Immunotargeting of Integrin α6β4 for Single-Photon Emission Computed Tomography and Near-Infrared Fluorescence Imaging in a Pancreatic Cancer Model

Abstract

To explore suitable imaging probes for early and specific detection of pancreatic cancer, we demonstrated that α6β4integrin is a good target and employed single-photon emission computed tomography (SPECT) or near-infrared (NIR) imaging for immunotargeting. Expression levels of α6β4were examined by Western blotting and flow cytometry in certain human pancreatic cancer cell lines. The human cell line BxPC-3 was used for α6β4-positive and a mouse cell line, A4, was used for negative counterpart. We labeled antibody against α6β4with Indium-111 ((111)In) or indocyanine green (ICG). After injection of(111)In-labeled probe to tumor-bearing mice, biodistribution, SPECT, autoradiography (ARG), and immunohistochemical (IHC) studies were conducted. After administration of ICG-labeled probe, in vivo and ex vivo NIR imaging and fluorescence microscopy of tumors were performed. BxPC-3 tumor showed a higher radioligand binding in SPECT and higher fluorescence intensity as well as a delay in the probe washout in NIR imaging when compared to A4 tumor. The biodistribution profile of(111)In-labeled probe, ARG, and IHC confirmed the α6β4specific binding of the probe. Here, we propose that α6β4is a desirable target for the diagnosis of pancreatic cancer and that it could be detected by radionuclide imaging and NIR imaging using a radiolabeled or ICG-labeled α6β4antibody.

Keywords: 111In SPECT; ICG; NIR imaging; integrin α6β4; pancreatic cancer.

Figure 1.

A, Expression of β₄ and α₆ integrins in 4 human pancreatic cancer cell lines, namely BxPC-3 (lane 2), AsPC-1 (lane 3), MIAPaCa-2 (lane 4), and PANC-1 (lane 5), were examined by Western blotting. Mouse cell line A4 (lane 1) was used as a negative control. Expression of β₄ and α₆ was normalized to actin band, and relative cell line expression standardized against to A4 cell lysate were shown under the panels. B, Expression of α₆β₄ in these cell lines was compared by flow cytometric analysis. Percentages of α₆β₄-positive cells (numbers in the upper-right quadrant) are presented in a bar graph. C, BxPC-3 and A4 cells were incubated with fluorescein- or ICG-labeled anti-α₆β₄ antibody (ITGA6B4) for 18 hours and examined under a fluorescent microscope. Intense fluorescence signals in membrane and intracellular signals indicated the localization of each probe and confirmed the high α₆β₄ expression in BxPC-3 but not in the negative control A4 cells (scale bar = 100 µm). ICG indicates indocyanine green. DAPI, 4 ,6-diamidino-2-phenylindole.

Figure 2.

In vitro assay of radiolabeled anti-α₆β₄ antibody (ITGA6B4) in BxPC-3 cells. A, ¹¹¹In-DTPA-ITGA6B4 binding increased with BxPC-3 cell concentration, however, no binding to A4 cells was observed. Data are presented as mean ± SD (n = 3 for each group; *P < .01 vs A4 cells). B, BxPC-3 cell binding was blocked by unlabeled intact ITGA6B4 (black circles) or DTPA-ITGA6B (white cycles) in the competitive inhibition assay, in a dose-dependent manner. No difference was observed in the K_d value. Data are presented as mean ± SD (n = 6 for each group). DTPA indicates N-[(R)-2-amino-3-(p-isothiocyanato-phenyl)propyl]-trans-(S, S)-cyclohexane-1,2-diamine-N, N, N′, N″, N″-pentaacetic acid; SD, standard deviation.

Figure 3.

Biodistribution of ¹¹¹In-DTPA-ITGA6B4 in tumor-bearing mice. Radioactivity of samples was measured at 1.5 hours (white bar), 24 hours (pale gray bar), 48 hours (gray bar), 72 hours (dark gray bar), and 96 hours (black bar) after receiving intravenous injection of ¹¹¹In-DTPA-ITGA6B4. Injected dose of ¹¹¹In-DTPA-ITGA6B4 (26 kBq) was adjusted to 5 μg per mouse by the addition of unlabeled antibody. Data are presented as mean ± SD (n = 5 for each group; *P < .02 vs radioactivity of A4 tumor at each time point). DTPA indicates N-[(R)-2-amino-3-(p-isothiocyanato-phenyl)propyl]-trans-(S, S)-cyclohexane-1,2-diamine-N, N, N′, N″, N″-pentaacetic acid; SD, standard deviation

Figure 4.

A, Serial SPECT/CT images of a mouse bearing xenografted target tumor (BxPC-3, pink arrowhead) and nontarget tumor (A4, white arrowhead) obtained at 1.5, 24, 48, 72, and 96 hours after injection of ¹¹¹In-DTPA-ITGA6B4. Injected dose of ¹¹¹In-DTPA-ITGA6B4 (1.85 MBq) was adjusted to 50 μg per mouse by the addition of unlabeled antibody. B, Time courses of radioactivity accumulation in both tumors were compared. C, At 96 hours, frozen BxPC-3 tumor section showed high radioactivity on ex vivo autoradiographic image, whereas A4 tumor showed no specific radioactivity (scale bar = 1000 µm). D, α₆β₄ immunohistochemical staining of adjacent sections displayed strong α₆β₄ staining on low- and high-power magnification in BxPC-3 tumor cells but not in A4 tumor cells (scale bar = 1000 µm at low magnification, 100 µm at high magnification). DTPA indicates N-[(R)-2-amino-3-(p-isothiocyanato-phenyl)propyl]-trans-(S, S)-cyclohexane-1,2-diamine-N, N, N′, N″, N″-pentaacetic acid; SPECT, single-photon emission computed tomography; CT, computed tomography.

Figure 5.

A, Serial NIR fluorescence imaging of a representative mouse bearing xenografted target tumor (BxPC-3, pink arrowhead) and nontarget tumor (A4, white arrowhead) was obtained before and after injection of 50 μg ICG-ITGA6B4 (1.5, 24, 48, 72, and 96 hours). The upper panel shows the overlayed image of the ICG spectrum image at 820 nm (middle panel) and the white light image (lower panel). BxPC-3 tumor FI is significantly higher than that of the A4 tumor. B, Time courses of the ICG specific FI of the 2 tumors were compared. Data are presented as mean ± SD (n = 3; *P < .03 vs FI of A4 tumor at each time point). C, Ex vivo fluorescence image of tumors and tissues obtained from euthanized mice at 96 hours after injection showed that BxPC-3 tumor emitted the highest fluorescence signal. Significantly higher FI was measured for BxPC-3 tumors. Data are presented as mean ± SD (n = 3) (*P < .03 vs FI of ex vivo A4 tumor. D, Fluorescence microscopy examination of subsequently frozen tumor section confirmed the α₆β₄-specific binding of the probe in BxPC-3 tumor. Overlayed image (left panel) was obtained by fusing the ICG fluorescence image (middle panel) and DAPI nuclear staining (right panel; scale bar = 200 µm). Inj indicates injection; NIR, near-infrared; ICG, indocyanine green; SD, standard deviation; FI, fluorescence intensity; DAPI, 4′,6-diamidino-2-phenylindole.