Research ArticleHEALTH AND MEDICINE

Covalent chemistry on nanostructured substrates enables noninvasive quantification of gene rearrangements in circulating tumor cells

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Science Advances  31 Jul 2019:
Vol. 5, no. 7, eaav9186
DOI: 10.1126/sciadv.aav9186
  • Fig. 1 Schematic illustrating the combined use of bioorthogonal ligation (i.e., the reaction between Tz and TCO) and disulfide cleavage–driven by DTT to enable capture and release of CTCs using Click Chips.

    The purified CTCs can then be subjected to RT-ddPCR to detect ALK or ROS1 rearrangements in NSCLC samples with single-molecule precision. cDNA, complementary DNA.

  • Fig. 2 Surface modification, characterization, and verification of Tz-grafted SiNWS.

    (A) Schematic summary of the stepwise functional group transformation developed for the preparation of Tz-grafted SiNWS. (B to D) X-ray photoelectron spectra of stepwise functionalized SiNWS: bare SiNWS (blue), SH-grafted SiNWS (orange), NH2-grafted SiNWS (green), and Tz-grafted SiNWS (red): (B) survey scans, (C) high-resolution XPS spectra in the energy range of the S 2p signals, and (D) high-resolution spectra in the energy range of the N 1s signals. (E) Scheme for verification of the successful preparation of Tz-grafted SiNWS via bioorthogonal ligation with Cy5-labeled TCO (2.6 mM) and disulfide cleavage by DTT (50 mM), respectively. Fluorescent images were captured through fluorescence microscopy (Nikon 90i, λex = 620/60) for (F) Tz-grafted SiNWS, (G) Cy5-grafted SiNWS, and (H) disulfide-cleaved SiNWS by DTT treatment. a.u., arbitrary units.

  • Fig. 3 Validation and optimization of Click Chips using artificial NSCLC samples.

    (A) Schematic representation of the mechanisms for bioorthogonal ligation–mediated capture and the disulfide cleavage–driven release of CTCs from Click Chips’ Tz-grafted SiNWS. (B) Schematic illustrating the conventional anti-EpCAM–mediated CTC capture process of NanoVelcro assays. (C) Comparison of CTC capture efficiency and specificity (i.e., nonspecifically captured WBCs) observed for Click Chips and NanoVelcro assays in the presence of 0.1, 20, and 200 ng of the respective anti-EpCAM capture agents (n = 3). (D) CTC capture efficiency of Click Chips was studied at flow rates of 0.1, 0.2, 0.5, 1.0, 2.0, and 5.0 ml hour−1 (n = 3). (E) CTC capture efficiency observed for the four different control groups: flat Si substrates without the Tz motif, Tz-grafted flat Si substrates, SiNWS without the Tz motif, and Click Chips based on Tz-grafted SiNWS (n = 3). (F) Comparison of capture efficiencies and specificity of bioorthogonal ligation–mediated CTC capture on Click Chips and magnetic beads. (G) CTC release efficiency was measured for Click Chips at flow rates of 0.1, 0.2, 0.5, 1.0, 2.0, and 5.0 ml hour−1 (n = 3). (H) General applicability of Click Chips for CTC capture and release was validated using artificial samples containing different NSCLC cell lines, i.e., HCC78, H2228, HCC827, H1975, and WBCs (n = 3). (I) Dynamic ranges observed for CTC capture and release using Click Chips. Spiked CTC numbers range from 5 to 200 cells ml−1. (J) A double parameter scatter plot showing the HCC78/WBC cell distribution observed for CTC capture in a Click Chip. (K) A double parameter scatter plot showing the cell distribution observed for CTC release, following the above experiment (J).

  • Fig. 4 Click Chips combined with RT-ddPCR analysis can be used to monitor dynamic changes in CTC count and CTC-derived ALK/ROS1 rearrangements in patients with NSCLC over the course of crizotinib (ALK/ROS1-TKI) treatments.

    (A) Schematic illustrating the general workflow developed for conducting CTC enumeration and CTC-based ALK/ROS1 rearrangement quantification. (B) The dynamic changes (0 to 129 days) of CTC counts and rearranged ALK transcripts (per 2 ml of blood) observed for a patient with NSCLC harboring the EML4-ALK rearrangement (A07) before and after crizotinib treatment. (C) CT images of patient A07 taken at days 0, 77, and 129, after crizotinib treatment. (D) Representative fluorescent micrographs of CTCs (DAPI+/CK+/CD45) captured from patient A07’s blood samples using Click Chips. (E) Dynamic change (0 to 75 days) of CTC counts and rearranged ROS1 transcripts (per 2 ml of blood) observed for a patient with NSCLC with the CD74-ROS1 rearrangement (R05) before and after crizotinib treatment. (F) Chest CT scans of patient R05 taken at days 0, 30, and 75 after crizotinib treatment. (G) Representative fluorescent micrographs of the CTC populations obtained from patient R05.

  • Table 1 Clinical characteristics of patients with NSCLC (adenocarcinoma) and healthy donors (HD) enrolled in our study.

    N/A, not applicable.

    Patient
    number
    GenderAge (years)Smoking
    history
    (years)
    Tumor gradeClinical
    stage
    CTC counts*ALK/
    ROS1
    status
    (tissue)
    ALK/
    ROS1
    status
    (CTCs)
    Copy numbers of
    ALK/ROS1
    rearrangements
    in CTCs*
    A01Male53None3IV18EML4-ALKEML4-ALK333
    A02Male38None3IV15EML4-ALKEML4-ALK603
    A03Male65None3IV32EML4-ALKEML4-ALK198
    A04Female65None3IV27EML4-ALKEML4-ALK1728
    A05Male53None3IV16EML4-ALKEML4-ALK252
    A06Male58None3IV26EML4-ALKEML4-ALK207
    A07-1Male39None2IV15EML4-ALKEML4-ALK108
    A07-2Male39None2IV11EML4-ALKEML4-ALK81
    A07-3Male39None2IV9EML4-ALKEML4-ALK54
    A07-4Male39None2IV0EML4-ALKEML4-ALK0
    A07-5Male39None2IV0EML4-ALKEML4-ALK0
    R01Female62None3IIIB28CD74-ROS1CD74-ROS1504
    R02Male41None2IIIB22CD74-ROS1CD74-ROS1594
    R03Male61352IV17CD74-ROS1CD74-ROS1684
    R04Male34None3IV27CD74-ROS1CD74-ROS1963
    R05-1Male32None3IV28CD74-ROS1CD74-ROS11773
    R05-2Male32None3IV6CD74-ROS1CD74-ROS1162
    R05-3Male32None3IV36CD74-ROS1CD74-ROS12061
    HD01Male30NoneN/AN/A0N/AN/A0
    HD02Male26NoneN/AN/A0N/AN/A0
    HD03Male29NoneN/AN/A0N/AN/A0
    HD04Male46NoneN/AN/A0N/AN/A0
    HD05Female36NoneN/AN/A0N/AN/A0
    HD06Male32NoneN/AN/A0N/AN/A0

    *Per 2 ml of blood.

    Supplementary Materials

    • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/7/eaav9186/DC1

      Fig. S1. High-resolution XPS characterization.

      Fig. S2. Deconvolution of x-ray photoelectron spectra of stepwise functionalized SiNWS.

      Fig. S3. Fluorescence imaging for exploring bioorthogonal ligation.

      Fig. S4. Dynamic changes of Cy5 fluorescence intensity upon disulfide cleavage by DTT.

      Fig. S5. Scheme of CTC capture on Tz-grafted magnetic beads.

      Fig. S6. Scheme of RT-ddPCR analysis of ALK/ROS1 rearrangements in CTCs.

      Fig. S7. Dynamic ranges for RT-ddPCR quantification of ALK/ROS1 rearrangements.

      Fig. S8. Comparison of cell lysis on-chip with cell lysis of DTT-released cells.

      Table S1. CTC purity during capture and release processes.

    • Supplementary Materials

      This PDF file includes:

      • Fig. S1. High-resolution XPS characterization.
      • Fig. S2. Deconvolution of x-ray photoelectron spectra of stepwise functionalized SiNWS.
      • Fig. S3. Fluorescence imaging for exploring bioorthogonal ligation.
      • Fig. S4. Dynamic changes of Cy5 fluorescence intensity upon disulfide cleavage by DTT.
      • Fig. S5. Scheme of CTC capture on Tz-grafted magnetic beads.
      • Fig. S6. Scheme of RT-ddPCR analysis of ALK/ROS1 rearrangements in CTCs.
      • Fig. S7. Dynamic ranges for RT-ddPCR quantification of ALK/ROS1 rearrangements.
      • Fig. S8. Comparison of cell lysis on-chip with cell lysis of DTT-released cells.
      • Table S1. CTC purity during capture and release processes.

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