The exploitation of polarized light for the investigation of light-tissue interaction has evolved to a powerful approach for the analysis of various biological samples with potential to be established for rapid and reliable in vivo measurement in life sciences and medicine in future. We report on our work on the development and application of polarization-modulated spectroscopic ellipsometry and wide-field multi-wavelength Mueller matrix polarimetry for use cases as versatile as the detection of E.coli bacteria in microfluidic cells, the identification of multiple bacterial colonies in biofilms or the analysis of microstructural changes occurring in skin cancer or neurodegenerative disorders. We present our recent results along these lines and discuss the challenges with respect to miniaturization of the systems as well as towards in vivo and clinical application. Based on the results obtained so far, it is evident that the developed approaches open the potential for further application in medicine and the life science in future, in particular when combined with concepts from machine learning and artificial intelligence.

Significance:Given the rapidly ageing global population and the projected rise in dementia cases, research into Alzheimer's disease (AD) has become an urgent scientific and medical priority. Continued investigation into the molecular mechanisms and early detection of AD is therefore essential to mitigate its growing personal and societal impact. Most current AD therapies in advanced phases of development target amyloid β -peptide ( A β ) production, aggregation, or accumulation. Aim:Mueller Matrix Polarimetry (MMP) has evolved into a prominent research subject, with a focus on identifying microstructural changes in biotissues. This is done by investigating light properties, which is especially useful in the early detection of brain cell degradation, among others. We set up and employed experimental MMP at three illuminating laser wavelengths (i.e., 445, 532, and 632 nm). Approach:We investigated the application of MMP to mouse brain tissue containing A β plaques. The investigated samples on glass slides consisted of paraffin-embedded brain and paraffin-embedded tissue slices. The tissues were taken at various stages of ageing, i.e., 75, 100, 125, 150, 175, 200, and 225 days. Paraffin tissue blocks were used as an additional sample set for comparison. Results:We performed a comparative analysis based on the Mueller matrix elements for each age category and highlighted the importance of certain elements, e.g., m 44 , for further analysis. We also compared the trends of decomposition parameters and could correlate them with the ageing. Contrary to previous studies, we also report on retardation, diattenuation, and polarizance changes for later AD changes. Conclusions:From the higher-order statistics, we concluded that the mean and standard deviation remained constant across the ages. Skewness values were positive and increased as the age progressed, whereas kurtosis decreased with age. The large available dataset opens the possibility of implementing machine learning methods to assist clinical diagnosis in the future.

Significance:Bacterial biofilm agglomerates are the cause of hard-to-treat implant-associated infections but currently can only be distinguished using sophisticated microbiological or molecular biological methods. Optical methods can potentially provide a label-free, noncontact approach to detect the presence of bacterial species associated with implant infections that could aid in the early diagnosis of implant-associated diseases. Aim:Our aim is to measure the polarization signal from implant-associated bacteria biofilms using Mueller matrix polarimetry. Furthermore, we present an analysis of the Mueller matrix element to detect and distinguish the different bacterial biofilm species. Approach:Several biofilms formed by bacterial species associated with orthopedic (Staphylococcus aureus and Staphylococcus epidermidis) and dental implants (Streptococcus oralis, Streptococcus mutans, and Porphyromonas gingivalis) were grown on titanium, a typical implant material. Polarization signals were acquired in a reflection mode using a calibrated polarimetry setup. Results:The results show that different biofilms could be qualitatively distinguished using the Mueller matrix element analysis. The values derived from bacterial species measurements were distinctly different from those of the bare titanium discs. From the Lu-Chipman decomposition, parameters such as polarizance and diattenuation were calculated for each of the species. Conclusions:The results provide deeper insight into the interaction of polarized light with bacterial microcolonies. The physiologically growing biofilms form the basis of their polarimetric response signal. Our approach has potential for fast and nondestructive investigation for implant infection detection, potentially in situ and in vivo.

1562 Background: Traditional clinicogenomic analysis workflows in oncology require substantial bioinformatics expertise and custom coding, creating a bottleneck where clinical researchers must rely on analysts for data interpretation. This dependency hinders hypothesis development, exploration and discovery, as researchers cannot directly interact with their data in real-time. DeepSeal addresses these challenges by providing a user-friendly, chat-based interface that enables immediate analysis of clinicogenomic data and seamless generation of results, empowering clinical researchers to independently explore and validate hypotheses. Methods: DeepSeal, an integration of a large language model with clinical and molecular databases and bioinformatics tools, was evaluated by replicating the findings of Riaz et al. (Cell, 2017), in advanced melanoma patients treated with nivolumab. Through natural language prompts, DeepSeal performed multiple analyses including differential gene expression analysis and Gene Set Enrichment Analysis (GSEA) to generate comprehensive molecular profiles of responders versus non-responders and to identify molecular signatures associated with treatment response. Results: DeepSeal successfully replicated the key findings of Riaz et al., identifying significant differential expression of immune-related genes in treatment responders. GSEA executed through DeepSeal’s chat interface further revealed enrichment of immune-related pathways critical for response, including B cell activation (GO:0042113), T cell activation (GO:0042110), and regulation of adaptive immune response (GO:0002819). The chat interface enabled rapid hypothesis testing and visualization generation, with analyses completed in minutes without the need for programming expertise. Conclusions: DeepSeal demonstrates the feasibility of enabling clinical researchers to independently analyze complex clinicogenomic data through natural language interaction. By successfully replicating and validating findings from Riaz et al., it generates reliable insights without programming expertise, offering a transformative approach to accelerate translational research. This removal of technical barriers between researchers and their data has the potential to substantially speed hypothesis testing and discovery in oncology, ultimately enhancing the pathway from molecular insights to improved patient care.

Electrospun polymer fiber mats feature versatile applications in tissue engineering, drug delivery, water treatment and chemical processes. The orientation of fibers within these mats is a crucial factor that significantly influences their properties and performance. However, the analysis of fiber samples using scanning electron microscopy (SEM) has limitations such as time consumption, fixed assembly, and restricted field of vision. Therefore, a fast and reliable method for qualitative measurements of fiber orientation is required. Mueller matrix polarimetry, a well-established method for measuring orientation of chemical and biological species, was employed in this case. We investigated the effect of four important parameters of the electrospinning process, namely collector speed, applied voltage, needle-to-collector distance, and solution concentration, on fiber orientation using Mueller matrix polarimetry thus extending the range of parameters analyzed. Measurements were performed using two extreme values and a central optimized value for each fabrication parameter. Changes in matrix values were observed for each fabrication parameter, and their correlation with fiber orientation was analyzed based on the Lu-Chipman decomposition. The results were compared with SEM images, which served as the ground truth, and showed overall good agreement. In the future, the analysis of electrospun polymer fibers can be done by using Mueller matrix polarimetry as alternative to current technology and fabrication parameters, including solution concentration for the first time in this context and the production can quickly be adjusted based on the outcome of the measurements.

Supplementary Data from Entinostat Decreases Immune Suppression to Promote Antitumor Responses in a HER2+ Breast Tumor Microenvironment

Cyclin dependent kinase 4 and 6 inhibitors such as abemaciclib are routinely used to treat metastatic estrogen receptor positive (ER+) breast cancer. However, adaptive mechanisms inhibit their effectiveness and allow for disease progression. Using ER+ breast cancer cell models, we show that acquired resistance to abemaciclib is accompanied by increase in metastatic potential. Mass spectrometry-based proteomics from abemaciclib sensitive and resistant cells showed that lysosomal proteins including CTSD (cathepsin D), cathepsin A and CD68 were significantly increased in resistant cells. Combination of abemaciclib and a lysosomal destabilizer, such as hydroxychloroquine (HCQ) or bafilomycin A1, resensitized resistant cells to abemaciclib. Also, combination of abemaciclib and HCQ decreased migration and invasive potential and increased lysosomal membrane permeability in resistant cells. Prosurvival B cell lymphoma 2 (BCL2) protein levels were elevated in resistant cells, and a triple treatment with abemaciclib, HCQ, and BCL2 inhibitor, venetoclax, significantly inhibited cell growth compared to treatment with abemaciclib and HCQ. Furthermore, resistant cells showed increased levels of Transcription Factor EB (TFEB), a master regulator of lysosomal-autophagy genes, and siRNA mediated knockdown of TFEB decreased invasion in resistant cells. TFEB was found to be mutated in a subset of invasive human breast cancer samples, and overall survival analysis in ER+, lymph node-positive breast cancer showed that increased TFEB expression correlated with decreased survival. Collectively, we show that acquired resistance to abemaciclib leads to increased metastatic potential and increased levels of protumorigenic lysosomal proteins. Therefore, the lysosomal pathway could be a therapeutic target in advanced ER+ breast cancer.

Severe insulin resistance syndromes result from primary insulin signaling defects, adipose tissue abnormalities or other complex syndromes. Mutations in TBC1D4 lead to partial insulin signaling defects, characterized mainly by postprandial insulin resistance. We describe an individual with severe insulin-resistant diabetes unresponsive to multiple therapies, in whom exome and genome analyses identified a complex rearrangement in TBC1D4 . The rearrangement was of the pattern DUP-TRP/INV-DUP, with mutational signatures suggestive of replicative repair and Alu-Alu recombination as the underlying mechanisms. TBC1D4 encodes the TBC1D4/AS160 RabGTPase activating protein (RabGAP) involved in the translocation of glucose transporter 4 (GLUT4) from the cytosol to the cell membrane. Although the precise functional mechanism underlying insulin resistance in the proband is yet to be determined, this case provides further support for the link between TBC1D4 and hereditary insulin-resistant diabetes.

Triple-negative breast cancer (TNBC) is an aggressive subtype associated with early metastatic recurrence and worse patient outcomes. TNBC tumors express molecular markers of the epithelial-mesenchymal transition (EMT), but its requirement during spontaneous TNBC metastasis in vivo remains incompletely understood. We demonstrated that spontaneous TNBC tumors from a genetically engineered mouse model (GEMM), multiple patient-derived xenografts, and archival patient samples exhibited large populations in vivo of hybrid E/M cells that lead invasion ex vivo while expressing both epithelial and mesenchymal characteristics. The mesenchymal marker vimentin promoted invasion and repressed metastatic outgrowth. We next tested the requirement for five EMT transcription factors and observed distinct patterns of utilization during invasion and colony formation. These differences suggested a sequential activation of multiple EMT molecular programs during the metastatic cascade. Consistent with this model, our longitudinal single-cell RNA analysis detected three different EMT-related molecular patterns. We observed cancer cells progressing from epithelial to hybrid E/M and strongly mesenchymal patterns during invasion and from epithelial to a hybrid E/M pattern during colony formation. We next investigated the relative epithelial versus mesenchymal state of cancer cells in both GEMM and patient metastases. In both contexts, we observed heterogeneity between and within metastases in the same individual. We observed a complex spectrum of epithelial, hybrid E/M, and mesenchymal cell states within metastases, suggesting that there are multiple successful molecular strategies for distant organ colonization. Together, our results demonstrate an important and complex role for EMT programs during TNBC metastasis.

Significance: Mueller matrix (MM) polarimetry is a promising tool for the detection of skin cancer. Polarimetric in vivo measurements often suffer from misalignment of the polarimetric images due to motion, which can lead to false results. Aim: We aim to provide an easy-to-implement polarimetric image data registration method to ensure proper image alignment. Approach: A feature-based image registration is implemented for an MM polarimeter for phantom and in vivo human skin measurements. Results: We show that the keypoint-based registration of polarimetric images is necessary for in vivo skin polarimetry to ensure reliable results. Further, we deliver an efficient semiautomated method for the registration of polarimetric images. Conclusions: Image registration for in vivo polarimetry of human skin is required for improved diagnostics and can be efficiently enhanced with a keypoint-based approach. (C) The Authors. Published by SPIE under a Creative Commons Attribution 4.0 International License.

Dermoscopy is the main tool for early detection of skin cancer. Non-contact dermoscopes often suffer from a small depth of field leading to images of skin topographies with regions that are not in focus. We aim to provide an easy-to-implement focus stacking-based approach to ensure all-in-focus images from a non-contact dermoscope. Further, we aim to extract additional information about the skin topography from the image stacks. The focus stacking procedure itself is implemented in a non-contact dermoscope with an electrically adjustable focus realized by using a tunable liquid lens. We show that all-in-focus imaging is possible for non-contact dermoscopy and deliver a method to extract topographical information for dermatologists from the acquired image stacks. Our finding indicate that the approach can be valuable for non-contact dermoscopic examination as well as for the early detection of skin diseases such as cancer as it possible to derive hyperfocus images and information on the skin topography. With this, we were able to develop a software for the acquisition of the raw image data and its processing into a high resolution hyperresolution dermoscopic image. In the next steps, we plan to apply the approach in the clinical environment for skin cancer diagnostics or imaging of inflammatory skin diseases.

The emergence of antibiotic-resistant strains of Helicobacter pylori necessitates the development of novel therapeutic strategies to fight against its infection. Recently, the enzyme inosine-5 '-monophosphate dehydrogenase (IMPDH) has emerged as a promising target to treat bacterial infections due to its crucial role in the de novo purine biosynthesis pathway. The differences between the prokaryotic and eukaryotic IMPDHs, in the NAD(+) binding domain and flap region, allow the identification of pathogen-specific inhibitors. In the present study, seven point mutants of wild type Helicobacter pylori IMPDH are constructed by site-directed mutagenesis, and characterized using in silico and kinetic studies. Point mutations in the NAD(+) binding domain and the flap region are shown to impart significant changes in the enzyme's structure and function. In addition, the product inhibition characteristics of the Arg396-Tyr397 dyad (RY dyad) show that both the residues are important for water activation in the reaction. The results obtained are beneficial for the design and development of small-molecule inhibitors, capable of species-specific inhibition.

e15532 Background: Patients (pts) with BRAF V600E mutant (mt) metastatic colorectal cancer (mCRC) still have a poor prognosis even when treated with encorafenib plus cetuximab. We reported that eribulin that inhibits G2-M cell cycle had limited activity for these pts in the phase II BRAVERY study (Masuishi T, et al. WCGC 2020). Barras D, et al. (2017) reported that BRAF V600E mt mCRCs were classified into BM1 and BM2 characterized by KRAS/AKT pathway activation and deregulation of the cell cycle with G2-M phase activation, respectively, based on gene expression. Methods: Whole transcriptome RNA-seq of FFPE pre-treatment tumor samples was performed using NovaSeq 6000 in the BRAVERY study. Molecular features were extracted using the Txome.ai platform (Ocean Genomics Inc., PA, USA) which included transcript- and gene-level expression quantification, expression-based clustering, and structural variant calling. Efficacy of eribulin was classified as “good” if pts had a tumor reduction and/or progression-free survival (PFS) of more than 6 months, and “poor” otherwise. The differential gene expression analysis was performed between pts with “good” and “poor” using Txome.ai. In addition, BM and consensus molecular subtype (CMS) classification were performed using the model developed by Barras D, et al. (2017) and Guinney J, et al. (2015), respectively. Results: Among 27 pts, 26 tumor samples were available to perform RNA-seq and analyze gene expression despite low mapping rates. Patient characteristics were as follows: median age of 58.5 (range, 33–71) years; ECOG PS of 0/1 (16/10); primary tumor location of right/left (11/15); and all 26 pts had MSS/pMMR. Four and 22 pts were classified into “good” and “poor” groups, respectively. Among 52 differentially expressed genes (GENCODE v31) with false discovery rate-adjusted P- value < 0.05, the top 5 genes with the lowest P-values are provided in the table. All 4 pts in the “good” group were classified into BM2 and pts in the “poor” group were classified into BM1 (8/22) and BM2 (14/22) (p = 0.07). In addition, all but 2 pts were classified into CMS4. These two pts belong to the “poor” group with one of them classified into CMS1 and the other into CMS2. Conclusions: These gene expression analyses suggest that BM2 subtype could be a predictive marker for the efficacy of eribulin and some genes could be novel targets with the goal to improve prognosis of pts with BRAF V600E mt mCRC. This is the first finding for a potential biomarker in this subgroup using RNA-seq analysis tools. These findings will require additional validation. Clinical trial information: UMIN000031552. [Table: see text]

Corneal collagen cross-linking is an established procedure for the treatment of certain eye diseases which is applied to enhance the mechanical stability of such biotissue without deteriorating its functionality. However, being transparent, the optical analysis of the outcome of such treatments is cumbersome and relies on relatively expensive experimental equipment. We aim to apply the Mueller matrix polarimetry for the detection of photo-induced collagen cross-linking in transparent biotissue after treatment with riboflavin and UV irradiation. A simple Mueller matrix polarimetry setup could provide a fast and non-invasive analysis of transparent media to sensitively detect small photo-induced cross-linking effects in biotissue. We demonstrated the current capabilities of the approach on non-planar porcine cornea samples ex vivo. We reported the distinction between untreated and riboflavin-treated samples. The differences observed were correlated with the variation of certain Mueller matrix elements and parameters derived from the decomposition. The measurement data show variation in the cross-linked and non-cross-linked samples, although the effect of the UV treatment on the riboflavin-treated samples was not at the same level of significance yet and needs further investigation. The Mueller matrix measurement represents a promising approach for the detection of the effects of corneal collagen cross-linking. Further studies with a larger sample number are required to validate this approach. In the future, this could enable the reliable and non-invasive detection of photo-induced effects in biotissue and open the possibility for in vivo application, e.g., in eye disease treatment or the detection of scar collagen development.

by Gayathri Purushothaman, Gaurav Sharma, Aishwarya Menon, Kapil Juvale, Vijay Thiruvenkatam and Sivapriya Kirubakaran