Single Staining Research Articles

Abstract 2040: Invisible chromogens expand brightfield multiplexing and enable combined protein expression and morphological analysis

Abstract Anatomical pathology relies upon visual evaluation of clinical specimens using brightfield microscopy. This necessarily utilizes histological stains and immunohistochemical (IHC) chromogens that absorb visible light, between 430 and 690 nm. To complement the visible stains, we developed chromogens for use in IHC that absorb light outside the visible range, and imaging methods for detecting dyes absorbing from the ultraviolet (UV) through the near infrared (NIR). Invisible chromogens partnered with conventional visible stains on the same slide provides complementary information, enabling pathologists to gain deeper insights from a patient’s tissue. Methods: Invisible IHC was performed using conventional antibodies and antibody-enzyme conjugates utilizing covalently deposited chromogens absorbing in the UV and NIR. This was combined with IHC using visible chromogens or histological stains. Resulting stained specimens were evaluated interactively in real-time using a dual-camera microscope system comprising color and monochrome cameras, or by recording single microscope fields using multispectral imaging and the monochrome camera. Results: Multiplex IHC was performed with different combinations of visible and invisible chromogens, with the highest level of multiplexing - 7 biomarkers + hematoxylin counterstain (5 visible IHC chromogens plus a UV and an NIR chromogen) - demonstrated on a formalin-fixed paraffin-embedded (FFPE) prostate tumor. Images of each stain were recorded using multispectral imaging, and staining patterns were concordant with single stain DAB analysis on serial sections. In addition to increased IHC multiplexing capacity, multiplexing of IHC with histological stain was demonstrated by combining an NIR absorbing chromogen, identifying MART-1 expression, with H&E staining on FFPE melanoma specimens. The dual-camera microscope presented video of the visible H&E staining (color) next to the NIR IHC staining (monochrome) on the computer monitor as the specimen was manually scanned. Archiving was afforded by multispectral imaging which also enabled quantitative analysis. An additional advantage of the NIR chromogen was the suppression of melanin pigment, the absorbance of which was significantly reduced in the NIR. Other conventional stains multiplexed with invisible IHC on colon, lung, pancreas, and tonsil FFPE tissues also showed good separation of conventional and IHC staining. Conclusion: Taking advantage of invisible chromogens, IHC multiplexing capacity can be increased and conventional histological stains can be combined with IHC on a single slide. Clinical benefits include saving precious specimen, enabling differentiation of cell populations by simultaneous expression of multiple biomarkers, and providing biomarker expression within the cellular and morphological context of conventional stains. Citation Format: Larry E. Morrison, Mark R. Lefever, Lauren J. Behman, Monesh J. Kapadia, Daniel R. Bauer. Invisible chromogens expand brightfield multiplexing and enable combined protein expression and morphological analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2040.

Establishment of multiplex IHC staining panel and immune subpopulation quantificationsystem for cancer prognosis.

e20525 Background: Tumor microenvironment (TME) is now an important direction for cancer prognosis research. For analyzing the immune component within TME, illustrating the distribution of the immune cells in patient tissue samples by immunohistochemistry (IHC) and image quantification analysis are now been applied in many cancer types. However, most of the TME studies only used the tumor microarray to investigate the composition of immune cells in TME, in which the views of immune component are restricted. In addition, with single biomarker staining to stratify therapy strategy shows limited efficacy in treatment since the complicated interactions of the immune components in TME. Based on these shortages, to increase the efficiency and identify the scarce immune subsets, a more systemic analysis approach for immune subpopulations quantification is now imperative. Methods: In this study, we first established an immune cell multiplex IHC staining panel (Opal Polaris, Akoya Bioscience) in non-small cell lung cancer (NSCLC) to investigate the immune components in all the tumor blocks of each patient. In this panel, we applied markers of infiltrating immune cells (CD8, CD68), regulatory markers (PD-1, PD-L1,CD163), and tumor marker (pan-cytokeratin) to explore the TME landscape in NSCLC. Then, with an in-house built quantitative software: SIMPiE, we could analyze the staining signal intensity of each cell markers generated from in Form software to define each lymphocytic immune subsets by phenotyping and quantifying. In addition, SIMPiE (Spatial Image cytometry Multiplex IHC analysis by phyton and in Form based Elements) software allowed us to generate image cytometry data to further explore the spatial context to map the organization and interactions of immune and tumor cells in the TME, and we can define thresholds for the graph to distribute the plurality of the mark points into a plurality of quadrants. Finally, we performed the survival analysis according to the clinicopathological and immune parameters of the patients, and to examine the prognostic significance of each immune subtypes. Results: We have built up an automation quantitative image analysis software to efficiently quantity the signal density and spatial context of all the immune subpopulations in all the tissue sections of each patient, and we further performed the integrative survival analysis to explore the prognostic significance of infiltrating immune cells and immunoregulatory molecules within the TME in NSCLC. Finally, in our study, we found that M1 macrophage patamars showed the most significantly regulatory effect to the patient’s overall survival rate, and M2 macrophage showed less effect compared with M1 macrophage. Conclusions: By this way, we can generate image cytometry data to further explore the spatial context to map the organization of immune and tumor cells in the TME for prognosis analysis.

Prevalence of T-cell antigen losses in mycosis fungoides and CD30-positive cutaneous T-cell lymphoproliferations in a series of 153 patients

Mycosis fungoides (MF) and primary cutaneous CD30-positive T-cell lymphoproliferative disorders (CD30LPD) are the most frequent primary cutaneous T-cell lymphomas. Our objective was to study pan-T-cell antigens and PD-1 expression in a large cohort of MF and CD30LPD with a special interest in antigen losses as a diagnostic tool. We retrospectively reviewed 160 consecutive samples from 153 patients over a 3 year period, including 104 with MF and 49 with CD30LPD. As controls, benign inflammatory dermatoses (BID, n=19) were studied. A semi-quantitative evaluation of CD2, CD3, CD4, CD5, CD7, CD8 expression was performed. PD-1 and double stainings (CD3+CD7 and PD-1+CD7) were performed in a subset of MF cases. CD8+ MF was frequent (23%) and CD7 was the most frequently lost antigen in both MF (45%) and CD30LPD (86%), while no significant T-cell antigen loss was observed in BID. CD7 loss was less frequent in folliculotropic MF (p<0.001). PD-1 was variably expressed in MF with no differences with BID. The CD3+/CD7- and PD-1+/CD7- neoplastic lymphocytes were highlighted by the use of chromogenic double staining experiments in MF with a CD7 loss identified with single staining. Multiple pan T-cell antigen losses were mostly seen in CD30LPD with CD2 being the most frequently preserved marker (90%). While PD-1 does not discriminate between MF and BID, CD7 is frequently lost in MF infiltrates as well as other pan-T-cell antigens in CD30LPD, which can be used as routine markers for diagnosis. We recommend the use of CD7 in addition to CD3, CD4 and CD8 as a minimal immunohistochemical panel for MF assessment, and the use of double stainings for CD3 and CD7 in difficult cases.

Research on Mechanism of FePt Nanoparticles at Varied Concentrations in Regulating DNA Injury and Inactivation of Hela Cells Through Mitogen-Activated Protein Kinase Signal Pathway

FePt nanoparticles were in this study prepared at varied concentrations to assess their mechanism in regulating DNA injury and inactivation of Hela cells through mitogen-activated protein kinase (MAPK) signal pathway. The cells were divided into following 5 group sets: 1st, 2nd, 3rd, 4th and 5th, which were randomly intervened by following varied concentrations of FePt nanoparticles; 0, 5, 10, 15 and 20 μg/mL. Their action on cellular toxicity, proliferation and inactivation of cells was detected with CCK-8 method, while apoptotic condition was detected with flow cytometry (FCM). The degree of DNA double chains fracture was detected with neutral single cell gel electrophoresis and immunofluorescence staining. The protein expressions of of EKR1/2, p-ERK1/2, p38 MAPK and p-p38 MAPK in every set were detected with Western Blot assay, while proliferative activity in the 4th and 5th sets was lower than in the 1st set, and inhibiting action in the 4th set was best. There was no notable difference between 4th and 5th sets, and apoptotic rate in the 1st set was lower than in the other sets. The DNA percentage in the Hela cells was reduced and Tial length was lengthened along with increased concentration of adopted FePt nanoparticles. The proliferation of Hela cells was restrained by FePt nanoparticles through restraining of the MAPK signal pathway, but growth of Hela cells was not affected, and DNA double-strand break (DSB) was increased. The FePt nanoparticles could therefore be adopted as one kind of effective anti-tumor drug through increased DNA DSB in tumor cells based on killing effect of tumor cells and good compatibility for normal cells.

Co‐expression of Zeb‐1 and K17 provides new insights into the role of EMT in pancreatic cancer progression

INTRODUCTIONDespite improvements in screening and therapeutic options, pancreatic ductal adenocarcinoma (PDAC) has the third‐worst mortality rate, highlighting the need for better diagnostic and therapeutic approaches. Epithelial‐mesenchymal transition (EMT) has been conferred with a greater likelihood of metastasis in multiple cancers, specifically identified through markers like Zeb‐1 and Twist‐1. Zeb‐1 (Zinc Finger E‐Box Binding Homeobox 1) was shown to repress E‐cadherin expression, consequently inducing EMT.Our lab has previously identified K17 (Keratin 17) as an independent prognostic biomarker of poor outcomes in PDAC patients. While increasing evidence has suggested that EMT drives pancreatic cancer migration and metastasis, there have not been any well‐established connections between K17 expression and EMT. This study aims to provide new insights into the role of K17 in EMT by evaluating the co‐expression of K17 and Zeb‐1 in human PDAC biopsies. We hypothesized that tumor cells co‐express K17 and Zeb‐1 as a hallmark of EMT in PDAC. In addition, our study aims to establish a novel method of scoring Zeb‐1 expression in stromal fibroblasts via immunofluorescence to remove variability in the stromal constituency.METHODSPDAC tissues obtained from patients who underwent surgical resection between 2013 and 2017 at Stony Brook University (n=20) were selected for serial sectioning. Double immunofluorescence and single immunohistochemical staining were achieved by incubation with Zeb‐1 and K17 antibodies followed by incubation of fluorescent or biotin conjugated secondary antibodies, respectively. Histologic examination was performed using immunofluorescence staining along with H&amp;E and immunohistochemical staining (IHC) to determine concordance.RESULTS● IHC for Zeb‐1 was detected predominantly in the nuclei of centroacinar cells, benign proliferative ducts, and stromal fibroblasts. Focal nuclear staining in tumor epithelial was also detected in some cases.● High levels of K17 and but decreased expression of Zeb‐1 were detected in diffusely infiltrative cells with morphologic features of EMT.● Both high Zeb‐1 and high K17 protein expression were associated with poor survival of PDAC patients.● Using confocal immunofluorescence microscopy, we detected co‐localization of Zeb‐1 and K17 in PDAC.● We quantified Zeb‐1 protein expression in PDAC samples from 20 patients and confirmed significantly higher expression in the tumor‐associated stroma of high‐grade tumors compared to stromal fibroblasts in areas devoid of tumor.CONCLUSIONOur results provide evidence that K17 is upregulated in tumor epithelial cells that show morphologic immunohistochemical properties of EMT. The loss of Zeb‐1 in diffusely infiltrative, K17‐positive tumor cells suggests that EMT upregulates K17, driving tumor aggression. Our results suggest that K17 may enhance PDAC aggression by mediating EMT. Hence, novel therapeutic agents targeting K17 may improve clinical care for this biologically distinct patient subgroup.

Characteristics of SOX9-positive progenitor-like cells during cholestatic liver regeneration in biliary atresia

BackgroundThe progression of Biliary Atresia (BA) is associated with the number of reactive ductular cells (RDCs) whose heterogeneity in origin and evolution in humans remains unknown. SOX9-positive liver progenitor-like cells (LPLCs) have been shown to participate in RDCs and new hepatocyte formation during cholestatic liver regeneration in an animal model, which implies the possibility that hepatocyte-reprogrammed LPLCs could be a source of RDCs in BA. The present study aimed to elucidate the characteristics of SOX9-positive LPLCs in BA for exploring new possible therapeutic targets by manipulating the bi-differentiation process of LPLCs to prevent disease progression.MethodsTwenty-eight patients, including 24 patients with BA and 4 patients with Congenital Choledochal Cyst as the control group, were retrospectively recruited. Liver biopsy samples were classified histologically using a 4-point scale based on fibrosis severity. LPLCs were detected by SOX9 and HNF4A double positive staining. Single immunohistochemistry, double immunohistochemistry, and multiple immunofluorescence staining were used to determine the different cell types and characteristics of LPLCs.ResultsThe prognostic predictors of BA, namely total bile acid (TBA), RDCs, and fibrosis, were correlated to the emergence of LPLCs. SOX9 and HNF4A double-positive LPLCs co-stained rarely with relevant markers of portal hepatic progenitor cells (portal-HPCs), including CK19, CK7, EPCAM, PROM1 (CD133), TROP2, and AFP. Under cholestasis conditions, LPLCs acquired superior proliferation and anti-senescence ability among hepatocytes. Moreover, LPLCs arranged as a pseudo-rosette structure appeared from the periportal parenchyma to the portal region, which implied the differentiation from hepatocyte-reprogrammed LPLCs to RDCs with the progression of cholestasis.ConclusionsLPLCs are associated with disease progression and prognostic factors of BA. The bipotent characteristics of LPLCs are different from those of portal-HPCs. As cholestasis progresses, LPLCs appear to gain superior proliferation and anti-senescence ability and continually differentiate to RDCs.

Open Reading Frame-3a gene of the 2019 novel coronavirus inhibits the occurrence and development of colorectal cancer

Intestinal microecology is composed of bacteria, fungi and viruses. As a part of intestinal microecology, viruses participate in the occurrence and development of colorectal cancer. The 2019-nCoV was detected in stool samples from patients during COVID-19, suggesting that the 2019-nCoV may be associated with intestinal microecology. However, the relationship of the 2019-nCoV and CRC is unclear. The aim of this study is to explore the role of Open Reading Frame-3a (ORF3a) of the 2019-nCoV in CRC. After the pCDH-CMV-MCS-EF1-Puro vector that provides high expression of ORF3a was transfected into the SW480 CRC cell line, immunofluorescence was used to determine the localization of ORF3a in SW480 cells. The proliferation, migration, invasion, apoptosis, and cell cycle progression of SW480 cells were measured using the Cell Counting Kit-8 (CCK-8), wound healing, Transwell assay, flow cytometry, the TUNEL assay, and propidium iodide single staining. The results showed that ORF3a inhibited the proliferation, invasion, and migration of SW480 cells and induced their apoptosis after 24, 48, 72 h. Meanwhile, ORF3a inhibited the cell cycle and blocked SW480 CRC cells in the G1 phase. In in vivo experiments, high ORF3a expression was associated with decreased tumor volume, tumor weight, relative tumor volume, and tumor activity. ORF3a inhibited the growth and induced apoptosis and necrosis of tumor tissues. Based on this, we demonstrated that ORF3a might play a role in CRC, providing a new direction for the prevention and treatment of CRC.

Identification of Breast Cancer Stem Cells Using a Newly Developed Long-acting Fluorescence Probe, C5S-A, Targeting ALDH1A1.

Aldehyde dehydrogenase (ALDH) 1A1 is a well-known marker for cancer stem cells (CSCs), characterized by self-renewal capacity and multidrug resistance in breast cancer. We developed a near-infrared turn-on fluorescence probe for ALDH1A1, C5S-A, which is suitable for observing and analyzing viable cells. Here, we demonstrated the utility of C5S-A in CSC research using breast cancer cell lines. To evaluate concordance between C5S-A and conventional stem cell markers, breast cancer cells sorted for ALDEFLUOR-positive cells and for CD44+/CD24- cell populations were stained with C5S-A. Tumorigenicity of C5S-A-positive cells was examined by mammosphere formation assay and subcutaneous transplantation to immunodeficient mice. Additionally, to determine how long fluorescence from a single staining remained observable, we cultured breast cancer cells for 5 days after C5S-A staining. We then evaluated whether C5S-A-positive cells possessed resistance to cytotoxic drugs by chronological imaging. C5S-A staining showed good concordance with conventional breast CSC markers, and good utility for research into CSC characteristics in breast cancer cell lines, including tumorigenesis. Additionally, C5S-A was observable for more than 3 days with a single staining. Using this property, we then confirmed that C5S-A-positive cells possessed resistance to cytotoxic drugs, which is one of the characteristics of CSCs. We showed that C5S-A is suitable for CSC research using breast cancer cell lines, and confirmed its utility in observing cells over time.

Distinguishing tissue structures via polarization staining images based on different combinations of Mueller matrix polar decomposition parameters

Mueller matrix polarimetry is gaining wide attention in the field of biophotonics due to its great potential in detecting the microstructures and optical properties of tissue samples label-freely. Individual Mueller matrix derived parameters were often used to characterize certain kind of tissue structure. However, it is found that the individual Mueller matrix parameters only contain partial structural information. Thus, it is difficult to accurately and comprehensively indentify different structures using a single polarization parameter image. Here we introduce an image fusion method based on color spaces to combine different Mueller matrix derived parameters to provide multi-dimensional structural information pixel by pixel in a single polarization staining image. The results of rat back skin tissue specimens indicate that different fibrous structures can be easily distinguished using the polarization staining image. Then, in order to quantitatively analyze the texture characteristics of the polarization staining images for different structures, the Tamura image processing and the gray level co-occurrence matrix (GLCM) methods are adopted to provide various evaluation indices after the images segmentations. The experimental results confirm that the information provided by the polarization staining images based on different Mueller matrix derived parameters can be used for accurate tissue structures discrimination. Combining with machine recognition systems and fast developing artificial intelligence techniques, the strategy proposed in this study can be very helpful for precise abnormal tissues detection and pathological diagnoses.

Methods for Assessment of Viability and Germination of Plasmodiophora brassicae Resting Spores.

Clubroot caused by the obligate biotrophic parasite Plasmodiophora brassicae is a destructive soil borne disease of cruciferous crops. Resting spores of P. brassicae can survive in the soil for a long period without hosts or external stimulants. The viability and germination rate of resting spores are crucial factors of the inoculum potential in the field. The accurate assessment of viability and germination rate is the foundation to evaluate the effect of control methods. In this study, we evaluated several methods for the assessment of viability and germination rate of P. brassicae resting spores. Dual staining with calcofluor white-propidium iodide (CFW-PI) or single stain with Evans blue showed reliable accuracy in estimating viability. CFW-PI was capable of reliably determining the viability within 10 min, while Evans blue required overnight incubation to obtain accurate results. Due to DNA degradation of heat treatments, acetone was selected to evaluate the efficiency of propidium monoazide (PMA)–quantitative PCR (qPCR) used for the quantification of DNA from viable cells. The staining with 4,6-Diamidine-2-phenylindole dihydrochloride (DAPI) and the use of differential interference contrast microscopy were suitable for the determination of resting spore germination rates. The latter method also allowed recording individual germination states of spores. Alternatively, dual staining with CFW-Nile red was successfully used to assess the germination rate of resting spores with a lethal pre-treatment. This study evaluates and confirms the suitability of various microscopic and molecular genetic methods for the determination of viability and germination of P. brassicae resting spores. Such methods are required to study factors in the soil regulating survival, dormancy and germination of P. brassicae resting spores causing clubroot disease in Brassicaceae hosts and therefore are fundamental to develop novel strategies of control.

Stain transfer using Generative Adversarial Networks and disentangled features

With the digitization of histopathology, machine learning algorithms have been developed to help pathologists. Color variation in histopathology images degrades the performance of these algorithms. Many models have been proposed to resolve the impact of color variation and transfer histopathology images to a single stain style. Major shortcomings include manual feature extraction, bias on a reference image, being limited to one style to one style transfer, dependence on style labels for source and target domains, and information loss. We propose two models, considering these shortcomings. Our main novelty is using Generative Adversarial Networks (GANs) along with feature disentanglement. The models extract color-related and structural features with neural networks; thus, features are not hand-crafted. Extracting features helps our models do many-to-one stain transformations and require only target-style labels. Our models also do not require a reference image by exploiting GAN. Our first model has one network per stain style transformation, while the second model uses only one network for many-to-many stain style transformations. We compare our models with six state-of-the-art models on the Mitosis-Atypia Dataset. Both proposed models achieved good results, but our second model outperforms other models based on the Histogram Intersection Score (HIS). Our proposed models were applied to three datasets to test their performance. The efficacy of our models was also evaluated on a classification task. Our second model obtained the best results in all the experiments with HIS of 0.88, 0.85, 0.75 for L-channel, a-channel, and b-channel, using the Mitosis-Atypia Dataset and accuracy of 90.3% for classification.

Towards Inline Prediction of Color Development for Wood Stained with Chemical Stains Using Near-Infrared Spectroscopy

Abstract The chemical composition of wood determines the color development when applying chemical stains to the surface of wood. However, different species and individuals from the same species can show variations in the chemical composition, resulting in the risk of nonuniform color development in industrial staining processes between different batches of wood. In the present study, near-infrared (NIR) models were developed to predict wood specimen color development after applying three different concentrations of the chemical stains iron acetate and sodium bicarbonate. The modeling dataset included the NIR spectra of the untreated wood, stain treatment, concentration, and the International Commission on Illumination (CIE) L*a*b* color value before stain application for 210 specimens from five commercial wood species, including red oak (Quercus rubra), white oak (Quercus alba), yellow poplar (Liriodendron tulipifera), southern yellow pine (Pinus spp.), and western red cedar (Thuja plicata). The models were developed by partial least squares regression (PLSR), using 13 different mathematical transformations on the NIR spectra as well as the raw spectral data. Models with single stains and global-species/stain models were developed and compared. The models for iron acetate showed promising results in predicting the color development with the coefficient of determination for cross-validation ( ≥ 0.92), while the models for sodium bicarbonate showed acceptable results with of 0.71 to 0.89. However, a global model including both stains resulted in an unsatisfying prediction of the CIE L*a*b* color values, with of 0.46 to 0.76. The NIR models can be useful for online predictions of color development in industrial staining processes of wood with chemical stains.

Single Organoids Droplet-Based Staining Method for High-End 3D Imaging of Mammary Organoids.

In the last decade, organoids became a tremendously popular technique in developmental and cancer biology for their high pathophysiological relevance to in vivo models with the advantage of easier manipulation, real-time observation, potential for high-throughput studies, and reduced ethical issues. Among other fundamental biological questions, mammary organoids have helped to reveal mechanisms of mammary epithelial morphogenesis, mammary stem cell potential, regulation of lineage specification, mechanisms of breast cancer invasion or resistance to therapy, and their regulation by stromal microenvironment. To exploit the potential of organoid technology to the fullest, together with optimal organoid culture protocols, visualization of organoid architecture and composition in high resolution in three dimensions (3D) is required. Whole-mount imaging of immunolabeled organoids enables preservation of the 3D cellular context, but conventional confocal microscopy of organoid cultures struggles with the large organoid sample size and relatively long distance from the objective to the organoid due to the 3D extracellular matrix (ECM) that surrounds the organoid. We have overcome these issues by physical separation of single organoids with their immediate stroma from the bulk ECM. Here we provide a detail protocol for the procedure, which entails single organoid collection and droplet-based staining and clearing to allow visualization of organoids in the greatest detail.

Sample Preparation for Multicolor STED Microscopy.

Stimulated emission depletion (STED) microscopy is one of the optical superresolution microscopy (SRM) techniques, more recently also referred to as nanoscopy, that have risen to popularity among biologists during the past decade. These techniques keep pushing the physical boundaries of optical resolution toward the molecular scale. Thereby, they enable biologists to image cellular and tissue structures at a level of almost molecular detail that was previously only achievable using electron microscopy. All the while, they retain the advantages of light microscopy, in particular with regards to sample preparation and flexibility of imaging. Commercially available SRM setups have become more and more available and also increasingly sophisticated, both in terms of optical performance and, importantly, ease of use. Institutional microscopy core facilities now offer widespread access to this type of systems. However, the field has grown so rapidly, and keeps growing, that biologists can be easily overwhelmed by the multitude of available techniques and approaches. From this vast array of SRM modalities, STED stands out in one respect: it is essentially an extension to an advanced confocal microscope. Most experienced users of confocal microscopy will find the transition to STED microscopy relatively easy as compared with some other SRM techniques. This also applies to STED sample preparation. Nonetheless, because resolution in STED microscopy does not only depend on the wavelength of the incident light and the numerical aperture of the objective, but crucially also on the square root of the intensity of the depletion laser and, in general, on the photochemical interaction of the fluorophore with the depletion laser, some additional considerations are necessary in STED sample preparation. Here we describe the single color staining of the somatostatin receptor subtype 2A (SSTR2A) and dual color staining of the trans-Golgi-network protein TGN 38 and the t-SNARE syntaxin-6 for STED in the endocrine cell line AtT20 and STED imaging of the samples, providing the protocols in as general a form as possible. The protocols in this chapter are used in this way in an institutional microscopy core facility.

A shared oligodendrocyte activation state associated with neuroinflammation and neurodegeneration.

Alzheimer's disease (AD) is a devastating neurodegenerative disease. Despite intensive research, all current therapeutic attempts have failed, highlighting the need for new therapeutic approaches. While most research traditionally attributed cognitive impairment to neuronal damage, recent studies have highlighted the role of non-neuronal cells, mainly the brain innate-immune cells, the microglia. However, the contribution of non-immune glial cells remains elusive.Oligodendrocytes are the central nervous system myelin-forming cells and are crucial for proper neuronal signaling and brain function. However, little is known about their fate in AD. In our study, we aimed to reveal cellular heterogeneity among non-immune glia, and particularly oligodendrocytes, in AD. To this end, we used the 5xFAD model of AD and performed single cell RNA-sequencing, bulk RNA-sequencing, computational meta-analysis and immunofluorescence stainings in mouse and human AD brain. We found that oligodendrocytes are the most affected cell type and unveiled a novel oligodendrocyte cell-state, which we termed 'Disease-associated OLigodendrocytes' (DOLs). DOLs were found in the AD mouse brains but not in the wild-type (WT), spatially associated with amyloid-beta plaques and increased with disease progression. DOL signature points at possible interactions with the immune system and immune-related signaling, plaque propagation, and blood-brain-barrier interference. Meta-analysis across other neuroinflammatory and neurodegenerative conditions revealed a conserved oligodendrocyte response similar to DOLs. Bulk RNA-sequencing of oligodendrocytes following different stimulations revealed partial similarity between DOL signature and oligodendrocyte response to specific inflammatory cues. Lastly, we showed that DOLs are present also in human AD brains but not in healthy controls. Overall, this study demonstrates that oligodendrocytes have a common response to neuroinflammation and neurodegeneration, which is recapitulated in humans, and can serve as a potential therapeutic target in multiple pathologies.

The Effect of Icariin on Human Hepatoellular Carcinomas Cell Apoptosis and Cell Cycle Arrest

Background: Natural products often have novel frameworks and unique mechanisms, It is an important way to develop new anti-tumor drugs.The paper explored the effect of Icariin on HepG2 cell apoptosis and cell cycle arrest. Material and methods: Human liver cancer HepG2 cells were studied. The biological activity of Icariin on HepG2 cells was comprehensively investigated. The mechanism was preliminarily explored from the aspects of proliferation, cell cycle, and apoptosis. Results: Icariin showed significant inhibitory effect on cell proliferation after administration, and the effect was time-and-concentration-dependent. Annexin V-PI detection showed that, after 48 hours of administration of Icariin at different concentrations, the apoptosis rate of HepG2 cells increased in a concentration-dependent manner. The results of Hoechst 33342 staining showed that, after 48 hours of intervention with Icariin at different concentrations, HepG2 cells appeared densely stained and granular fluorescence, characterizing apoptosis. In the JC-1 mitochondrial membrane potential experiment, Icariin was found to destroy cell mitochondrial membrane potential and induce HepG2 cell apoptosis. After 48 h administration of Icariin at different concentrations, Bcl-2 and survivin proteins were down-regulated, and Bax was up-regulated, both in a concentration-dependent manner. PI single staining combined with flow cytometry to detect cell cycle results showed that, Icariin can induce G2/M phase arrest of HepG2 cells, and is time-and-concentration-dependent. Western Blot detection revealed that Icariin can down-regulate the cycle-related proteins Cyclin B and CDK1 in a concentration-dependent manner, and also significantly down-regulate the expressions of p-AKT, AKT, p-ERK and ERK proteins. Conclusion: Icariin is a selectively potential active compound that can treat liver cancer. The paper provided theoretical basis and experimental support for the clinical application of Icariin in the drug treatment of liver cancer. It is necessary to further study the antitumor effect of Icariin, explore and find the target, and provide higher selectivity for the treatment of liver cancer by Icariin.

The Effectiveness of Dual-Staining Immunohistochemistry in the Detection of Mantle Cell Lymphoma in the Bone Marrow.

Immunohistochemistry (IHC) is a useful method for mantle cell lymphoma (MCL) detection in the bone marrow (BM). However, recognition of the neoplastic B cells can be challenging, especially when there is low-level disease. We examined BM from 105 patients with MCL. IHC was performed using cyclin D1/CD79a and PAX5/CD5 dual stains, which were compared with single stains that included CD20, CD79a, cyclin D1, and CD5 and with multiparameter flow cytometry (FC). Based on the FC data, the overall sensitivity of the dual IHC stains was 95.6%. Both dual IHC stains showed better efficacy for detecting MCL cells compared with the aggregated single stains (P = .012). While three cases were positive by FC analysis but negative for dual staining, four cases showed cells positive for cyclin D1/CD79a and PAX5/CD5 dual staining that were not detected by FC. Two of these latter cases were in patients with minimal or focal disease involvement. Cyclin D1/CD79a and PAX5/CD5 dual IHC staining is an efficient procedure for the detection of MCL in the marrow and is particularly helpful in low-level or focal involvement by MCL. This approach can be particularly useful when marrow aspirates are inadequate or unavailable.

Association of GMF-β expression with Ki-67 and its significance in the prognostic evaluation of astrocytoma

Objective: To clarify the correlation of the expression of glia maturation factor-β (GMF-β) with Ki-67 in astrocytoma, and to investigate the prognostic implications of combined detection of GMF-β and Ki-67. Methods: One hundred and forty human astrocytoma samples (WHO Ⅱ-Ⅳ grade) were collected at Southwest Hospital, Army Medical University (the Third Military Medical University), China from 2006 to 2009. Clinicopathological information and 3-year follow-up data were collected. Expression of GMF-β and Ki-67 was detected by single and double immunohistochemical staining, then the association of GMF-β expression with Ki-67 and its significance in prognostic evaluation of astrocytoma were statistically analyzed. Results: GMF-β expression in astrocytoma cells was correlated to both tumor grade and Ki-67 (both P<0.05); Kaplan-Meier survival analysis showed that GMF-β and Ki-67 expression were negatively correlated to the 3 year-survival rates, respectively (both P<0.01). Further analysis demonstrated that the two factors were co-influenced on survival, showing a trend of "GMF-βlow Ki-67low>GMF-βhigh Ki-67low>GMF-βlow Ki-67high>GMF-βhigh Ki-67high" in 3-year survival rate with significant intergroup differences (P<0.05, P<0.01). Conclusions: GMF-β expression is positively associated with Ki-67 in astrocytoma. Combined detection of GMF-β and Ki-67 can predict prognosis of patients with glioma.

Immune Correlates of Non-Necrotic and Necrotic Granulomas in Pulmonary Tuberculosis: A Pilot Study

A granuloma, a pathologic hallmark of tuberculosis (TB), is a complex cellular structure that develops at the site of Mycobacterium tuberculosis (Mtb) infection and is comprised of different immune cell types. Severe pulmonary TB in humans is characterized by the presence of heterogeneous granulomas, ranging from highly cellular to solid/non-necrotic and necrotic lesions, within the lungs. The host-Mtb interactions within the granulomas dictate the containment of Mtb infection or its progression into a necrotic, cavitary disease. However, the immune environment in various granulomas is poorly understood. The myeloid-derived suppressor cells (MDSCs) are key immune cells that regulate the protective versus permissive host responses against Mtb infection. However, their contexture within the lung granulomas remains unclear. In this study, using single and multiplex immunohistochemical staining, we analyzed the distribution of MDSCs, macrophages, CD4+ T cells and their immunometabolic and effector function states in the solid/non-necrotic and necrotic granulomas in patients with active pulmonary TB. We found increased MDSCs with elevated expression of immunosuppressive molecules in the solid/non-necrotic granulomas. In contrast, cells in the solid and necrotic granulomas produced similar levels of IL-6 and IL-10. Our findings suggest that MDSCs are present in solid/non-necrotic granuloma, which may play an essential role in the progression into a necrotic lesion, thus exacerbating disease pathology and transmission.

A novel 5x multiplex immunohistochemical staining reveals PSMA as a helpful marker in prostate cancer with low p504s expression.

The ability to combine multiple immunohistochemical (IHC) markers within a single tissue section facilitates the evaluation and detection of co-expressions, while saving tissue. A newly developed 5x multiplex (MPX) IHC staining of five different IHC markers (Basal cell cocktail (34βE12 + p63), p504s (SP116), ERG (EPR3864), Ki-67 (30−9), PSMA (EP192)) was applied on whole sections of n = 37 radical prostatectomies (RPE) including normal and cancerous tissue. Four different colors including brown, magenta, yellow and teal coded for different stainings, whereas magenta was used twice for nuclear Ki-67 and cytosolic / membranous PSMA. The staining of multiplex IHC was compared to single stains of ERG, PSMA and p504s. The proper staining of the basal cell cocktail and Ki-67 could be assessed by internal positive controls in the multiplex staining. The proportion of PSMA and p504s expression revealed a significant correlation between multiplex and single stains (p < 0.01) as well as a concordant staining pattern for ERG (n = 14 prostate cancers were identified ERG positive with both methods). Our proof of concept study demonstrates a robust staining pattern of all five different antibodies with this newly developed 5x MPX IHC. This approach facilitates the recognition of prostate cancer, in particular by adding PSMA in cases with low p504s expression.