WO2011025976A2 - Methods of detaching and collecting cells - Google Patents

Abstract

The abundance of circulating tumor cells (CTCs) indicates patient prognosis. Molecular characterization of CTCs may add additional information about a patient's disease. However, currently available methods are limited by contamination with blood cells. We describe a study using a modified CTC-chip to capture CTCs from an orthotopic xenograft model. Using laser capture microscopy to collect CTCs from the chip, we compared transcripts from purified CTCs to those from primary and metastatic tissue. Transcriptional profiles showed strong concordance among primary, metastatic and CTC sources. Moreover, cells captured on the chip were viable, and could be expanded in culture. We conclude that the CTC-chip is a useful tool to further characterize animal models of cancer, and that viable CTCs can be isolated and show transcriptional similarity to solid tumors.

Description

METHODS OF DETACHING AND COLLECTING CELLS

CROSS-REFERENCE

[0001] This application claims benefit of priority to U.S. Provisional Application No. 61/238,048, filed August 28, 2009, which is incorporated herein by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

[0002] Early detection is critical for favorable clinical outcome of a cancer. In certain cancer, for example, metastatic breast cancer, tumor cells are present in the blood. These cells are called circulating tumor cells (CTCs). High number of CTCs has been correlated with the poor prognosis of a cancer. However, CTCs are rare in the blood, presenting a difficulty in evaluating them. Therefore, there exists a need for streamlined methods for investigating rare cells such as CTCs.

INCORPORATION BY REFERENCE

[0003] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, and patent application was specifically and individually indicated to be incorporated by reference.

SUMMARY OF THE INVENTION

[0004] The present invention relates to methods for isolation, quantitation, and molecular investigation of rare cells, such as CTCs or circulating stem cells. The methods may also include rare cell enrichment steps if such step would provide convenience in handling the sample.

[0005] The isolation methods in the invention relate to the use of unique configurations of PALM laser system, which will permit laser microdissection and laser catapult to cells placed in laboratory materials for handling a cellular sample, such as a blood sample. The laboratory material can be a plastic container, a glass container, or a cell-trapping module designed to capture the rare cells. A cellular sample, whether previously subject to enrichment or not, can be used for a source of heterogeneous population of cells from which rare cells can be isolated. The methods can be used for the isolation of single rare cells or a number of rare cells existing as a cluster.

[0006] Various enrichment methods may be utilized to obtain rare cells. For example, red blood cells may be lysed with a hypotonic buffer if it is deemed necessary. If the volume of blood sample is too large to handle for subsequent isolation and quantitation process, a centrifugation step may be employed to separate cells from the plasma and resuspend the cells in a volume suitable for subsequent steps. The enrichment may be achieved by labeling cells with fluorescence-conjugated antibodies either to leukocytes in the blood, or to rare cells, which will allow the separation of rare cells by fluorescent- activated cell sorting (FACS). Another example can be the use of size-based separation modules. The blood sample can be flown through an array of obstacles placed on a lattice with a fixed interval. By manipulating the placement of the obstacles on the lattice, cells falling within a defined range of volume can be directed to one direction while cells do not fall within the range can be directed away to another direction. The size-based separation module can be used in combination with rare cell-capturing antibodies.

[0007] Isolated rare cells can be numbered. For example, if single rare cell is captured, the total number of individually captured rare cells in a defined volume of blood sample can be used to estimate the total number of rare cells in a human subject. If a cluster of rare cells is captured, the cluster can be treated to be dispersed in an appropriate buffer and be manually counted. If counting individual rare cells is nonviable, for example in the case of clumping, the clump of rare cells can be processed for genomic DNA PCR in which the cell numbers can be estimated by quantitating the presence of chromosome-specific markers. Total rare cell counts can be used in correlation with cancer staging, prognosis, or as an indicative measure for drug response in human subject undergoing chemotherapy.

[0008] Several methods can be used to obtain molecular characteristics of isolated rare cells. For example, rare cells can be pooled in 5 to 10 cells and be processed for RNA isolation and cDNA synthesis. The cDNAs can be applied to BioMark Dynamic Array (Fluidigm Co.). BioMark Dynamic Array is a convenient tool for performing multiple PCR conditions on multiple, heterogeneous population of cDNAs. When used for the analysis of cDNAs obtained from rare cells, the array can yield an expression profile for a sleuth of cancer markers in single run. In another example, the rare cells can be process for genomic DNA isolation. The genomic DNA can be applied to the array for the detection of genetic karyotypes known to be associated with cancer or certain stem-cell related diseases. In some instances, individual rare cells from a single patient are assayed in the BioMark Dynamic Array separately to analyze heterogeneity of rare cells in a patient. In some instances, rare cells from a patient are pooled an assayed collectively. Such assays can be compared to controls (rare cells from healthy patients). The invention also provides for running samples from multiple patients simultaneously.

[0009] The invention provides for a method of catapulting cells from a solid substrate comprising: applying a laser beam at a focal offset greater than 0 μm above a target cell immobilized on a solid substrate; and collecting the resulting catapulted cell in a collection device. The solid substrate can comprise a staggered array of obstacles.

[0010] The focal offset can be greater than 0 μm and less than 2 μm above the target cell. The focal offset can be greater than or equal to 2 μm and less than 4 μm. The focal offset can be greater than or equal to 4 μm and less than 8 μm. The focal offset can be greater than or equal to 8 μm and less than 12 μm.

[0011] In some embodiments, the solid substrate can comprise glass. The solid substrate can comprise plastic.

[0012] In other embodiments, the laser energy is between 45 and 50 μJ. The laser energy can be between 50 and 55 μJ. The laser energy can be between 55 and 60 μJ. The laser energy can be between 60 and 65 μJ. The laser energy can be between 65 and 75 μJ. The laser energy can be between 75 and 85 μJ. [0013] The target cells can be circulating tumor cells. The target cells can be circulating stem cells.

[0014] The solid substrate can be optimally hydrated. The cells can be catapulted after the fluid bathing such cells has been allowed to evaporate 15-20 minutes.

[0015] The laser can be an infra-red laser. The laser can be an ultraviolet laser.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0017] Figure 1 illustrates one embodiment of isolation configuration.

[0018] Figure 2 illustrates one embodiment of rare cell isolation and quantitation of the cell number by

PCR.

[0019] Figure 3 illustrates one embodiment in which gene expression profile of multiple cancer markers is obtained from isolated rare cells.

[0020] Figure 4A shows a table of biomarkers.

[0021] Figure 4B shows a continued table of biomarkers.

[0022] Figure 4C shows a continued table of biomarkers.

[0023] Figure 4D shows a continued table of biomarkers.

[0024] Figure 4E shows a continued table of biomarkers.

[0025] Figure 4F shows a continued table of biomarkers.

[0026] Figure 4G shows a continued table of biomarkers.

[0027] Figure 5 illustrates CTCs recovered from PC-3 tumor xenograft mice compared to (A) weight of primary tumor and (B) extent of metastatic disease. CTC number is plotted versus tumor weight in grams or the observed extent of metastasis from open cavity examination. (B) Box and whiskers plot representing animals with no detected metastasis (n=3, 2 represented by dots, had no CTCs),

dissemination to only the proximal lymph node (n=4) or to the mesentery and more (n=10).

[0028] Figure 6 illustrates efficiency of enumeration techniques and CTC capture from the PC-3 in vivo murine model. (A) Prestained PC-3 cells were titrated into blood to generate 10, 50 and 100 cells per

10 μl aliquot. Cells counted are indicated by squares in the scatter plot. A best fit line is graphed. The average of the data points is represented by horizontal bars. (B) Counts of GFP+/Hoechst+ cells from

10 μl aliquots of a cardiac bleed compared to counts from 109 - 540 μl of the same bleed run over a CTC- chip. CTC counts were normalized to 10 μl. Thirteen mice are represented in this graph.

[0029] Figure 7 illustrates imaging of cells on a CTC-chip. (A) Fluorescence imaging of PC-3 CTCs immediately after capture. Cells are stained with Hoechst, and GFP is visible only in CTCs. Mouse leukocytes are smaller and only stain blue. (B) Cytokeratin stained CTC captured on the chip. (C) CTCs cultured on the chip after 12 days. Propidium iodide (pink) indicates a dead cell. (D) Wright-Giemsa staining of cultured CTCs. Scale bars indicate 100 μm.

[0030] Figure 8 illustrates PC-3 cells collected by LPC from the CTC-chip are transcriptionally similar to unprocessed PC-3 cells. PC-3 cells were cultured, harvested and fixed. Duplicate aliquots of approximately 45 cells were used as templates for molecular analysis. PC-3 cells were also run across the

CTC-chip and 30 cells were collected in duplicate by LPC and used as templates for molecular analysis.

All templates were subject to described transcript analysis and standard deviation between duplicate RT-

PCRs is indicated. Transcripts with Cts less than 15 are plotted with a standard curve best fit line.

[0031] Figure 9 illustrates transcript intensities are correlated for primary tumors (PT), metastatic lymph nodes (met LN), metastatic liver (met liver), and CTCs. Tissue RNA (500 pg) and CTCs (50 cells) were subject to our described RT-PCR preamplification and qPCR. Scatter plots display transcript levels with normalized Cts less than 15 and a standard curve best fit line. Unless otherwise noted, standard deviation corresponds to duplicate RT-PCR reactions for tissue RNA and triplicate qPCR reactions for CTCs. (A)

Correlation of primary tumors and metastatic lymph nodes as averaged across five experimental mice (P-

01, 3, 4, 6, 8) with standard deviation across the five listed mice. (B) Transcripts from primary tumor of mouse P-08 compared to metastatic lesions from liver in that mouse. (C, D) Transcripts from primary tumor of mouse P-08 (R² = .56) or P-04 (R² = .45) respectively compared to CTCs.

[0032] Figure 10 illustrates LPC of cells enables comparison of purified CTC transcripts to tumor tissues. A heat map was generated by auto-scaling each sample to a scale of 0 to 1 (approximate Ct values of 0 to undetectable) for 23 transcripts for five mice to enable intra-experimental comparisons.

Samples include primary or metastatic tumor tissue and CTCs as labeled.

[0033] Figure 11 illustrates Wright-Giemsa staining shows wide range of CTC size. Whole blood collected from mouse P-04 was spread on a glass slide and stained with Wright-Giemsa. CTCs were identified based on the morphologic features including cell size and high nucleus to cytoplasmic ratio.

Bar denotes a distance of 10 μm.

[0034] Figure 12 illustrates Gene names, probes, forward and reverse primers used in qPCR analysis.

Probe numbers are from the Roche Human Universal ProbeLibrary Set except the EGFR probe from

Applied Biosystems (ABI) as listed.

[0035] Figure 13 shows microfluidic device having a lid and removable threaded screw ports attached to the inlet and to the outlet.

[0036] Figure 14 shows a cross-sectional view of the microfluidic device of Figure IA having a lid and removable screw ports, cut along line B-B of Figure 13.

[0037] Figure 15 illustrates a system of three microfluidic devices wherein two devices are configured to flow a single sample in parallel, and wherein the third micrfluidic device is configured to flow the sample in series through the device after the sample has flowed through the first two devices, whereby the outlets of the first two devices flow to the inlet of the third device, and wherein a peristaltic pump is adapted and configured to flow the sample through the system. DETAILED DESCRIPTION OF THE INVENTION

[0038] The present invention provides methods to isolate, quantitate and evaluate the molecular characteristics of rare cells in a blood sample. In particular, the invention discloses specific configurations for isolating rare cells from cellular sample and performing nucleic acid analysis on such cells.

[0039] Cellular samples, such as blood samples can be obtained from a healthy human subject or a non- healthy human subject. Samples of rare cells can be obtained from a patient in need of diagnosis, prognosis or from a patient that needs to determine a course of treatment. To obtain the blood sample, any technique acknowledged by a licensed medical practitioner as an acceptable procedure can be used. The use of commercially available anti-coagulant may also be employed.

[0040] The present invention contemplates rare cell isolation using a laser for catapulting rare cell into a proper receptacle. In some embodiments, the laser can be a PALM laser or any laser that can be used for microdissection. The laser beam can have a diameter of about, no more than about, or greater than about 0.01, 0.1, 0.5, 1, 5, 10, 20, 25, 50, 100, 250, 500, or 1000 microns. Such isolation technique maintains cell viability. A receptacle can be an eppendorf microcentrifuge tube. The rare cells are catapulted from a clear platform. Such platform may be made out of a polymeric material, e.g., plastic, coated plastic, glass or treated glass. Plastic coating may include materials, such as poly-D-lysine, that enhances cell attachment to the plastic. The walls of a device for capturing cells, which may be plastic, polymeric, glass, or any other material described herein, may be coated with a thin film coating (e.g., a monolayer) of commercial non-stick reagents, such as those used to form hydrogels. Additional examples chemical species that may be used to modify walls include oligoethylene glycols, fluorinated polymers, organosilanes, thiols, poly-ethylene glycol, hyaluronic acid, bovine serum albumin, poly-vinyl alcohol, mucin, poly-HEMA, methacrylated PEG, and agarose. Charged polymers may also be employed to repel oppositely charged species. The walls may be coated with L-Lysine, poly-L-lysine, or any other amino acid or polymers of amino acids. The type of chemical species used for repulsion and the method of attachment to the channel walls will depend on the nature of the species being repelled and the nature of the walls and the species being attached. Such surface modification techniques are well known in the art. The walls may be functionalized before or after the device is assembled. The channel walls may also be coated in order to capture materials in the sample, e.g., membrane fragments or proteins.

[0041] In some embodiments, the platform is a flat substrate. In some instances, the platform is an enrichment device, such as a microfluidic device comprising an array of obstacles with microfluidic gaps between obstacles. An example of a device with an array of obstacles is shown in Figures 13 and 14. Figure 13 shows a microfluidic device 100 having an array 102 of obstacles 104, a lid 106 and removable threaded screw ports 108A, 108B attached to the inlet 110 and to the outlet 112. Some portion of the array 102, the base layer 114, or the lid 106 may be coated with one or more binding moieties for capture of one or more rare cells. Alternatively, or in addition, the geometry and features of the device 100 and the flow of sample 118 and buffer through the device 100 may result in capture of one or more rare cells based on size. Depicted in Figure 14 is a cross-sectional view of the micro fluidic device 100 of Figure 13 having a lid 106 and removable screw ports 108A, 108B, cut along line B-B of Figure 13. The device 100 and the array 102 may be transparent, and the lid 106 may also be transparent for rare cell analysis or enumeration directly on the device 100. Removing the screw ports 108A, 108B allow for enumeration, processing, or analysis of the captured one or more rare cells directly on the device 100 using methods and processes provided herein. The device 100 may be made from various materials, including, but not limited to, glass, plastic or silicon.

[0042] Such an enrichment device can be used to enrich rare cells from a blood sample prior to catapulting them into the receptacle. Multiple devices may be placed in series, parallel, or any combination thereof to enrich cells.

[0043] An array of obstacles can be used to separate rare cells based on size, shape, and/or deformability. An example of a system utilizing multiple arrays of obstacles is shown in Figure 15. Figure 15 depicts is a system 216 of three microfluidic devices 200A, 200B, 200C having arrays 202A, 202B, 202C of obstacles 204A, 204B, 204C, wherein two devices 200A, 200B are configured to flow a single sample 218 in parallel, and wherein the third microfluidic device 200C is adapted and configured to flow the sample in series through the device 200C after the sample has flowed through the first two devices 200A, 200B, whereby the outlets 212A, 212B of the first two devices 200A, 200B, flow to the inlet 210C of the third device 21 OC, and wherein a peristaltic pump 220 is adapted and configured to flow the sample 218 through the system 216. The direction of flow is shown by arrows W, X, Y, and the direction that the peristaltic pump, for example, turns is shown by arrow Z. Also shown is a sample reservoir 222, which may include a rocker or another preprocessing system as described herein. Further shown is a container 224 for capturing the sample 218 which has been flowed through the system 216.

[0044] Further continuing with Figure 15, there are multiple variations of this system 216 and in a single device 200. For example, other flow generators and placements are contemplated, the flow of the sample or the buffer or both may be continuous or intermittent, the number and the arrangement of devices may be varied, the direction of flow may be varied, the size of the arrays may be varied, the existence and types of binding moieties may be varied, the size, shapes, and arrangements of the obstacles may be varied, the number of times the sample is run through a device or multiple devices may be varied, the amount or existence of a buffer introduced in the system, as well as its flow rate may be varied, the amount and flow rates of the sample may be varied, among other non- limiting variations discussed herein.

[0045] An example of an array of obstacles is shown in Figure 7B-D. Figure 7B-D show top views of an array of obstacles, each obstacle appearing like a circle in the image. The spacing of the obstacles can be regular or irregular. As shown in Figure 7B, the spacing between some obstacles can be narrowed. For example, a microfluidic device comprising an array of obstacles can function to inhibit rare cells from migrating to an outlet based on their larger than average size and/or unique affinity. Arrays of obstacles with microfluidic gaps can have at least a subset of microfluidic gaps with a length that is smaller than the average hydrodynamic size of the rare cells. Arrays of obstacles can have at least a subset of obstacles that is covered with binding moieties that selectively bind rare cells, e.g., anti-Ep-CAM, anti-cytokeratin, and anti-EGFR antibodies. In some embodiments, the arrays of obstacles or the capture devices, can have surfaces binding moieties that are specific antibodies to any biomarker listed in Figures 4A-4G. The binding moieties may be specific antibodies to more than one biomarker. Specific antibodies can be spatially organized on a capture device. For example, antibodies to Ep-CAM can be located in a first region and antibodies to cytokeratin can be located in a second region. Examples of devices that can be used to capture cells is described, e.g., in US Appl. Publ. 20070026417 and 20070026413, which are each herein incorporated by reference in their entirety.

[0046] Regardless of the type of platform in which rare cells are placed, rare cells are immersed in liquid. The liquid can be buffer, serum, or blood plasma. The rare cells are allowed to reach an optimal hydration level on the platform prior to catapulting. This can be achieved by allowing the cells to hydrate or pre-incubation for a period between 1 and 59 minutes, between 5 and 45 minutes, between 20 and 50 minutes, between 10 and 30 minutes, between 15 and 25 minutes, or between 12 and 20 minutes. The hydration or pre-incubation time can be about, no more than about, or greater than about 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 90, 120, 150, or 180 minutes. Variables considered for the determination of incubation period comprise the volume of rare cell-containing liquid, the depth of liquid measured from the surface of the liquid to the bottom solid support in which the liquid is contained, the type of liquid, the humidity of the room, the heat conductivity of the material holding rare cells, or the combinations of aforementioned variables. For example, if an atmospheric environment has high humidity, cells may hydrate more quickly and a lower incubation time may be sufficient. Also, if in combination with high humidity, the depth of the liquid or amount of liquid for hydrating the cells is high, then the time for incubation can be further reduced. In preferred embodiments, cells enriched on a plastic microfluidic device are washed with a PBS buffer and allowed to remain on the platform for 5-30, 10-20 or 12-15 minutes. The cells enriched using the capture devices described herein can be hydrated or incubated prior to being catapulted for about, no more than about, or greater than about 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 90, 120, 150, or 180 minutes. The enriched cells can be hydrated prior being catapulted for between 1 and 59 minutes, between 5 and 45 minutes, between 20 and 50 minutes, between 10 and 30 minutes, between 15 and 25 minutes, or between 12 and 20 minutes.

[0047] The platform containing rare cells is then mounted on a sample stage of a LCM, such as the Zeiss PALM Microbeam model for rare cell isolation. An embodiment of this invention is illustrated in Figure 1. The sample stage has a clear window which allows the laser beam located underneath the sample stage to access the cells in the platform (Fig. 1, 1). The focal plane (Fig. 1, 2) of the laser is then adjusted to a positive offset value such that it brings the focal plane above the surface (Fig. 1, 3) where the cell is laid out. In one embodiment, the focus offset is adjusted to positive values which may be up to plus 40, 30, 22, 18, 16, 14, 12, 10 or 8 μm above the surface where the cell is laid out. This depends on the size of rare cell, the size of a rare cell cluster, or the location of the cell on the post. In some embodiments, the focus offset is adjusted to positive values which may be greater than 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 30, and 40 μm above the surface where the cell is laid out. As the laser is focused above and away from rare cells, less energy reaches the rare cells.

[0048] To catapult the cells, such energy lost is compensated by increasing the energy output level of the laser between by 1% up to 120%, or more preferably between 10% up to 90%, or 20% up to 80%. In some instances, the laser energy is measured as a percentage of maximum energy of the device, e.g. % max. The laser energy may be at least 40% max; 50% max, 60% max, 70% max, 80% max, 90% max, or

100% max.

[0049] The laser energy may be 10-120, microjoules (μJ); 20-100 μJ, 30-80 μJ, or 40-60 μJ. Other embodiments involve applying a laser energy that is greater than 10, 20, 30, 40, 50, 60 70, 80, 90, or 100 μJ.

[0050] The energy applied to the cell region may be delivered in a pulse having a length of up to 2 nanoseconds (ns); 2-4 ns; or 4-6 ns.

[0051] The laser beam may be a pulsed ultraviolet A (UVA) laser beam. The laser beam may have a repetition rate of 100 Hz. The repetition rate can be about, no more than about, or greater than about 1,

10, 25, 50, 60, 75, 100, 125, 150, 200, 250, 500, 750, or 1000 Hz. The power of the laser beam may be

20 mW. The power of the laser beam can be about, no more than about, or greater than about 1, 2, 3, 4, 5,

7.5, 10, 12.5, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 125, 150, or 200 mW. The beam profile may be Gaussian.

[0052] In some embodiments, the target cells are hydrated by submersion in fluid from 25-30 minutes prior to the application of the laser beam and then the fluid is allowed to evaporate from around the cell.

In some embodiments, such rehydration is effected by immersing the cells in a 10% glycerol solution.

The amount of evaporation time may be shorter than 30 minutes. In some embodiments, the evaporation time is up to 20 minutes, 15 minutes or 10 minutes. The evaporation time can be about, no more than about, or greater than about 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 90, 120, 150, or

180 minutes. The hydration liquid can be evaporated for between 1 and 59 minutes, between 5 and 45 minutes, between 20 and 50 minutes, between 10 and 30 minutes, between 15 and 25 minutes, or between

12 and 20 minutes

[0053] The invention may comprise delivery of a pulsed (3 ns) ultraviolet A (uvA) laser beam, impacting the sample at high energy density. The high energy density may be 10MW/cm . The condensed UV radiation (1=337 nm) within the focal spot absorbed by the sample. In certain embodiments, energy transferred to the targeted object will suffice to break the molecular bonds resulting in fragmentation without mechanical contact.

[0054] The energy imparted to the cell may be focused energy. The energy imparted to the target cell may be diffuse in nature. Such energy may be diffuse when compared to energy focused directly at the cell, i.e. comes to a point at the cell. [0055] Following, or contemporaneously with, the energy transfer to the targeted object, the cell is detached from its position on the platform and may either be transported downstream to another location on the platform or be catapulted into a receptacle.

[0056] In some embodiments, the cells are from a patient suffering from a disease. The disease may be cancer. In some embodiments, the method comprises enriching cells from peripheral blood of a patient.

[0057] The method may comprise enumerating analytes in a sample, such as circulating tumor cells (CTCs). The method comprises applying a sample with analytes of interest to a clear platform, immersing the analytes, allowing the analytes to evaporate for a period of time, and applying laser energy above the analytes to catapult them into a receptacle. The analytes, e.g., CTCs can then be enumerated, and used to determine the percentage of CTCs within a sample.

[0058] The isolated rare cells can be counted and be analyzed for their molecular characteristics. Manual counting methods using a hemocytometer can be employed for the counting, if rare cells are individually collected. For a rare cell cluster, a mild cell dissociation treatment can be applied to separate the cluster into single cell suspension and be counted. If rare cells are formed in a tightly associated clump, or if rare cells are tightly associated in a clump of heterogeneous cells, other quantitation methods can be employed to account for the number of rare cells in the clump. In one embodiment where individual counting of rare cell is not viable, quantitative RT-PCR or genomic PCR can be used to count for the number of rare cells. For RT-PCR, level of rare cell-specific transcripts can be obtained and compared to the level of housekeeping gene and the cell numbers can be estimated. For genomic PCR, chromosome specific amplifications can be employed to account for the number of chromosomes in order to estimate the number of cells present in the sample.

[0059] To obtain molecular characteristics of rare cells, quantitative RT-PCR can be used. If the number of rare cells available presents a limitation for molecular analysis, amplification steps may be employed. To ensure contamination- free, hands-free amplification operation, and detection of multiple cancer- related transcripts, a system that allows the user to perform automated multiplex PCR can be used. Such system may be BioMark Dynamic Array (Fluidigm Co). In one embodiment, analysis of rare cell transcripts using Fluidigm can be performed to obtain information useful for cancer diagnosis. Such information can also be used to evaluate the progression of certain cancer, for example, staging of a cancer using commercially available stage-specific marker to detect the expression of such transcripts, to evaluate drug response of tumor cells for patients undergoing chemotherapy, or to evaluate certain genotypes known for increased propensity toward tumor formation. In one embodiment, quantitative RT- PCR results of EGF (epidermal growth factor), EGFR (EGF receptor), EpCAM, GA733-2, MUC-I Her- 2, or Claudin-7 can be performed. Other genes whose analysis can be performed using the methods herein are listed in Figures 4A-4G. The identity of rare cells can be re-confirmed by the presence or EpCAM transcripts. The detection may be performed in combination with primers specifically amplifying certain mutations of EGF or EGFR known for their correlation with epithelial cancer, therefore providing further information on rare cells. Level of Her-2 transcripts can provide useful prognostic information as its expression and mutation are linked to breast cancer propensity. Other stem cell markers or metastatic markers may also be employed to investigate the heterogeneity of rare cells, for example, percentage of cells beginning to assume metastatic phenotype. Other methods and system for catapulting cells may also be employed, for example, those described in Langer et al., "Live cell catapulting and recultivation does not change the karyotype of HCTl 16 tumor cells," Cancer Genet Cytogenet. 2005 Sep 161(2); 174-7, Helzer et al., "Circulating Tumor Cells Are Transcriptionally Similar to the Primary Tumor in a Murine Prostate Model," Cancer Res October 1, 2009 69; 7860, Burgemeister, "New Aspects of Laser

Microdissection in Research and Routine," Journal of Histochemistry and Cytochemistry Volume 53 (3): 409-412, 2005, and Stich et al., "Live cell catapulting and recultivation," Pathol Res Pract.

2003 ;199(6) :405-9, which are each incorporated by reference herein.

EXAMPLES EXAMPLE 1

[0060] An example of the current invention may be capturing cells GFP labeled PC3 cells and obtaining gene expression profiles. PC3 cells are established from human prostate cancer. GFP labeled PC3 cells are engineered to constitutively express GFP in the cytoplasm. PC3 cells are orthotopically implanted to mice in order to establish primary tumor site. After a period of time, metastasis of PC3 cells occurs, which can be observed my monitoring GFP under fluorescence scope. A small amount of blood can be drawn from mice, for example 500 μl, and be directly applied to The microfluidic device. After the application of blood, wash is performed with PBS buffer. The microfluidic device contains

microfabricated posts coated with anti-EpCAM antibody. PC3 cells are captured in the chip by their size and their affinity to anti-EpCAM antibody. Other cells are removed during the wash step. Captured PC3 cells are visually located under a fluorescence microscope due to their expression of GFP. In Zeiss PALM Microbeam system, identified PC3 cells are laser microdissected and catapulted into eppendorf tubes by using 60% laser energy output and plus 8 μm focus offset. The cells are counted and diluted in a buffer. RNA is isolated from each cell and reverse transcribed to synthesize first strand of cDNA. The resulting pre-amplified DNA is diluted and loaded into a BioMark Dynamic Array system (Fluidigm Co.) for amplification and detection of transcripts. Quantitative RT-PCR is performed in the BioMark Dynamic Array with primers specific for experimental controls and cancer-associated transcripts. For experimental control, beta-actin and GAPDH are used. In figure 2, cells captured by a microfluidic device are visualized by cell tracker green staining (Fig. 2, 4) or by Hoechest dye (Fig. 2, 5). Beta-actin PCR shows a single rare cell (Fig. 2, 6), 5 rare cells (Fig. 2, 7), and 10 rare cells (Fig. 2, 8) captured by the device. GAPDH PCR also shows a single rare cell (Fig. 2, 9), 5 rare cells (Fig. 2, 10), and 10 rare cells (Fig. 2,

H)

[0061] The resulting gene expression profile from enriches cells as shown in Figure 3 shows that circulating PC3 cells originated from the implanted PC3 cells express characteristic markers of prostate cancer, such as CEACAM (carcinoembryonic antigen related cell adhesion molecule), TACSTDl (tumor associated calcium signaling transducer 1), AGR2 (anterior gradient 2, a breast cancer membrane protein), and keratin 19. When 3 populations of PC3 cells are examined, 10 isolated PC3 cells per each population, molecular heterogeneity among different populations are observed, such as the expression of

Bcl-2 being identified in group but not in other groups of PC3 cells.

EXAMPLE 2: Isolating of CTCs from blood cells

[0062] Circulating tumor cells (CTCs) can be released into the blood stream from tumors of epithelial origin (1). The number of CTCs can be shown to predict patient outcome for metastatic breast, metastatic colorectal and metastatic prostate cancer (2, 3). In addition to indicating patient prognosis, it may be possible to use isolated CTCs to interrogate the current biology of a patient's cancer (4).

[0063] A lack of consensus on how similar CTCs are to parent tumors currently precludes their use as a surrogate for solid tumor biopsies. Published reports of genetic mutations (4) and mRNA profiles (5) used enriched CTCs in a background of leukocytes. These studies were somewhat limited by the use of enriched, not purified, CTC molecular templates as signal from non-target cells can overwhelm molecular signatures from the rare CTCs.

[0064] Recent publications suggest that certain xenograft models generate CTCs. In particular, specific reports of prostate cancer cells implanted into the prostate of immune compromised mice resulted not only in tumor formation, but in metastases and the generation of viable CTCs (5, 6). A similar study surveyed breast cancer cell lines and reported one model from which CTCs were observed (6).

[0065] Using a plastic version of the previously described CTC-chip, we demonstrated isolation and analysis of CTCs from a xenograft model of prostate cancer. Cells captured on the CTC-chip are viable and can be expanded in culture. Using a laser pressure catapulting procedure we showed that CTCs can be isolated away from contaminating blood cells and that purified CTC populations were similar to primary tumor and tested metastatic sites within the dynamic range of measured transcripts.

[0066] More on use of lasers to catapult cells from a CTC-chip is described in Helzer et al., "Circulating

Tumor Cells Are Transcriptionally Similar to the Primary Tumor in a Murine Prostate Model," Cancer

Res October 1, 2009 69; 7860, which is incorporated by reference in its entirety for all purposes.

EXAMPLE 3: Materials and Methods

[0067] Cell lines and orthotopic mouse model. PC-3 cells (ATCC) were grown in RPMI

supplemented with 10% FBS and penicillin-streptomycin (Invitrogen).

[0068] The orthotopic model has been previously described (7, 8). Cardiac blood (0.20- 1.0 ml) was collected upon sacrifice in EDTA tubes (BD Biosciences) and stored at room temperature up to 2 hours until processed. Whole body and open cavity imaging was performed as previously described (7).

Primary and metastatic tumors were discriminated from adjacent normal tissue via their GFP

fluorescence, selectively isolated, and flash frozen in liquid nitrogen. RNA from tissue was isolated using the RNeasy Plus RNA Kit (Qiagen).

[0069] For expansion of captured CTCs, mouse blood was run across a CTC-chip and washed as described below. The tape was removed from the chip and placed in a dish of RPMI/10% FBS/penicillin- streptomycin under 5.0% CO₂ and grown for 12 days. Cells were imaged by fluorescence microscopy and phase contrast microscopy.

[0070] Histochemical analysis. For histochemical analysis, dried blood smears or cultured CTCs were fixed for 1 minute in 100% methanol (Sigma) and stained with Wright-Giemsa stain (Sigma) per manufacturer's protocol. For bright field immunohistochemical analysis, the Vectastain Elite ABC kit and DAB Substrate Kit for Peroxidase (Vector Laboratories) were used. Reagents were flowed through the CTC-chip at 50 μl/min. Cells captured on the chip were fixed with 4% paraformaldehyde, permeabilized with 0.2 % Triton X-100 (Sigma) and blocked with normal goat serum. Mouse anti- cytokeratin monoclonal antibody (clone AE1/AE3) (Invitrogen) was used at a 1 :200 concentration followed by biotin-labeled anti-mouse IgG, HRP-labeled avidin and then DAB.

[0071] Preparation of CTC capture chip. The design and conjugation of antibodies to a CTC-chip has been described previously (9) with the following modifications. The chips were made from plastic using an embossing process. Chips were cleaned and activated with oxygen plasma, incubated with 4%

1 l-(succinimidyloxy)undecycldimethylethoxysilane (Gelest) in ethanol, washed with ethanol, incubated with 10 μg/ml Neutravidin (Pierce), washed with PBS (Cellgro) and then incubated with 10 μg/ml biotinylated goat anti-EpCAM antibody (R&D Systems). Stability was extended by washing the antibody coated chips in a sugar buffer composed of 2 mM L-histidine and 60 mM trehalose (Sigma). Chips were then sealed with adhesive tape and Tygon® tubing attached to ports. Chips were stored desiccated at 4° C until use (up to 1 month).

[0072] CTC capture, imaging, and enumeration on the CTC-chip. Sealed chips were attached via 2- stop Pharmed BPT tubing (Cole Parmer) to IPC peristaltic pumps (Ismatec) which allowed simultaneous runs of four samples per pump. Chips were placed on elevated, level platforms and primed with 1.0 ml

CAS buffer (Invitrogen). Blood was run at 16.7 μl/minute across the chip and washed with 1% BS A/PBS and Hoechst 33342 (Invitrogen) at a flow rate of 50.0 μl /min. In some instances when transcriptional analysis was not to be performed cells were fixed on the chip using 1% paraformaldehyde (EMS) and washed before imaging.

[0073] Samples used for transcriptional analysis were pre-processed. Specifically, blood samples were fixed by adding paraformaldehyde to a final concentration of 1% and incubating for 15 minutes at room temperature. An equal volume of l%BSA/PBS/5 mM EDTA was added followed by centrifugation at

40Og for 2 minutes. Plasma and buffer were removed and blood was resuspended to the original volume with l%BSA/PBS/5 mM EDTA.

[0074] All chips were imaged on a BioView scanning microscope using a 5X objective (Nes Ziona,

Israel). Events that were GFP and Hoechst positive were tagged by a BioView script as putative CTCs.

Visual review of putative events was used for final enumeration of validated true CTCs as

morphologically distinct GFP positive cells with a Hoechst stained nucleus.

[0075] Validation of blood smear enumeration and calculation of CTC-chip capture efficiency.

PC-3 cells were stained with both CellTracker Green (Invitrogen) and Hoechst 33342 (Invitrogen). Cells were then fixed by adding paraformaldehyde to a final concentration of 1% and incubating for 15 minutes at room temperature. Stained cells were counted and spiked into 1 ml mouse blood. PC-3 cells were titrated to final concentrations of 10,000, 5,000 and 1,000 cells per ml of blood. For each titration, 10 μl was pipetted onto slides in triplicate and scanned using a LeicaDM fluorescent microscope. Celltracker Green positive cells were counted using scanned images. Co-incident Hoechst/CellTracker Green staining was also verified.

[0076] Having validated the accuracy of CTC enumeration using blood smears, we were able to demonstrate the efficiency of the CTC-chip by comparing CTCs captured on the chip to CTCs counted in blood smears. To enumerate CTCs in whole blood, 10 μl of blood was spotted onto a glass slide with Hoechst 33342. Slides were scanned by Bio View for CTC enumeration. CTC counts from the whole blood were then compared to counts from the same blood sample run over a CTC-chip.

[0077] Laser Pressure Catapulting (LPC) collection of CTCs. To isolate CTCs, pre-processed mouse blood was run across a CTC-chip as described above. Following enumeration, the tape was removed from the chip to allow fluid to evaporate. LPC protocols were modified from the manufacturer (Zeiss PALM MB IV) to use 72% power and a focal offset of a few microns to account for differences between the CTC-chip and a glass slide. LPC was used to collect 50 cells per chip in duplicate. Cells were captured in the lid of PALM microfuge tubes and immediately subjected to molecular analysis.

[0078] TaqMan® assay development. Numerous gene-specific intron spanning TaqMan® assays were designed using the Human Universal ProbeLibrary (UPL) Set and the UPL Assay Design software (Roche). The EGFR TaqMan assay was purchased from Applied Biosystems (see online supplement for sequences). A final panel of to 42 gene-specific TaqMan® assays was selected based on individual and multiplex performance with template RNAs from various tissues or cell lines, as well as with PC-3 cells.

[0079] RNA isolation and amplification from CTCs, tissues and controls. Control PC-3 RNA was isolated from tissue culture cells with the Rneasy Plus Mini Kit (Qiagen). Samples of CTCs, PC-3 cells, 500 pg tissue RNA or 150 pg control RNAs were collected and/or lysed with targeted cDNA

preamplification via 1^st step RT-PCR: 50 ^°C 15', 95 ^°C T, and 18 cycles of 95 ^°C 15', 60 ^°C 4'. cDNAs were generated with a 5 μl master mix containing 0.19 μM primers (Integrated DNA Technologies) and 0.05 μM TaqMan® probes (Roche) from frozen aliquots, and 0.1 μl Superscript® III RT/Platinum® Taq mix (with RNaseOUT™ ribonuclease inhibitor) and IX Reaction Buffer from the CellsDirect™ One-Step qRT-PCR Kit (Invitrogen). Controls included prostate tissue RNA from a control non-tumor bearing nude mouse, blood leukocyte RNA (UniChain Biosystems, Inc.), PC-3 RNA, and LPC or water negative controls. Preamplified cDNAs were routinely stored at -20C overnight and then diluted 1 :5 with water for qPCR analysis.

[0080] cDNAs were subject to triplicate qPCR across the panel (42 genes with GAPDH and ACTB duplicated and 3 no-assay controls) using the standard BioMark™ Real-Time PCR protocol for 48.48 dynamic array chips (Fluidigm) with the following modification. Individual gene-specific TaqMan assays containing 9.45 μM primers, 2.35 μM probes, and 0.5x assay loading reagent, were frozen in aliquots for use in qPCR.

[0081] TaqMan® assay data analysis. Statistical analysis was completed in Excel and Spotfire Decision Site 9.1.1 for Microarray Analysis (Tibco Software, Inc). Gene expression levels are reported as Ct_norm values following normalization of Ct values for each transcript to GAPDH. In cases where amplification was not detected during the 40 cycle qPCR, an arbitrary cycle number of Ct 50 was used for normalization purposes. Next, Ct_nO0n values from triplicate qPCR reactions were averaged, excluding any undetectable transcripts. Duplicate RT-PCR reactions were performed for all samples other than tissue. Respective Ct_norm values of duplicate RT-PCR reactions were subsequently averaged, excluding any undetectable transcripts. To examine correlations between tissues, Ct_norm values from respective RT- PCRs of primary tumor or lymph node metastatic tissue were averaged as a set. As we found Ct_norm values less than 15 to be reproducible we graphed only the 22-24 transcripts from our panel with these higher gene expression levels.

[0082] Multiple experimental data sets are presented in "Heat Map" format. In the process of generating the heat map, normalization between experiments was accomplished by auto-scaling Ct_norm values within a single sample type, on a scale of zero to one, where zero represents the lowest Ct_norm value and one represents undetected transcripts.

EXAMPLE 4: Results

[0083] Generation of circulating tumor cells in a model system. To study circulating tumor cell generation in a mouse model, we chose a mode with cells that expressed GFP in order to monitor tumor growth and extent of disease using non-invasive whole body imaging. Whole body imaging and examination of the viscera after sacrifice allowed us to group the animals as having no metastatic spread from the primary tumor, spread only to adjacent lymph nodes, or disseminated into other organs of the body including the mesentery and in some animals, to the diaphragm, liver and pancreas. Necropsies did not include an analysis of the bones so the full extent of metastases is not known although metastatic spread in this model has been described in detail elsewhere (7) and did include metastasis to the bones. While the numbers of CTCs did not correlate with the weight of the primary tumor, there was a trend, though not statistically validated, towards greater CTCs with the extent of gross metastasis in the abdominal cavity (Fig. 5A,B). We attempted to obtain CTCs from peripheral site bleeds; foot pad, tail vein and retro-orbital, as described elsewhere (6) and were likewise unable to consistently detect CTCs in these samples. Blood collected via terminal cardiac punctures generated an average of over 8 CTCs per μl across the 18 mice tested in this study.

[0084] Measuring the efficiency of the CTC-chip. The CTC-chip technology has been previously described and evaluated (9). To validate the ability of the CTC-chip to capture in vivo derived CTCs, we first demonstrated the accuracy of enumerating fluorescently labeled cells in 10 μl blood smears (Fig. 6A). When 10 cells were expected per smear (n=3 samples) we counted 11, 12 or 13 cells, within the range of error for spike-in dilutions at this concentration. The process was linear for the tested 10, 50 and 100 cells per sample (R =0.9529) validating this simple blood smear method. Subsequent comparison of the number of CTC-chip captured cells to CTCs in blood smears revealed an average capture efficiency of 71% with a range from 44% to 125% for 14 mice tested on 6 different days (Fig. 6B).

[0085] Imaging of CTCs using fluorescence, histochemical and cytokeratin analysis. Cancer cells captured by the chip were differentiated from mouse leukocytes based on GFP expression using fluorescent microscopy (Fig. IA). CTCs appeared bright green with a Hoechst positive nucleus and ranged in size from approximately 5 microns to over 20 microns in diameter. The cells were observed as both single cells and as clusters (supplementary Fig. 5). To confirm cells were in fact CTCs, we used an antibody recognizing cytokeratin, an epithelial marker broadly used in pathology labs. Captured CTCs were strongly cytokeratin positive and were easily identified and imaged using bright field microscopy (Fig. 8B).

[0086] Capture of viable circulating tumor cells. The CTC-chip has been shown to capture live cells from patient blood (9). We wanted to test the ability to both capture live cells and expand them in culture. We therefore used captured cells from mouse P-04 for ex vivo growth. The culture was monitored periodically by phase contrast microscopy. Cells spread out and exhibited a flattened morphology after one day in culture. After 5 days, individual colonies of cells were observed. After 12 days in culture, colonies were expanding and cells were growing on top of the posts of the chip. Hoechst 33342 was added to visualize nuclei and propidium iodide was added to identify dead cells. Nearly all cells (>99%) were both GFP positive and nucleated, and very few cells were dead (<1%) (Fig. 1C). The cultured cells were also stained with Wright-Giemsa (Fig. ID) to permit clearer examination of the cellular morphology.

[0087] Validation of transcript panel and CTC collection process. We evaluated 42 transcripts selected for their relevance in cancer biology (5, 10-12) and abundance in an aliquot of approximately 50 PC-3 cells. Our analysis identified 24 transcripts in our panel that could reliably be measured below a Ct of 15 in cultured PC-3 cells and xenograft tumors (Fig. 8, 9A,B). Adjacent normal mouse tissue did not cross-react with our TaqMan Assays (data not shown).

[0088] Published methods for CTC enrichment do not completely isolate CTCs from blood cells which can interfere with accurate measurement of transcript levels in CTCs (5). We used Laser Pressure Catapulting (LPC) to specifically isolate CTCs already greatly enriched by the CTC-chip (9). To examine how the entire CTC collection process affects transcript measurements, we compared fixed tissue culture cells to fixed cells captured by the CTC-chip and isolated by LPC (Fig. 8). This process decreased the number of evaluable transcripts to 22 which ranged from normalized Ct values of approximately 0 for GAPDH to 12 for CEAC AM5. The correlation between the samples has an R² of 0.87 with even greater similarity occurring in transcripts with a normalized Ct below 7. This indicates a dynamic range which extends approximately 2 orders of magnitude below the expression levels of GAPDH when approximately 50 cells are measured with this transcription panel. [0089] Transcriptional analysis of tumor tissue and CTCs. We next compared transcript levels in primary tumor tissue to those in metastatic tissue collected from the lymph nodes from five mice (Fig. 9A). We found similar gene expression profiles for primary tumor and lymph node metastases, with a best fit correlation of R² of 0.98 for the transcripts analyzed. Moreover, the standard deviation for individual normalized Ct values is low for the group of five mice when either primary tumor or metastatic lymph node tissues is examined. We also collected metastatic tissue from the liver of mouse P-08 and compared transcript levels to those of the primary tumor (Fig. 9B) and again observed high concordance with a best fit correlation of R² of 0.97.

[0090] Since we had determined that LPC collection of cells from the CTC-chip retained gene expression profiles, we compared the transcript levels from CTCs isolated from five mice to those from each respective primary tumor. Individual plots from mouse P-08 and P-04 (Fig. 9C,D) show the changes in CTC transcripts relative to the primary tumor. We represented 24 transcript measurements from five mice in a scaled heat map to evaluate the variability (Fig. 10). Within the 2 orders of magnitude dynamic range of the LPC procedure for samples of 50 CTCs, transcripts are strikingly similar across the various mouse tissues and CTCs. BCL2α transcripts do show differential expression in CTCs relative to malignant tissue.

EXAMPLE 5: Discussion

[0091] Many cancer mouse models have been developed in an effort to better recapitulate cancer disease progression. We chose to use the PC-3 xenograft model since it illustrated aspects of the clinical pattern of metastatic spread of advanced clinical prostate cancer (13) and yielded reliably detectable CTCs in pilot experiments. Using a plastic version of the CTC-chip we showed that fluorescent as well as brightfield analysis was possible. Brightfield analysis enabled the use of LPC to collect CTCs away from contaminating blood cells. Our comparison of a select set of transcripts from primary, metastatic tissues and CTCs shows a strong concordance. We also demonstrate culturing of CTCs.

[0092] Eliane et al. describe a survey of breast cancer models and observed CTC generation in animals injected orthotopically with MDA-MB-23 1 cells (6). They serially measured CTCs from the same animal via cardiac punctures to compare tumor progression with CTC generation. We used cardiac bleeds at termination to compare the transcription profile of CTCs to that of tumor material. A less invasive peripheral bleed technique for mice that reliably collects CTCs has not to our knowledge been described. However, the high levels of CTCs collected from the model presented here should support serial bleeds for investigators examining aspects of CTCs with tumor progression.

[0093] Using GFP expressing cells allowed for the monitoring of disease progression by whole-body fluorescent imaging and cells shed from the tumor retained GFP-expression allowing unequivocal enumeration. GFP expression is not a prerequisite for CTC imaging. Others have described in detail using fluorescent anti-cytokeratin antibodies to detect CTCs from cancer patients using the CTC-chip (4, 9). We also show that captured cells stain positive for cytokeratin (Fig. 1C) using brightfield

immunohistochemical analysis of captured CTCs, a feature made possible by using a plastic CTC-chip. The plastic CTC-chip demonstrated an average recovery efficiency of 71% with CTCs derived from the mouse model, which compares favorably with spike-in studies performed on the silicon CTC-chip (9).

[0094] It had previously been shown that CTC numbers do not correlate with the tumor size in a xenograft model (6). Our study confirms this observation. It seems plausible that a trend exists towards increased CTCs with the extent of metastasis as Figure 5A suggests. Such a trend would normally be difficult to determine due to the difficulty of accurately measuring tumor burden throughout the animal. However, strategies for accurate quantification of tumor metastasis have been published using GFP positive tumors and could be applied in future studies (14). It is intriguing to think that CTCs are an indicator of metastatic activity. Understanding the etiology of CTCs and the initiation of metastasis can allow for the treatment and control of cancer. Correlations between metastatic activity and CTC counts can increase our understanding of the biology of CTCs and how they affect disease outcome.

[0095] We confirmed that viable CTCs can be captured by the CTC-chip and showed that the cells could be grown directly on the chip. Since most techniques do not isolate enough CTCs to perform multiple analyses, the possibility that a patient's CTCs can be cultured ex vivo and used to characterize the original tumor greatly expands the range of genetic and chemotherapy testing options that otherwise could not be accomplished without a biopsy.

[0096] The transcriptional profile of the primary tumor and metastatic tumors from our xenograft model was similar (Fig. 9A,B; Fig. 10). This may be explained by the aggressive nature of the model: PC-3 cells were originally isolated from metastatic prostate cancer and may already harbor the corresponding molecular changes necessary for metastatic spread. The fidelity with which the CTC-chip and LPC method maintained the transcript levels of tissue culture cells (Fig. 8) encourages us to believe that the differences observed between transcripts in CTCs compared to primary or metastatic lesions, while modest, are representative of the biological changes that a cell undergoes as it leaves the solid tumor and enters circulation. The methods we describe enable the ability to profile transcripts from patient CTCs. If patient CTCs are also transcriptionally similar to solid tumors they can be an ideal source of mutational information (4, 9) and a general surrogate for analysis of the primary tumor.

[0097] When a patient has detectible CTCs, they range widely in abundance (4, 9, 15, 16). In particular, patients with metastatic prostate cancer have been reported to have a mean CTC count of 75 (15). We found that working with 35-50 cells (Fig. 8) was sufficient to determine whether the CTC-chip could be used with transcriptional analysis. The mid to low abundance transcripts (e.g. ERCCl, GUS) can be measured reliable with approximately 30 cells. Highly expressed transcripts (e.g. GAPDH) can be detected from lower cell inputs (data not shown). Since this is within the range of CTCs reportedly isolated from prostate cancer patients, analysis of at least some transcripts from purified patient CTCs is feasible and can provide useful information. CTCs can be surrogates for the primary biopsy in human patients.

[0098] There were some notable changes in transcript abundance. As an example, we observed an increase in the BCL2α gene expression levels of CTCs relative to primary tumor or metastatic tissue (Fig. 9C,D; Fig. 10). BCL2α in an anti-apoptotic gene involved in chemoresistance, and is often found upregulated in cancers (1). Its upregulation in CTCs may indicate that it is also involved in one of the processes thought to be required for cancer spread, be it escape from the primary tumor, entrance into the blood stream or survival upon entering circulation.

[0099] The demonstrated advantages of the plastic CTC-chip we present include the ability to perform brightfield analysis, including immunohistochemical and Wright-Giemsa staining, and visual monitoring of CTC growth. In addition, the plastic surface enables LPC collection of CTCs away from background cells which in turn increases the molecular data gathered from CTCs since signal from the background is all but eliminated. Together, this approach greatly expands the information that can be gleaned from fluorescent analysis commonly used in the CTC field.

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Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method of catapulting cells from a solid substrate comprising:

applying a laser beam at a focal offset greater than 0 μm above a target cell immobilized on a solid substrate; and

collecting the resulting catapulted cell in a collection device.

2. The method of claim 1 , wherein the solid substrate comprises a staggered array of obstacles.

3. The method of claim 1, wherein the focal offset is greater than 0 μm and less than 2 μm above the target cell.

4. The method of claim 1 , wherein the focal offset is greater than or equal to 2 μm and less than 4 μm.

5. The method of claim 1, wherein the focal offset is greater than or equal to 4 μm and less than 8 μm.

6. The method of claim 1, wherein the focal offset is greater than or equal to 8 μm and less than 12 μm.

7. The method of claim 1, wherein the solid substrate comprises glass.

8. The method of claim 1, wherein the solid substrate comprises plastic.

9. The method of claim 1, wherein the laser energy is between 45 and 50 μJ.

10. The method of claim 1, wherein the laser energy is between 50 and 55 μJ.

11. The method of claim 1, wherein the laser energy is between 55 and 60 μJ.

12. The method of claim 1, wherein the laser energy is between 60 and 65 μJ.

13. The method of claim 1, wherein the laser energy is between 65 and 75 μJ.

14. The method of claim 1, wherein the laser energy is between 75 and 85 μJ.

15. The method of claim 1, wherein the target cells are circulating tumor cells.

16. The method of claim 1, wherein the target cells are circulating stem cells.

17. The method of claim 1, wherein the solid substrate is optimally hydrated.

18. The method of claim 1, wherein said cells are catapulted after the fluid bathing such cells has been allowed to evaporate 15-20 minutes.

19. The method of claim 1, wherein the laser is an infra-red laser.

20. The method of claim 1, wherein the laser is an ultraviolet laser.