Electron Microscopy in Human Diseases: Diagnostic and Research Perspectives

Keywords::

• asbestos
• breast cancer
• electron microscopy
• heavy metal
• nanomedicine
• oncology
• personalized medicine

In the era of personalized medicine, the correct management of oncological patients requires a multidisciplinary approach able to provide information about morphological, molecular and genetic characteristics of neoplastic lesions [1]. In this context, new techniques associated with electron microscopy (EM) such as cryo-EM, energy-dispersive x-ray (EDX) microanalysis and immunogold labeling can expand the diagnostic equipment available to oncologists. EM is a technique that uses an electron beam for the observation of samples with both magnifications and resolution up to 1000-times higher than optical microscopy [2]. The advantage of combining high magnification and, at the same time, high resolution, wide amplitude of the focus and a simple sample preparation makes EM the preferred technique to: obtain information on the morphology and the chemical–physical composition of the sample; analyze the bioaccumulation of contaminants in the tissues, and evaluate the protein expression at subcellular level [2]. Due to its features, EM lends itself to a wide window of application both in research and in diagnostics.

The recent spectacular advance in resolution of a single particle cryo-EM is mainly due to the development of direct electron detectors [3]. Indeed, now that high-resolution 3D reconstructions of purified macromolecular complexes can be obtained, we can envisage the recovery of high-resolution information from structures inside the cell by cryo-EM tomography [4]. In the oncological field, the identification of protein structure and protein interaction by cryo-EM is emerging with new and surprising possibilities for comprehension of the molecular mechanisms involved in cancer development. In a very recent study, Chen et al. elucidated the structure of the target of rapamycin (mTOR) complex 2 (mTORC2), a protein complex involved in many human tumors [5]. Aberrant mTORC2 signaling has been shown to be involved in cell proliferation and mTORC2 detection may serve as a potential anticancer drug target [6]. Thus, higher resolution cryo-EM results will be used to identify potential new anticancer targets, providing key information for development of specific drugs. Another important transmission EM ancillary technique that can implement the diagnostic approach in the oncological field is the EDX microanalysis. It is a technique of elemental analysis based on the generation of characteristic x-ray that reveals the presence of elements in tissues [7]. The spectrum of EDX microanalysis contains both semiqualitative and semiquantitative information and it is used in the study of environmental pollution and in the characterization of mineral bioaccumulation [7–9]. In agreement with this, EDX microanalysis approach allowed the identification of heavy metals, or more general toxic elements, that accumulated in the human tissue. The EDX technology, as reported in a recent investigation, is particularly efficient in the study of lung tissue, where it was possible to find heavy metals, such as cobalt (Co), chromium (Cr), manganese (Mn) and lead (Pb) [7]. The use of ultrastructural microanalysis demonstrated a putative correlation between the bioaccumulation of Co, Cr and Pb and the occurrence of lung cancer [7]. Also, the transmission EM associated to EDX microanalysis has been used for the study of elemental composition of breast microcalcifications [10,11]. In an original paper published in 2014, EDX was fundamental in demonstrating, for the first time, the presence of three different types of breast calcifications: calcium oxalate, hydroxyapatite and magnesium-substituted hydroxyapatite [10]. It is important to note that the discrimination of the breast calcifications can provide important prognostic information for the oncologists. In fact, we know that calcium oxalate is generally present in the benign breast lesions, whereas it is possible to detect magnesium-substituted hydroxyapatite only in the cancerous breast tissue [11].

A very important application of the EM concerns the study of the accumulation of nanoparticles (NPs) in human tissues. They are microscopic particles with at least one dimension less than 100 nm [12]. Nowadays, NP research is an area of intense investigation, due to a wide variety of potential applications in biomedical, optical and electronic fields [12]. Specifically, several studies characterized the cytotoxic properties of specific NPs also demonstrating their role in the pathophysiogenesis of several human diseases such as mesothelioma [13]. In this scenario, EDX microanalysis can be considered an extraordinary tool for detection of nanoasbestos fiber in lung cancer. Our group performed EDX microanalysis studies to associate asbestos nanofibers bioaccumulation and lung cancer development [14]. In particular, we applied transmission EM–EDX microanalysis to show the presence of asbestos nanofibers in histological lung specimens (paraffin blocks) of patients with possible occupational exposure to asbestos [14]. Thus, we showed the frequent bioaccumulation of specific types of nanoasbestos fibers in lung cancer patients. Recently, interest for the potential toxicological impact of consumer-relevant engineered NPs on the population has dramatically increased. In a study by Campagnolo et al., authors investigated whether inhaled silver nanoparticles (AgNPs) were able to both reach and cross the mouse placental barrier, inducing adverse effects [15]. The presence of AgNPs was identified and quantitated in maternal tissues, placentas and fetuses by transmission EM coupled with EDX-ray spectroscopy and single particle inductively coupled plasma mass spectrometry. Through using this technology, authors demonstrated that inhalation of AgNPs results in an increased number of resorbed fetuses associated with reduced estrogen plasma levels, in the 4  h/day exposed mothers. Increased expression of pregnancy-relevant inflammatory cytokines is also detected in the placentas of exposed mice. These results suggest that NPs are able to reach and cross the mouse placenta and suggest that precaution should be taken with respect to acute exposure to NPs during pregnancy [15].

Finally, a very promising EM technique is immunogold labeling. It combines the localization of a defined protein with fine structural details of the cell or tissue. Application of gold labeling has been demonstrated by pre-embedment and post-embedment protocols and applied both for transmission and scanning EM [16]. The ultrastructural localization of proteins in human cancer has been successfully applied for the study of epithelial-to-mesenchymal transition phenomenon. Specifically, vimentin detection by immunogold has made it possible to describe the ultrastructural characteristics of breast cancer cells which underwent epithelial to mesenchymal transition [17]. Also, in a recent study, Kijanka et al. developed a novel immunogold labeling protocol for nanobody-based detection of HER2 in breast cancer cells [16]. The application of immunogold labeling in the study of HER2 expression can shed new light on the mechanisms of anti-HER2 therapies. Indeed, nonresponder patients could be characterized by high rate of submembrane HER2 expression.

The dissertation here reported highlights the essential role of EM and its ancillary techniques in the field of nanomedicine, an interdisciplinary field, where nanoscience, nanoengineering and nanotechnology interact with the life sciences [18]. EM can provide an essential contribution for the development of new nanomedical approaches for the management of patients. In this context, the ultrastructural investigations NPs can be considered the most promising application in the field of nanomedicine. Indeed, NPs can be used as delivery vehicles for pharmaceutical agents, as bioactive materials or as important components in implants [12,13]. In conclusion, the desirable dissemination of EM technologies in clinical facilities, and their use for the development of new medical approaches based on nanomedicine, can ‘nourish the dream’ of a personalized medicine that takes into account the genetic and molecular variability of each human individual.

Author contributions

All authors designed the study and drafted the manuscript. Also, all authors have read and approved the final manuscript.

Financial & competing interest disclosure

The study is original and the manuscript has not been published yet and is not being considered for publication elsewhere in any language either integrally or partially except as an abstract. All authors have agreed with the submission in its present (and subsequent) forms. The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

Additional information

Funding

The study is original and the manuscript has not been published yet and is not being considered for publication elsewhere in any language either integrally or partially except as an abstract. All authors have agreed with the submission in its present (and subsequent) forms. The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.