Quantitative Phase Imaging as Sensitive Screening Method for Nanoparticle-Induced Cytotoxicity Assessment.

The assessment of nanoparticle cytotoxicity is challenging due to the lack of customized and standardized guidelines for nanoparticle testing. Nanoparticles, with their unique properties, can interfere with biochemical test methods, so multiple tests are required to fully assess their cellular effects. For a more reliable and comprehensive assessment, it is therefore imperative to include methods in nanoparticle testing routines that are not affected by particles and allow for the efficient integration of additional molecular techniques into the workflow. Digital holographic microscopy (DHM), an interferometric variant of quantitative phase imaging (QPI), has been demonstrated as a promising method for the label-free assessment of the cytotoxic potential of nanoparticles. Due to minimal interactions with the sample, DHM allows for further downstream analyses. In this study, we investigated the capabilities of DHM in a multimodal approach to assess cytotoxicity by directly comparing DHM-detected effects on the same cell population with two downstream biochemical assays. Therefore, the dry mass increase in RAW 264.7 macrophages and NIH-3T3 fibroblast populations measured by quantitative DHM phase contrast after incubation with poly(alkyl cyanoacrylate) nanoparticles for 24 h was compared to the cytotoxic control digitonin, and cell culture medium control. Viability was then determined using a metabolic activity assay (WST-8). Moreover, to determine cell death, supernatants were analyzed for the release of the enzyme lactate dehydrogenase (LDH assay). In a comparative analysis, in which the average half-maximal effective concentration (EC50) of the nanocarriers on the cells was determined, DHM was more sensitive to the effect of the nanoparticles on the used cell lines compared to the biochemical assays.

Durable 3D murine ex vivo retina glaucoma models for optical coherence tomography.

Durable and standardized phantoms with optical properties similar to native healthy and disease-like biological tissues are essential tools for the development, performance testing, calibration and comparison of label-free high-resolution optical coherence tomography (HR-OCT) systems. Available phantoms are based on artificial materials and reflect thus only partially ocular properties. To address this limitation, we have performed investigations on the establishment of durable tissue phantoms from ex vivo mouse retina for enhanced reproduction of in vivo structure and complexity. In a proof-of-concept study, we explored the establishment of durable 3D models from dissected mouse eyes that reproduce the properties of normal retina structures and tissue with glaucoma-like layer thickness alterations. We explored different sectioning and preparation procedures for embedding normal and N-methyl-D-aspartate (NMDA)-treated mouse retina in transparent gel matrices and epoxy resins, to generate durable three-dimensional tissue models. Sample quality and reproducibility were quantified by thickness determination of the generated layered structures utilizing computer-assisted segmentation of OCT B-scans that were acquired with a commercial HR-OCT system at a central wavelength of 905 nm and analyzed with custom build software. Our results show that the generated 3D models feature thin biological layers close to current OCT resolution limits and glaucoma-like tissue alterations that are suitable for reliable HR-OCT performance characterization. The comparison of data from resin-embedded tissue with native murine retina in gels demonstrates that by utilization of appropriate preparation protocols, highly stable samples with layered structures equivalent to native tissues can be fabricated. The experimental data demonstrate our concept as a promising approach toward the fabrication of durable biological 3D models suitable for high-resolution OCT system performance characterization supporting the development of optimized instruments for ophthalmology applications.

Dysregulated Stem Cell Markers Musashi-1 and Musashi-2 are Associated with Therapy Resistance in Inflammatory Breast Cancer.

BACKGROUND AND AIM: While preliminary evidence points to pro-tumorigenic roles for the Musashi (MSI) RNA-binding proteins Musashi-1 (MSI1) and Musashi-2 (MSI2) in some breast cancer subtypes, no data exist for inflammatory breast cancer (IBC). METHODS: MSI gene expression was quantified in IBC SUM149PT cells. We then used small interfering RNA-based MSI1 and MSI2 double knockdown (DKD) to understand gene expression and functional changes upon MSI depletion. We characterized cancer stem cell characteristics, cell apoptosis and cell cycle progression via flow cytometry, mammospheres via spheroid assays, migration and proliferation via digital holographic microscopy, and cell viability using BrdU assays. Chemoresistance was determined for paclitaxel and cisplatin with MTT assays and radioresistance was assessed with clonogenic analyses. In parallel, we supported our in vitro data by analyzing publicly available patient IBC gene expression datasets. RESULTS: MSI1 and MSI2 are upregulated in breast cancer generally and IBC specifically. MSI2 is more commonly expressed compared to MSI1. MSI DKD attenuated proliferation, cell cycle progression, migration, and cell viability while increasing apoptosis. Stem cell characteristics CD44(+)/CD24(-), TERT and Oct4 were associated with MSI expression in vivo and were decreased in vitro after MSI DKD as was ALDH expression and mammosphere formation. In vivo, chemoresistant tumors were characterized by MSI upregulation upon chemotherapy application. In vitro, MSI DKD was able to alleviate chemo- and radioresistance. CONCLUSIONS: The Musashi RNA binding proteins are dysregulated in IBC and associated with tumor proliferation, cancer stem cell phenotype, chemo- and radioresistance. MSI downregulation alleviates therapy resistance and attenuates tumor proliferation in vitro.

Single capture bright field and off-axis digital holographic microscopy: publisher's note.

This publisher's note contains corrections to Opt. Lett.48, 876 (2023)10.1364/OL.478674.

Quantitative Phase Imaging Using Digital Holographic Microscopy to Assess the Degree of Intestinal Inflammation in Patients with Ulcerative Colitis.

Ulcerative colitis (UC) is characterized by chronic inflammation of the colorectum. Histological remission has emerged as a potential future treatment goal; however, the histopathological assessment of intestinal inflammation in UC remains challenging with a multitude of available scoring systems and the need for a pathologist with expertise in inflammatory bowel disease (IBD). In previous studies, quantitative phase imaging (QPI) including digital holographic microscopy (DHM) was successfully applied as an objective method for stain-free quantification of the degree of inflammation in tissue sections. Here, we evaluated the application of DHM for the quantitative assessment of histopathological inflammation in patients with UC. In our study, endoscopically obtained colonic and rectal mucosal biopsy samples from 21 patients with UC were analyzed by capturing DHM-based QPI images that were subsequently evaluated using the subepithelial refractive index (RI). The retrieved RI data were correlated with established histological scoring systems including the Nancy index (NI) as well as with endoscopic and clinical findings. As a primary endpoint, we found a significant correlation between the DHM-based retrieved RI and the NI (R2 = 0.251, p < 0.001). Furthermore, RI values correlated with the Mayo endoscopic subscore (MES; R2 = 0.176, p < 0.001). An area under the receiver operating characteristics (ROC) curve of 0.820 confirms the subepithelial RI as a reliable parameter to distinguish biopsies with histologically active UC from biopsies without evidence of active disease as determined by conventional histopathological examination. An RI higher than 1.3488 was found to be the most sensitive and specific cut-off value to identify histologically active UC (sensitivity of 84% and specificity of 72%). In conclusion, our data demonstrate DHM to be a reliable tool for the quantitative assessment of mucosal inflammation in patients with UC.

Tumor suppressor miR-218 directly targets epidermal growth factor receptor (EGFR) expression in triple-negative breast cancer, sensitizing cells to irradiation.

PURPOSE: MicroRNA-218 (miR-218) is a key regulator of numerous processes relevant to tumor progression. In the present study, we aimed to characterize the relationship between miR-218 and the Epidermal Growth Factor Receptor (EGFR) as well as to understand downstream effects in triple-negative breast cancer (TNBC). METHODS: We assessed miR-218 and EGFR expression in cell lines and publicly available primary breast cancer gene expression data. We then overexpressed miR-218 in two TNBC cell lines and investigated effects on EGFR and downstream mitogen-activated protein (MAP) kinase signaling. Luciferase reporter assay was used to characterize a direct binding interaction between miR-218 and EGFR mRNA. Digital holographic microscopy helped investigate cell migration and dry mass after miR-218 overexpression. Cell division and invasion were assessed microscopically, while radiation response after miR-218 overexpression alone or combined with additional EGFR knockdown was investigated via clonogenic assays. RESULTS: We found an inverse correlation between EGFR expression and miR-218 levels in cell lines and primary breast cancer tissues. MiR-218 overexpression resulted in a downregulation of EGFR via direct binding of the mRNA. Activation of EGFR and downstream p44/42 MAPK signaling were reduced after pre-miR-218 transfection. Cell proliferation, motility and invasiveness were inhibited whereas cell death and mitotic catastrophe were upregulated in miR-218 overexpressing cells compared to controls. MiR-218 overexpressing and EGFR siRNA-treated cells were sensitized to irradiation, more than miR-218 overexpressing cells alone. CONCLUSION: This study characterizes the antagonistic relationship between miR-218 and EGFR. It also demonstrates downstream functional effects of miR-218 overexpression, leading to anti-tumorigenic cellular changes.

Application of Digital Holographic Microscopy to Analyze Changes in T-Cell Morphology in Response to Bacterial Challenge.

Quantitative phase imaging (QPI) is a non-invasive, label-free technique used to detect aberrant cell morphologies caused by disease, thus providing a useful diagnostic approach. Here, we evaluated the potential of QPI to differentiate specific morphological changes in human primary T-cells exposed to various bacterial species and strains. Cells were challenged with sterile bacterial determinants, i.e., membrane vesicles or culture supernatants, derived from different Gram-positive and Gram-negative bacteria. Timelapse QPI by digital holographic microscopy (DHM) was applied to capture changes in T-cell morphology over time. After numerical reconstruction and image segmentation, we calculated single cell area, circularity and mean phase contrast. Upon bacterial challenge, T-cells underwent rapid morphological changes such as cell shrinkage, alterations of mean phase contrast and loss of cell integrity. Time course and intensity of this response varied between both different species and strains. The strongest effect was observed for treatment with S. aureus-derived culture supernatants that led to complete lysis of the cells. Furthermore, cell shrinkage and loss of circular shape was stronger in Gram-negative than in Gram-positive bacteria. Additionally, T-cell response to bacterial virulence factors was concentration-dependent, as decreases in cellular area and circularity were enhanced with increasing concentrations of bacterial determinants. Our findings clearly indicate that T-cell response to bacterial stress depends on the causative pathogen, and specific morphological alterations can be detected using DHM.

Single capture bright field and off-axis digital holographic microscopy.

We report on a single capture approach for simultaneous incoherent bright field (BF) and laser-based quantitative phase imaging (QPI). Common-path digital holographic microscopy (DHM) is implemented in parallel with BF imaging within the optical path of a commercial optical microscope to achieve spatially multiplexed recording of white light images and digital off-axis holograms, which are subsequently numerically demultiplexed. The performance of the proposed multimodal concept is firstly determined by investigations on microspheres. Then, the application for label-free dual-mode QPI and BF imaging of living pancreatic tumor cells is demonstrated.

Interlaboratory evaluation of a digital holographic microscopy-based assay for label-free in vitro cytotoxicity testing of polymeric nanocarriers.

State-of-the-art in vitro test systems for nanomaterial toxicity assessment are based on dyes and several staining steps which can be affected by nanomaterial interference. Digital holographic microscopy (DHM), an interferometry-based variant of quantitative phase imaging (QPI), facilitates reliable proliferation quantification of native cell populations and the extraction of morphological features in a fast and label- and interference-free manner by biophysical parameters. DHM therefore has been identified as versatile tool for cytotoxicity testing in biomedical nanotechnology. In a comparative study performed at two collaborating laboratories, we investigated the interlaboratory variability and performance of DHM in nanomaterial toxicity testing, utilizing complementary standard operating procedures (SOPs). Two identical custom-built off-axis DHM systems, developed for usage in biomedical laboratories, equipped with stage-top incubation chambers were applied at different locations in Europe. Temporal dry mass development, 12-h dry mass increments and morphology changes of A549 human lung epithelial cell populations upon incubation with two variants of poly(alkyl cyanoacrylate) (PACA) nanoparticles were observed in comparison to digitonin and cell culture medium controls. Digitonin as cytotoxicity control, as well as empty and cabazitaxel-loaded PACA nanocarriers, similarly impacted 12-h dry mass development and increments as well as morphology of A549 cells at both participating laboratories. The obtained DHM data reflected the cytotoxic potential of the tested nanomaterials and are in agreement with corresponding literature on biophysical and chemical assays. Our results confirm DHM as label-free cytotoxicity assay for polymeric nanocarriers as well as the repeatability and reproducibility of the technology. In summary, the evaluated DHM assay could be efficiently implemented at different locations and facilitates interlaboratory in vitro toxicity testing of nanoparticles with prospects for application in regulatory science.

A JAM-A-tetraspanin-αvß5 integrin complex regulates contact inhibition of locomotion.

Contact inhibition of locomotion (CIL) is a process that regulates cell motility upon collision with other cells. Improper regulation of CIL has been implicated in cancer cell dissemination. Here, we identify the cell adhesion molecule JAM-A as a central regulator of CIL in tumor cells. JAM-A is part of a multimolecular signaling complex in which tetraspanins CD9 and CD81 link JAM-A to αvß5 integrin. JAM-A binds Csk and inhibits the activity of αvß5 integrin-associated Src. Loss of JAM-A results in increased activities of downstream effectors of Src, including Erk1/2, Abi1, and paxillin, as well as increased activity of Rac1 at cell-cell contact sites. As a consequence, JAM-A-depleted cells show increased motility, have a higher cell-matrix turnover, and fail to halt migration when colliding with other cells. We also find that proper regulation of CIL depends on αvß5 integrin engagement. Our findings identify a molecular mechanism that regulates CIL in tumor cells and have implications on tumor cell dissemination.

Investigating Morphological Changes of T-lymphocytes after Exposure with Bacterial Determinants for Early Detection of Septic Conditions.

Sepsis is a leading cause of morbidity and mortality, annually affecting millions of people worldwide. Immediate treatment initiation is crucial to improve the outcome but despite great progress, early identification of septic patients remains a challenge. Recently, white blood cell morphology was proposed as a new biomarker for sepsis diagnosis. In this proof-of-concept study, we aimed to investigate the effect of different bacteria and their determinants on T-lymphocytes by digital holographic microscopy (DHM). We hypothesize that species- and strain-specific morphological changes occur, which may offer a new approach for early sepsis diagnosis and identification of the causative agent. Jurkat cells as a model system were exposed to different S. aureus or E. coli strains either using sterile determinants or living bacteria. Time-lapse DHM was applied to analyze cellular morphological changes. There were not only living bacteria but also membrane vesicles and sterile culture supernatant-induced changes of cell area, circularity, and mean phase contrast. Interestingly, different cellular responses occurred depending on both the species and strain of the causative bacteria. Our findings suggest that investigation of T-lymphocyte morphology might provide a promising tool for the early identification of bacterial infections and possibly discrimination between different causative agents. Distinguishing gram-positive from gram-negative infection would already offer a great benefit for the proper administration of antibiotics.

Label-Free Digital Holographic Microscopy for In Vitro Cytotoxic Effect Quantification of Organic Nanoparticles.

Cytotoxicity quantification of nanoparticles is commonly performed by biochemical assays to evaluate their biocompatibility and safety. We explored quantitative phase imaging (QPI) with digital holographic microscopy (DHM) as a time-resolved in vitro assay to quantify effects caused by three different types of organic nanoparticles in development for medical use. Label-free proliferation quantification of native cell populations facilitates cytotoxicity testing in biomedical nanotechnology. Therefore, DHM quantitative phase images from measurements on nanomaterial and control agent incubated cells were acquired over 24 h, from which the temporal course of the cellular dry mass was calculated within the observed field of view. The impact of LipImage™ 815 lipidots® nanoparticles, as well as empty and cabazitaxel-loaded poly(alkyl cyanoacrylate) nanoparticles on the dry mass development of four different cell lines (RAW 264.7, NIH-3T3, NRK-52E, and RLE-6TN), was observed vs. digitonin as cytotoxicity control and cells in culture medium. The acquired QPI data were compared to a colorimetric cell viability assay (WST-8) to explore the use of the DHM assay with standard biochemical analysis methods downstream. Our results show that QPI with DHM is highly suitable to identify harmful or low-toxic nanomaterials. The presented DHM assay can be implemented with commercial microscopes. The capability for imaging of native cells and the compatibility with common 96-well plates allows high-throughput systems and future embedding into existing experimental routines for in vitro cytotoxicity assessment.

Digital Holographic Microscopy for Label-Free Detection of Leukocyte Alternations Associated with Perioperative Inflammation after Cardiac Surgery.

In a prospective observational pilot study on patients undergoing elective cardiac surgery with cardiopulmonary bypass, we evaluated label-free quantitative phase imaging (QPI) with digital holographic microscopy (DHM) to describe perioperative inflammation by changes in biophysical cell properties of lymphocytes and monocytes. Blood samples from 25 patients were investigated prior to cardiac surgery and postoperatively at day 1, 3 and 6. Biophysical and morphological cell parameters accessible with DHM, such as cell volume, refractive index, dry mass, and cell shape related form factor, were acquired and compared to common flow cytometric blood cell markers of inflammation and selected routine laboratory parameters. In all examined patients, cardiac surgery induced an acute inflammatory response as indicated by changes in routine laboratory parameters and flow cytometric cell markers. DHM results were associated with routine laboratory and flow cytometric data and correlated with complications in the postoperative course. In a subgroup analysis, patients were classified according to the inflammation related C-reactive protein (CRP) level, treatment with epinephrine and the occurrence of postoperative complications. Patients with regular courses, without epinephrine treatment and with low CRP values showed a postoperative lymphocyte volume increase. In contrast, the group of patients with increased CRP levels indicated an even further enlarged lymphocyte volume, while for the groups of epinephrine treated patients and patients with complicative courses, no postoperative lymphocyte volume changes were detected. In summary, the study demonstrates the capability of DHM to describe biophysical cell parameters of perioperative lymphocytes and monocytes changes in cardiac surgery patients. The pattern of correlations between biophysical DHM data and laboratory parameters, flow cytometric cell markers, and the postoperative course exemplify DHM as a promising diagnostic tool for a characterization of inflammatory processes and course of disease.

Shot-noise limited, supercontinuum-based optical coherence tomography.

We present the first demonstration of shot-noise limited supercontinuum-based spectral domain optical coherence tomography (SD-OCT) with an axial resolution of 5.9 µm at a center wavelength of 1370 nm. Current supercontinuum-based SD-OCT systems cannot be operated in the shot-noise limited detection regime because of severe pulse-to-pulse relative intensity noise of the supercontinuum source. To overcome this disadvantage, we have developed a low-noise supercontinuum source based on an all-normal dispersion (ANDi) fiber, pumped by a femtosecond laser. The noise performance of our 90 MHz ANDi fiber-based supercontinuum source is compared to that of two commercial sources operating at 80 and 320 MHz repetition rate. We show that the low-noise of the ANDi fiber-based supercontinuum source improves the OCT images significantly in terms of both higher contrast, better sensitivity, and improved penetration. From SD-OCT imaging of skin, retina, and multilayer stacks we conclude that supercontinuum-based SD-OCT can enter the domain of shot-noise limited detection.

Unexpected localization of AQP3 and AQP4 induced by migration of primary cultured IMCD cells.

Aquaporin-2-4 (AQP) are expressed in the principal cells of the renal collecting duct (CD). Beside their role in water transport across membranes, several studies showed that AQPs can influence the migration of cells. It is unknown whether this also applies for renal CD cells. Another fact is that the expression of these AQPs is highly modulated by the external osmolality. Here we analyzed the localization of AQP2-4 in primary cultured renal inner medullary CD (IMCD) cells and how osmolality influences the migration behavior of these cells. The primary IMCD cells showed a collective migration behavior and there were no differences in the migration speed between cells cultivated either at 300 or 600 mosmol/kg. Acute increase from 300 to 600 mosmol/kg led to a marked reduction and vice versa an acute decrease from 600 to 300 mosmol/kg to a marked increase in migration speed. Interestingly, none of the analyzed AQPs were localized at the leading edge. While AQP3 disappeared within the first 2-3 rows of cells, AQP4 was enriched at the rear end. Further analysis indicated that migration induced lysosomal degradation of AQP3. This could be prevented by activation of the protein kinase A, inducing localization of AQP3 and AQP2 at the leading edge and increasing the migration speed.

Refractive Index Changes of Cells and Cellular Compartments Upon Paraformaldehyde Fixation Acquired by Tomographic Phase Microscopy.

Three-dimensional quantitative phase imaging is an emerging method, which provides the 3D distribution of the refractive index (RI) and the dry mass in live and fixed cells as well as in tissues. However, an insufficiently answered question is the influence of chemical cell fixation procedures on the results of RI reconstructions. Therefore, this work is devoted to systematic investigations on the RI in cellular organelles of live and fixed cells including nucleus, nucleolus, nucleoplasm, and cytoplasm. The research was carried out on four different cell lines using a common paraformaldehyde (PFA)-based fixation protocol. The selected cell types represent the diversity of mammalian cells and therefore the results presented provide a picture of fixation caused RI changes in a broader context. A commercial Tomocube HT-1S device was used for 3D RI acquisition. The changes in the RI values after the fixation process are detected in the reconstructed phase distributions and amount to the order of 10-3 . The RI values decrease and the observed RI changes are found to be different between various cell lines; however, all of them show the most significant loss in the nucleolus. In conclusion, our study demonstrates the evident need for standardized preparation procedures in phase tomographic measurements. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC. on behalf of International Society for Advancement of Cytometry.

HS2ST1-dependent signaling pathways determine breast cancer cell viability, matrix interactions, and invasive behavior.

Heparan sulfate proteoglycans (HSPGs) act as signaling co-receptors by interaction of their sulfated glycosaminoglycan chains with numerous signaling molecules. In breast cancer, the function of heparan sulfate 2-O-sulfotransferase (HS2ST1), the enzyme mediating 2-O-sulfation of HS, is largely unknown. Hence, a comparative study on the functional consequences of HS2ST1 overexpression and siRNA knockdown was performed in the breast cancer cell lines MCF-7 and MDA-MB-231. HS2ST1 overexpression inhibited Matrigel invasion, while its knockdown reversed the phenotype. Likewise, cell motility and adhesion to fibronectin and laminin were affected by altered HS2ST1 expression. Phosphokinase array screening revealed a general decrease in signaling via multiple pathways. Fluorescent ligand binding studies revealed altered binding of fibroblast growth factor 2 (FGF-2) to HS2ST1-expressing cells compared with control cells. HS2ST1-overexpressing cells showed reduced MAPK signaling responses to FGF-2, and altered expression of epidermal growth factor receptor (EGFR), E-cadherin, Wnt-7a, and Tcf4. The increased viability of HS2ST1-depleted cells was reduced to control levels by pharmacological MAPK pathway inhibition. Moreover, MAPK inhibitors generated a phenocopy of the HS2ST1-dependent delay in scratch wound repair. In conclusion, HS2ST1 modulation of breast cancer cell invasiveness is a compound effect of altered E-cadherin and EGFR expression, leading to altered signaling via MAPK and additional pathways.

Paired Basic Amino Acid-cleaving Enzyme 4 (PCSK6): An Emerging New Target Molecule in Human Melanoma.

Although recent therapeutic developments raise hope, melanoma remains a devastating disease with a need for new treatment targets. In other tumours prohormone convertases have been shown to be pro-tumourigenic as they are involved in processing preforms of matrix-metalloproteinases, growth factors and adhesion molecules. The aim of this study was to look for new treatment options for melanoma, by investigating the role of the prohormone convertase Paired basic Amino acid-Cleaving Enzyme 4 (PACE4/PCSK6) in melanoma cell lines and human melanoma tissue. PACE4-transfected A375 melanoma cells displayed significantly increased proliferation, MMP-2 production, gelatinase activity and migratory capacity in vitro compared with sham-transfected cells. In vivo, elevated PACE4 expression resulted in significantly increased tumour growth on immunodeficient mice. In the majority of 45 human primary melanomas and melanoma metastases ex vivo PACE4 immunoreactivity was detectable, while it was absent in in situ melanomas. These results indicate PACE4 as a regulator of melanoma cell aggressiveness.

Knockdown of Musashi RNA Binding Proteins Decreases Radioresistance but Enhances Cell Motility and Invasion in Triple-Negative Breast Cancer.

The therapeutic potential of Musashi (MSI) RNA-binding proteins, important stemness-associated gene expression regulators, remains insufficiently understood in breast cancer. This study identifies the interplay between MSI protein expression, stem cell characteristics, radioresistance, cell invasiveness and migration. MSI-1, MSI-2 and Notch pathway elements were investigated via quantitative polymerase chain reaction (qPCR) in 19 triple-negative breast cancer samples. Measurements were repeated in MDA-MB-231 cells after MSI-1 and -2 siRNA-mediated double knockdown, with further experiments performed after MSI silencing. Flow cytometry helped quantify expression of CD44 and leukemia inhibitory factor receptor (LIFR), changes in apoptosis and cell cycle progression. Proliferation and irradiation-induced effects were assessed using colony formation assays. Radiation-related proteins were investigated via Western blots. Finally, cell invasion assays and digital holographic microscopy for cell migration were performed. MSI proteins showed strong correlations with Notch pathway elements. MSI knockdown resulted in reduction of stem cell marker expression, cell cycle progression and proliferation, while increasing apoptosis. Cells were radiosensitized as radioresistance-conferring proteins were downregulated. However, MSI-silencing-mediated LIFR downregulation resulted in enhanced cell invasion and migration. We conclude that, while MSI knockdown results in several therapeutically desirable consequences, enhanced invasion and migration need to be counteracted before knockdown advantages can be fully exploited.

Quantitative Phase Imaging Using Digital Holographic Microscopy Reliably Assesses Morphology and Reflects Elastic Properties of Fibrotic Intestinal Tissue.

Intestinal strictures are a frequent complication in patients with Crohn's Disease (CD) and the presence of fibrosis within strictures impacts the therapeutic treatment approach. Here, we evaluate quantitative phase imaging (QPI) using digital holographic microscopy (DHM) for the evaluation of fibrosis within CD strictures. 30 full thickness resection specimens were obtained from non-stenotic and stenotic tissue areas of 15 CD patients. Cryostat sections were analyzed by DHM to measure the spatial distribution of the refractive index (RI) to quantify tissue density. Complementary, histopathological evaluation of H&E staining and immunofluorescence (IF) targeting fibrosis markers served as the gold standard. Moreover, tissue stiffness was evaluated by elastography. RI values assessed by DHM were significantly higher in stenotic compared to non-stenotic tissue areas (p < 0.001). Histopathological analysis using H&E staining and IF confirmed the elevated expression of fibrosis markers in stenotic compared to non-stenotic tissue (all p < 0.001). The RI retrieved by DHM strongly correlated with the amount of fibrosis as determined by IF (p < 0.001; R2 = 0.48). Furthermore, elastography detected a significantly higher tissue stiffness in stenotic as compared to non-stenotic tissue sections (p < 0.001). In conclusion, QPI using DHM accurately assesses fibrotic properties of CD-associated strictures and may improve the characterization of CD strictures.