(171e) Unnatural Killer Cells: TRAIL-Coated Leukocytes That Kill Cancer Cells in the Circulation

Introduction:
Hematogenous metastasis contributes to a poor patient
prognosis in many types of cancer, with over 90% of cancer-related death
attributed to metastasis (1). For metastasis to occur, cancer cells detach from
the primary tumor, invade through the basement membrane, and intravasate into the peripheral circulation as circulating
tumor cells (CTCs) (2). It is believed that adhesive interactions between
selectins on the blood vessel wall and selectin ligands on the CTC surface
facilitate metastatic progression, in a manner similar to the leukocyte
adhesion cascade essential in the inflammatory response (3). Here, we describe
a novel approach to functionalize human and murine leukocytes with tumor
necrosis factor-related apoptosis-inducing ligand (TRAIL) on their surface,
along with E-selectin (ES), to capture and induce cancer cell apoptosis both in vitro in human blood and in vivo in mouse circulation.

Materials and
Methods: ES/TRAIL nanoscale liposomes and COLO
205 or PC-3 cancer cells suspended in buffer or spiked in peripheral human
blood were subjected to shear flow in a cone-and-plate viscometer. Cancer cells
and mononuclear cells were isolated, cultured overnight, and analyzed for viability
using flow cytometry (FC). To determine leukocyte
functionalization with ES/TRAIL, fluorescent (FL) liposomes were exposed to
human blood under shear flow, and leukocytes were isolated and analyzed for
ES/TRAIL attachment using FC and confocal microscopy. To examine cancer cell
capture and killing in vivo, mice
were injected with ES/TRAIL retro-orbitally, followed
by injection of FL-labeled COLO 205 cells via tail vein injection. Mouse blood
was removed via cardiac puncture, and viable and apoptotic cancer cells were
analyzed using FC. Viable and apoptotic cancer cells lodged within the mouse
lung, liver, and lymph nodes were quantified using multiphoton
microscopy.

Results and
Discussion: Upon exposure to shear flow, ES/TRAIL liposomes in human blood
were functionalized to the surface of leukocytes, as confirmed by confocal
microscopy (Fig. 1A) and FC. Exposure of functionalized ES/TRAIL leukocytes
into cancer cell-spiked blood successfully targeted and killed cancer cells
under shear flow, compared to controls (Fig. 1B). Mouse studies revealed that
ES/TRAIL-functionalized leukocytes capture and induce apoptosis in cancer cells
in the peripheral circulation in vivo
(Fig. 1C). Cancer cells lodged within the mouse lung were apoptotic after
treatment with ES/TRAIL leukocytes, as determined by multiphoton
microscopy.

Conclusions:  We have shown that leukocytes functionalized with ES/TRAIL can capture and
induce cancer cell apoptosis in vitro in
human blood and in vivo in mouse
experiments. ES/TRAIL-functionalized leukocytes can form the basis of a novel
method to target CTCs in the bloodstream as a means to prevent cancer
metastasis. Clinically, this therapeutic strategy can serve as preventive
measure upon diagnosis of metastatic hematogenous cancers.

Acknowledgements:
The work described was supported by
the Cornell Center on the Microenvironment and Metastasis through Award Number
U54CA143876 from the National Cancer Institute.

Figure 1: (A) Functionalized ES/TRAIL leukocytes after
ES/TRAIL liposome exposure to human blood under shear flow. Green = FL ES/TRAIL
liposomes. Blue = leukocyte nuclei. Scale bar = 5 um. (B) Viable COLO 205
cancer cells (gated FC population) after exposure leukocytes functionalized
with ES or ES/TRAIL. Untreated: viable COLO 205 control sample. (C) COLO 205
cells (yellow arrow) and functionalized leukocytes (red arrow) isolated from
mouse blood after exposure to ES/TRAIL or ES liposomes, sTRAIL, or buffer
injections in vivo. Scale bar = 20
um.

References:

1.    
Mitchell MJ, King MR. Computational and experimental
models of cancer cell response to fluid shear stress. Frontiers in Oncology.
2013 Mar 2;:1–11.

2.     Hughes
AD, King MR. Nanobiotechnology for the capture and
manipulation of circulating tumor cells. WIREs Nanomed
Nanobiotechnol. 2011. Dec 7;4(3):291–309.

3.     Geng Y, Marshall JR, King MR. Glycomechanics
of the Metastatic Cascade: Tumor Cell–Endothelial Cell Interactions in
the Circulation. Ann Biomed Eng. 2011 Nov 19;40(4):790–805.