Topology-Based Prediction of Pathway Dysregulation Induced by Intense Terahertz Pulses in Human Skin Tissue Models
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The strong interaction between terahertz (THz) radiation and biological systems has motivated the development of several biomedical technologies, including imaging and spectroscopy applications with promising potential for improved disease diagnosis. This interaction mechanism also implies that external excitation with intense pulses of THz energy could couple to important biological structures and induce significant downstream phenotypic effects. In this study, we expose human skin tissue models to a prolonged train of high-intensity THz pulses and measure the resulting global differential gene expression. From these data, signal pathway perturbation analysis identified pathways that are predicted to be significantly dysregulated, including the cytokine-cytokine receptor interaction and glioma pathways, and further identified the gene-level mechanisms predominantly responsible. These results indicate that induction of an inflammatory-like response and suppression of division/differentiation in cancer are possible. These effects could be further explored and characterized in different types of normal and cancerous tissues to determine potential novel clinical applicability of intense THz pulses.
KeywordsTerahertz Intense terahertz pulses Global gene expression Skin Bioinformatics Pathway dysregulation Inflammatory response Glioma Cancer
Terahertz (THz) frequencies strongly couple to natural low-frequency oscillations of many important biomolecules, and the stretching/twisting modes of hydrogen bond networks that are ubiquitous in biological systems (e.g., water, proteins, and DNA) . This interaction mechanism makes THz a highly sensitive probe of molecular structure in biological systems, which has led to the development of diagnostic imaging/spectroscopy technologies with excellent contrast between healthy and diseased tissue resulting in high sensitivity/specificity in diagnosing several human cancers [2, 3, 4, 5, 6, 7]. However, several studies have also shown that THz interactions can induce significant biological changes [8, 9]. Echchgadda et al. have investigated thermal effects induced in vitro for varying CW THz frequencies and observed frequency-dependent genomic expression responses nearly entirely distinct when compared to equivalent uniform bulk heating in human keratinocytes [10, 11]. Studies investigating non-thermal effects have found that THz pulses alter gene expression at the transcript and protein level in human skin tissue models , increase membrane permeation , affect cellular differentiation in mouse stem cells , or induce severe forms of genotoxic stress at the DNA  or chromosome  level in skin cells. These effects may indicate the potential for currently unknown health risks, for which safe exposure levels should be determined, or lead to phenotypic effects that could be exploited for novel diagnostic/therapeutic clinical application.
In this paper, we outline the technical details and results of an investigation of the dysregulation in gene networks and signaling pathways in human skin models induced by prolonged exposure to a train of intense THz pulses. Illumina Whole Genome BeadChip arrays were used to analyze global gene expression changes at the transcript (mRNA) level in THz-exposed samples. Since mRNA is a precursor to a functional protein, biological effects are predicted based on the known interaction characteristics of the corresponding proteins. Bioinformatic analyses allow for prediction of the possible phenotypic endpoint by calculation of the magnitude and direction (i.e., activation vs. inhibition) of the expected perturbation in known gene interaction networks. The pathways corresponding to the greatest amount of predicted dysregulation are identified and discussed.
2 Experimental Methods
2.1 Generation and Detection of Intense THz Pulses
Several methods of generating high-intensity THz pulses are available with varying capabilities depending on the experimental design : plasma sources are capable of producing intense broadband pulses at high THz frequencies (< 30 THz), which allow for shorter, more tightly focused pulses and greater coupling efficiency to faster biological dynamics. However, the low spectral density results in weak coupling to a wider range of modes, and the small spot size exposes a proportionately reduced cell population. Organic crystals (e.g., DAST) are capable of achieving very large THz fields at intermediate frequencies (~ 1–10 THz), but the limited repetition rate (< 10–100 Hz) requires extended exposure times for equivalent effect.
2.2 Sample and Exposure Details
3D human skin models were obtained from the MatTek corporation (EpidermFT, www.mattek.com). These are full-thickness 3D artificial models of human skin, derived from co-cultures of human-derived epidermal keratinocytes and dermal fibroblasts in a collagen matrix. The stratified growth and the metabolic/mitotic properties closely model the biological activity of human skin in vivo . Tissues were stored and handled according to the manufacturer’s instructions. Briefly, upon delivery, samples were transferred to wells containing fresh warm media such that the top surface was exposed to air, and the bottom surface absorbed media through a thin porous membrane. All wells were placed in an incubator overnight to equilibrate. For exposures, a sample holder was centered over the THz focus by maximizing energy/field through an attached pinhole iris, and samples were positioned such that the THz beam focused to the epidermal layer. Each sample was individually exposed for 10 min (44.4 J/cm2 total accumulated energy density). Following THz exposure, samples were returned to wells with fresh media, incubated (37 °C, 5% CO2) for 30 min, and fixed by snap-freezing in liquid nitrogen. Sham-exposed controls went through the exact same process, but with the THz beam fully blocked. Each set of exposure parameters was repeated in quadruplicate.
2.3 Measurement of Global Differential Gene Expression
The central exposed region (1.5-mm diameter) of the frozen tissue samples was excised with a sterile punch tool aligned using the same pinhole iris used for exposure alignment, and total RNA was isolated as per the manufacturers’ instructions (Zymo Research, Irvine, CA). Following elution in DNase/RNase-free ultrapure water, the quality was quantified with UV spectroscopy (NanoDrop, Wilmington, DE) and assessed for RNA integrity (Agilent 2100 Bioanalyzer, Santa Clara, CA). cDNA was amplified (Ovation FFPE WTA), labeled (Encore BiotinIL), and hybridized to the HumanHT-12_v4 Whole Genome Expression BeadChip arrays according to the manufacturer’s instructions (Illumina, San Diego, CA). BeadChips were imaged, the fluorescence signal for each probe was quantified using the iScan platform (Illumina), and data were normalized/analyzed using the Illumina BeadStudio software. Differential expression (fold-change) was quantified, and corresponding p values were corrected for multiple hypothesis testing via the false discovery rate (FDR) method. Data were corrected for batch effects via the ComBat algorithm (http://www.bu.edu/jlab/wp-assets/ComBat/Abstract.html).
2.4 Topology-Based Signal Pathway Perturbation Analysis
Corresponding p values are determined via a bootstrapping simulation as described in  and adjusted for multiple hypothesis testing with the FDR method. Significantly dysregulated pathways for which Atot is positive are predicted to be “activated,” and pathways for which Atot is negative are predicted to be “suppressed.” This procedure allows for prediction of the relative magnitudes by which pathways are affected, the genes most sensitive to THz exposure, and the genes that are predominantly responsible for driving the predicted pathway dysregulation.
3.1 Global Differential Gene Expression Induced by Intense THz Pulses
These data were used in combination with information from the KEGG database of pathway network information to calculate the predicted perturbative effects in 184 distinct gene interaction networks as described in Section 2.
3.2 Pathway Perturbation Analysis
The pathways predicted to be most significantly dysregulated by intense THz pulses are (1) cytokine-cytokine receptor interaction pathway and (2) glioma pathway.
The total accumulated perturbation calculated for the cytokine-cytokine receptor interaction pathway was Atot = + 23.3 (p = 0.004) and so is predicted to be activated by exposure to intense THz pulses. Cytokines are secreted by cells in response to some stimuli, usually as an innate or adaptive inflammatory defense . The interaction with cytokine receptors activates cellular processes involved in inflammatory responses to external stimuli.
The total accumulated perturbation calculated for the glioma pathway was Atot = − 36.8 (p = 0.004) and is therefore predicted to be suppressed by exposure to intense THz pulses. This pathway describes the gene-level mechanisms involved in initiation, progression, and metastatic activity of glioma, a type of brain cancer affecting glial cells . As will be discussed, although the glioma pathway describes molecular signaling in neural tissue cell types, the genes that are affected are ubiquitous in a wide variety of eukaryotic cell/tissue types, including skin.
- chemokines (CXCL family, CCL family)
Released in several cell types undergoing inflammatory stimuli
Involved in mediating inflammatory responses by guiding neutrophils to target site
- interleukins (IL6)
Wide variety of biological functions
Primarily produced at acute inflammation sites and induces inflammatory response (required for generation of helper T cells)
- interferons (IL24)
Over-expression correlates with apoptosis induction
Anti-proliferative properties in melanoma cells
The upregulation of these genes and predicted activation of the corresponding pathway indicate that prolonged exposure to intense THz pulses may induce an inflammatory-like response in human skin.
- calmodulin family (CALM/CALML, CAMK subfamilies)
Encode for calcium-binding messenger proteins expressed in eukaryotic cells
Active in signal transduction pathways regulating a diverse set of biological functions, including regulating cell growth/proliferation in the glioma pathway
- KRAS and ERK
Encode for proteins in the canonical mitogen-activated protein kinase (MAPK) pathway, which regulates proliferation/differentiation and is one of the most commonly implicated pathways in human cancer .
These results report gene-/pathway-level information that regulate the effects observed in reference , in which a downregulation of genes was predicted to largely suppress epidermal differentiation. It is recognized that glioma (and the initiation/progression regulated by the glioma pathway) is a cancer affecting cell types corresponding to neural tissue, whereas these predictions are based on observations in skin tissue models. However, the gene-level mechanisms responsible for the negative perturbation are genes that encode for proteins involved in calcium and MAPK signaling, and these are ubiquitous and well-conserved across many different cell types including skin and neural cells [32, 33]. The downregulation of these genes and predicted suppression of the glioma pathway indicate that THz exposures may play a role in suppression of division, differentiation, and progression of this cancer if this effect is found to occur in the relevant cell type.
To pursue these results, future studies should focus on an investigation of these potential effects in glial cells (or other neural tissue cells), for both normal and cancerous phenotypes, which may elucidate presently unknown clinical applications of intense THz pulses. Furthermore, these results were obtained at a relatively early time-point (30-min following exposure), and so a characterization of later time-points (several hours to days following exposure) probing prolonged effects on division and differentiation would be of significant interest.
In this paper, we show that high-intensity pulses of THz radiation induce a large genomic expression response in human skin tissue models, and this effect tends to logically scale when compared to THz intensities utilized in similar studies (Table 1). Furthermore, pathway perturbation analysis using recently developed bioinformatics analysis tools allows for the phenotypic endpoint to be predicted from the profile of differentially expressed genes [23, 24, 25]. The pathways corresponding to the largest significant dysregulation were identified in order to predict the dominant effect of prolonged exposure to intense THz pulses. In the considered pathways, the largest positive perturbation (i.e., activation) was observed in the cytokine-cytokine receptor interaction pathway, indicating that an acute inflammatory response may be induced by the strong external stimulus of the very high peak electric field. The pathway corresponding to the largest significant negative perturbation (i.e., inhibition) was the glioma pathway, indicating a potential suppression of growth, division, and differentiation in molecular signaling pathways that drive glioma progression. The genes measured to be predominantly responsible for these effects were identified, and the observations are consistent with results of studies utilizing similar experimental designs [12, 29]. A relevant pursuit of these findings involves investigation of the effect of intense THz pulses on division/differentiation dynamics in neural cell types. If suppression of growth is observed in these (or other) systems, especially in cancerous tissues, intense THz pulses may potentially find novel clinical application with the goal of targeted inhibition of mitotic activity in cancerous cells.
We acknowledge support from NSERC, CFI, and the AITF Strategic Chairs Program, and technical assistance from Beipei Shi, Greg Popowich, Matt Reid, Rocio Rodriguez-Juarez, Rommy Rodriguez-Juarez, Andrey Golubov, and Yaroslav Ilnytskyy.
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