Nontoxic Fluorescent Nanoprobes for Multiplexed Detection and 3D Imaging of Tumor Markers in Breast Cancer

Abstract

Multiplexed fluorescent immunohistochemical analysis of breast cancer (BC) markers and high-resolution 3D immunofluorescence imaging of the tumor and its microenvironment not only facilitate making the disease prognosis and selecting effective anticancer therapy (including photodynamic therapy), but also provides information on signaling and metabolic mechanisms of carcinogenesis and helps in the search for new therapeutic targets and drugs. The characteristics of imaging nanoprobe efficiency, such as sensitivity, target affinity, depth of tissue penetration, and photostability, are determined by the properties of their components, fluorophores and capture molecules, and by the method of their conjugation. Regarding individual nanoprobe components, fluorescent nanocrystals (NCs) are widely used for optical imaging in vitro and in vivo, and single-domain antibodies (sdAbs) are well established as highly specific capture molecules in diagnostic and therapeutic applications. Moreover, the technologies of obtaining functionally active sdAb–NC conjugates with the highest possible avidity, with all sdAb molecules bound to the NC in a strictly oriented manner, provide 3D-imaging nanoprobes with strong comparative advantages. This review is aimed at highlighting the importance of an integrated approach to BC diagnosis, including the detection of biomarkers of the tumor and its microenvironment, as well as the need for their quantitative profiling and imaging of their mutual location, using advanced approaches to 3D detection in thick tissue sections. The existing approaches to 3D imaging of tumors and their microenvironment using fluorescent NCs are described, and the main comparative advantages and disadvantages of nontoxic fluorescent sdAb–NC conjugates as nanoprobes for multiplexed detection and 3D imaging of BC markers are discussed.

Invasive papillary carcinoma of the breast

“…It is more sensitive and specific for the myoepithelium, which is not expressed by vascular endothelium or myofibroblasts. Both CK14 and CK5 demonstrate the presence of myoepithelial cells and show varying levels of positivity in the hyperplastic epithelium of benign papillary neoplasms ( 72 ). However, staining is reduced in intraductal papillary carcinomas.…”

Shark IgNAR: The Next Broad Application Antibody in Clinical Diagnoses and Tumor Therapies?

“…Antibody fragments with a smaller molecular weight, which have been shown to have a more uniform distribution than mAbs in tissues, can improve diffusivity and vascular permeability [ 5 ]. In diagnostic and therapeutic applications, smaller and more flexible therapeutic antibody formats with exciting therapeutic potential have been considered and developed for optical imaging both in vitro and in vivo [ 6 ], viruses [ 7 , 8 ], tumors [ 9 ], and other human disease therapies [ 10 ].…”

Estimating the point-spread-function using 1 µm diameter microspheres for image restoration in biomedical multiphoton microscopy

“…The current strategy for the estimation of PSF consists in the creation of a point source produced by fluorescent beads with dimensions smaller than the resolution and which can emit light at a single point. Usually, microspheres with sub-resolution diameter of less than 200 nm are often chosen [5][6][7]. However, their detection is not really easy.…”