SELECTIVE DELIVERY OF MATERIAL TO CELLS

Abstract

Isolating or identifying a cell based on a physical property of said cell can include providing a cell suspension; passing said suspension through a microfluidic channel that includes a constriction; passing the cell suspension through the constriction; and, contacting said cell suspension solution with a compound. The constriction can be sized to preferentially deform a relatively larger cell compared to a relatively smaller cell.

Claims

1-19. (canceled)

20. A method for delivering a compound into a cell based on a physical property of said cell comprising: providing a cell suspension comprising a first group of cells and a second group of cells, wherein the first group of cells have a relatively different size, diameter, and/or membrane stiffness than the second group of cells; passing said cell suspension through and out of a microfluidic channel that includes a constriction, said constriction being sized to deliver a compound to the first group of cells to a greater extent than the second group of cells, wherein passage through the constriction disrupts the cell membranes of the first group of cells such that the compound is delivered into the first group of cells to a greater extent than the second group of cells in the cell suspension that passes through the microfluidic channel; and contacting said cell suspension with the compound.

21. The method of claim 20, wherein the first group of cells have a relatively different size than the second group of cells.

22. The method of claim 20, wherein the first group of cells have a relatively different diameter than the second group of cells.

23. The method of claim 20, wherein the constriction has a diameter smaller than the size of cell in the cell suspension to which the compound is delivered.

24. The method of claim 20, wherein the constriction has a diameter smaller than the size of the smallest cell of the first group of cells or the smallest cell of the second group of cells.

25. The method of claim 20, wherein the constriction has a diameter smaller than the size of the largest cell of the first group of cells or the second group of cells.

26. The method of claim 20, wherein the cell suspension is contacted with the compound after the suspension is passed through the microfluidic channel comprising the constriction.

27. The method of claim 20, wherein the cell suspension comprises whole blood.

28. The method of claim 20, wherein the cell suspension comprises peripheral blood mononuclear cells (PBMCs).

29. The method of claim 20, wherein the cell suspension comprises an erythrocycte-depleted population of peripheral blood cells.

30. The method of claim 20, wherein the compound has a molecular mass of 0.5 kDa to 5 MDa or a molecular mass of 3 kDa to 10 kDa.

31. The method of claim 20, wherein the compound comprises one or more of a protein, a nucleic acid, a detectable marker, an active biomolecule, and a toxin.

32. The method of claim 20, wherein the compound comprises a detectable marker.

33. The method of claim 20, wherein: (a) the constriction has a width from 4 μm-10 μm; (b) the constriction has a length of 1 μm-100 μm; and/or (c) the microfluidic channel has 1-10 constrictions in series.

34. The method of claim 20, wherein the speed of the cells traversing the constriction ranges from 10 mm/s to 10 m/s.

35. The method of claim 20, further comprising applying a pressure to the cell suspension to drive the cell suspension through the constriction of the microfluidic channel.

36. The method of claim 20, wherein the first group of cells and/or the second group of cells comprise a cell selected from the group consisting of: tumor cells, PBMCs, and erythrocytes.

37. The method of claim 36, wherein the first group of cells are PBMCs.

38. The method of claim 36, wherein the PBMCs comprise leukocytes.

39. The method of claim 38, wherein the leukocytes comprise neutrophils, eosinophils, basophils, lymphocytes, and/or monocytes.

40. The method of claim 38, wherein the leukocytes are lymphocytes.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG. 1 is a diagram of a system for size selective tagging of CTCs by rapid mechanical deformation.

[0026] FIG. 2 is a bar graph showing that combining size selective delivery of the microfluidic platform with antibody staining for CD45 produces a sample enrichment factor over an order of magnitude better than either technique independently.

[0027] FIG. 3A is a schematic diagram of cell labeling. Red blood cells (RBCs) were depleted from whole blood by RBC lysis using standard erythrocyte lysis reagents such as eBioscience RBC lysis buffer (Cat. No. 00-4333). The resulting suspension flowed through the constriction channel microfluidics device incubated with a fluorescent dye (and optionally other compounds). The suspension was then labeled for CD45 and processed on a fluorescence-activated cell sorter (FACS) machine to collect the non-CD45+ cells that have been labeled with the fluorescent dye.

[0028] FIG. 3B is a series of flow cytometry plots of cascade blue conjugated 3 kDa dextran delivered by CellSqueeze devices to PBMCs (30-6 chip at 50 psi), HT-29 (30-6 chip at 50 psi), SK-MEL-5 (10-7 chip at 50 psi), and PANC-1 (10-7 chip at 50 psi).

[0029] FIG. 3C is a series of transmitted light and fluorescence micrographs of Panc-1 tumor cells and blood cells before and after passing through the constriction channel. The pre-delivery cells are incubated in the presence of dye to correct for background endocytosis. The post-delivery images were taken 24 h after delivery to demonstrate retention of dye and ability of the cells to adhere and grow following delivery. Although large blood cells can also get labeled in the process, these data demonstrate selective labeling of tumor cells.

[0030] FIG. 4 is a plot of PBMC delivery versus percent PBMC in PBMC and lymphoma mixture showing selective delivery of dyes to lymphoma cells vs. healthy PBMCs. Even when the suspension is 99.9% healthy PBMCs by number, in some implementations up to 8 times specificity in delivery can be achieved. In other implementations, greater specificity can be achieved.

[0031] FIG. 5A is a FACS plot of tetramethylrhodamine dextran-labeled Panc-1-GFP cells spiked into whole blood (40 cells/ml) and processed with a CD45 counter stain (APC).

[0032] FIG. 5B is a FACS plot of GFP versus CD45, demonstrating how PANC-1 GFP tagging could be verified independently based on GFP fluorescence. The P5 gate would be used as a basis for sorting candidate CTCs, P4 is used to verify the identity of PANC-1 GFP cells. Green dots are accurate hits (P4 & P5), red dots are false positives (P5 only), blue dots are misses (P4 only).

[0033] FIG. 5C is an image of histopathology of HTB1760's primary tumor confirms pancreatic ductal adenocarcinoma.

DETAILED DESCRIPTION

[0034] CTCs are tumor cells that are found in the bloodstream, and are believed to be responsible for the dissemination of cancer to distant organs. CTCs are regarded as minimally-invasive, “liquid biopsies” for cancer patients and are useful as prognostic indicators for patient outcome and treatment efficacy. Comprehensive characterizations of these single cells provide a better understanding of metastatic dissemination, treatment resistance, and tumor biology.

[0035] A typical human erythrocyte has a disk diameter of approximately 6.2-8.2 μm and a thickness at the thickest point of 2-2.5 μm and a minimum thickness in the center of 0.8-1 μm, being much smaller than most other human cells. Leukocytes (white blood cells) include neutrophils (12-14 μm diameter), eosinophils (12-17 μm diameter), basophils (14-16 μm diameter), lymphocytes (average 6-9 μm in diameter for resting, and 10-14 μm diameter for activated), and monocytes, the largest type of white blood cells that can be up to 20 μm in diameter. As shown in FIG. 1, the size difference between CTCs and hematologic cells generally permits distinguishing CTCs from other cells in circulating blood (CTCs˜9-20 μm; RBC˜8 μm discoid; leukocytes˜7-12 μm). See FIG. 1. Subsequent tumor cell specific labeling using antibodies (or cell-specific fragments thereof) or other tumor cell specific ligands increase the selectivity of the method.

[0036] Since CTCs are present as one in 10.sup.6-10.sup.7 mononuclear cells in the bloodstream, high-sensitivity enrichment techniques are used that rely on immunological or morphological differences in CTCs from the blood cells. Immunological approaches often target epithelial cell surface markers (such as EpCAM) and tumor-specific proteins (such as Her2-neu, MUC I/MUC2, carcinoembryonic antigen (CEA), mammaglobulin, and alpha-fetoprotein) or aim to deplete CD45+ cells. Microfilters, density-gradient separations, and microfluidics platforms are examples of morphology-based methods. All of these approaches have inherent biases, suffer from low enrichment efficiencies and a significant number of CTCs may down-regulate surface antigens or exhibit varying morphological features. These biases pose a significant challenge in the field as it is still largely unknown which subset of CTCs are responsible for metastasis or are reliable prognostic markers. Thus, it is important to develop techniques that can ensure high sensitivity isolation of all candidate CTC sub-types to screen for the most clinically relevant candidates. The devices and methods described herein permit the isolation and enumeration of the CTC subtype of interest.

[0037] A combined enrichment method integrates both immunological and morphologic-based approaches to tag and isolate pure CTCs with less bias and based on tunable parameters. The method combines microfluidic intracellular delivery (FIG. 1) and antibody staining to yield robust, high sensitivity purification of circulating tumor cells from whole blood (FIG. 2) comprises a width from 4μ-10 μm, length of 1 μm-100 μm, and 1-10 constrictions in series. The estimated speed of the cells can range from 10 mm/s to 10 m/s. The specific device parameters chosen are dictated by the target tumor cell type, e.g., a different device design is used to select CTCs for a melanoma patient vs. a colon cancer patient. Examples of tumor cell sizes/diameters include; melanoma˜15 um, colon cancer˜11 um, and pancreatic cancer˜15 um.

[0038] In this approach, a rapid mechanical deformation delivery system exploits the inherent size difference between many CTCs and the surrounding blood cells to selectively deliver fluorescent, magnetic and/or other distinguishing materials to the tumor cells. In further processing, antibody-based fluorescent and/or magnetic tagging is used to enhance the contrast between the candidate CTCs and the surrounding blood cells. By uniquely combining size-based and immunological approaches to CTC isolation, this technology has demonstrated utility for the non-biased isolation of candidate tumor cells from patient samples for analysis. In some implementations, both smaller and larger cells are deformed but the smaller cells membrane is not deformed to the point that the membrane becomes compromised. For example, to selectively delivering to 15 μm tumor cells in whole blood where most healthy white blood cells are ˜8 μm in size, a 6 um width constriction can be used. Such a constriction would deform both cell types but would very preferentially disrupt the membrane of the 15 μm tumor cells not the 8 μm blood cells.

[0039] Clinical/Translation Relevance

[0040] CTCs are being explored as surrogates for tumor biopsies for understanding mechanisms of resistance and guiding the selection of targeted therapies. Measures of the number and composition of CTCs before and after treatment indicate treatment efficacy and prognosis. The approach utilizes a robust, high-throughput, disposable device for the tagging of CTCs based on cell size and surface antigens. Moreover, the ability to deliver a diversity of macromolecules also enables one to deliver molecular probes (such as antibodies, quantum dots, carbon nanotubes, and molecular beacons) that respond to the intracellular environment and thus provide further information on the intracellular properties of the target cell. This combinatorial approach provides a robust platform capable of enriching CTC populations that would have been missed by alternative methods that rely solely on immunological or morphological separation. The technique is useful to isolate patients' CTCs.

Example 1

[0041] Whole blood or other cell suspensions are processed using both unlabeled and/or antibody-coated magnetic beads. These cells are then isolated using a high-fidelity, magnetic enrichment system for rare cells. A nanowell technology may also be used to achieve high purity isolations by imaging and robotically-retrieving single cells of interest from an elastomeric array of 84,672 subnanoliter wells.

[0042] Obtaining single, live, pure, intact CTCs of diverse phenotypes allows a host of characterization efforts from the genomic to functional levels with immediate clinical and translational relevance. The methods permit a highly sensitive and specific enrichment of live, diverse CTCs with reduced bias.

Example 2

[0043] Magnetic nanoparticles are delivered to tumor cell lines & PBMCs. Nanoparticle delivery to EpCAM-expressing, epithelial cancer cell lines, e.g., HT-29, LNCaP, and SK-BR-3, is compared to bulk peripheral blood mononuclear cell (PBMC) suspensions derived from human blood.

[0044] 10 nm iron-oxide nanoparticles with a polyethylene glycol (PEG) surface coating are delivered to cancer cells mixed with whole blood, and the resulting mixture of tagged cells are processed using the cell separation system described above. For example, the microfluidic delivery system was used to induce a rapid mechanical deformation of a cell to generate transient pores in the cell membrane (FIG. 1). The approach has demonstrated an ability to deliver a range of materials, including proteins, RNA, DNA and nanoparticles to a variety of cell types and works with whole blood, a medium that often poses problems for microfluidic systems.

[0045] Exemplary tagging molecules, e.g., 3 kDa and 70 kDa, fluorescently-labeled, dextran polymers as model molecules, were used to discriminate between PBMCs and two different cancer cell lines based on size alone. The results also indicate the utility of the system for the selective delivery of magnetic particles to tumor cells in the blood. PEG coated iron-oxide particles are used to magnetically tag colon cancer (e.g., as exemplified by the cell line HT-29). Further enrichment is accomplished using conjugation of FITC to the iron-oxide nanoparticle surface to directly measure nanoparticle uptake.

[0046] PEG coated 10 nm iron-oxide nanoparticles are delivered to cell suspensions that are suspected of containing or are known to contain CTCs, e.g., a patient-derived blood sample, or cell lines HT-29, LNCaP, and SK-BR-3 cells, separately mixed with whole blood. The resulting mixture of tagged cells are then purified, e.g., using a high fidelity magnetic separator. The separator accurately discriminates between the model CTCs with high iron-oxide content and less-effectively labeled PBMCs. Optionally, red blood cells are lysed prior to treatment, nanoparticle concentration increased, their size altered, or incorporating multiple treatment steps.

Example 3

[0047] A combined immunological and morphologic-based method is carried out as follows. After cell size-based processing by the device, cells are treated with an antibody or other tumor cell specific ligand such as fluorescently labeled anti-CD45 antibodies. The sensitivity and specificity of three different separation approaches were compared: 1) device only 2) anti-CD45 antibody only 3) device+anti-CD45 antibody. Morphologic tagging (device)+immunological tagging (e.g., anti-CD45 antibodies) was found to show superior sensitivity (and specificity) relative to either of the individual techniques (FIG. 2). For example, a 2-5× increase in sensitivity and/or a 2-5× increase in specificity relative to anti-CD45 antibodies alone is observed. Enrichment factor of over an order of magnitude was observed (FIG. 2).

Example 4

[0048] In one example, the devices are fabricated out of silicon and glass. Alternatively, the device is fabricated using a polymer such as silicone, PDMS, polycarbonate, acrylic, polypropylene, polystyrene. Either device is sterilized (heat or gamma radiation) and disposable. Performance of the devices is validated for various cell types using materials and parameters. For example, performance at a range of flow speeds (100 mm/s-10,000 mm/s) using PEG coated quantum dots (ranging from 10-50 nm in size) is used to determine if the delivery efficiency of nanoparticles and cell viability. Exemplary device are described in PCT/US2012/060646, hereby incorporated by reference.

[0049] Advantages

[0050] When compared to existing approaches this method has the following advantages. Relative to antibody-based methods, this approach provides a non-biased isolation procedure that is generalizable to most cancer types and is independent of any particular cell surface marker. The device and method accomplishes the identification of CTCs that could not be isolated by existing markers and thus, has significant diagnostic and prognostic implications.

[0051] Relative to existing size-based isolation methods, the device and methods described herein provide far higher throughput and are tunable by varying “W” (FIG. 1) to capture specific CTC size ranges. For example, a 6 μm width constriction is suitable for the capture of colon cancer cells whereas a 7 μm, and 8 μm width are suitable for the capture of pancreatic cancer and melanoma cells respectively. Moreover, unlike existing technologies, this system is combined with antibody-based technologies to enhance isolation sensitivity and/or enable multi-parametric isolation of subsets of CTCs (for example by isolating CTCs of a certain size+surface marker).

[0052] By enabling the effective, robust isolation of CTCs from a range of cancer types this technology would be a valuable platform in the fight against cancer. The prognostic and diagnostic potential of this technology could enable the identification of new genes that are critical to cancer progression and thus enable the development of novel therapeutics. It may also provide a more accurate prediction of patient life-expectancy and treatment efficacy.

[0053] The CTC isolation methods described herein combines immunological and size-based isolation to yield a high enrichment factor/recovery rate and adjustable bias (marker specific vs. size specific).

[0054] Although a few variations have been described in detail above, other modifications are possible. For example, the implementations described above can be directed to various combinations and subcombinations of the disclosed features and/or combinations and subcombinations of several further features disclosed above. In addition, the logic flows described herein do not require the particular order described, or sequential order, to achieve desirable results. Other embodiments may be within the scope of the following claims.