Heat-induced Antigen Retrieval Research Articles

Abstract 2744: Exploring the role of the MSLN/MUC16 interaction in cancer progression via the in situ proximity ligation technique

Abstract Introduction: Mesothelin (MSLN) is a glycoprotein present on the surface of mesothelial cells, sometimes used as a tumor marker. Its overexpression in various cancers, including mesothelioma, ovarian, pancreatic, and lung, is linked to increased tumor aggressiveness, evasion of apoptosis, metastasis, and resistance to treatments. Mucin16 (MUC16) belongs to a family of proteins that play a crucial role in cell adhesion, signaling, and immune responses. In cancer, alterations in mucin expression and structure may contribute to tumor progression and metastasis by enhancing cell survival and immune evasion. Mesothelin is known to interact with mucins, influencing tumor cell behavior and metastatic potential. More specifically, the MSLN/MUC16 interaction facilitates tumor cell invasion and peritoneal spread in ovarian cancer. However, reliable molecular tools to study protein interactions have been scarce. Here, we relied on the Naveni® in situ proximity ligation technology to identify MSLN/MUC16 in various cancer samples. The method detects proteins located within interaction range (<40 nm) by means of antibodies conjugated to oligonucleotides that create amplified fluorescent signal. Since the technique does not compromise the structural integrity of tissues, communication between proteins can be studied in their native environment. Materials & Methods: Human FFPE tissue microarrays (TMA) with ovarian primary carcinoma and matched metastatic cancer (80 cores) and ovarian cancer tissue with normal tissue (100 cores) were deparaffinized and subjected to standard heat-induced antigen retrieval and blocking. Staining against the MSLN/MUC16 interaction was performed with the NaveniFlex Tissue MR Atto647N kit (Navinci Diagnostics). Co-staining antibodies against pan-Cytokeratin and Ki67 were included in the detection mix. Immunofluorescence (IF), using the same MSLN and MUC1 primary antibodies, was completed on an additional slide of each TMA, and then secondary antibodies conjugated to Alexa Fluor™ 488 and 594 were used for detection. Imaging was performed on an Olympus scanner VS200. Results & Discussion: In this study, we showed that the interaction is abundant in ovarian cancer, with a distinguishable difference in the signal presence between cancerous and normal adjacent tissue. Furthermore, the additional tumor marker co-stainings give insights into the tumor invasion and growth. When comparing the Naveni® signal of MSLN/MUC16 against IF staining of each, the Naveni® signal shows true interactions, as opposed to expression patterns. Understanding the roles of mesothelin and mucin in cancer, both individually and in interaction, is crucial for developing targeted therapies and diagnostic tools. Further research on their interaction and specific molecular pathways triggered by it is essential for devising effective strategies to combat cancer progression. Citation Format: Sara Bodbin, Doroteya Raykova, Agata Zieba-Wicher. Exploring the role of the MSLN/MUC16 interaction in cancer progression via the in situ proximity ligation technique [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2744.

Comparing Heat-Induced Antigen Retrieval Methods for ER Expression in Invasive Breast Carcinoma

In the realm of cancer diagnosis and treatment, immuno-histochemical staining has become a pivotal tool for discerning molecular markers within tissue samples. Objective: To comprehensively compare different heat-induced antigen retrieval methods for evaluating estrogen receptor (ER) expression in female invasive breast carcinoma of no special type. Methods: A cross-sectional design was employed utilizing 250 formalin-fixed paraffin-embedded (FFPE) tissue samples from confirmed cases of invasive breast carcinoma. The study was conducted within Histopathology Department in CMH Peshawar. The study spanned from January 6, 2022 to June 30, 2023. Three heat-induced antigen retrieval methods were evaluated: conventional microwave-based retrieval, pressure cooking retrieval, and water bath heating. ER expression was assessed through immune-histo-chemical staining, and scores were analyzed using descriptive statistics and ANOVA. A post hoc Tukey's Honestly Significant Difference (HSD) test was conducted to identify which specific retrieval methods exhibited significant differences in ER expression scores. Results: The pressure-cooking retrieval method exhibited significantly higher mean ER expression scores (7.52) compared to the conventional microwave-based (6.87) and water bath heating methods (6.65). Post hoc analysis confirmed the substantial differences. Standard deviations and interquartile ranges were consistent with each method. Conclusions: The study underscores the pivotal role of retrieval techniques in accurate ER expression assessment in female invasive breast carcinoma of no special type. The pressure cooking method emerges as a potent enhancer of ER expression, advocating for standardized protocols to ensure consistent diagnostic outcomes and informed therapeutic decisions. Despite limitations, this research contributes to the refinement of patient care in the management of invasive breast carcinoma.

Advancements in 3D spheroid imaging: Optimised cryosectioning and immunostaining techniques

This article presents a modified protocol for embedding and sectioning spheroids and organoids, which are increasingly used in research due to their ability to emulate living tissue. The modifications aim to reduce the distortion and damage of these fragile structures during the embedding and sectioning process. The new method involves using optimized embedding containers, a modified embedding protocol, and optimized temperatures for cryosectioning. A heat-induced antigen retrieval protocol was tested and found to significantly increase immunostaining intensity without compromising spheroid integrity. The combined approach allowed for the creation of thinner cryosections, leading to clearer and more detailed images. The results suggest that the modified protocol could be widely adopted to enhance the imaging of spheroids and organoids.•Paraformaldehyde fixation of spheroids•Antigen retrieval treatment of spheroids•Embedding in freezing medium and cryosectioning

Effect of liquid-based cytology fixing solution on immunocytochemistry: Efficacy of antigen retrieval in cytologic specimens.

Immunocytochemistry (ICC) is an indispensable technique to improve diagnostic accuracy. ICC using liquid-based cytology (LBC)-fixed specimens has been reported. However, problems may arise if the samples are not fixed appropriately. We investigated the relationship between the LBC fixing solution and ICC and the usefulness of antigen retrieval (AR) in LBC specimens. Specimens were prepared from five types of LBC-fixed samples using cell lines and the SurePath™ method. ICC was performed using 13 antibodies and analyzed by counting the number of positive cells in the immunocytochemically stained specimens. Insufficient reactivity was observed using ICC without heat-induced AR (HIAR) in nuclear antigens. The number of positive cells increased in ICC with HIAR. The percentage of positive cells was lower in CytoRich™ Blue samples for Ki-67 and in CytoRich™ Red and TACAS™ Ruby samples for estrogen receptor and p63 than in the other samples. For cytoplasmic antigens, the percentage of positive cells for no-HIAR treatment specimens was low in the three antibodies used. In cytokeratin 5/6, the number of positive cells increased in all LBC specimens with HIAR, and the percentage of positive cells in CytoRich™ Red and TACAS™ Ruby samples was significantly lower (p < .01). For cell membrane antigens, CytoRich™ Blue samples had a lower percentage of positive cells than the other LBC-fixed samples. The combination of detected antigen, used cells, and fixing solution may have different effects on immunoreactivity. ICC using LBC specimens is a useful technique, but the staining conditions should be examined before performing ICC.

Disease-Specific α-Synuclein Seeding in Lewy Body Disease and Multiple System Atrophy Are Preserved in Formaldehyde-Fixed Paraffin-Embedded Human Brain.

Recent studies have been able to detect α-synuclein (αSyn) seeding in formaldehyde-fixed paraffin-embedded (FFPE) tissues from patients with synucleinopathies using seed amplification assays (SAAs), but with relatively low sensitivity due to limited protein extraction efficiency. With the aim of introducing an alternative option to frozen tissues, we developed a streamlined protein extraction protocol for evaluating disease-specific seeding in FFPE human brain. We evaluated the protein extraction efficiency of different tissue preparations, deparaffinizations, and protein extraction buffers using formaldehyde-fixed and FFPE tissue of a single Lewy body disease (LBD) subject. Alternatively, we incorporated heat-induced antigen retrieval and dissociation using a commercially available kit. Our novel protein extraction protocol has been optimized to work with 10 sections of 4.5-µm-thickness or 2-mm-diameter micro-punch of FFPE tissue that can be used to seed SAAs. We demonstrated that extracted proteins from FFPE still preserve seeding potential and further show disease-specific seeding in LBD and multiple system atrophy. To the best of our knowledge, our study is the first to recapitulate disease-specific αSyn seeding behaviour in FFPE human brain. Our findings open new perspectives in re-evaluating archived human brain tissue, extending the disease-specific seeding assays to larger cohorts to facilitate molecular subtyping of synucleinopathies.

Heat-induced antigen retrieval utilizing modified Tris-EDTA buffer for reprobing of Western blots on nitrocellulose paper

The development of heat-induced antigen retrieval technologies with Tris-EDTA buffer has dramatically improved immunostaining of specific antigens for routine immunohistochemical detection (Krenacs et al., 2010) [1]. However, little evidence exists on whether heat-Induced antigen retrieval utilizing Tris-EDTA buffer can strip western blot (WB) membranes and allow sequential reprobing. Here, we serendipitously discover that ∼95 °C Tris-EDTA buffer with 0.01% Tween 20 could repeatedly strip the Nitrocellulose membranes (NC). After electroblotting, NC blots were soaked into Tris-EDTA stripping buffer (∼95 °C, 10–25min) and we could perform at least five rounds (the following antibodies used: Vinculin, Atg7, Caspase-3, UBA5, JNK and ERK1/2) stripping in sequential chemiluminescent detections. The NC membranes also show clear western signals and background without losing transferred proteins during the reprobing process of WB. Hence, this study report additional new roles of the heat-Induced antigen retrieval Tris-EDTA buffer with 0.01% Tween 20. The method is simpler, more affordable and harmless for the nitrocellulose paper, which will be helpful for effective reprobing in western blotting applications.

Direct Immunofluorescence of IgG on Formalin-Fixed Paraffin-Embedded Tissue by Heat-Induced Antigen Retrieval as a Sensitive Method for the Diagnosis of Pemphigus.

Direct immunofluorescence (DIF) on frozen sections (DIF-F) plays a key role in the identification and differential diagnosis of bullous dermatoses, which are a group of critical autoimmune diseases that include pemphigus, bullous pemphigoid (BP), and epidermolysis bullosa acquisita (EBA). However, this technique requires specialized laboratory equipment conditions, sample acquisition and sample preservation. In this study, the application value of DIF on paraffin-embedded tissue sections (DIF-P) detecting IgG using heat-induced antigen retrieval (HIAR) in the diagnosis of bullous dermatosis was explored. Samples from 12 patients with pemphigus vulgaris (PV), 10 patients with pemphigus foliaceus (PF), 17 patients with BP, and 4 patients with EBA were retrospectively studied for DIF-P IgG detection. Formalin-fixed, paraffin-embedded tissue (FFPE) was used, and the antigen retrieval method used in the experiment was HIAR. All patients were diagnosed with the autoimmune bullous disease (AIBD) based on clinical presentation, histopathology, DIF-F, and enzyme-linked immunosorbent assay (ELISA). Intercellular staining for IgG in the epidermis was successful in paraffin-embedded tissue sections in 11 of 12 PV samples and in all 10 PF samples. IgG at the basement membrane zone (BMZ) was not detected by immunofluorescent staining in 17 BP samples and 4 EBA samples. The detection of IgG by DIF-P using HIAR can be used for the diagnosis of pemphigus as an alternative method to DIF-F.

Abstract P2-26-15: CCR7 expression in Inflammatory breast cancer

Abstract Background: Inflammatory breast cancer is an aggressive breast cancer characterized by florid congestion of lymphovascular spaces by tumor emboli. CCR7 is an immune cell receptor that mediates traffic of immune cells into lymphatics that can be expressed on tumor cells. An RNA-seq screen of tumor promoting mammary glands in mice identified CCR7 as an upregulated signal in mammary glands that promoted IBC-like skin invasion. We examined the expression of CCR7 in IBC cell lines and patients to determine the prevalence of this receptor in IBC. Methods: Protein lysates from IBC and non-IBC cell lines were subjected to immunoblotting using anti CCR7 (R&amp;D systems). An IBC tissue microarray from post-chemotherapy mastectomy specimens of 39 patients, each with three replicates was subjected to immunohistochemical staining for CCR7 (Invitrogen, catalog number MA5-31992) performed using a Leica Bond RX autostainer with an incubation time of 60 minutes at 1:15,000 after 20 minutes of heat-induced antigen retrieval at pH 6.0. In 15 cases there were no tumor cells observed in the cores. Staining was scored by an expert breast pathologist for intensity and percent tumor stained. Staining patterns were scored. Descriptive statistics were examined for representation by receptor subtype. Results: CCR7 staining was strong in all lines examined including MCF7, SUM149, SUM190, MDA-IBC, and SUM159. Among 24 IBC patient cores with tumor in the tissue, 23 (96%) expressed CCR7 in tumor, 15 with complete membranous staining and 9 with incomplete membranous staining. In one case with LVSI the emboli were CCR7 positive. Among the 24 positive cases, 22 were 3+ intensity while two were 2+. Nine CCR7+ cases were estrogen receptor (ER) ER+, 8 ER-, and 6 unknown. HER2 status is pending. Conclusions: CCR7 expression is present across tumor subtypes in IBC cell lines and in both ER+ and ER- patient tumors. Given developing novel pharmacologic targeting of CCR7, this target warrants further investigation in IBC and other breast cancers. Citation Format: Wintana Balema, Savitri Krishnamurthy, Alison Lawrence, Megan Rodriguez, Richard Larson, Natalie Fowlkes, Naoto T. Ueno, Wendy Woodward. CCR7 expression in Inflammatory breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-26-15.

The expression of HER2/neu in patients with lung cancer and its associated factors.

Lung cancer is the most common cause of cancer-related death worldwide in both sexes. Evidence suggests the role of genetic factors in lung cancer. Studying of such factors can help understand the cancer prognosis. Overexpression of the human epidermal growth factor receptor-2 (HER2/neu) protein is considered an important prognostic factor in breast cancer, but its role has not been confirmed in lung cancer. Therefore, the present study aimed to investigate the role of its expression in patients with lung cancer. In this cross-sectional study, patients aged >18 years who were referred to Afzalipoor Hospital, Kerman, Iran, from 2016 to 2017, and were diagnosed with lung cancer were enrolled into the study if they had a pathological sample of their cancerous lung. Their demographics were recorded, and the sample was sectioned and stained to measure HER2/neu gene expression according to DAKO instructions using heat-induced antigen retrieval (HIER) enzyme marker. The samples of 100 patients with lung cancer were evaluated (84% men and 16% women) with a mean age of 61.34 years (standard deviation of 12.51 years). HER2/neu expression was significantly associated with the type of cancer and was highest in adenocarcinoma and zero in small cell carcinoma (P < 0.001), but not with patients' sex, age, smoking status and family history of cancer (P > 0.05). These results emphasized the overexpression of HER2/neu in different types of lung cancer, which can be used further for therapeutic purposes. The results showed that HER2/neu was overexpressed not only in adenocarcinoma but also in other types, like squamous cell carcinoma. Therefore, all subtypes of non-small cell lung carcinoma should be considered for anti-HER2 therapies, and further research is required for small cell lung carcinoma.

Multiplex Immunochromogenic Tissue Staining Employing Primary Antibodies from the Same Species.

Chromogenic immunohistochemistry (IHC) serves as an essential assay for the diagnoses of many diseases including cancer. Single-marker IHC detection is the standard used for clinical diagnostic assays. A technology to stain multiple biomarkers chromogenically on a single tissue will also yield contextual biomarker information. Methods to chromogenically stain multiple biomarkers simultaneously employing antibodies from the same species are limited and require complex protocols. Here we describe both manual and automated protocols using the UltraPlex™ mxIHC technology that allows simultaneous detection of up to three biomarkers on a single tissue using a single heat-induced antigen retrieval step in formaldehyde-fixed paraffin-embedded (FFPE) tissue and using primary antibodies from any species.

Optimized Mouse-on-mouse Immunohistochemical Detection of Mouse ESR2 Proteins with PPZ0506 Monoclonal Antibody.

Despite the physiological significance of ESR2, a lack of well-validated detection systems for ESR2 proteins has hindered progress in ESR2 research. Thus, recent identification of a specific anti-human ESR2 monoclonal antibody (PPZ0506) and its specific cross-reactivity against mouse and rat ESR2 proteins heightened momenta toward development of appropriate immunohistochemical detection systems for rodent ESR2 proteins. Building upon our previous optimization of ESR2 immunohistochemical detection in rats using PPZ0506, in this study, we further aimed to optimize mouse-on-mouse immunohistochemical detection using PPZ0506. Our assessment of several staining conditions using paraffin-embedded ovary sections revealed that intense heat-induced antigen retrieval, appropriate blocking, and appropriate antibody dilutions were necessary for optimization of mouse-on-mouse immunohistochemistry. Subsequently, we applied the optimized immunostaining method to determine expression profiles of mouse ESR2 proteins in peripheral tissues and brain subregions. Our analyses revealed more localized distribution of mouse ESR2 proteins than previously assumed. Moreover, comparison of these results with those obtained in humans and rats using PPZ0506 revealed interspecies differences in ESR2 expression. We expect that our optimized methodology for immunohistochemical staining of mouse ESR2 proteins will help researchers to solve multiple lines of controversial evidence concerning ESR2 expression.

Development of monoclonal antibodies specific to Marek disease virus-EcoRI-Q (Meq) for the immunohistochemical diagnosis of Marek disease using formalin-fixed, paraffin-embedded samples.

Marek disease (MD) is a viral disease characterized by the development of lymphoma in poultry. Although morphologic confirmation of lymphoma is used to diagnose MD, immunohistochemical detection of MD virus-EcoRI-Q (Meq), which is a viral protein that is expressed exclusively in MD tumor cells, would further improve the accuracy of diagnosis. We developed monoclonal antibodies (mAbs) that specifically detect Meq by immunohistochemistry (IHC) using formalin-fixed, paraffin-embedded (FFPE) sections. We evaluated the sensitivity and specificity of 14 mAbs that we produced, using FFPE samples of MDCC-MSB1 cells, MD tumor tissues, and tissues of uninfected chickens. Four different antigen retrieval conditions were investigated. Thirteen mAbs reacted with Meq in FFPE sections, but immunohistochemical reactivity and specificity varied depending on the mAb and antigen retrieval condition; heat-induced antigen retrieval (HIAR) was more effective at detecting Meq than the other tested conditions. HIAR pH 9 tended to increase immunoreactivity and decrease specificity. Of the 5 mAbs that immunoreacted strongly with Meq without nonspecific reactions under the optimal antigen retrieval conditions, 3 mAbs (1C1-121, 3A3-112, 5F7-82) did not produce background staining of tumor or non-tumor tissues; 2 mAbs (2C5-11, 4A5-54) produced background staining. The mAb 6B5-128 reacted moderately with Meq without nonspecific reactions and background staining. The remaining mAbs showed weak immunoreactivity or problematic nonspecific reactions. Our results suggest that some of our developed mAbs can be used in IHC to detect Meq in FFPE sections with high specificity, and that the use of IHC may greatly improve the diagnosis of MD.

Antibody Staining for Nematodes with Heat-induced Antigen Retrieval (HIAR).

Immunocytochemistry remains a valuable and necessary tool for biologists working with nematodes, even those nematode model organisms with advanced molecular genetic tools and transgenics. Because of the highly idiosyncratic nature of successful immunostaining procedures, innovations can still be found for this long-established technique. Heat-induced antigen retrieval (HIAR) is well known from other systems, but seems not to have been applied to antibody staining in nematodes. For some antigens, adding HIAR to an established antibody staining protocol for nematodes can reveal strong and reliable staining that without HIAR is poor or completely absent.

Heat-induced antigen retrieval in fluorescence in situ hybridization: An effective approach enhancing signal intensity in poor-quality FFPE sections.

Fluorescence in situ hybridization (FISH) serves as an ancillary tool for assessing chromosomal abnormalities and is important in differential diagnoses and treatment decisions. In clinical practice, pathologists encounter unsatisfactory formalin-fixed paraffin-embedded (FFPE) sections exhibiting weak fluorescence signals, mostly due to inappropriate tissue processing or preservation, leading to interpretation difficulties. For the present study, FFPE samples for which conventional FISH failed were collected. Instead of a pretreatment step using a commercial kit, heat-induced antigen retrieval (HIAR) was introduced using either citrate buffer or Tris-EDTA buffer, while the subsequent experimental workflow remained unchanged. After HIAR-assisted FISH, the hybridization efficiency and signal intensity were markedly enhanced and no difference in signal adequacy was observed when comparing the effect of the two AR solutions. The present study demonstrated that HIAR is a reliable tool for FISH, particularly for poor-quality FFPE sections yielding weak or no fluorescence signals in the conventional analysis.

Sequential Indirect Dual Immunohistochemistry with Primary Rabbit Antibodies on Cochlear Sections Using an Intermediate Heat-Denaturation Step.

Advanced immunohistochemical (IHC) protocols aim to visualize different molecules in situ simultaneously. These techniques are of utmost importance as a first step in studying possible interactions of proteins at the subcellular level. Colocalized stains in tissue sections indicate proximity of two proteins of interest. Frequently, double staining protocols are restricted by the lack of primary antibodies generated in different animal species for indirect IHC visualization. Here, we present a detailed protocol for mouse inner ear tissue using two different primary rabbit antibodies directed against transmembrane ion channel proteins of cochlear neurons. The two antibodies are combined for fluorescence (confocal) as well as dual multiplex colorimetric visualization in two sequential single IHC stainings. A heat-denaturation step is performed in between. Primary antibody specificity is tested by preadsorption with the immunogenic peptide, and positive and negative tissue controls are performed to confirm the reliability of the antibody detection. We describe the whole procedure in detail beginning with tissue extraction of the mouse inner ear and continuing with chemical fixation, cryoembedding, and preparation for manual and fully automated immunostaining, including steps for heat-induced antigen retrieval. The potential to use antibodies from the same host species for single and double IHC staining opens up multiple possibilities for detecting different targets in the same tissue section using resources and materials that are widely available. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Tissue preparation, cryoembedding, and sectioning Basic Protocol 2: Double colorimetric immunostaining with an automatic immunostainer Basic Protocol 3: Double manual fluorometric immunostaining with fluorescence.

Defective HLA Class I Expression and Patterns of Lymphocyte Infiltration in Chordoma Tumors.

There are no effective systemic therapies for chordoma. The recent successes of immunotherapeutic strategies in other cancers have resulted in a resurgence of interest in using immunotherapy in chordoma. These approaches rely on a functional interaction between the host's immune system and the expression of tumor peptides via the human leukocyte antigen (HLA) Class I antigen. It is not known whether chordoma cells express the HLA Class I antigen. (1) Do chordoma tumors exhibit defects in HLA Class I antigen expression? (2) What is the pattern of lymphocyte infiltration in chordoma tumors? Patients with chordoma treated at Massachusetts General Hospital between 1989 and 2009 were identified with permission from the institutional review board. Of the 75 patients who were identified, 24 human chordoma tumors were selected from 24 distinct patients based on tissue availability. Histology slides from these 24 formalin-fixed paraffin-embedded chordoma tissue samples were deparaffinized using xylene and ethanol and underwent heat-induced antigen retrieval in a citrate buffer. Samples were incubated with monoclonal antibodies directed against HLA Class I antigen processing machinery components. Antibody binding was detected via immunohistochemical staining. Staining intensity (negative, weakly positive, strongly positive) was assessed semiquantitatively and the percentage of chordoma cells stained for HLA Class I antigen subunits was assessed quantitatively. Hematoxylin and eosin-stained histology slides from the same 24 chordoma samples were assessed qualitatively for the presence of tumor-infiltrating lymphocytes and histologic location of these lymphocytes. Immunohistochemical staining with monoclonal antibodies directed against CD4 and CD8 was performed in a quantitative manner to identify the lymphocyte subtype present in chordoma tumors. All results were scored independently by two investigators and were confirmed by a senior bone and soft tissue pathologist. Seven of 24 chordoma samples exhibited no staining by the anti-HLA-A heavy chain monoclonal antibody HC-A2, two had weak staining intensity, and eight had a heterogeneous staining pattern, with fewer than 60% of chordoma cells exhibiting positive staining results. Four of 24 samples tested were not stained by the anti-HLA-B/C heavy chain monoclonal antibody HC-10, five had weak staining intensity, and 11 displayed a heterogeneous staining pattern. For the anti-β-2-microglobulin monoclonal antibody NAMB-1, staining was detected in all samples, but 11 had weak staining intensity and four displayed a heterogeneous staining pattern. Twenty-one of 24 samples tested had decreased expression in at least one subunit of HLA Class I antigens. No tumors were negative for all three subunits. Lymphocytic infiltration was found in 21 of 24 samples. Lymphocytes were primarily found in the fibrous septae between chordoma lobules but also within the tumor lobules and within the fibrous septae and tumor lobules. Twenty-one of 24 tumors had CD4+ T cells and 11 had CD8+ T cells. In chordoma tissue samples, HLA Class I antigen defects commonly were present, suggesting a mechanism for escape from host immunosurveillance. Additionally, nearly half of the tested samples had cytotoxic CD8+ T cells present in chordoma tumors, suggesting that the host may be capable of mounting an immune response against chordoma tumors. The resulting selective pressure imposed on chordoma tumors may lead to the outgrowth of chordoma cell subpopulations that can evade the host's immune system. These findings have implications in the design of immunotherapeutic strategies for chordoma treatment. T cell recognition of tumor cells requires HLA Class I antigen expression on the targeted tumor cells. Defects in HLA Class I expression may play a role in the clinical course of chordoma and may account for the limited or lack of efficacy of T cell-based immunity triggered by vaccines and/or checkpoint inhibitors.

Tropism of Severe Acute Respiratory Syndrome Coronavirus 2 for Barrett’s Esophagus May Increase Susceptibility to Developing Coronavirus Disease 2019

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Severe Acute Respiratory Syndrome Coronavirus 2 Attachment Receptor Angiotensin-Converting Enzyme 2 Is Decreased in Crohn’s Disease and Regulated By Microbial and Inflammatory Signaling

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Spatially resolved analysis of FFPE tissue proteomes by quantitative mass spectrometry.

Bottom-up mass spectrometry-based proteomics relies on protein digestion and peptide purification. The application of such methods to broadly available clinical samples such as formalin-fixed and paraffin-embedded (FFPE) tissues requires reversal of chemical crosslinking and the removal of reagents that are incompatible with mass spectrometry. Here, we describe in detail a protocol that combines tissue disruption by ultrasonication, heat-induced antigen retrieval and two alternative methods for efficient detergent removal to enable quantitative proteomic analysis of limited amounts of FFPE material. To show the applicability of our approach, we used hepatocellular carcinoma (HCC) as a model system. By combining the described protocol with laser-capture microdissection, we were able to quantify the intra-tumor heterogeneity of a tumor specimen on the proteome level using a single slide with tissue of 10-µm thickness. We also demonstrate broader applicability to other tissues, including human gallbladder and heart. The procedure described in this protocol can be completed within 8 d.

Identification of Neutrophil Extracellular Traps in Paraffin-Embedded Feline Arterial Thrombi using Immunofluorescence Microscopy.

Neutrophil extracellular traps (NETs), composed of cell-free DNA (cfDNA) and proteins like histones and neutrophil elastase (NE), are released by neutrophils in response to systemic inflammation or pathogens. Although NETs have previously been shown to augment clot formation and inhibit fibrinolysis in humans and dogs, the role of NETs in cats with cardiogenic arterial thromboembolism (CATE), a life-threatening complication secondary to hypertrophic cardiomyopathy, is unknown. A standardized method to identify and quantify NETs in cardiogenic arterial thrombi in cats will advance our understanding of their pathological role in CATE. Here, we describe a technique to identify NETs in formaldehyde-fixed and paraffin-embedded thrombi within the aortic bifurcation, extracted during necropsy. Following deparaffinization with xylene, aortic sections underwent indirect heat-induced antigen retrieval. Sections were then blocked, permeabilized, and ex vivo NETs were identified by colocalization of cell-free DNA (cfDNA), citrullinated histone H3 (citH3), and neutrophil elastase (NE) using immunofluorescence microscopy. To optimize the immunodetection of NETs in thrombi, autofluorescence of tissue elements was limited by using an autofluorescence quenching process prior to microscopy. This technique could be a useful tool to study NETs and thrombosis in other species and offers new insights into the pathophysiology of this complex condition.