Quantitative Stain-free and Continuous Multimodal Monitoring of Wound Healing in vitro with Digital Holographic Microscopy

Impaired epithelial wound healing has significant pathophysiological implications in several conditions including gastrointestinal ulcers, anastomotic leakage and venous or diabetic skin ulcers. Promising drug candidates for accelerating wound closure are commonly evaluated in in vitro wound assays. However, staining procedures and discontinuous monitoring are major drawbacks hampering accurate assessment of wound assays. We therefore investigated digital holographic microscopy (DHM) to appropriately monitor wound healing in vitro and secondly, to provide multimodal quantitative information on morphological and functional cell alterations as well as on motility changes upon cytokine stimulation. Wound closure as reflected by proliferation and migration of Caco-2 cells in wound healing assays was studied and assessed in time-lapse series for 40 h in the presence of stimulating epidermal growth factor (EGF) and inhibiting mitomycin c. Therefore, digital holograms were recorded continuously every thirty minutes. Morphological changes including cell thickness, dry mass and tissue density were analyzed by data from quantitative digital holographic phase microscopy. Stimulation of Caco-2 cells with EGF or mitomycin c resulted in significant morphological changes during wound healing compared to control cells. In conclusion, DHM allows accurate, stain-free and continuous multimodal quantitative monitoring of wound healing in vitro and could be a promising new technique for assessment of wound healing

Early Dynamics of Quantitative SEPT9 and SHOX2 Methylation in Circulating Cell-Free Plasma DNA during Prostate Biopsy for Prostate Cancer Diagnosis

Background: The methylation status of Septin 9 (SEPT9) and short stature homeobox 2 (SHOX2) in circulating cell-free DNA (ccfDNA) are validated pan-cancer biomarkers. The present proof-of-concept study aimed to investigate the potential and dynamics of quantitative SEPT9 and SHOX2 methylation in prostate cancer (PCa) patient tissue and ccfDNA during prostate biopsy as a diagnostic tool. Methods: The methylation patterns of SEPT9 and SHOX2 in prostate tissue were analyzed using The Cancer Genome Atlas data set (n = 498 PCa and n = 50 normal adjacent prostate tissue (NAT)). Next, dynamic changes of ccfDNA methylation were quantified in prospectively enrolled patients undergoing prostate biopsy (n = 72), local treatment for PCa (n = 7; radical prostatectomy and radiotherapy) as well as systemic treatment for PCa (n = 6; chemotherapy and 177-Lu-PSMA-therapy). Biomarker levels were correlated with clinicopathological parameters. Results: SEPT9 and SHOX2 were hypermethylated in PCa tissue (p < 0.001) and allowed discrimination of PCa and non-tumor prostate tissue (mSEPT9: AUC 0.87, 95%CI [0.82–0.92]; mSHOX2: AUC 0.89, 95%CI 0.84–0.94). SHOX2 methylation and mRNA levels were significantly higher in PCa tissue and increased with tumor stage and grade, as well as in patients suffering from biochemical recurrence following radical prostatectomy. SEPT9 and SHOX2 ccfDNA methylation allowed distinguishing patients with localized and metastatic disease (p < 0.001 for both). In addition, methylation levels increased shortly after prostate biopsy only in patients with PCa (ΔmSEPT9: p < 0.001 and ΔmSHOX2: p = 0.001). Conclusions: The early dynamics of methylated SEPT9 and SHOX2 in ccfDNA allow differentiation between PCa patients and patients without PCa and is a promising marker for tumor monitoring in the metastatic stage to determine tumor burden under systemic therapy

Visualization of epithelial wound healing by white light microscopy and DHM.

<p>(<b>A</b>) Conventional white light microscopy is hardly able to visualize outer borders of Caco-2 cells. (<b>B</b>) Phase contrast images provided by DHM (upper row) enable recognition of cell outlines, which are depicted by segmented DHM phase contrast images (lower row).</p

Simultaneous monitoring of cellular key characteristics during epithelial wound closure illustrated by results from a single measurement.

<p>(<b>A</b>) The cell covered area <i>S</i>_c after start of the wound healing assay is markedly decreased after stimulation with mitomycin c as compared to untreated control cells, and EGF-treated cells. (<b>B</b>) Over the period of 40 h, the slope of the cellular dry mass Δ<i>DM</i> of EGF-stimulated cell in the wound gap is slightly decreased in comparison to unstimulated control cells whereas mitomycin c-treated cell only reveal a modest increase in cellular dry mass. (<b>C</b>) The average cell layer thickness as well as temporal thickness increase of mitomycin c-treated and unstimulated control was comparable while EGF-stimulated cell show a dramatically increased cell layer thickness that slightly decreased during the observation period. (<b>D</b>) The cellular volume <i>V</i> of mitomycin c-stimulated cells was constant during the 40 h observation period. In contrast, <i>V</i> of unstimulated control cells and EGF stimulated cells increased continuously and were almost doubled after 40 h. (<b>E</b>) The quotient of total dry mass in the gap and mean dry mass of single cells for each condition reveals the absolute cell number <i>n</i> in the wound. Unstimulated and EGF stimulated cells indicated a marked increase while mitomycin c treatment resulted in an almost constant cell number.</p

Alteration of cellular thickness of stimulated Caco-2 cells during wound healing.

<p>(<b>A</b>) Averaged profiles <i>S</i> through the cell layer thickness <i>d</i> of control cells and (<b>B</b>) cell layers after treatment with mitomycin c (left) and EGF (right). (<b>C,D</b>) False color-coded pseudo 3D plots of corresponding representative quantitative DHM phase contrast images.</p

Refractive index, dry mass and cellular volume of stimulated and unstimulated Caco-2 single cells in suspension.

<p>(<b>A–C</b>) Representative quantitative DHM phase images of suspended single Caco-2 cells (coded to 256 gray levels), (<b>A</b>) untreated control cells, (<b>B</b>) after treatment with either epidermal growth factor (EGF) or (<b>C</b>) mitomycin c. (<b>D–F</b>) The me an cellular radius <i>r</i> as assessed by false color coded pseudo 3D representations of quantitative phase images was slightly increased after EGF stimulation and markedly more enhanced after mitomycin c treatment. (<b>G</b>) The refractive index <i>n</i>_cell of mitomycin c-stimulated cells was significantly decreased as compared to EGF-stimulated cells and untreated control cells. (<b>H, I</b>) Dry mass <i>DM</i> and cellular volume <i>V</i> of EGF-treated Caco-2 cells were significantly increased as compared to untreated cells but were reduced as compared to mitomycin c-stimulated cells. Data are means ±SE; <i>N</i> = 89, ***<i>P</i><0.001 (the numerical data of diagrams G, H, I are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107317#pone.0107317.s001" target="_blank">Table S1</a>).</p

Time constants of key cellular characteristics during epithelial wound closure.

<p>(<b>A</b>) While the change of cell-covered area per minute of mitomycin c-stimulated cells was significantly diminished as compared to untreated control cells, no significant difference was detected between EGF- and untreated control cells. (<b>B</b>) The temporal dry mass change of mitomycin c-stimulated cells was significantly decreased as compared to EGF-stimulated and untreated control cells. (<b>C,D</b>) No significant differences in the temporal changes of cell layer thickness and cellular volume were detected between EGF or mitomycin c-stimulated cells and untreated control cells. Data are means ±SE; *, <i>P</i><0.05; (the numerical data of the diagrams A–D are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107317#pone.0107317.s002" target="_blank">Table S2</a>).</p

Utilized off-axis setup for digital holographic microscopy (DHM).

<p>A laser beam is divided by a beam splitter into an object wave, illuminating the specimen through a condenser and an undisturbed reference wave. The object wave interferes with the slightly tilted reference wave on a charge coupled device sensor (off-axis geometry). Morphological changes of the biological specimen lead to changes of the optical path length of the object wave, which are coded in the resulting interference pattern (digital hologram).</p