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Liquid biopsy and therapeutic response: Circulating tumor cell cultures for evaluation of anticancer treatment

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Science Advances  13 Jul 2016:
Vol. 2, no. 7, e1600274
DOI: 10.1126/sciadv.1600274
  • Fig. 1 Schematic overview depicting the procedure for anticancer drug screening via conventional methods and the CTC cluster method.

    In the case of conventional methods, cancer cells are derived from commercialized cell lines or patient-derived CTCs and tumors. Establishment of CTC cell lines requires more than 6 months, and tumor sampling can only be carried out as a single sampling. In addition, pre-enrichment of CTCs is required before they can be cultured. Conversely, CTC clusters can be generated within 2 weeks, and the blood samples do not require pre-enrichment before culture. In this procedure, blood samples are first lysed briefly to remove red blood cells (RBCs), and the resultant nucleated cell fraction is seeded into an integrated microwell-based microfluidic assay. Drugs can be introduced directly in situ, and a microfluidic component helps to efficiently distribute a range of drug concentrations.

  • Fig. 2 Establishment of CTC cluster assay for routine drug screening.

    (A) Three-dimensional layout of drug assay displaying the layers for the gradient generator, barrier, and microwells. (B) Gradient distribution of input reagents demonstrated by blue and red dyes. (C) (Left) Representative images of negative and positive samples. Bright-field images of microwells comprising a negative sample at ×10 magnification. Scale bar, 100 μm. (Middle) Hoechst staining of clusters in situ. Negative samples generated debris with some residual white blood cells (WBCs). Scale bar, 50 μm. (Right) Combined scatterplots of gray values, which reflected the density of cells, across each microwell. Values were normalized to the highest count for a particular microwell. Microwells with sparse groups of cells or debris demonstrated high gray values within the microwell region. (D) (Left) Bright-field images of microwells comprising a positive sample at ×10 magnification. Scale bar, 100 μm. (Middle) Nuclei staining using Hoechst on cell clusters in situ. Positive samples generated clusters with some residual WBCs. Scale bar, 50 μm. (Right) Combined scatterplots of gray values, with values normalized to the highest count for a particular microwell. Microwells with dense cell clusters demonstrated consistently low gray values (<0.5) within the microwell region.

  • Fig. 3 Comparison of custom tapered microwells fabricated using diffuser back-side lithography for CTC cluster assay and conventional cylindrical microwells.

    (A) Fabrication procedure: elliptical openings defining the size and position of the wells created on a soda-lime optical mask blank by laser direct writing. Subsequent Cr etching and stripping of the remaining resist were followed by coating with a layer of SU-8 2100 resist. Ultraviolet (UV) exposure to obtain pillar-like structures with the elliptical footprint and elliptical cross-shaped structures. Postbaking, ultrasound bath, and hard baking resulted in a template ready for PDMS molding. (B) (Left) Culture of MCF-7 in custom tapered microwells and cylindrical microwells. Single clusters were consistently established with tapered microwells. Scale bars, 50 μm. (C) (Left) Culture of clinical blood samples in custom tapered microwells and cylindrical microwells. Only debris was formed in cylindrical microwells. Scale bars, 50 μm. (Right) Combined scatterplots of gray values, with values normalized to the highest count for a particular microwell. Cylindrical microwells did not generate clusters, whereas tapered microwells led to a single dense cell cluster as observed from the region of low gray value. (D) Bar graph presenting results from a viability assay using trypan blue staining. Percentage of cells negative for trypan blue (viable cells) was significantly lower in the sample portion cultured in cylindrical microwells. All error bars represent SD of triplicate cultures from different samples. **P < 0.01.

  • Fig. 4 Assay validation with controls.

    (A) Screening of doxorubicin in microwell assay using MCF-7 cancer cell line. Cultures were imaged in situ after staining with LIVE (calcein-AM; green) and DEAD [ethidium bromide (EtBr); red] under 72 hours of exposure to doxorubicin. Clusters under high drug concentrations are mostly nonviable (red), whereas clusters under low drug concentrations are mostly viable (green). (B) Dose-response curve and corresponding IC50 value (0.78 ± 0.02 μM) of MCF-7 generated from viability results. Representative image shows an MCF-7 cell cluster within a microwell (inset). Scale bars, 50 mm. (C) Scatterplot demonstrating overall high gray values that reflect the absence of clusters from cultures of blood from healthy volunteers. Representative image shows cell debris generated within a microwell from culture of a healthy sample (inset). Scale bars, 50 mm.

  • Fig. 5 Screening of doxorubicin in microwell assay using clinical human primary cancer cells cultured from clinical samples at serial time points (before and after treatment).

    (A) Imaging of clusters generated from the pretreatment sample in situ after staining with LIVE (calcein-AM; green) and DEAD (EtBr; red) after 72 hours of exposure to doxorubicin. Clusters under high drug concentrations were mostly nonviable (red), whereas clusters under low drug concentrations were mostly viable (green). Scale bars, 100 μm. (B) Dose-response curve and corresponding IC50 value (0.94 ± 0.04 μM) of samples obtained from the same patient at different treatment time points. Monitoring IC50 values of a patient could reveal onset of drug tolerance or resistance. All error bars represent SD of triplicate cultures from different samples.

  • Fig. 6 Proposed workflow of routine anticancer treatment evaluation with clinical human CTC cultures.

    Cluster formation potential correlates inversely with overall patient survival, and increased IC50 values suggest possible onset of drug tolerance or resistance. The procedure can be completed within 2 weeks and will aid the clinician’s decision of maintaining or altering a patient’s drug regimen.

  • Table 1 Comparison of the sensitivity of the CTC cluster assay and conventional CTC expansion techniques. N.D., not determined.
    Cancer
    type
    Samples
    validated
    DurationCulture typePre-
    enrichment
    Efficiency in cultureCorrelation to
    treatment
    Reference
    Breast36>6 monthsCell lines, long-termYes16.70%N.D.(8)
    Breast8<1 month or
    ≧1 month
    Colonies (short-term) or
    cell lines (long-term)
    Yes37.50%N.D.(7)
    Colon71>2 monthsCell lines, long-termYes2.80%N.D.(52)
    Prostate17>6 monthsOrganoid lines, long-termYes~15–20%N.D.(9)
    Breast732 weeksPrimary CTC cluster (short-
    term)
    No19.6–59.3% (depending on
    treatment time point)
    YesThis work
  • Table 2 IC50 values for the samples from breast cancer patients that yielded clusters.

    Time point of blood withdrawal is provided.

    SampleIDTime pointIC50 value (μM)
    1CTB039Pretreatment0.85
    2CES021Posttreatment>1
    3CES053Posttreatment0.34
    4P2B28Posttreatment>1
    5P2B29Pretreatment0.94
    6P2B29Posttreatment0.86

Supplementary Materials

  • Supplementary materials for this article are available at http://advances.sciencemag.org/cgi/content/full/2/7/e1600274/DC1

    fig. S1. Microfabricated molds for the assay.

    fig. S2. Schematics of the gradient generator design.

    fig. S3. Flow rate of the device and the dynamics within the channel.

    fig. S4. Simulated flow conditions of the assay.

    fig. S5. Consistency of gradient concentration in channels over time.

    fig. S6. Estimation of cell counts after influence of flow.

    fig. S7. Validation of integrated assay for proliferation with MCF-7 cell lines.

    fig. S8. Optimization of cancer cell line culture parameters.

    fig. S9. Phase-contrast images of 2-week cultures under different culture conditions.

    fig. S10. Proportion of CTCs before culture affected the potential of cluster formation.

    fig. S11. Fluorescence in situ hybridization of cultured cells.

    fig. S12. Epithelial CTC counts before and after culture.

    fig. S13. Screening of doxorubicin in microwell assay using clinical human primary cancer cells cultured from blood samples.

    fig. S14. Characterization of cultures.

    fig. S15. Representative images of residual WBC populations.

    fig. S16. Scanning electron microscopy images demonstrating the densely packed array of microwells to maximize surface for capturing CTCs for culture.

    fig. S17. Representative image of a microwell containing cells contaminated by RBCs due to inadequate RBC lysis.

    fig. S18. Optimization of culture medium conditions.

    table S1. Concentration of a single reagent at each serpentine.

    table S2. Samples for preliminary validation of procedure.

    table S3. Samples evaluated for drug screening.

    table S4. Average percentage of viable CD45 cells in clinical samples.

    table S5. Disease evaluation of three sets of serial samples with at least one positive culture generated.

    table S6. CTC counts per milliliter as reported by notable CTC enrichment methods (non–culture-based).

    movie S1. Illustration of seeding, growth, and drug flow.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Microfabricated molds for the assay.
    • fig. S2. Schematics of the gradient generator design.
    • fig. S3. Flow rate of the device and the dynamics within the channel.
    • fig. S4. Simulated flow conditions of the assay.
    • fig. S5. Consistency of gradient concentration in channels over time.
    • fig. S6. Estimation of cell counts after influence of flow.
    • fig. S7. Validation of integrated assay for proliferation with MCF-7 cell lines.
    • fig. S8. Optimization of cancer cell line culture parameters.
    • fig. S9. Phase-contrast images of 2-week cultures under different culture conditions.
    • fig. S10. Proportion of CTCs before culture affected the potential of cluster formation.
    • fig. S11. Fluorescence in situ hybridization of cultured cells.
    • fig. S12. Epithelial CTC counts before and after culture.
    • fig. S13. Screening of doxorubicin in microwell assay using clinical human primary cancer cells cultured from blood samples.
    • fig. S14. Characterization of cultures.
    • fig. S15. Representative images of residual WBC populations.
    • fig. S16. Scanning electron microscopy images demonstrating the densely packed array of microwells to maximize surface for capturing CTCs for culture.
    • fig. S17. Representative image of a microwell containing cells contaminated by RBCs due to inadequate RBC lysis.
    • fig. S18. Optimization of culture medium conditions.
    • table S1. Concentration of a single reagent at each serpentine.
    • table S2. Samples for preliminary validation of procedure.
    • table S3. Samples evaluated for drug screening.
    • table S4. Average percentage of viable CD45 cells in clinical samples.
    • table S5. Disease evaluation of three sets of serial samples with at least one positive culture generated.
    • table S6. CTC counts per milliliter as reported by notable CTC enrichment methods (non–culture-based).
    • Legend for movie S1

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    Other Supplementary Material for this manuscript includes the following:

    • movie S1 (.mp4 format). Illustration of seeding, growth, and drug flow.

    Files in this Data Supplement:

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