Visualization of melanoma tumor with lectin-conjugated rare-earth doped fluoride nanocrystals

Abstract

Aim: To develop specific fluorescent markers for melanoma tumor visualization, which would provide high selectivity and reversible binding pattern, by the use of carbohydrate-recognizing proteins, lectins, combined with the physical ability for imaging deep in the living tissues by utilizing red and near infrared fluorescent properties of specific rare-earth doped nanocrystals (NC).

Methods: B10F16 melanoma cells were inoculated to C57BL/6 mice for inducing experimental melanoma tumor. Tumors were removed and analyzed by lectin-histochemistry using LABA, PFA, PNA, HPA, SNA, GNA, and NPL lectins and stained with hematoxylin and eosin. NPL lectin was conjugated to fluorescent NaGdF4:Eu(3+)-COOH nanoparticles (5 nm) via zero length cross-linking reaction, and the conjugates were purified from unbound substances and then used for further visualization of histological samples. Fluorescent microscopy was used to visualize NPL-NaGdF4:Eu(3+) with the fluorescent emission at 600-720 nm range.

Results: NPL lectin selectively recognized regions of undifferentiated melanoblasts surrounding neoangiogenic foci inside melanoma tumor, PNA lectin recognized differentiated melanoblasts, and LCA and WGA were bound to tumor stroma regions. NPL-NaGdF4:Eu(3+) conjugated NC were efficiently detecting newly formed regions of melanoma tumor, confirmed by fluorescent microscopy in visible and near infrared mode. These conjugates possessed high photostability and were compatible with convenient xylene-based mounting systems and preserved intensive fluorescent signal at samples storage for at least 6 months.

Conclusion: NPL lectin-NaGdF4:Eu(3+) conjugated NC permitted distinct identification of contours of the melanoma tissue on histological sections using red excitation at 590-610 nm and near infrared emission of 700-720 nm. These data are of potential practical significance for development of glycans-conjugated nanoparticles to be used for in vivo visualization of melanoma tumor.

Figure 1

Melanoma growth was accompanied by progression of undifferentiated melanoblasts (umb) surrounding neoangiogenic vessels (v). Subsequently umb differentiated to melanoblasts (dmb) that produce melanin (m) as well as to melanocytes (mc) that form tumor stroma. (A) an entire (intact) tumor 8 days after inoculation, a prevalence of undifferentiated cells can be clearly seen. (B,C) a differentiated melanin-rich tissue was formed (micrographs on different magnifications were taken 15 days after cell inoculation).

Figure 2

Lectin-histochemical analysis of different tumor regions. Lectins used for staining are indicated at top left. Lectin signal is in brown color. Hematoxylin and eosin staining is shown for the same tumor region as for NPL (subsequent slices were used). Objective ×10.

Figure 3

Properties of NPL- NaGdF₄:Eu³⁺ nanocrystal (NC). (A) NC fluorescence is induced by 395 nm wavelength; (B) HRTEM image of NaGdF₄:Eu³⁺ crystals revealed the diameter of 5 nm; (C) excitation and emission spectra of NaGdF₄:Eu³⁺ NC; (D) scheme of NC conjugation with protein via zero-length cross linking reaction. (E) agarose gel electrophoresis of NPL protein and its conjugates with NC after extensive dialysis. No unbound protein was detected.

Figure 4

Visualization of undifferentiating neovascularized melanoma regions with NPL-NaGdF4:Eu³⁺ nanocrystals (NC). Melanoma slice stained with hematoxylin and eosin (A) is compared with sequential slice stained with NC conjugates, using near-infrared (NIR) fluorescence detection mode (B). NC provided two fluorescence peaks and were imaged using ex. 546/12nm, em. 610/60nm (a typical red fluorescent signal) with exposure of 500 ms (C); the same samples emitting at 690/50 nm (a NIR fluorescence, imaging using Carl Zeiss filter set 50) with exposure of 1100 ms were imaged at (D); cell nuclei of the sample in (C) and (D) were counterstained with 4',6-diamidino-2-phenylindole dihydrochloride (E); (F) merged images D and E.