buffers, tubes, and other desired apparatus. In some embodiments, the kit may include components for preparing columns, such as syringes, buffers, and tubes.

[0114] In some embodiments, the kits described herein may additionally or alternatively include reagents and devices for downstream processing or analysis of captured cancer cells. For example, the kits may include reagents or devices for culturing isolated cells, identifying isolated cells using cancer cell biomarkers, or extracting and analyzing nucleic acids from captured cancer cells.

[0115] The kit may also contain one or more of the following: wash buffers and / or reagents, hybridization buffers and / or reagents, labeling buffers and / or reagents, and detection tools. The buffers and / or reagents are typically optimized for the amplification / detection technology for which the kit is intended. The kit may also include protocols for different steps of the procedure using these buffers and reagents.

[0116] This disclosure further provides columns for use in the disclosed methods. The column may comprise a plurality of beads or particles containing positively charged surfaces, for example, functionalized with positively charged portions such as one or more amines, polyethyleneimine (PEI), and / or guanidine groups. Alternatively, this disclosure provides capillaries comprising positively charged surfaces, for example, functionalized with positively charged portions such as one or more amines, polyethyleneimine (PEI), and / or guanidine groups.

[0117] Examples of Implementation

[0118] Example 1. A method for removing cancer cells from a biological sample, the method comprising: a) passing a biological sample containing cancer cells through a separation matrix comprising functionalized microspheres; and b) collecting the biological sample flowing through the separation matrix in a first aliquot, wherein the cancer cells are bound to the separation matrix.

[0119] Example 2. The method according to Example 1, wherein the functionalized microspheres are functionalized with positively charged functional groups.

[0120] Example 3. The method according to Example 1 or 2, wherein the functionalized microspheres are functionalized with amines, polyethyleneimine (PEI) and / or guanidine groups.

[0121] Example 4. The method according to any one of Examples 1 to 3, wherein the microspheres comprise glass, polymer or resin.

[0122] Example 5. The method according to any one of Examples 1 to 4, wherein the diameter of the microsphere is between 500 µm and 600 µm.

[0123] Example 6. The method according to any one of Examples 1 to 5, wherein the biological sample comprises blood.

[0124] Example 7. The method according to any one of Examples 1 to 6, wherein the biological sample is obtained from a subject who has or is suspected of having cancer.

[0125] Example 8. The method according to Example 7, wherein the cancer is a hematologic cancer or includes a solid tumor.

[0126] Example 9. The method according to any one of Examples 1 to 8 further includes: eluting the cancer cells bound to the separation matrix into a second aliquot sample.

[0127] Example 10. The method according to any one of Examples 7 to 9, wherein the first aliquot of the sample is administered back to the subject.

[0128] Example 11. The method according to any one of Examples 1 to 9 further includes: detecting the presence or absence of cancer cells in the biological sample.

[0129] Example 12. The method according to any one of Examples 7 to 11 further includes: lysing the cancer cells to obtain cancer cell lysates.

[0130] Example 13. The method according to Example 12 further includes incorporating the cancer cell lysate into a cancer vaccine.

[0131] Example 14. The method according to Example 12 further includes: determining the mRNA copy number from the cancer cell lysate.

[0132] Example 15. The method according to Example 14, wherein the mRNA copy number is determined by quantitative RT-PCR.

[0133] Example 16. The method according to any one of Examples 1 to 15, further comprising: calculating the number of cancer cells bound to the functionalized microspheres.

[0134] Example 17. A cancer vaccine prepared by means of the following process: 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 cancer cell lysate; and c) incorporating the cancer cell lysate into yeast cell wall particles (YCWP).

[0135] Example 18. A cancer vaccine comprising: a) yeast cell wall particles (YCWP); and b) cancer cell lysate as described in Example 12.

[0136] Example 19. The vaccine according to Example 17 or 18, wherein the YCWP is modified by end capping with silicate.

[0137] Example 20. The vaccine according to Example 19, wherein the silicate ester is selected from the group consisting of tetraethyl orthosilicate, tetramethyl orthosilicate, tetrapropyl orthosilicate and tetrabutyl orthosilicate.

[0138] Example 21. The vaccine according to any one of Examples 17 to 20, further comprising one or more adjuvants, excipients and preservatives.

[0139] Example 22. A method for delivering a vaccine to a subject, the method comprising: administering to the subject the vaccine according to any one of Examples 17 to 21.

[0140] Example 23. A method for treating or preventing cancer, the method comprising: administering a vaccine according to any one of Examples 17 to 21 to a subject in need of the treatment.

[0141] Example 24. The method according to Example 22 or 23, wherein the vaccine is administered subcutaneously, orally or intravenously.

[0142] Example 25. The method according to Example 22 or 23, wherein the vaccine is applied to the dermis of the subject.

[0143] Example 26. A method for treating a patient's cancer, the method comprising: extracting cancer cells from the patient's blood by passing blood through a separation matrix comprising functionalized microspheres that bind to cancer cells.

[0144] Example 27. The method according to Example 26, wherein the functionalized microspheres are functionalized with positively charged functional groups.

[0145] Example 28. The method according to Example 26 or 27, wherein the functionalized microspheres are functionalized with amines, polyethyleneimine (PEI) and / or guanidine groups.

[0146] Example 29. The method according to any one of Examples 26 to 28, wherein the microspheres comprise glass, polymer or resin.

[0147] Example 30. The method according to any one of Examples 26 to 29, wherein the diameter of the microsphere is between 500 µm and 600 µm.

[0148] Example 31. The method according to any one of Examples 26 to 30, wherein the patient has hematologic cancer or malignant cancer.

[0149] Example 32. The method according to any one of Examples 26 to 31, further comprising: eluting bound cancer cells from the separation matrix.

[0150] Example 33. The method according to Example 32 further includes: lysing the eluted cells to obtain lysates and incorporating the lysates into a cancer vaccine for use in treating the patient.

[0151] Example 34. A method for detecting cancer cells in a patient, the method comprising: a) passing a biological sample from the patient through a separation matrix of functionalized microspheres, wherein the functionalized microspheres bind cancer cells; b) eluting the cancer cells from the matrix; and c) detecting the presence or absence of cancer cells in the biological sample.

[0152] Example 35. The method according to Example 34, wherein the patient has a hematologic cancer or malignant cancer.

[0153] Example 36. The method according to Example 34 or 35, wherein the functionalized microspheres are functionalized with amines, polyethyleneimine (PEI) and / or guanidine groups.

[0154] Example 37. The method according to any one of Examples 34 to 36, wherein the microspheres comprise glass, polymer, or resin.

[0155] Example 38. The method according to any one of Examples 34 to 37, wherein the diameter of the microsphere is between 500 µm and 600 µm.

[0156] Example 39. A cancer cell isolation matrix comprising functionalized microspheres containing positively charged functional groups, wherein the functionalized microspheres bind to cancer cells.

[0157] Example 40. Cancer cell isolation matrix according to Example 39, wherein the functionalized microspheres are functionalized with amines, polyethyleneimine (PEI) and / or guanidine groups.

[0158] Example 41. A cancer cell isolation matrix according to Example 39 or 40, wherein the microspheres comprise glass, polymer, or resin.

[0159] Example 42. Cancer cell isolation matrix according to any one of Examples 39 to 41, wherein the diameter of the microspheres is between 500 µm and 600 µm.

[0160] Example 43. A kit for purifying cancer cells from a biological sample, the kit comprising functionalized microspheres wherein the functionalized microspheres bind cancer cells from the biological sample.

[0161] Example 44. A method for preparing a column for cancer cell isolation, the method comprising: a) preparing an isolation matrix, wherein the isolation matrix comprises functionalized microspheres; and b) depositing the isolation matrix into a container, wherein the container has an inlet and an outlet.

[0162] Example 45. The method according to Example 44, wherein the functionalized microspheres are functionalized with positively charged functional groups.

[0163] Example 46. The method according to Example 44 or 45, wherein the functionalized microspheres are functionalized with amines, polyethyleneimine (PEI) and / or guanidine groups.

[0164] Example 47. The method according to any one of Examples 44 to 46, wherein the microspheres comprise glass, polymer, or resin.

[0165] Example 48. The method according to any one of Examples 44 to 47, wherein the diameter of the microsphere is between 500 µm and 600 µm.

[0166] Example 49. The method according to any one of Examples 44 to 48, wherein the preparation of the functionalized microspheres comprises coating the microspheres with a 5% 3-aminopropyltriethoxysilane solution.

[0167] Example 50. The method according to any one of Examples 44 to 49, wherein the preparation of the functionalized microspheres includes coating the microspheres with 5% silane coupling agent.

[0168] Example 51. The method according to any one of Examples 44 to 50, wherein the preparation of the functionalized microspheres includes coating the microspheres with polyethyleneimine (PEI).

[0169] Example 52. The method according to any one of Examples 44 to 50, wherein the preparation of the functionalized microspheres includes coating the microspheres with aminoguanidine.

[0170] Example 53. The method according to any one of Examples 44 to 51, wherein depositing the separation matrix comprises depositing functionalized microspheres of 0.1 ml to 1 ml into the container.

[0171] Example 54. A method for removing cancer cells from a biological sample, the method comprising: a) passing a biological sample containing cancer cells through a functionalized capillary; and b) collecting the biological sample flowing through the capillary in a first aliquot, wherein the cancer cells are bound to the capillary.

[0172] Example 55. The method according to Example 54, wherein the capillary is functionalized with an amine, polyethyleneimine (PEI) and / or a guanidine group.

[0173] Example 56. A method for removing cancer cells from a biological sample, the method comprising: contacting a biological sample containing cancer cells with a positively charged surface, wherein the cancer cells are bound to the positively charged surface.

[0174] Example 57. The method according to Example 56, wherein the positively charged surface is functionalized with amines, polyethyleneimine (PEI) and / or guanidine groups.

[0175] Example 58. The method according to Example 56 or 57, wherein the positively charged surface is made of glass, polymer or resin.

[0176] Example 59. The method according to any one of Examples 56 to 58, wherein the positively charged surface is selected from beads, microparticles, capillaries, blood collection tubes, microscope slides, and microscope slide covers.

[0177] Example 60. The method according to any one of Examples 56 to 59, wherein the biological sample comprises blood.

[0178] Example 61. The method according to any one of Examples 56 to 60, wherein the biological sample is obtained from a subject who has or is suspected of having cancer.

[0179] Example 62. The method according to Example 61, wherein the cancer is a hematologic cancer or includes a solid tumor.

[0180] Example 63. The method according to any one of Examples 56 to 62, further comprising: detecting the presence or absence of cancer cells in the biological sample.

[0181] Example 64. The method according to any one of Examples 56 to 62, further comprising: lysing the cancer cells to obtain cancer cell lysates.

[0182] Example 65. The method according to Example 64 further includes: incorporating the cancer cell lysate into a cancer vaccine.

[0183] Example 66. The method according to any one of Examples 56 to 65, further comprising: calculating the number of cancer cells bound to the positively charged surface.

[0184] Example 67. A cancer vaccine comprising: a) yeast cell wall particles (YCWP); and b) cancer cell lysate according to Example 64.

[0185] Example 68. The vaccine according to Example 67, wherein the YCWP is modified by end capping with a silicate ester optionally selected from tetraethyl orthosilicate, tetramethyl orthosilicate, tetrapropyl orthosilicate and tetrabutyl orthosilicate.

[0186] Example 69. The vaccine according to Example 67 or 68 further comprises one or more adjuvants, excipients and preservatives.

[0187] Example 70. A method for delivering a vaccine to a subject, the method comprising: administering to the subject the vaccine according to any one of Examples 67 to 69.

[0188] Example 71. A method for treating or preventing cancer, the method comprising: administering a vaccine according to any one of Examples 67 to 69 to a subject in need of the treatment.

[0189] Example 72. The method according to Example 70 or 71, wherein the vaccine is administered subcutaneously, orally or intravenously.

[0190] Example 73. The method according to Example 70 or 71, wherein the vaccine is applied to the dermis of the subject.

[0191] Example 74. A method for treating a patient's cancer, the method comprising: extracting cancer cells from the patient's blood by contacting the patient's blood with a positively charged surface, wherein the cancer cells are bound to the positively charged surface; and returning the blood to the patient after contacting the positively charged surface.

[0192] Example 75. The method according to Example 75, wherein the positively charged surface is functionalized with amines, polyethyleneimine (PEI) and / or guanidine groups.

[0193] Example 76. The method according to Example 74 or 75, wherein the positively charged surface is made of glass, polymer or resin.

[0194] Example 77. The method according to any one of Examples 74 to 76, wherein the positively charged surface is selected from beads, microparticles, capillaries, blood collection tubes, microscope slides, and microscope slide covers.

[0195] Example 78. The method according to any one of Examples 74 to 77, wherein the patient has a hematologic cancer or malignant cancer.

[0196] Example 79. A method for detecting cancer cells in a subject, the method comprising: removing cancer cells from a biological sample according to any one of Examples 56 to 60; and detecting the presence or absence of cancer cells in the biological sample.

[0197] Example 80. The method according to Example 79, wherein the patient has a hematologic cancer, including solid tumors, or malignant cancer.

[0198] Example 81. An apparatus for isolating cancer cells, the apparatus comprising a positively charged surface containing amine groups, polyethyleneimine (PEI), guanidine groups, or any combination thereof.

[0199] Example 82. The device according to Example 81, wherein the positively charged surface is made of glass, polymer or resin.

[0200] Example 83. The apparatus according to Example 81 or 82, wherein the positively charged surface is selected from beads, microparticles, capillaries, blood collection tubes, microscope slides, and microscope slide covers.

[0201] Example

[0202] The following examples are provided to provide a description of how the compositions and methods claimed herein can be performed, prepared, and evaluated to those skilled in the art, and are intended to be entirely exemplary and not to limit the scope of this disclosure.

[0203] Method: The following materials and methods are used in the examples.

[0204] Microsphere functionalization

[0205] Amine-coated glass beads

[0206] 5% 3-Aminopropyltriethoxysilane Beads: Weigh 10 g of MO-SCI OL-GLO0191B5-2338 500-600µm glass beads into a 50 ml tube. Etch the beads by rotating them in 30 ml of 30% NaOH solution (prepared by adding 10 ml of 1M NaOH to 20 ml of ddH2O) for 1 h. Prepare a 4% aqueous ethanol solution by mixing 4 ml of ddH2O with 96 ml of ethanol and adjusting the pH to 4.5-5.5 with acetic acid. Prepare a 50 ml 5% 3-aminopropyltriethoxysilane solution as follows: Dissolve 2.5 ml of 3-aminopropyltriethoxysilane in 50 ml of 4% aqueous ethanol solution and rotate the solution in a plastic tube at room temperature for 15 min to allow hydrolysis and formation of reactive silanols. Then wash the etched glass beads 5 times with 30 ml of ddH2O to ensure removal of NaOH. The beads were then washed three times in 30 ml of ethanol to ensure removal of ddH₂O. The beads were then reacted with 35 ml of 5% 3-aminopropyltriethoxysilane solution by gentle rotation at room temperature for 2 hours. The beads were then washed three times with 30 ml of ethanol to ensure removal of the 3-aminopropyltriethoxysilane solution, and then three times with 30 ml of ddH₂O to ensure removal of ethanol. The beads were then frozen at -84°C and then freeze-dried to remove water and form siloxane bonds. The same procedure can be used to coat the inside of capillaries or the surface of other devices (such as coverslips), but instead of gentle rotation, the capillaries or other devices are placed in a 5% 3-aminopropyltriethoxysilane solution for 2 hours while frequently moving the capillaries or devices up and down to mix the solution.

[0207] 10% 3-Aminopropyltriethoxysilane Beads: Prepare 10% 3-aminopropyltriethoxysilane beads using the same procedure as for 5% 3-aminopropyltriethoxysilane beads, except that 5 ml of 3-aminopropyltriethoxysilane is dissolved in a 4% aqueous ethanol solution. All other steps are the same. The same procedure can be used to coat the inside of capillaries or the surface of other devices (such as coverslips), but instead of gently rotating, immerse the capillary or other device in the 10% 3-aminopropyltriethoxysilane solution for 2 hours while frequently moving the capillary or device up and down to mix the solution.

[0208] 5% 3-Aminopropyltrimethoxysilane Beads: Weigh 10 g of MO-SCI OL-GLO0191B5-2338 500-600µm glass beads into a 50 ml tube. Etch the beads by rotating them in 30 ml of 30% NaOH solution (prepared by adding 10 ml of 1M NaOH to 20 ml of ddH2O) for 1 h. The 50 ml 5% 3-aminopropyltrimethoxysilane solution is prepared as follows: Dissolve 2.5 ml of 3-aminopropyltrimethoxysilane in 50 ml of methanol and rotate the solution in a plastic tube at room temperature for 15 min to allow hydrolysis and the formation of reactive silanols. Then wash the etched glass beads 5 times with 30 ml of ddH2O to ensure removal of NaOH. Then wash the beads 3 times with 30 ml of methanol to ensure removal of ddH2O. The beads were then reacted by gently rotating them in 35 ml of 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, followed by three washes in 30 ml of ddH₂O to ensure removal of methanol. The beads were then frozen at -84°C and freeze-dried to remove water and form siloxane bonds. The same procedure can be used to coat the inside of capillaries or the surface of other devices (such as coverslips), but instead of gently rotating, the capillary or device is placed in a 5% 3-aminopropyltrimethoxysilane solution for 2 hours while frequently moving the capillary or device up and down to mix the solution.

[0209] 10% 3-Aminopropyltrimethoxysilane Beads: Prepare 10% 3-Aminopropyltriethoxysilane beads using the same procedure as for preparing the 5% 3-Aminopropyltrimethoxysilane beads described above, except that 5 ml of 3-Aminopropyltrimethoxysilane is dissolved in methanol. All other steps are the same. The same procedure can be used to coat the inside of capillaries or the surface of other devices (such as coverslips), but instead of gently rotating, immerse the capillary or other device in the 10% 3-Aminopropyltrimethoxysilane solution for 2 hours while frequently moving the capillary or device up and down to mix the solution.

[0210] acetaldehyde-coated glass beads

[0211] Beads were coated in acetaldehyde using the silane coupling agent triethoxysilylbutyraldehyde. First, 10 g of 500-600 µm glass beads (MO-SCIOL-GLO0191B5-2338) were weighed into a 50 ml tube. The beads were etched by rotating them in 30 ml of 30% NaOH solution (prepared by adding 10 ml of 1M NaOH to 20 ml of ddH2O) for 1 h. A 4% aqueous ethanol 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 silane coupling agent solution was prepared by dissolving 2.5 ml of triethoxysilylbutyraldehyde in 50 ml of the 4% aqueous ethanol solution and rotating the solution in a plastic tube at room temperature for 5 min to allow hydrolysis and formation of reactive silanols. The etched glass beads were then washed five times with 30 ml of ddH2O to ensure removal of NaOH. The beads were then washed three times in 30 ml of ethanol to ensure removal of ddH₂O. The beads were then reacted by gently rotating them for 2 hours at room temperature using 35 ml of 5% silane coupling agent solution. The beads were then washed three times with 30 ml of ethanol to ensure removal of the silane coupling agent solution, and then three times with 30 ml of ddH₂O to ensure removal of ethanol. The beads were then frozen at -84°C and then freeze-dried to remove water and form siloxane bonds. The same procedure 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 placed in a 5% silane coupling agent solution for 2 hours while frequently moving the capillaries or devices up and down to mix the solution.

[0212] Polyethylene imide (PEI) coated glass beads

[0213] Preparation via chemical reaction (high pH): A 10 mg / ml amine-containing protein solution was prepared by dissolving 2.5 g of branched PEI in 50 ml of 0.1 M sodium borate (pH 9.5). 10 g of acetaldehyde-coated beads prepared as described above were weighed into a 50 ml tube and washed three times in PBS to neutralize the pH of the beads. 35 ml of the PEI solution and 350 µl of 5 M sodium cyanoborohydride in 1 N NaOH solution were added to the beads, and the mixture was rotated for 2 hours at room temperature. The beads were then washed five times in 30 ml of PBS to ensure the removal of unreacted PEI.

[0214] Preparation via chemical reaction (high-low pH): Two amine-containing protein solutions with 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, 0.15 M NaCl (pH 7.2) to generate high-pH and low-pH PEI solutions, respectively. 10 g of acetaldehyde-coated beads prepared as described above were weighed into a 50 ml tube and washed three times in PBS to neutralize the pH of the beads. The beads were first resuspended in 35 ml of high-pH PEI solution and gently swirled for 15 min. The supernatant was then removed, and the beads were resuspended in 35 ml of low-pH PEI solution. Then, 350 µl of 5 M sodium cyanoborohydride in 1 N NaOH solution was added to the beads, and swirled for 2 hr at room temperature. The beads were then washed five times in 30 ml of PBS to ensure removal of unreacted PEI.

[0215] Preparation was performed via electrostatic reaction: 10 g of MO-SCI OL-GLO0191B5-2338 500-600 µm glass beads were weighed into a 50 ml tube. The beads were etched by rotating in 30 ml of 30% NaOH solution (prepared by adding 10 ml of 1M NaOH to 20 ml of ddH2O) for 1 h. The beads were then washed 5 times with 30 ml of ddH2O to ensure removal of NaOH, and resuspended in 35 ml of PEI solution prepared by dissolving 5 g of branched polyethyleneimine (PEI) in 35 ml of PBS and rotated at room temperature for 2 h. The beads were then washed 5 times with 30 ml of PBS to ensure removal of unreacted PEI.

[0216] Guanidine-coated glass beads

[0217] Preparation was performed via electrostatic reaction: 10 g of MO-SCI OL-GLO0191B5-2338 500-600 µm glass beads were weighed into a 50 ml tube. The beads were etched by rotating in 30 ml of 30% NaOH solution (prepared by adding 10 ml of 1M NaOH to 20 ml of ddH2O) for 1 h. The beads were then washed 5 times with 30 ml of ddH2O to ensure removal of NaOH. An aminoguanidine solution was then prepared by dissolving 2 g of aminoguanidine hydrochloride in 10 ml of DMSO. Once 40 ml of MCF was completely dissolved, a pH 6.0 buffer was added to bring the total volume to 50 ml. The 40 ml aminoguanidine solution was then added to the washed beads and rotated at room temperature for 2 h. The beads were then washed 3 times with 30 ml of PBS to ensure removal of unreacted aminoguanidine.

[0218] Histidine-coated glass beads

[0219] Preparation is achieved via a chemical reaction: 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 solution prepared above is prepared by dissolving 250 mg of EDC in 5 ml of the N-acetyl-L-histidine MES buffer solution. 10 g of 10% 3-aminopropylmethoxysilane-coated beads prepared as described above are weighed into a 50 ml tube. Alternatively, any of the amine-coated beads can be used. The N-acetyl-L-histidine-containing EDC in MES buffer is added to the tube containing the amine beads, and they are rotated at room temperature for 2 hours to allow the reaction to proceed. After the reaction time, the beads are washed three times with 30 ml of ddH2O to ensure the removal of unreacted solution. The same procedure 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 placed in a MES buffer solution containing N-acetyl-L-histidine EDC for 2 hours while frequently moving the capillaries or devices up and down to mix the solution.

[0220] Cell culture

[0221] Cells were cultured in T75 or T25 flasks in DMEM medium containing 10% fetal bovine serum. Cells were incubated at 37°C in a 5% CO2 incubator. The medium was changed every 2–3 days, and cells were reseeded into new T75 or T25 flasks when the cell population became too large or when cells were needed for experiments. All cell lines except CCRF-SB were adherent cell lines, and to collect these cells, the medium was removed, the cell layer was briefly washed with PBS, and then 1–2 ml of trypsin-EDTA was added to the flask to disperse the cells. The cells were then collected, and the flasks were washed again with PBS to collect any excess cells. The cells were then centrifuged at 500 x g for 5 min. The supernatant was discarded, and the cells were resuspended in a volume sufficient for cell counting using a hemocytometer. Based on the cell count, 1.0 x 10⁶ cells were counted. 5 One and 1.0x10 6The remaining cells were retained for further procedures described below, and the remaining cells were centrifuged at 500 x g for 5 min, then resuspended in culture medium and replate into new flasks. The CCRF-SB cell line is a suspension cell line, and to collect these cells, a sufficient amount of culture medium was removed; the cells were centrifuged at 500 x g for 5 min. The supernatant was then discarded, and the cells were resuspended in a volume sufficient for cell counting using a hemocytometer. Based on the cell count, 1.0 x 10⁻⁶ cells were plated. 5 One and 1.0x10 6 The remaining cells were retained for the further procedures described below, and the remaining cells were centrifuged at 500xg for 5 min and then resuspended in the culture medium and placed in a new flask.

[0222] Red blood cell lysis and whole blood washing

[0223] Collect 5 ml of human whole blood in a 15 ml centrifuge tube and wash with 5 ml of PBS to remove any serum. Centrifuge the blood at 500 x g for 5 min and remove the supernatant. Add 5 ml of ACK lysis buffer to the washed blood and allow incubation at room temperature for 7–10 min. Add 5 ml of PBS and centrifuge the sample at 500 x g for 5 min. Remove the supernatant and resuspend the precipitate in 5 ml of ACK lysis buffer and allow incubation for another 7–10 min. Add 5 ml of PBS and centrifuge the sample again at 500 x g for 5 min. Repeat the lysis, incubation, and centrifugation steps until the precipitate is white. Resuspend the precipitate in 5 ml of PBS. In some embodiments, human whole blood is simply washed to remove plasma and serum, which are negatively charged and can interfere with charged particles or capillaries. Collect 5 ml of human whole blood in a 15 ml centrifuge tube and wash with 5 ml of PBS to remove any serum and / or plasma. Centrifuge the blood at 500 x g for 5 min and remove the supernatant. This washing step is repeated twice. Use PBS to bring the final washed blood to a volume of 5 ml and prepare it for adding cells for use on microparticle columns or capillaries as described in this disclosure.

[0224] Cell staining

[0225] Collect 1.0 x 10⁻⁶ from the culture. 5 One and 1.0x10 6Cells were collected and resuspended in 1 ml PBS. They were stained with a pre-prepared solution of 12 µl calcein AM dye in 100 µl DMSO and incubated on ice in the dark for 1–3 hours. Cells were then centrifuged at 500 x g for 5 min. The supernatant was discarded, and the cells were washed in 1 ml PBS and centrifuged again at 500 x g for 5 min. The supernatant was discarded, and the cells were resuspended in 1 ml PBS.

[0226] The cells were mixed with nickel-coated particles before separation.

[0227] In some sections, cells are treated with positively charged nickel-coated microparticles or nanoparticles before being applied to microparticle columns or single capillaries for separation or capture. 1.0 x 10⁻⁶ cells are collected from the culture. 5 One and 1.0x10 6 Cells were collected and resuspended in 1 ml of PBS. The cells were then stained using the method described above. 100 µl (approximately 1.0 x 10⁻⁶ cells) was used to stain the cells. 5 Add 1-100 µl of nickel-coated nanoparticles or microparticles to a test tube, so that each cell has approximately 100-1000 nickel particles. Make the mixture to 500 ml with PBS and rotate for 15 min to allow the nickel particles to react with the cells. After incubation, the cells with the nickel particle solution are ready for use on microparticle columns or capillaries as described in this disclosure.

[0228] Column preparation

[0229] The column was prepared by placing a 40 µm cell filter between a 1 ml syringe and a 23-gauge needle. The filter prevents beads from getting stuck in the needle tip. Using a funnel, a suitable amount (e.g., 0.5 ml) of dried functionalized glass microspheres was added to an Eppendorf tube, and the volume was brought to 1.5 ml with PBS. The tip of a plastic pipette was cut off, and the microspheres in PBS were carefully transferred into the column until the 0.5 ml mark on the syringe was reached. Air bubbles and gaps between the beads were removed as much as possible. The column was washed five times with 500 µl PBS to ensure it was thoroughly washed. The column was allowed to run until it stopped dripping.

[0230] Capillary preparation and procedures

[0231] As previously described, capillaries were prepared by treating them with different functional groups. Capillaries were used in each run. Cells were collected and stained as previously described. 100 µl (approximately 1.0 x 10⁻⁶ cells) was used. 5Add 100 µl of stained cells to a test tube (“Initial Cell Solution”) and bring the volume to 500 µl with 400 µl PBS. Thoroughly mix the cells and add them to the capillary as follows: Carefully transfer the cells into the interior of the capillary using a micropipette with a long tip, ensuring the cell solution contacts the sides of the capillary. Add 100 µl of cells at a time, ensuring to avoid air bubbles, and collect the effluent in a second tube (“Pass”). Once the capillary stops running, wash the capillary with 500 µl PBS, adding 100 µl at a time, ensuring to avoid air bubbles, and collect the effluent in a third tube (“First Wash”). Once the capillary stops running, wash the capillary a second time with 500 µl PBS, adding 100 µl at a time, ensuring to avoid air bubbles, and collect the effluent in a fourth tube (“Second Wash”). Once the capillary stops running, analyze the capillary under a microscope to observe the adhesion of green fluorescent cells to the inner surface of the capillary.

[0232] Single bead diagnosis

[0233] In some embodiments, a single functionalized bead is used in the assay described herein. For example, a single bead, functionalized as described above and having a sufficiently large diameter for handling with tweezers, is used. The bead has a diameter greater than 1 mm, or more specifically between 1.7 mm and 2.5 mm. The bead is placed in a tube containing a patient's blood sample and rotated at room temperature for 1 hour. The bead is then removed from the tube, washed in PBS, and the cells are lysed and used for downstream analysis, as described below.

[0234] Cancer cell lysis

[0235] Tumor cells bind exceptionally tightly to the positively charged beads or matrix disclosed herein, and the cells cannot be easily and completely removed. Therefore, tumor cell lysates are generated by exposing the matrix containing bound circulating tumor cells (CTCs) to lysis conditions (such as rapid freeze-thaw) or by chemical lysis of tumor cells after capture from a patient's blood for all diagnostic and therapeutic steps.

[0236] Example 1. Isolation of colon cancer cells

[0237] Using the method described above, approximately 1.0 x 10 6 100 µl (approximately 1.0 x 10⁻⁶) of SW620 colon cancer cells were stained. 5Add 100 µl of stained cells to a test tube (“Initial Cell Solution”) and bring the volume to 500 µl with 400 µl PBS. Thoroughly mix the cells and add them to a column of 0.5 ml 5% 3-aminopropyltriethoxysilane or 10% 3-aminopropyltrimethoxysilane beads prepared as described above. Add 100 µl of cells at a time, ensuring to avoid air bubbles, and collect the effluent in a second tube (“Pass”). Once the column has stopped dripping, wash the column with 500 µl PBS, adding 100 µl at a time, ensuring to avoid air bubbles, and collect the effluent in a third tube (“First Wash”). Once the column has stopped dripping, wash the column a second time with 500 µl PBS, adding 100 µl at a time, ensuring to avoid air bubbles, and collect the effluent in a fourth tube (“Second Wash”). Once the column has stopped dripping, cap the needle and analyze the cells from each of the four tubes under a microscope.

[0238] Figure 1 Bright-field and green fluorescence views of 10 µl initial cell solutions (A and E), through samples (B and F), first-wash samples (C and G), and second-wash samples (D and H) of colon cancer cells passed through a column of 5% 3-aminopropyltriethoxysilane beads are shown. Live colon cancer cells are visualized as green fluorescence under a microscope when observed with green light due to calcein AM staining. Multiple cells are visible in the initial cell solutions, but no cells are seen in the through samples and in each of the first and second wash samples, indicating that live colon cancer cells are retained in the column and captured by the positively charged 5% 3-aminopropyltriethoxysilane beads. Repeated experiments yielded similar results.

[0239] Figure 2 Bright-field and green fluorescence views of 10 µl initial cell solutions (A and E), through samples (B and F), first-wash samples (C and G), and second-wash samples (D and H) of colon cancer cells passed through a column of 10% 3-aminopropyltrimethoxysilane beads are shown. Live colon cancer cells are visualized as green fluorescence under a microscope when observed with green light due to calcein AM staining. Multiple cells are visible in the initial cell solutions, but no cells are seen in the through samples and in each of the first and second wash samples, indicating that live colon cancer cells are retained in the column and captured by the positively charged 10% 3-aminopropyltriethoxysilane beads.

[0240] Each of the 5% 3-aminopropyltriethoxysilane or 10% 3-aminopropyltrimethoxysilane bead columns demonstrated similar results in capturing colon cancer cells.

[0241] Example 2. Isolation of breast cancer cells

[0242] Using the method described above, approximately 5.0 x 10 5 Stain 100 µl (approximately 1.0 x 10⁻⁶) of T47D cancer cells. 5 Add 100 µl of stained cells to a test tube (“Initial Cell Solution”) and bring the volume to 500 µl with 400 µl PBS. Thoroughly mix the cells and add them to a column of 0.5 ml 5% 3-aminopropyltriethoxysilane or 10% 3-aminopropyltrimethoxysilane beads prepared as described above. Add 100 µl of cells at a time, ensuring to avoid air bubbles, and collect the effluent in a second tube (“Pass”). Once the column has stopped dripping, wash the column with 500 µl PBS, adding 100 µl at a time, ensuring to avoid air bubbles, and collect the effluent in a third tube (“First Wash”). Once the column has stopped dripping, wash the column a second time with 500 µl PBS, adding 100 µl at a time, ensuring to avoid air bubbles, and collect the effluent in a fourth tube (“Second Wash”). Once the column has stopped dripping, cap the needle and analyze the cells from each of the four tubes under a microscope.

[0243] Figure 3 Bright-field and green fluorescence views of 10 µl initial cell solutions (A and E), through samples (B and F), first-wash samples (C and G), and second-wash samples (D and H) of breast cancer cells passed through a column of 5% 3-aminopropyltriethoxysilane beads are shown. Live breast cancer cells are visualized as green fluorescence under a microscope when observed with green light due to calcein AM staining. Multiple cells are visible in the initial cell solutions, but the fewest cells are seen in the through samples, and none are seen in the first and second-wash samples. This indicates that live breast cancer cells are retained in the column and captured by the positively charged 5% 3-aminopropyltriethoxysilane beads.

[0244] Figure 4 Bright-field and green fluorescence views of 10 µl initial cell solutions (A and E), through samples (B and F), first-wash samples (C and G), and second-wash samples (D and H) of breast cancer cells passed through a column of 10% 3-aminopropyltriethoxysilane beads are shown. Live breast cancer cells are visualized as green fluorescence under a microscope when observed with green light due to calcein AM staining. Multiple cells are visible in the initial cell solutions, but the fewest cells are seen in each of the through samples and the first and second wash samples, indicating that live breast cancer cells are retained in the column and captured by the positively charged 10% 3-aminopropyltriethoxysilane beads.

[0245] Each of the 5% 3-aminopropyltriethoxysilane or 10% 3-aminopropyltrimethoxysilane beads demonstrated similar results in capturing breast cancer cells.

[0246] Example 3. Isolation of lung cancer cells

[0247] Using the method described above, approximately 1.0 x 10 5 A549 lung cancer cells were stained. 100 µl (approximately 1.0 x 10⁻⁶) of A549 lung cancer cells were stained. 5 Add 100 µl of stained cells to a test tube (“Initial Cell Solution”) and bring the volume to 500 µl with 400 µl PBS. Thoroughly mix the cells and add them to a column of 0.5 ml 5% 3-aminopropyltriethoxysilane or 10% 3-aminopropyltrimethoxysilane beads prepared as described above. Add 100 µl of cells at a time, ensuring to avoid air bubbles, and collect the effluent in a second tube (“Pass”). Once the column has stopped dripping, wash the column with 500 µl PBS, adding 100 µl at a time, ensuring to avoid air bubbles, and collect the effluent in a third tube (“First Wash”). Once the column has stopped dripping, wash the column a second time with 500 µl PBS, adding 100 µl at a time, ensuring to avoid air bubbles, and collect the effluent in a fourth tube (“Second Wash”). Once the column has stopped dripping, cap the needle and analyze the cells from each of the four tubes under a microscope.

[0248] Figure 5 Bright-field and green fluorescence views of 10 µl initial cell solutions (A and E), through-samples (B and F), first-wash samples (C and G), and second-wash samples (D and H) of lung cancer cells passed through a column of 5% 3-aminopropyltriethoxysilane beads are shown. Live lung cancer cells are visualized as green fluorescence under a microscope when observed with green light due to calcein AM staining. Multiple cells are visible in the initial cell solutions, but the fewest cells are seen in the through-samples, first-wash samples, and second-wash samples. This indicates that most live lung cancer cells remain in the column and are captured by the positively charged 5% 3-aminopropyltriethoxysilane beads, but some cells are not captured. Repeated experiments yielded similar results.

[0249] Figure 6Bright-field and green fluorescence views of 10 µl initial cell solutions (A and E), through-samples (B and F), first-wash samples (C and G), and second-wash samples (D and H) of lung cancer cells passed through a column of 10% 3-aminopropyltrimethoxysilane beads are shown. Live lung cancer cells are visualized as green fluorescence under a microscope when observed with green light due to calcein AM staining. Multiple cells are visible in the initial cell solutions, but the fewest cells are seen in the through-samples, first-wash samples, and second-wash samples. This indicates that most live lung cancer cells remain in the column and are captured by the positively charged 10% 3-aminopropyltrimethoxysilane beads, but some cells are not captured.

[0250] Both 5% 3-aminopropyltriethoxysilane and 10% 3-aminopropyltrimethoxysilane bead columns demonstrated similar results in capturing lung cancer cells. However, because the number of uncaptured cells was greater than in any other cell line examined, the 5% 3-aminopropyltriethoxysilane column was repeated once with more beads and the same number of cells, and once with the same number of beads and fewer cells. These experiments were established to observe whether the cell line required columns with increased capacity via bead addition.

[0251] For experiments using more beads and the same number of cells, the experiments were performed in the same manner as described above, except that the column was prepared using 0.8 ml of 5% 3-aminopropyltriethoxysilane beads. Results can be obtained from... Figure 7 The results showed that multiple cells were visible in the initial cell solution, but fewer cells were observed in the sample, first and second washes, compared to a 0.5 ml column. This suggests that the cell line may require a higher volume to improve cell binding.

[0252] For experiments using the same number of beads and fewer cells, except using 30 µl, approximately 3.0 x 10 4 The cells were then subjected to the same experiments as described above, with the volume increased to 500 µl using 470 µl PBS. Results can be obtained from... Figure 8 The results showed that while multiple cells were visible in the initial cell solution, fewer cells were observed in the samples after passing through the sample, and in the first and second washes, compared to experiments using more cells. This again suggests that the cell line may require a higher capacity to improve cell binding.

[0253] Example 4. Isolation of acute lymphoblastic leukemia cells

[0254] Using the method described above, approximately 1.0 x 10 6 Stain 100 µl (approximately 1.0 x 10⁻⁶) of CCRF-SB acute lymphoblastic leukemia cells. 5Add 100 µl of stained cells to a test tube (“Initial Cell Solution”) and bring the volume to 500 µl with 400 µl PBS. Thoroughly mix the cells and add them to a column of 0.5 ml 5% 3-aminopropyltriethoxysilane or 10% 3-aminopropyltrimethoxysilane beads prepared as described above. Add 100 µl of cells at a time, ensuring to avoid air bubbles, and collect the effluent in a second tube (“Pass”). Once the column has stopped dripping, wash the column with 500 µl PBS, adding 100 µl at a time, ensuring to avoid air bubbles, and collect the effluent in a third tube (“First Wash”). Once the column has stopped dripping, wash the column a second time with 500 µl PBS, adding 100 µl at a time, ensuring to avoid air bubbles, and collect the effluent in a fourth tube (“Second Wash”). Once the column has stopped dripping, cap the needle and analyze the cells from each of the four tubes under a microscope.

[0255] Figure 9 Bright-field and green fluorescence views of 10 µl initial cell solutions (A and E), through samples (B and F), first-wash samples (C and G), and second-wash samples (D and H) of acute lymphoblastic leukemia (ALL) cancer cells passed through a column of 5% 3-aminopropyltriethoxysilane beads are shown. Live ALL cancer cells are visualized as green fluorescence under a microscope when viewed with green light due to calcein AM staining. Multiple cells are visible in the initial cell solutions, but the fewest cells are seen in the through samples, and no cells are seen in the first and second-wash samples. This indicates that live ALL cancer cells are retained in the column and captured by the positively charged 5% 3-aminopropyltriethoxysilane beads.

[0256] Figure 10 Bright-field and green fluorescence views of 10 µl initial cell solutions (A and E), through samples (B and F), first-wash samples (C and G), and second-wash samples (D and H) of acute lymphoblastic leukemia (ALL) cancer cells passed through a column of 10% 3-aminopropyltrimethoxysilane beads are shown. Live ALL cancer cells are visualized as green fluorescence under a microscope when observed with green light due to calcein AM staining. Multiple cells are visible in the initial cell solutions, but the fewest cells are seen in each of the through samples and the first and second wash samples, indicating that the live ALL cancer cells remain in the column and are captured by the positively charged 10% 3-aminopropyltriethoxysilane beads.

[0257] Each of the 5% 3-aminopropyltriethoxysilane or 10% 3-aminopropyltrimethoxysilane bead columns demonstrated similar results in capturing acute lymphoblastic leukemia cancer cells.

[0258] Example 5. Isolation of cancer cells in the presence of non-cancerous white blood cells (WBCs)

[0259] To determine whether a positively charged bead matrix could separate cancer cells from a mixture of cancer cells and non-cancer cells, SW620 colon cancer cells were mixed with purified non-cancer leukocytes and the mixture was passed through a column.

[0260] Using the method described above, approximately 1.0 x 10 6 SW620 colon cancer cells were stained, and WBCs were purified using the blood lysis protocol described above. 100 µl (approximately 1.0 x 10⁻⁶) of WBCs were added. 5 Stained tumor cells were mixed with 200 µl of WBC in PBS in a test tube (“Initial Cell Solution”), and the volume was brought to 500 µl with 200 µl of PBS. The cells were thoroughly mixed and added to a column of 0.5 ml of 5% 3-aminopropyltriethoxysilane or 10% 3-aminopropyltrimethoxysilane beads prepared as described above. 100 µl of cells were added at a time, ensuring air bubbles were avoided, and the effluent was collected in a second tube (“Pass”). Once the column had stopped dripping, the column was washed with 500 µl of PBS, adding 100 µl at a time, ensuring air bubbles were avoided, and the effluent was collected in a third tube (“First Wash”). Once the column had stopped dripping, the column was washed a second time with 500 µl of PBS, adding 100 µl at a time, ensuring air bubbles were avoided, and the effluent was collected in a fourth tube (“Second Wash”). Once the column had stopped dripping, the needle was capped and the cells from each of the four tubes were analyzed under a microscope.

[0261] Figure 11 Bright-field and green fluorescence views of 10 µl initial cell solutions (A and E), 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 column of 5% 3-aminopropyltriethoxysilane beads are shown. Both colon cancer cells and white blood cells are visible in the bright-field views. Live colon cancer cells are visualized as green fluorescence under a microscope when observed with green light due to calcein AM staining. Multiple live colon cancer cells are visible in the initial cell solutions, but none are seen in the through samples, first-wash samples, and second-wash samples. On the other hand, WBCs are visible in the bright-field views of each through sample, first-wash sample, and second-wash sample. This indicates that live colon cancer cells are retained in the column and captured by the positively charged 5% 3-aminopropyltriethoxysilane beads, while non-cancerous WBCs are not captured.

[0262] The experiment was repeated as the number of white blood cells in the mixture increased. The experiment was performed in the same manner as above, except for the 400 µl WBC PBS solution. Results can be obtained from... Figure 12 Similarly, no live colon cancer cells were observed in the through sample, the first wash, and the second wash sample, and WBCs were visible in the bright-field view of each of the through sample, the first wash, and the second wash sample. This confirms that live colon cancer cells were retained in the column and captured by the positively charged 5% 3-aminopropyltriethoxysilane beads, while non-cancerous WBCs were not captured.

[0263] Example 7. Isolation of cancer cells using microcolumns

[0264] A single capillary containing 10% positively charged 3-aminopropyltrimethoxysilane beads (prepared as previously described) was used as a “microcolumn” for each run. Cells were collected and stained as previously described. 100 µl (approximately 1.0 x 10⁻⁶) was used. 5 Add 100 µl of stained cells to a test tube (“Initial Cell Solution”) and bring the volume to 500 µl with 400 µl PBS. Thoroughly mix the cells and add them to the microcolumn using a micropipette with a long tip. Add 100 µl of cells at a time, ensuring to avoid air bubbles, and collect the effluent in a second tube (“Pass”). Once the microcolumn has stopped running, wash the microcolumn with 500 µl PBS, adding 100 µl at a time, ensuring to avoid air bubbles, and collect the effluent in a third tube (“First Wash”). Once the microcolumn has stopped running, wash the microcolumn a second time with 500 µl PBS, adding 100 µl at a time, ensuring to avoid air bubbles, and collect the effluent in a fourth tube (“Second Wash”). Once the microcolumn has stopped running, analyze the microcolumn under a microscope to observe the adhesion of green fluorescent cells to the positively charged glass beads.

[0265] Figure 13 A green fluorescent view of breast cancer cells captured on positively charged glass beads within a 1.5 mm microcolumn of clear glass is shown. The narrow diameter of the capillary allows for clear observation, analysis, and evaluation of the cells via fluorescently labeled antibody markers to determine the tissue of origin.

[0266] Example 8. Measuring the capacity of beads

[0267] To determine the required number of beads for downstream applications (such as vaccine preparation), for example, bead capacity was measured. One amine-coated glass bead prepared as described above and having a diameter between 1.7 mm and 2.5 mm was used with 1 x 10... 6Cells were incubated in 1 ml PBS for 1 hour. After incubation, each bead was washed three times in PBS. 40 µl of 1% SDS was added to the beads to lyse the cells and incubated for 30 min. After incubation, the beads and lysed cells were centrifuged at 10,000 x g for 5 min. The absorbance of the supernatant was read at 280 nm on a Nanodrop and compared with a standard curve prepared as described below. This procedure was then repeated using 5 beads.

[0268] A standard curve was established using the CCRF-SB cell line (human acute lymphoblastic leukemia). 20 µl A set number of cells in PBS, 1x10 3 2.5x10 3 5x10 3 1x10 4 and 1.5x10 4 Lysis was performed using 20 µl of 1% SDS. After incubation for 30 minutes, the tube was centrifuged at 10,000 x g for 5 min. The absorbance of the supernatant was read at 280 nm on a nanodrop, and a graph of µg / µl protein versus cell number was plotted. Figure 15 ).

[0269] result

[0270] The cell lysate generated from one bead had an absorbance of 0.02725 at 280 nm. The cell number was determined using an equation generated from the standard curve: Y = 1.165e-005*X + 0.009881. Therefore, one bead had a capacity of 1490.90 cells.

[0271] The absorbance of 5 beads at 280 nm is 0.0876. The equation generated from the curve is used to determine the cell number. Therefore, 5 beads have a capacity of 6676.88, and thus 1 bead has a capacity of 1335.38 cells.

[0272] in conclusion

[0273] This data leads to the conclusion that one amine-coated charged bead binds approximately 1400 cells. This information can be used for downstream applications, for example, where 1x10⁻⁶ cells are needed. 6 One cell is needed to prepare sufficient lysate for the tumor lysate vaccine described herein. Therefore, approximately 750 beads are required to capture 1 x 10 cells. 6 A single cell, and a column containing 750 or more beads, is sufficient to capture enough leukemia or other blood cancer cells from a patient's blood to produce an effective therapeutic cancer vaccine.

[0274] Example 9. Quantitative analysis of circulating cancer cells

[0275] For diagnostic purposes, 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.

[0276] Amine-coated glass beads were prepared as described herein and functionalized with 10% 3-aminopropyltriethoxysilane. Ten million human PBMCs, ten million human PBMCs incorporating 160 human SKOV3 breast cancer cells, and 160 human SKOV3 cells were prepared, each with a final volume of 50 µl in PBS. Cells were transferred to U-bottom wells in 96-well plates, with each well containing a single glass bead with a diameter between 1.7 mm and 2.5 mm. The plates were shaken at 100 rpm for one hour at room temperature. Beads were picked up with tweezers and washed three times with PBS. The beads were then placed in PCR tubes and lysed with 50 µl of lysis buffer. 10 µl of cell lysate was used for reverse transcription, resulting in a final volume of 20 µl. 1 µl of reverse transcription was used for quantitative PCR analysis.

[0277] The number of amplification cycles required to observe the PCR product was measured using β-actin PCR primers (housekeeping gene). Figure 16 A). Similarly, the number of amplification cycles required to observe the PCR products was measured using Her2 PCR primers. Figure 16 B).

[0278] A standard curve was created using the number of cycles. Figure 17 This demonstrates that individual breast cancer cells can be identified via quantitative PCR analysis within approximately 37 cycles. The standard curve can be used to determine the number of breast cancer cells in a sample. It can be followed... Figure 18 The flowchart (in white box) describes a scheme for creating similar curves for different cancer samples to quantify the number of cancer cells in the samples.

[0279] Example 10. Detection and identification of circulating cancer cells in patient samples

[0280] use Figure 18The protocol outlined in the flowchart (white and gray boxes) describes a method used to quantify, detect, and identify cancer cells in a patient. PBMCs from 7.5 ml of patient blood were resuspended in 200 µl of PBS. Circulating cancer cells were isolated using 10 µl of cells in 100 µl of PBS with two glass beads. After incubation at 100 rpm for 1 hour at room temperature, the glass beads were separated and washed three times with PBS. 10 µl of pristine PBMCs, PBMCs isolated from circulating cancer cells, and the glass beads were lysed with 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 in the 20 µl reaction for quantitative PCR amplification.

[0281] result

[0282] like Figure 19 As can be seen, these results clearly show that the circulating cancer cells captured by the beads from the patient's blood sample were breast cancer cells expressing Her2 and β-actin, and only breast cancer cells were captured, as the captured circulating cancer cells showed no contamination by lymphocytes (the vast majority of cells in the patient's blood sample), and because the highly sensitive CD45 PCR assay was absolutely negative. Figure 19 C).

Claims

1. A method for removing cancer cells from a biological sample, the method comprising: a) Passing biological samples containing cancer cells through a separation matrix comprising functionalized microspheres; as well as b) Collect the biological sample that has flowed through the separation matrix in the first aliquot. The cancer cells therein bind to the separated matrix.

2. The method according to claim 1, wherein the functionalized microspheres are functionalized with positively charged functional groups.

3. The method according to claim 1 or 2, wherein the functionalized microspheres are functionalized with amines, polyethyleneimine (PEI) and / or guanidine groups.

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

5. The method according to any one of claims 1 to 4, wherein the diameter of the microspheres is between 500 µm and 600 µm.

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

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

8. The method of claim 7, wherein the cancer is a hematologic cancer or includes a solid tumor.

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

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

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

12. The method according to any one of claims 7 to 11, further comprising: The cancer cells were lysed to obtain cancer cell lysates.

13. The method of claim 12, further comprising: The cancer cell lysate is incorporated into a cancer vaccine.

14. The method of claim 12, further comprising: The mRNA copy number was determined from the cancer cell lysate.

15. The method of 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: The number of cancer cells that have bound to the functionalized microspheres is calculated.

17. A cancer vaccine prepared by a process comprising: a) Contacting a biological sample containing cancer cells with a positively charged surface, wherein the cancer cells are bound to the positively charged surface; b) Lyse the cancer cells to obtain cancer cell lysate; as well as c) Incorporating the cancer cell lysate into yeast cell wall particles (YCWP).

18. A cancer vaccine comprising: a) Yeast cell wall granules (YCWP); and b) The cancer cell lysate according to claim 12.

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

20. The vaccine of claim 19, wherein the silicate ester is selected from the group consisting of 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 subject, the method comprising: The vaccine according to any one of claims 17 to 21 shall be administered to the subject.

23. A method for treating or preventing cancer, the method comprising: The vaccine according to any one of claims 17 to 21 shall be administered to subjects who require it.

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

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

26. A method for treating a patient's cancer, the method comprising: Cancer cells are extracted from the patient's blood by passing the blood through a separation matrix comprising functionalized microspheres that bind to cancer cells.

27. The method of claim 26, wherein the functionalized microspheres are functionalized with positively charged functional groups.

28. The method according to claim 26 or 27, wherein the functionalized microspheres are functionalized with amines, polyethyleneimine (PEI), and / or guanidine groups.

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

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

31. The method according to any one of claims 26 to 30, wherein the patient suffers from hematologic cancer or malignant cancer.

32. The method according to any one of claims 26 to 31, further comprising: The bound cancer cells were eluted from the separated matrix.

33. The method of claim 32, further comprising: The eluted cells are lysed to obtain lysates, which are then incorporated into a cancer vaccine for use in treating the patient.

34. A method for detecting cancer cells in a patient, the method comprising: a) Passing a biological sample from the patient through a separation matrix of functionalized microspheres, wherein the functionalized microspheres bind cancer cells; b) Elution of the cancer cells from the matrix; as well as c) Detect the presence or absence of cancer cells in the biological sample.

35. The method of claim 34, wherein the patient suffers from hematologic malignancy or cancer.

36. The method according to claim 34 or 35, wherein the functionalized microspheres are functionalized with amines, polyethyleneimine (PEI), and / or guanidine groups.

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

38. The method according to any one of claims 34 to 37, wherein the diameter of the microspheres is between 500 µm and 600 µm.

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

40. The cancer cell separation matrix according to claim 39, wherein the functionalized microspheres are functionalized with amines, polyethyleneimine (PEI) and / or guanidine groups.

41. The cancer cell separation matrix according to claim 39 or 40, wherein the microspheres comprise glass, polymer, or resin.

42. The cancer cell separation matrix according to any one of claims 39 to 41, wherein the diameter of the microspheres is between 500 µm and 600 µm.

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

44. A method for preparing a column for cancer cell isolation, the method comprising: a) Preparing a separation matrix, wherein the separation matrix comprises functionalized microspheres; as well as b) Deposit the separation matrix into a container, wherein the container has an inlet and an outlet.

45. The method of claim 44, wherein the functionalized microspheres are functionalized with positively charged functional groups.

46. ​​The method according to claim 44 or 45, wherein the functionalized microspheres are functionalized with amines, polyethyleneimine (PEI), and / or guanidine groups.

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

48. The method according to any one of claims 44 to 47, wherein the diameter of the microspheres is between 500 µm and 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 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 separation matrix comprises depositing functionalized microspheres of 0.1 ml to 1 ml into the container.

54. A method for removing cancer cells from a biological sample, the method comprising: a) Passing biological samples containing cancer cells through a functionalized capillary; as well as b) Collect the biological sample flowing through the capillary in the first aliquot. The cancer cells therein bind to the capillaries.

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

56. A method for removing cancer cells from a biological sample, the method comprising: A biological sample containing cancer cells is brought into contact with a positively charged surface, wherein the cancer cells bind to the positively charged surface.

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

58. The method of 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 cover slips.

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

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

62. The method of claim 61, wherein the cancer is a hematologic cancer or includes a solid tumor.

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

64. The method according to any one of claims 56 to 62, further comprising: The cancer cells were lysed to obtain cancer cell lysates.

65. The method of claim 64, further comprising: The cancer cell lysate is incorporated into a cancer vaccine.

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

67. A cancer vaccine comprising: a) Yeast cell wall granules (YCWP), and b) The cancer cell lysate according to claim 64.

68. The vaccine of claim 67, wherein the YCWP is modified by end capping with a silicate ester 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, the method comprising: The vaccine according to any one of claims 67 to 69 shall be administered to the subject.

71. A method for treating or preventing cancer, the method comprising: The vaccine according to any one of claims 67 to 69 shall be administered to subjects who require it.

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

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

74. A method for treating a patient's cancer, the method comprising: Cancer cells are extracted 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 the blood is returned to the patient after contact with the positively charged surface.

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

76. The method of 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 suffers from hematologic malignancy or malignant cancer.

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

80. The method of claim 79, wherein the patient suffers from hematologic malignancy, including solid tumors, or malignant cancer.

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

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

83. The apparatus 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