Multivalent Binding and Biomimetic Cell Rolling Improves the Sensitivity and Specificity of Circulating Tumor Cell Capture

Abstract

Purpose: We aimed to examine the effects of multivalent binding and biomimetic cell rolling on the sensitivity and specificity of circulating tumor cell (CTC) capture. We also investigated the clinical significance of CTCs and their kinetic profiles in patients with cancer undergoing radiotherapy treatment.Experimental Design: Patients with histologically confirmed primary carcinoma undergoing radiotherapy, with or without chemotherapy, were eligible for enrollment. Peripheral blood was collected prospectively at up to five time points, including before radiotherapy, at the first week, mid-point and final week of treatment, as well as 4 to 12 weeks after completion of radiotherapy. CTC capture was accomplished using a nanotechnology-based assay (CapioCyte) functionalized with aEpCAM, aHER-2, and aEGFR.Results: CapioCyte was able to detect CTCs in all 24 cancer patients enrolled. Multivalent binding via poly(amidoamine) dendrimers further improved capture sensitivity. We also showed that cell rolling effect can improve CTC capture specificity (% of captured cells that are CK⁺/CD45^-/DAPI⁺) up to 38%. Among the 18 patients with sequential CTC measurements, the median CTC decreased from 113 CTCs/mL before radiotherapy to 32 CTCs/mL at completion of radiotherapy (P = 0.001). CTCs declined throughout radiotherapy in patients with complete clinical and/or radiographic response, in contrast with an elevation in CTCs at mid or post-radiotherapy in the two patients with known pathologic residual disease.Conclusions: Our study demonstrated that multivalent binding and cell rolling can improve the sensitivity and specificity of CTC capture compared with multivalent binding alone, allowing reliable monitoring of CTC changes during and after treatment. Clin Cancer Res; 24(11); 2539-47. ©2018 AACR.

Figure 1

Schematic diagram of CapioCyte fabrication and its capture mechanism. (A, B) The surface of an epoxy-functionalized glass slide was defined with a patterned polydimethylsiloxane (PDMS) gasket. (C) The CapioCyte-S surface was then functionalized by a sequential immobilization of PEG, G7 PAMAM dendrimers, and antibody mixtures using the EDC/NHS chemistry. (D, E) For CapioCyte-D, the gasket was removed (D), followed by backfilling with E-selectin (E). (F) After flowing cells including leukocytes and CTCs were recruited onto the surface by E-selectin-medicated cell rolling, CTCs were isolated from the rolling cells using antibody-dendrimer-coated surface. (G) The 2-channel fluidic system fabricated in house was used for this clinical pilot study.

Figure 2

CTC capture in cancer patients undergoing RT. (A) Epithelial cell counts obtained using blood samples from healthy donors. Based on these counts falsely counted as CTCs, the thresholds of CTC counts using CapioCyte-S and CapioCyte-D were set at 9.7 ± 0.9 and 2.4 ± 1.5 cells per mL (mean ± standard error (SE)), respectively. (B) Significant CTC counts per mL blood from all patients (N=24) obtained using CapioCyte-S. The CTC counts from non-head and neck cancer patients are shown in gray (patient #02: colorectal, patient #05: cervical, and patient #12: prostate) bars. (C) Significant CTC counts per mL blood from patients (N=23) obtained using CapioCyte-D. Note that the CTC count for the first patient was not included as the blood sample was treated with EDTA, instead of heparin, destabilizing the rolling response of the cells.

Figure 3

Enhanced CTC capture sensitivity and specificity via multivalent binding and biomimetic cell rolling using CapioCyte-D. (A-B) Enhanced CTC capture sensitivity via multivalent binding and multi-antibody approach. (A) Schematic representation of the effect using antibody mixture (blue), G7 dendrimers (green), and combination of the two (red). (B) Fold enhancements of antibody mixture, G7 dendrimers, and combination of the two, relative to the CTC counts captured on the control surface coated with aEpCAM only (orange dotted line). (C-F) Enhanced CTC capture specificity via E-selectin-mediated cell rolling to CapioCyte-D, with addition of E-selectin-mediated cell rolling to CapioCyte-S. (C) Comparison of the CTC counts measured using CapioCyte-D and CapioCyte-S. (D) Significantly enhanced CTC capture purities (%) among all captured cells using CapioCyte-D (0.66 - 37.54%), compared to those using CapioCyte-S (0.05 -6.78%). This result indicates that the capture specificity of CapioCyte-D was dramatically enhanced via E-selectin-mediated cell rolling. (E, F) Representative fluorescence images for CTC count analysis. CTCs (shown in purple) were captured with significantly less leukocytes (green) using CapioCyte-D, compared to CapioCyte-S. An image (×63 magnification) of the captured CTCs on CapioCyte-S at ×63 magnification is inserted on the representative image of CapioCyte-S at ×20 magnification. All red average lines indicate the mean ± standard error (SE).

Figure 4

Therapeutic effect monitoring using CapioCyte-D. (A) In the 18 patients with complete CTC measurements during the course of RT, the CTC counts at the Pre-RT (median 113 cells/mL, ranging from 43 - 849 cells/mL) were statistically significantly decreased in response to RT (median 32 cells/mL at the End-RT, range of 2 - 150 cells/mL, p = 0.001). The gray line indicates the median values of the data for Pre-RT and End-RT. (B) Compared to the reported CTC counts per 7.5 mL of HNSCC patients’ detected using CellSearch™ (5 ± 2, n = 19) (–28), significantly higher numbers of CTCs were captured using CapioCyte-D (1,450 ± 350 cells, n = 20). All gray average lines indicate the mean ± SE. (C) Rectal cancer patient with initial complete response to RT, as the 1-month Post-RT nadir CTC count is 6 cells/mL, significantly decreased from Pre-RT level of 662 cells/mL. In the setting of clinical, radiographic and subsequent pathologic-proven local recurrence at > 6 months Post-RT, the CTC count increased to 50 cells/mL. (D) Baseline PET/CT scan for this rectal cancer patient showed metabolic uptake in the rectum. (E) After completion of definitive chemoradiotherapy (CRT), 3-month PET/CT scan showed complete metabolic response of tumor in rectum. (F) Surveillance PET/CT scan at 6 months Post-RT showed increased metabolic uptake in rectum, concerning for cancer recurrence.