Methods and Devices for Capturing Circulating Tumor Cells

Abstract

A method of capturing a Circulating Tumor Cell (CTC) from a sample includes introducing a sample into a microfluidic device having a cell capture surface and a flow modification surface under conditions that allow a CTC to bind to a cell rolling-inducing agent and a capturing agent disposed on the cell capture surface. The flow modification surface induces a rotational flow within the sample as it flows through the microfluidic device.

Description

CROSS REFERENCE TO RELATED APPLICATION[0001]This application claims priority to U.S. Provisional Patent Application No. 61 / 172,454, filed Apr. 25, 2009, and U.S. Provisional Patent Application No. 61 / 174,602, filed May 1, 2009, the disclosures of which are incorporated herein by reference in their entirety.STATEMENT OF GOVERNMENT SUPPORT[0002]This invention was made with government support under Grant No. CBET-0931472 awarded by the National Science Foundation. The government has certain rights in the invention.BACKGROUND[0003]1. Field of the Invention[0004]The invention relates to a method of capturing Circulating Tumor Cells from a sample, and a microfluidic device for performing the method.[0005]2. Brief Description of Related Technology[0006]Cancer remains one of the world's most devastating diseases, with more than 10 million new cases every year. Although recent advances in diagnostic and therapeutic methods to treat primary tumors have resulted in a decrease in mortality of c...

AI Technical Summary

Benefits of technology

[0103]The recent development of nanotechnology has demonstrated many breakthroughs in a range of biomedical applications—particularly for cancer treatment. For a new design of effective targeted drug delivery / imaging vectors based on nanotechnology, multivalent effects are desirable as they dramatically enhance active targeting efficacy. The similar enhancement can be also achieved in specific capturing when the delivery vectors are immobilized on the surfaces.

[0104]Preparation of PAMAM dendrimer-based nanodevice: the PAMAM dendrimer-based folate receptor (FAR) targeting nanodevices were synthesized as summarized in FIG. 3. Briefly, G5 PAMAM dendrimers were partially acetylated (70 of the 110 total primary amines), resulting in G5-Ac70. The remaining 40 primary amine groups were used for reaction to further functionalize the dendrimers. Note that a G5 PAMAM dendrimer molecule has approximately 110 primary amine termini according to the previous titration measurement. To fluorescently label the dendrimers, AlexaFluor® 488 (AF488, Molecular Probes) dissolved in DMSO was added to the dendrimer / H 2 0 solution at a molar ratio of 5:1 (AF488:dendrimer) in the presence of 1 M NaHCO 3 and the reaction mixture was stirred at RT for 48 hr. The resulting mixture of the dendrimer conjugate (G5-Ac70-AF488) was then dialyzed in water for 2 days and lyophilized for 2 days, followed by 10 cycles of ultrafiltration with PBS (with Ca2+ and Mg2+) and water using a 10,000 molecular weight cut-off membrane at 21° C., 5000 rpm for 30 min each. G5-Ac 70-AF488 conjugate in H2O was then reacted with FA preactivated by 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide / HCI (EDC) in DMF / DMSO at different molar ratios (3:1, 6:1, 9:1, 12:1, 15:1) of FA to G5-Ac70-AF488. The same purification process was carried out as described in the AF488 conjugation. Lastly, full acetylation of the remaining primary amine group was completed, yielding our final products G5-Ac-AF488-FA0, G5-Ac-AF488-FA2.6, G5-Ac-AF488-FA4.7, G5-Ac-AF488-FA7.2, G5-Ac-AF488-FA11.5, and G5-Ac-AF488-FA13.7. Since all the nanodevices were conjugated with the same number of AF488, differences in fluorescence intensities from the nanodevices in later FACS data (FIG. 4a-red squares) can be regarded as a result of differences in nanodevice binding and / or uptake by KB cells. The nanodevices become more polydisperse and ultimately give a bimodal distribution as the number of attached FA increases. In the case of G5-Ac-AF488-FA13.7, the polydispersity index (PDI=Mw / Mn) is 12.41 which is significantly greater than PDIs of previously reported dendrimer conjugates.

[0105]Quantitative analysis of multivalent effect mediated by PAMAM dendrimers: The material properties required for maximal multivalent effects include: 1) flexibility for conformational deformation to increase interacting surface area at a low cost of entropy and 2) localized reactive groups for targeting agents to utilize receptor clustering effect for a maximal number of simultaneous binding events in the given area. A series of experiments to quantitatively measure the multivalent targeting has been conducted using PAMAM dendrimers that satisfy the two pre-requisite properties, yielding a substantial enhancement in binding avidity as high as ˜170,000 fold compared to the monovalent binding counterpart. To study the interaction of FA-conjugated G5 PAMAM-based nanodevices (G5-Ac-AF488-FAx: x=2.6, 4.7, 7.2, 11.5, or 13.7) with folate binding protein (FBP), the surface plasmon resonance (SPR) technique using BIAcore X (Pharmacia Biosensor AB, Uppsala, Sweden) was employed. FBP was immobilized on the sensor chip surface (channel 2) of a carboxylated dextran-coated gold film (CM 5 sensor chip) by amine coupling as described. The dendritic nanodevices (30 μl) were injected at concentrations of 500 nM, 1 mM, and 2 mM at a flow rate of 10 μl / min, allowing the nanodevices to flow in both channels (channel 1 for reference and channel 2 with FBP) for 3 min. The final SPR sensorgrams were obtained from the signals from channel 2 subtracted by those from channel 1. Binding parameters of free FA with FBP were evaluated by the same condition but at different concentrations (1 and 2 mM used for free FA). The binding curves were fit using the 1:1 Langmuir binding model in BIAevaluation software. Associations and dissociations were fit separately since there was turbulence in the curves between association and dissociation phases in the process of subtracting signals from the reference channel. Dissociation constants (KD) for each dendrimer were obtained by averaging at least three different sets of results which had χ2 values lower than 3.0. All runs were independently analyzed for errors associated with mass transport by exporting the data files to Excel and plotting dR / dt versus R following the analysis described by Glaser.

[0106]To compare the SPR results to in vitro cell level data, the KB cell line (ATCC, Manassas, Va.) was employed and grown continuously as a monolayer at 37° C. and 5% CO2 in RPMI 1640 medium (Mediatech, Herndon, Va.) supplemented with penicillin (100 units / ml), streptomycin (100 mg / ml), and 10% heat-inactivated fetal bovine calf serum (FBS) before use. KB cells were also cultured in RPMI 1640 medium without folic acid (Mediatech) for at least 4 days before experiments, resulting in the folic acid receptor overexpressing KB (FAR+ KB) cell line. For the FACS measurements, the FAR-KB cells were seeded on a 24-well plate for tissue culture at a concentration of 2×105 cells / well and at 37° C., 5% CO2 for 24 hr. The cells were then incubated with the series of the prepared nanodevices at 37° C. for 1 hr. After removal of supernatants, cells were trypsinized and collected into FACS tubes, followed by centrifugation at 1500 rpm for 5 min to obtain cell pellets. The pellets were washed with PBS (Ca2+, Mg2+) twice using a repetitive centrifugation and resuspension process and then finally resuspended in PBS with 0.1% bovine serum albumin. The FACS sample preparation was performed on ice to inhibit cellular reactions such as further uptake. Fluorescence signal intensities from the samples were measured using a Coulter EPICS / XL MCL Beckman-Coulter flow cytometer, and data were analyzed using Expo32 software (Beckman-Coulter, Miami, Fla.).

[0107]As shown in FIG. 4a, an optimum number of targeting molecules (folic acid (FA)) appeared to be ˜5 where the dendrimers showed an exponential increase in binding avidity and sustained their monodispersed properties. Note that conjugation of more than 10 FA molecules caused substantial deterioration of homogeneity of the materials. Based on this optimized design criteria, engineered dendritic anti-cancer nanodevices utilizing the multivalency have exhibited great efficacy in targeting and killing cancer cells both in vitro and in vivo without apparent harmful side effects. This study supports the idea that nanoparticle based drug delivery systems can be significantly improved in targeting efficacy if the optimization process is conducted to maximize the multivalent effect without compromising the material's properties.

[0108]The significance of these results regarding the multivalent effect is three-fold: 1) the ability of PAMAM dendrimer-based scaffolds to afford a functional multivalent effector system is demonstrated 2) the in vivo effect is demonstrated to arise from the substantial enhancement of KD, not an increased rate of endocytosis and 3) the on-rate, ka, increases linearly with the number of targeting agents and shows no cooperativity whereas the off-rate, kd, decreases exponentially with the number of targeting agents (4b).

Problems solved by technology

Although recent advances in diagnostic and therapeutic methods to treat primary tumors have resulted in a decrease in mortality of cancer for the past two years, metastasis of cancer still poses a great challenge as patients often relapse.

Detection of DTCs requires aspiration of BM—a process that is invasive, time-consuming, and often painful for the patients, precluding repeated samplings that are necessary for prognosis studies along with therapeutic treatments.

However, the clinical usage of CTCs has not yet been implemented for routine clinical practice.

Unlike DTCs in BM that are relatively easy to enrich using Ficoll-based assays or the OncoQuick approach, and other immunomagnetic enrichment procedures, CTCs are extremely rare (estimated to be in the range of one tumor cell in the background of 106-109 normal blood cells), presenting a tremendous challenge for efficient, clinically significant detection of CTCs.

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