Exploiting Serum Interactions with Cationic Biomaterials Enables Label-Free Circulating Tumor Cell Isolation

Exploiting Serum Interactions with Cationic Biomaterials Enables Label-Free Circulating Tumor Cell Isolation

Michael J. Mitchell¹, Carlos A. Castellanos², Michael R. King²

¹Department of Chemical Engineering, Koch Institute for Integrative Cancer Research, MIT, Cambridge, MA 02139

²Department of Biomedical Engineering, Cornell University, Ithaca, NY 14853

Introduction:  The ability to isolate circulating tumor cells (CTCs) from patient blood at high purity will facilitate the development of effective personalized medicine regimens for patients with metastatic cancer. Our lab has developed microscale flow devices with immobilized cell adhesion molecules to both isolate CTCs from patient blood and deliver therapeutics to cancer cells (1-3). Improving upon current isolation methods is challenged by (1) the lack of adhesion receptors common to a broad range of CTCs, and (2) low CTC capture due to leukocyte bio-fouling onto capture surfaces. Herein, we evaluated the effect of charged polymer coated surfaces on differential capture of CTCs under flow.

Materials and Methods: Microscale flow devices were functionalized with cationic polymer by incubation with 0.025 - 0.1% (w/v) aqueous poly-L-lysine (PLL). Functionalized surfaces were characterized using dynamic light scattering, AFM, TEM, circular dichroism spectroscopy, and contact angle goniometry.  Cells were suspended in PBS buffer containing up to 1% (w/v) human albumin and perfused through microscale devices at a flow rate of 0.008 mL/min for 10 minutes using a motorized syringe pump and monitored via an inverted microscope.  Micrographs and video of cell adhesion were used to measure cancer cell capture and purity.

Results and Discussion:  PLL-functionalized surfaces were able to differentially capture cancer cells and leukocytes suspended in albumin-containing buffers (Fig. 1A - B). Leukocyte capture significantly decreased on surfaces when cells were suspended in 1% (w/v) albumin solution, while the number of cancer cells captured remained unchanged.  Cell suspensions from metastatic prostate cancer patients were processed on PLL-coated surfaces and  putative CTCs identified on basis of DAPI, cytokeratin, and CD45 staining (Fig 1 C).

Figure 1: PLL functionalized surfaces differentially capture cancer cells and leukocytes under flow. Number of firmly adhered cancer cells (A) and leukocytes (B) per 180,000² um. ***P<0.0001. *P <0.01. NS: not significant. (C) Number of prostate cancer cells captured from blood of metastatic cancer patients and representative images of CTCs. CTCs were confirmed on basis of DAPI, cytokeratin (CK), and CD45 staining. Scale bar: 5 um.

Conclusions:  We have shown that PLL functionalized surfaces contribute to differential capture of cancer cells under flow.  While both cancer cells and leukocytes adhesively interact with polylysine, the addition of albumin to buffer solution acts to repel leukocytes while maintaining cancer cell capture, which has the potential to improve applications for the isolation of rare CTC populations of interest, for the development of effective personalized medicine regimens.

References:

1.  Mitchell MJ, Castellanos CA, King MR. Surfactant functionalization induces robust, differential adhesion of tumor cells and blood cells to charged nanotube-coated biomaterials under flow. Biomaterials. 2015; 56:179-186.

2.  Mitchell MJ, Wayne E, Rana K, Schaffer CB, King MR. TRAIL-coated leukocytes that kill cancer cells in the circulation. PNAS. 2014; 111: 930-935.

3. Mitchell MJ, Castellanos CA, King MR. Immobilized surfactant‐nanotube complexes support selectin‐mediated capture of viable circulating tumor cells in the absence of capture antibodies. Journal of Biomedical Materials Research Part A. 2015; 103:3407-3418.