Paraffin-embedded Sections Research Articles

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.

133 Anatomical evidence for kisspeptin and dopaminergic neuron interaction in the diestrous mare

Dopamine (DA), via dopaminergic neurons, modulates prolactin secretion from the pituitary by tonic inhibition of lactotropes. Previously, kisspeptin (Kp) attenuated the prolactin response to the DA antagonist, sulpiride, in diestrous mares and enhanced the prolactin response to sulpiride in estradiol-primed mares. Whether or not Kp can modulate prolactin by acting on DA neurons has yet to be described in horses; therefore, the objective of this study was to determine if Kp and kisspeptin receptor (Kiss1r) co-localize with DA neurons, thus providing evidence for a direct action of Kp on DA neurons. Hypothalami from 4 light-horse diestrous mares, aged 6 to 21, were obtained following euthanasia. Mares were determined to be diestrous based on presence of corpora lutea and progesterone concentrations. Immediately following euthanasia, the carotid artery was perfused with sodium nitrite and 4% paraformaldehyde before decapitation and dissection of hypothalami. Immunofluorescent double labeling of tyrosine hydroxylase (TH) and Kp, as well as TH and Kiss1r, was carried out on paraffin-embedded hypothalamic sections using validated antibodies. Whole slide scans were analyzed for double-labeled cells; percentage of DA neurons colocalized with Kp-ir cell bodies and fibers, as well as percentage of DA neurons colocalized with Kiss1r-ir cells, were calculated and averaged for multiple sectionsper horse from rostral to caudal throughout the hypothalamus. Similarly, percentage and number of Kp or Kiss1r-ir cell bodies co-colocalized with TH-ir cells were calculated for different hypothalamic regions: the arcuate nucleus (Arc), ventromedial nucleus (VMH), and dorsomedial nucleus (DMH), and compared using Kruskall Wallis with Dunn's multiple comparison test. DA neurons were observed lining the third ventricle and spread from the Arc to the DMH, with little to none present in the posterior nucleus. TH and Kp expressing cell bodies co-localized between 8 and 30%, with less in the caudal DMH (P ≤ 0.05) compared with the Arc. Kp fibers synapsed with TH-expressing cell bodies at a low percentage, between 0 and 16%, with less connections in the DMH (P ≤ 0.05) than in the ARC. TH and Kiss1r co-localized between 20 and 65%, with similar distribution throughout the Arc, VMH, and DMH in rostral sections. In caudal hypothalamic sections, co-localization was greater (P ≤ 0.05) in the Arc compared with other areas that express lower or no DA neurons. Co-localization of TH and Kiss1r as well as co-localization and synaptic attachment of Kp on TH positive cells suggests a direct effect of kisspeptin on DA neurons to modulate prolactin release.

Immunohistochemistry of Leukemia Inhibitory Factor and Integrin αVβ3 in Mouse Endometrium Following Kisspeptin-54 Ovulation Trigger.

Kisspeptin (KP) is a group of hypothalamic neuropeptides encoded by KISS-1 gene. KP-54, a 54-amino-acid peptide, helps regulate the hypothalamic-pituitary-ovarian axis and plays a potential role in implantation. C57BL/6J female mice were superovulated via intraperitoneal injection of 5 International Units (IU) pregnant mare serum gonadotrophin (day 1). Forty-eight hours later, mice (5/group) were injected with phosphate-buffered saline (PBS) (group A), 5IU human chorionic gonadotrophin (hCG) (group B), or 3nmol KP-54 (group C). On day 7, mice were euthanized and uteri excised to create paraformaldehyde-fixed paraffin-embedded sections that were immunostained for the implantation markers: leukemia inhibitory factor (LIF) and integrin αVβ3 (ITG αVβ3). Slides were scored for intensity of staining in endometrial glandular epithelium (GE) and stromal cells (SCs) via histoscore (H-score). Data were analyzed using the Kruskal-Wallis test followed by the Mann-Whitney U test for pairwise comparisons. LIF expression was significantly higher in GE and SCs of mice triggered with KP-54 compared to placebo (P = .009 for both), but only higher than hCG trigger group in SCs (P = .009). Meanwhile, ITG αVβ3 expression was significantly higher in SCs of mice triggered with KP-54 compared to placebo (P = .028). In conclusion, using KP-54 as an ovulation trigger resulted in higher expression of the implantation markers LIF and ITG αVβ3 in mice endometrium compared to hCG or placebo. This suggests a potential role for KP-54 trigger in improving embryo implantation in clinical IVF. However, further studies are needed to correlate these results with clinical implantation rates and pregnancy outcomes.

Instant Pot for antigen retrieval: a simple, safeand economical method for use in immunohistochemistry.

Here, the authors report a simple method to perform antigen retrieval using a commonly available commercial Instant Pot® for immunohistochemistry. It provides a validated alternative to previous antigen retrieval methods that employ water baths, microwave ovensor scientific-grade pressure cookers. The Instant Pot can be set to obtain a variety of desired temperatures and is straightforward to use, making it extremely amenable to optimization. The Instant Pot method is an easy, safeand inexpensive alternative means to perform immunohistochemistry on formalin-fixed paraffin-embedded sections. It has been validated using several different monoclonal antibodies including ones directed against cell surface or intracellular antigens. As a result, it should be useful for a variety of research labs as well as undergraduate laboratory courses.

Preantral follicle numbers and size in heifers carrying Trio, a bovine high fecundity allele.

The bovine high fecundity allele, Trio, results in the occurrence of multiple ovulations and is characterized by antral follicles that develop slower and acquire ovulatory capacity at smaller sizes. This study provides novel information on the effect of the Trio allele on early folliculogenesis. The bovine high fecundity allele, Trio, causes overexpression in granulosa cells (GCs) of SMAD6, an inhibitor of BMP15-activated SMAD signalling. Furthermore, the Trio allele results in antral follicles that develop slower, acquire ovulatory capacity at smaller sizes, and have three-fold greater ovulation rate compared to half-sib non-carriers. The present study was designed to determine preantral follicle numbers and size in Trio carrier and non-carrier cattle testing the hypothesis that inhibition of SMAD signalling would alter preantral follicle activation and/or growth. Ovarian tissues from Trio carrier (n = 12) and non-carrier (n = 12) heifers were obtained by laparotomy after follicle wave synchronization. Follicle numbers and dimensions were determined for each stage of development (primordial, transitional, primary, and secondary) from paraffin-embedded sections. There were no differences in the number of primordial, transitional, or secondary follicles or in antral follicle count, circulating AMH, or ovarian volume between carriers and non-carriers. Trio carriers had ~2.5-fold greater (P < 0.01) number of primary follicles than non-carriers, and transitional and primary follicles were larger (~1.2-fold; P < 0.1) in Trio carriers. Oocyte volume of primordial and transitional follicles was not different between genotypes; however, oocytes were larger (P < 0.05) in primary (~1.3-fold) and secondary (~1.8-fold) follicles for Trio carriers. Granulosa cell numbers were not different (P > 0.3) between carriers and non-carriers, irrespective of the stage of development. These results suggest that, after primordial follicle activation, follicles in Trio carrier cattle have slower progression through the primary stage, hence the larger oocyte and greater number of primary follicles.

3D Visualization, Skeletonization and Branching Analysis of Blood Vessels in Angiogenesis.

Angiogenesis is the process of new blood vessels growing from existing vasculature. Visualizing them as a three-dimensional (3D) model is a challenging, yet relevant, task as it would be of great help to researchers, pathologists, and medical doctors. A branching analysis on the 3D model would further facilitate research and diagnostic purposes. In this paper, a pipeline of vision algorithms is elaborated to visualize and analyze blood vessels in 3D from formalin-fixed paraffin-embedded (FFPE) granulation tissue sections with two different staining methods. First, a U-net neural network is used to segment blood vessels from the tissues. Second, image registration is used to align the consecutive images. Coarse registration using an image-intensity optimization technique, followed by finetuning using a neural network based on Spatial Transformers, results in an excellent alignment of images. Lastly, the corresponding segmented masks depicting the blood vessels are aligned and interpolated using the results of the image registration, resulting in a visualized 3D model. Additionally, a skeletonization algorithm is used to analyze the branching characteristics of the 3D vascular model. In summary, computer vision and deep learning is used to reconstruct, visualize and analyze a 3D vascular model from a set of parallel tissue samples. Our technique opens innovative perspectives in the pathophysiological understanding of vascular morphogenesis under different pathophysiological conditions and its potential diagnostic role.

Simple differential staining method of paraffin-embedded plant sections with safranin-alcian blue

Summary We present here a modified protocol for safranin-alcian blue staining of paraffin-embedded plant samples. After deparaffination, the double staining uses a 2:1 mix of 1% safranin and 1% alcian blue. This method is less expensive than previously published double staining protocols as it does not require absolute ethanol. Moreover, safranin-alcian blue staining creates a range of hues that allows good cell type differentiation, it gives reproducible results for a wide variety of plant tissues and is easy to use. We hope it will be found useful both for research and teaching purposes.

Mast Cells in Kidney Transplant Biopsies With Borderline T Cell-mediated Rejection and Their Relation to Chronicity.

Fourty-seven KTx biopsies (2009-2018) with borderline pathological evidence for T cell-mediated rejection according to the Banff'17 Update were retrospectively included and corresponding clinical data was collected. Immunohistochemistry for tryptase was performed on formalin-fixed paraffin-embedded sections. Cortical MCs were counted and corrected for area (MC/mm²). Interstitial fibrosis was assessed by Sirius Red staining and quantified using digital image analysis (QuPath). Increased MC number was correlated to donor age (spearman's r = 0.35, P = 0.022), deceased donor kidneys (mean difference = 0.74, t [32.5] = 2.21, P = 0.035), and delayed graft function (MD = 0.78, t [33.9] = 2.43, P = 0.020). Increased MC number was also correlated to the amount of interstitial fibrosis (r = 0.42, P = 0.003) but did not correlate with transplant function over time (r = -0.14, P = 0.36). Additionally, transplant survival 2 y post-biopsy was not correlated to MC number (mean difference = -0.02, t [15.36] = -0.06, P = 0.96). MC number in suspicious (borderline) for acute T cell-mediated rejection is correlated to interstitial fibrosis and time post-transplantation, suggesting MCs to be a marker for cumulative burden of tissue injury. There was no association between MCs and transplant function over time or transplant survival 2 y post-biopsy. It remains unclear whether MCs are just a bystander or have pro-inflammatory or anti-inflammatory effects in the KTx with minimal lesions.

The Effect of Induced Thyrotoxicosis on Avian Limb Development.

Thyroid hormone (TH) is essential for bone development. Altered TH levels such as hyperthyroidism, or thyrotoxicosis, results in the early maturation of the cartilaginous scaffold for long bone development, which can cause improper ossification. Maternal hyperthyroidism during embryonic development causes neonatal thyrotoxicosis and can therefore lead to stunted growth in infants. In our avian model of induced thyrotoxicosis, we injected saline (control) or 25ng thyroxine (T4) in 0.1 mL dose into fertilized chicken eggs on embryonic (E) days E11 and E15. On E19, chicken embryo length was measured and limbs were collected. The left limbs were whole-mount stained with Alizarin Red (bone) and Alcian Blue (cartilage). Limbs were then imaged and tibias were measured using ImageJ. The tibias from the right limbs were further dissected, embedded in paraffin, cut (5μM), and stained with Safranin O (cartilage) and Fast Green (bone). Sections were examined to determine morphological changes in the cartilaginous long bone scaffold. Body length was 4.8% shorter following thyroxine exposure (p&lt;0.01). Tibias were 2.4% shorter, though not statistically significant (p=0.36). Paraffin-embedded sections suggested increased chondrocyte hypertrophy, although further quantification is required. In conclusion, our data support thyroxine-induced changes in long bone morphology. Understanding how thyrotoxicosis can alter bone development in utero is important for developing potential targeted therapies for improving ossification in cases of neonatal thyrotoxicosis.&#x0D; &#x0D; Acknowledgment of NASA West Virginia Space Grant Consortium (Grant #80NSSC20M0055) and the Genomics Core Facility and WV-INBRE (NIH grant P20GM103434).

The relationship between Meckel's cartilage resorption and incisor tooth germ in mice.

Our understanding of the initiation and cellular mechanisms underlying endochondral resorption of Meckel's cartilage (MC) remains limited. Several studies have shown that the resorption site of MC and the mandibular incisor tooth germ are located close to each other. However, whether incisor tooth germ development is involved in MC resorption remains unclear. In this study, we aimed to elucidate the spatio-temporal interaction between the initiation site of MC resorption and the development of incisor tooth germs in an embryonic mouse model. To this effect, we developed a histology-based three-dimensional (3D) reconstruction technique using paraffin-embedded serial sections of various tissues in the jaw. The serial sections were cut in the frontal section and the tissue constituents (e.g., MC, incisor, and mineralized mandible) were studied using conventional and enzyme-based histochemistry. The outline of each component was marked on the frontal sectional images and 3D structures were constructed. To assess the vascular architecture at the site of MC resorption, immunohistochemical staining using anti-laminin, anti-factor VIII, and anti-VEGF antibodies was performed. MC resorption was first observed on the lateral incisor-facing side of the cartilage rods at sites anterior to the mental foramen on E16.0. The 3D analysis suggested that: (a) the posterior region of the clastic cartilage resorption corresponds to the cervical loop of the incisor; (b) the cervical portion of the tooth germ inflates probably due to temporal cellular congestion prior to differentiation into matrix-producing cells; (c) the incisor tooth germ tissue is present in close proximity to MC even in mouse with continuously growing tooth and determines the disappearance of MC as the tooth development.

Expression and localisation of MUC1 modified with sialylated core-2 O-glycans in mucoepidermoid carcinoma

Mucoepidermoid carcinoma (MEC) is the most frequent of the rare salivary gland malignancies. We previously reported high expression of Mucin 1 (MUC1) modified with sialylated core-2 O-glycans in MEC by using tissue homogenates. In this study, we characterised glycan structures of MEC and identified the localisation of cells expressing these distinctive glycans on MUC1. Mucins were extracted from the frozen tissues of three patients with MEC, and normal salivary glands (NSGs) extracted from seven patients, separated by supported molecular matrix electrophoresis (SMME) and the membranes stained with various lectins. In addition, formalin-fixed, paraffin-embedded sections from three patients with MEC were subjected to immunohistochemistry (IHC) with various monoclonal antibodies and analysed for C2GnT-1 expression by in situ hybridisation (ISH). Lectin blotting of the SMME membranes revealed that glycans on MUC1 from MEC samples contained α2,3-linked sialic acid. In IHC, MUC1 was diffusely detected at MEC-affected regions but was specifically detected at apical membranes in NSGs. ISH showed that C2GnT-1 was expressed at the MUC1-positive in MEC-affected regions but not in the NSG. MEC cells produced MUC1 modified with α2,3-linked sialic acid-containing core-2 O-glycans. MUC1 containing these glycans deserves further study as a new potential diagnostic marker of MEC.

The relationship between elastin cross linking and alveolar wall rupture in human pulmonary emphysema.

To better define the role of mechanical forces in pulmonary emphysema, we employed methods recently developed in our laboratory to identify microscopic level relationships between airspace size and elastin-specific desmosine and isodesmosine (DID) cross links in normal and emphysematous human lungs. Free DID in wet tissue (a biomarker for elastin degradation) and total DID in formalin-fixed, paraffin-embedded (FFPE) tissue sections were measured using liquid chromatography-tandem mass spectrometry and correlated with alveolar diameter, as determined by the mean linear intercept (MLI) method. There was a positive correlation between free lung DID and MLI (P < 0.0001) in formalin-fixed lungs, and elastin breakdown was greatly accelerated when airspace diameter exceeded 400 µm. In FFPE tissue, DID density was markedly increased beyond 300 µm (P < 0.0001) and leveled off around 400 µm. Elastic fiber surface area similarly peaked at around 400 µm, but to a much lesser extent than DID density, indicating that elastin cross linking is markedly increased in response to early changes in airspace size. These findings support the hypothesis that airspace enlargement is an emergent phenomenon in which initial proliferation of DID cross links to counteract alveolar wall distention is followed by a phase transition involving rapid acceleration of elastin breakdown, alveolar wall rupture, and progression to an active disease state that is less amenable to therapeutic intervention.NEW & NOTEWORTHY The current findings support the hypothesis that airspace enlargement is an emergent phenomenon in which initial proliferation of DID cross links to counteract alveolar wall distention is followed by a phase transition involving rapid acceleration of elastin breakdown, alveolar wall rupture, and progression to an active disease state that is less amenable to therapeutic intervention.

Abstract 6375: Transcriptomic signatures in melanoma lymph node metastasis that relate to immune oncology treatment

Abstract Background: Frequently, patients with primary cutaneous melanoma develop regional lymph node metastasis (LNM). The outcomes in melanoma patients with LNM can be poor, depending on clinicopathological features including the size and the number of the LNM. Newer immunotherapies including immune checkpoint inhibitors (ICI) have significantly improved outcomes in metastatic melanoma patients, and when tumors respond to ICI the durable responses are noteworthy. The aim of the current study is to identify transcriptomics changes in immune-related genes in LNM that correlate to outcomes in melanoma patients receiving adjuvant ICI therapy. Methods: The analysis were performed in formalin-fixed paraffin-embedded (FFPE) sections from LNM positive (LNM(+); n=17) and LNM negative (LNM(-); n=8) of patients who received surgery at SJHC. For each tissue, a single section was processed and analyzed for 2,002 genes using the direct (mRNA extraction-free) transcriptomic assay of the HTG EdgeSeq AutoImmune Panel (HTG-AI). The HTG-AI transcriptomic normalized data were compared between both groups. For verification of gene signatures, publicly available RNA-seq datasets were obtained from patients with clinical outcomes receiving anti-PD-1. All biostatistical analysis were performed using HTG Reveal and/or GraphPad Prism software. Results: Comparisons between melanoma LNM(+) and LNM(-) samples showed 229 DEGs (Log2fold-change ←2; &amp;gt;2 FDR&amp;lt;0.01), with 213 genes upregulated and 16 downregulated. Pathway analyses using Reactome indicated that the DEGs in LNM(+) were associated with cytokine, interferon (IFN), and chemokine signaling, as well as signaling by receptor tyrosine kinases (FDR&amp;lt;0.0001). The chemokines CXCL8, CXCL2, and CXCL10 were significantly upregulated in LNM(+) (p&amp;lt;0.001). LNM(+) showed significantly higher levels of IFN signaling mediators (STAT1, STAT2, IFI6, IFI30, IFI44) compared to LNM(-) specimens (p&amp;lt;0.001). Also, LNM(+) showed a significant increase in CD276 (B7-H3) levels (p&amp;lt;0.001). Then, we correlated the mRNA levels of IFN signaling (STAT1, STAT2, IFI6, IFI30, and IFI44) obtained from samples before anti-PD-1 therapy (Nivolumab or Pembrolizumab) to overall survival (OS). Of clinical importance, patients with elevated levels of the IFN signaling genes showed better OS for patients receiving adjuvant anti-PD-1 (HR=0.6, CI=0.42-0.86, Log-rank test, p=0.0044). Conclusions: We identified a distinctive transcriptomic profile in LNM(+). Several genes were upregulated and associated with specific pathways. Using publicly available data, we demonstrated that STAT1, STAT2, IFI6, IFI30, and IFI44 gene signature correlated with improved OS for patients receiving adjuvant ICI treatment. The STAT1, STAT2, IFI6, IFI30, and IFI44 genes may represent new biomarkers to assess in biopsies of patients that will receive adjuvant or neoadjuvant ICI therapies. Citation Format: Matias A. Bustos, Shodai Mizuno, Suyeon Ryu, Dave S. Hoon. Transcriptomic signatures in melanoma lymph node metastasis that relate to immune oncology treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6375.

Abstract 4293: Strategies and resources for applying a quantitative multiplex IHC imaging workflow to characterize immune contexture in solid tumors

Abstract Conventional immunohistochemistry (IHC) is a standardized diagnostic technique used in tissue pathology. However, the capacity to label only one marker per tissue section is a critical limitation. Multiplex immunohistochemistry (mIHC) is a powerful imaging technique used in basic, translational research and clinical settings to simultaneously detect the expression of multiple epitopes in a single formalin-fixed paraffin-embedded (FFPE) tissue section, allowing for characterization and quantification of cells while maintaining their spatial location. This technique allows for a comprehensive view of the immune milieu, expression patterns, and interactions between cell types that can help elucidate the complexity of the tumor-immune microenvironment (TiME). Multiplex imaging has emerged as a powerful tool to better understand tumor progression, response or resistance to therapy, and potentially identify predictive biomarkers. However, this technique requires careful tissue considerations and generates enormous amounts of hierarchical data, including single cell marker expressions, locations, and shape features, resulting in complex and challenging analyses. We previously published a validated platform using nine matched primary and recurrent head and neck squamous cell carcinoma (HNSCC) sections stained sequentially with a panel of 29 antibodies identifying malignant tumor cells, and 17 distinct leukocyte lineages and their functional states1. Here, we have optimized and consolidated important considerations, including tissue quality and QC steps necessary for generating high quality data from multiplex imaging studies. We demonstrate strategies for a streamline analytic pipeline using our quantitative workflow developed to characterize immune cells and infiltration patterns within spatial compartments of solid tumors in FFPE tissues, and herein, provide strategies and resources to practically adapt and utilize our analytic mIHC pipeline. 1. Banik, G., et al., Methods Enzymol., 2020 Citation Format: Shamilene Sivagnanam, Courtney M. Betts, Nell Kirchberger, Konjit Betre, Lisa M. Coussens. Strategies and resources for applying a quantitative multiplex IHC imaging workflow to characterize immune contexture in solid tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4293.

Abstract 1360: Spatial image analysis of tumor-infiltrating lymphocytes on gastric cancer to predict anti-PD1 inhibitor response

Abstract Background: The Tumor Immune microenvironment (TME) plays an important role in tumor development, progression, and treatment response. Recent studies show that spatial heterogeneity of Tumor-Infiltrating Lymphocytes (TILs), consisting of lymphocytic cell populations, is an important histopathologic feature and a powerful prognostic and predictive marker correlating with cancer progression and treatment response. Detecting and quantifying TIL density in Whole Slide Images (WSIs) is, however, labor-intensive, and it is difficult to assess differences in the composition of immune cells. Therefore, it remains a challenge to establish the analysis of TILs in the complex TME. We present an automated spatial image analysis of TILs on gastric cancer to address cancer immunotherapy. Method: 1,031WSIs of hematoxylin and eosin-stained (H&amp;E) formalin-fixed paraffin-embedded (FFPE) sections from four patient cohorts: TCGA-STAD (329), Yonsei-1 (620 patients), Yonsei-2 (45), and St. Mary (37), were analyzed. Two treatment groups in response were classified as CR + PR, while another two treatment groups in non-response were classified as PD + SD. H&amp;E stained WSIs were normalized using the Reinhard normalization method at 20X magnification for reducing the color variability of the stained tissue images. We used U-Net architecture to predict the normalized H&amp;E stained WSIs. Three U-Net models were trained for predicting tumor, TILs, and stroma, using Adam optimizer, 1e-4 learning rate, a binary loss function, L2 regularization, and 50 epochs. The normalized H&amp;E stained WSIs were predicted using the trained U-Net models. Spatial analysis of TILs was conducted on the predicted WSIs. Regional quantification of TILs was performed by two immune phenotype groups: immune-immersed (TILs enriched in tumor cells) and immune-excluded (TILs not enriched in tumor cells but in stromal cells). Kaplan-Meier with a log-rank test was used to evaluate the survival differences between the groups. Results: In TCGA-STAD, the proportion of immune-immersed composition is significantly different from the immune-excluded (p-value 0.05). In Yonsei-1, the immune-excluded composition is significantly different among the three risk groups (p-value 3e-05). In the Yonsei-2 and St. Mary cohorts, including anti-PD1 treated gastric cancer patients, the immune-immersed composition is significantly different between two response groups (p-value 0.007) and represented the diagnostic accuracy (AUROC: 0.78) on CR + PR groups. Conclusion: These findings suggest that the proposed TIL quantification in the complex TME is strongly associated with patient prognosis and anti-PD1 treatment response in gastric cancer. Citation Format: Sanghoon Lee, Sunho Park, Jae-Ho Cheong, Sam C. Wang, Matthew R. Porembka, In-Ho Kim, Sung Hak Lee, Tae Hyun Hwang. Spatial image analysis of tumor-infiltrating lymphocytes on gastric cancer to predict anti-PD1 inhibitor response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1360.

Abstract 5642: Validation of a novel multiplex immuno-fluorescence panel for the spatial analysis of the tumor microenvironment

Abstract IHC counterparts. Subsequently, the panel was transferred on a multi-organ TMA including several tumoral and non-tumoral specimens, showing robust performance across multiple tissue types. The protocol was optimized to achieve high staining quality for all 13 markers in terms of signal specificity Investigation of the tumor microenvironment (TME) by multiplex immunofluorescence (mIF) has accelerated the understanding of the spatial immune context in tumors. mIF has proven to be a powerful technique for the identification of new potential biomarkers and therapeutical targets. Despite increased application of mIF assays to characterize the TME, state-of-the art protocols remain technically challenging. Manual execution and use of dedicated reagents render them lengthy and costly. Moreover, their reproducibility is often questioned together with their transferability between different tissue types. Here, we show the development and validation of an Immuno-Oncology (IO) Core Panel of 13 clinically relevant biomarkers to enable spatial analysis of the immune TME on the COMET™ platform across various tissue types. Formalin-fixed paraffin-embedded human tissue sections from tonsil and a 24-cores multi-organ tissue microarray (TMA) were stained using the IO Core Panel from Lunaphore on the COMET™ platform by fully automated sequential immunofluorescence (seqIF™), which consists of cycles of staining, imaging, and elution. The panel allows for simultaneous detection of CD3, CD4, CD8, CD45, FoxP3, PD1, PD-L1, CD11c, CD20, CD56, CD68, aSMA and Ki-67 by indirect immunofluorescence using unlabeled primary antibodies and Alexa Fluor™ Plus secondary antibodies. The 13-plex IO Core panel was initially developed and validated on tonsil as positive control tissue. To compare immunofluorescent (IF) and immunohistochemistry (IHC) staining patterns, the sections retrieved from COMET™ after seqIF™, were stained by a histology facility with standard IHC established for routine pathological diagnosis. All markers demonstrate accurate detection with specific IF staining, comparably to gold-standard, sensitivity, ratio to background and dynamic range. The repeatability and reproducibility of the automated IO Core Panel on the COMET™ platform was verified by day-to-day tests on one platform and tests among multiple platforms, respectively. Our study demonstrated the robustness of the validated IO Core Panel across multiple tissue types with highly specific and reproducible results. The marker detection with standard indirect immunofluorescence on the COMET™ instrument allows for future, rapid expansion and customization of the panel including additional primary antibodies towards the need of the individual underlying scientific question. Citation Format: François Rivest, Victor de Gautard, Vytautas Navikas, Saska Brajkovic, Bastian Nicolai. Validation of a novel multiplex immuno-fluorescence panel for the spatial analysis of the tumor microenvironment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5642.

Abstract 4310: Ultra high-plex spatial proteogenomic investigation of giant cell glioblastoma multiforme immune infiltrates reveals distinct protein and RNA expression profiles

Abstract The advancement of spatially resolved, multiplex proteomic and transcriptomic technologies has revolutionized and redefined the approaches to complex biological questions pertaining to tissue heterogeneity, tumor microenvironments, cellular interactions, cellular diversity, and therapeutic response. Most spatial technologies yield single analyte proteomic or transcriptomic datasets from separate formalin-fixed paraffin-embedded (FFPE) tissues sections. Multiple studies have demonstrated a poor correlation between RNA expression and protein abundance owing to transcriptional and translational regulation, target turnover, and post-translational protein modifications. Therefore, a workflow that accurately measures RNA and protein simultaneously within a single tissue section with distinct spatial context is critical to a more complete biological understanding of cellular interactions and activities. Such multimodal omic datasets of protein and DNA or RNA have been termed “spatial proteogenomics”. Here we present a novel spatial proteogenomic (SPG) assay on the GeoMx® Digital Spatial Profiler platform with NGS readout that enables ultra high-plex digital quantitation of proteins (147-plex) and RNA (whole transcriptome, &amp;gt; 18,000-plex) from a single FFPE sample. We demonstrated high concordance, R &amp;gt; 0.85, and minor change in sensitivity (&amp;lt;11%) between the SPG assay and the single analyte GeoMx Whole Transcriptome Atlas and GeoMx NGS Protein assays. We used the SPG assay to interrogate 23 different glioblastoma multiforme samples across 4 pathologies. We observed clustering of both RNA and protein based on cancer pathology and anatomic location. The in-depth investigation of giant cell glioblastoma multiforme (gcGBM) revealed distinct protein and RNA expression profiles compared to that of glioblastoma multiforme (GBM). Spatial proteogenomics allowed simultaneous interrogation of critical protein post-translational modifications alongside whole transcriptomic profiles within the same distinct cellular neighborhoods. Within our dataset, we observed &amp;gt;2-fold higher protein expression levels of phospho-GSK3β (Ser9) in gcGBM compared to GBM. Inactivation of GSK3β through phosphorylation has been shown to enhance proliferation of GBM cells. We also observed differential protein expression phosphorylated Tau variants. Phospho-Thr231 Tau was &amp;gt;2-fold higher in GBM compared to gcGBM. Associated with neurodegenerative Alzheimer’s disease, changes in Tau expression and phosphorylation have also been observed in glioblastoma. Our study exemplifies the utility of the SPG assay in expanding our understanding of glioblastoma multiforme molecular pathology. Citation Format: Shilah Bonnett, Alyssa Rosenbloom, Giang Ong, Mark Conner, Daniel Newhouse, Felicia New, Hiromi Sato, Chi Pha, Saskia Ilcisin, John Lyssand, Gary Geiss, Joseph M. Beechem. Ultra high-plex spatial proteogenomic investigation of giant cell glioblastoma multiforme immune infiltrates reveals distinct protein and RNA expression profiles. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4310.

Abstract 4612: Microphysiological vascularized solid liver tumor model for drug and cell therapy

Abstract Development of efficacious therapeutic strategies against solid tumors is limited by the lack of pre-clinical models that can reliably predict treatment outcomes in patients. This is primarily because models often do not accurately reflect the complexity of the tumor microenvironment (TME). The TME consists of vasculature, stromal cells and immune cells that can promote tumor resistance or prevent targeted drug and cell therapy. Here, we developed an in vitro vascularized liver tumor model in a microfluidic device to evaluate drug delivery and immunotherapy approaches. The model consists of a tumor spheroid surrounded by a perfusable self-organized vasculature bed with physiologically relevant permeability. Sorafenib (Nexavar) efficacy was evaluated by assessing tumor spheroid viability, quantified by the loss of fluorescence signal. The presence of vasculature showed enhanced cytotoxicity, highlighting the importance of tumor-vascular interactions for testing therapeutic efficacy. Engineered immune cells (chimeric antigen receptor (CAR) T-cell and transiently expressing T-cell receptors (TCR)) were introduced into the model to demonstrate immunotherapy validation. Our results showed that the engineered immune cells were more effective at infiltrating the tumor spheroid without a vasculature but were more cytotoxic in the presence of the vasculature. By removing the cultured microtissue from the device and performing digital spatial transcriptomics on standard paraffin-embedded sections, we identified known and novel genes involved in creating a pro-tumorigenic microenvironment. This platform can be utilized to model critical features of the in vivo TME, providing better predictability of drug responses, with potential applications towards personalized medicine. Citation Format: Jyothsna Vasudevan, Ragavi Vijayakumar, Jose Antonio Reales-Calderon, Maxine Sin Lam, Jin Rong Ow, Joey Aw, Damien Tan, Giulia Adriani, Andrea Pavesi. Microphysiological vascularized solid liver tumor model for drug and cell therapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4612.

Abstract 3972: Novel renal cell carcinoma therapy targeting glutaminolysis

Abstract Objective: Metabolic reprogramming of cancer cells contributes to tumor development and provides a target for cancer therapy ASCT2 is a glutamine transporter on the plasma membrane. Glutamine is converted into the cell to glutamate by glutaminase (GLS) and utilized by the TCA cycle. This glutaminolysis has been implicated in tumor progression in various malignancies, including renal cell carcinoma. In the present study, we investigated whether the unique strategy that effectively targets glutamine addiction would be effective as a treatment for RCC. Methods: Tumor tissue (T) and normal renal tissue (N) from surgical specimens of 66 patients with RCC were collected, and metabolomic analysis using liquid chromatography-mass spectrometry (LC/MS) to calculate the T/N ratio of Gln and Glu, respectively, were performed. In addition, immunohistological (IHC) staining of paraffin-embedded sections of 106 metastatic RCC cases was used to compare GLS and ASCT2 expression in tumor and normal tissue. In vitro, cell viability of the human RCC cell lines 786-O and 769-P was assessed by WST-8 assays. The protein expression was detected by Western blotting. Intracellular concentrations of glutamine and glutamate were analyzed using LC/MS. V9302 was used as the specific ASCT2 inhibitor, and CB839 as the specific GLS inhibitor. As the xenograft model in vivo, athymic nude mice were injected subcutaneously with 786-O cells and given various doses of V9302 and CB839 intraperitoneally. Results: LC/MS assays using surgical specimens showed a notable increase in glutamine concentration in tumor tissue. IHC assays confirmed high expression of ASCT2 in tumor tissue, and patients with high expression of ASCT2 had a poorer prognosis than those with low expression. Treatment of V9302 with 786-O and 769-P resulted in a limited decrease in cell viability. LC/MS analysis confirmed that V9302 treatment reduced glutathione metabolites. V9302 administration was shown to increase intracellular glutamine levels, presumably by compensatory mechanisms, whereas the combination of V9302 and CB839 significantly decreased glutamine levels in 786-O cells. Western blot analysis showed that the expression levels of ASCT2 and GLS1 decreased after the combination therapy. The xenograft study confirmed that the combination of V9302 and CB839 significantly inhibited 786-O tumor growth compared with controls within three weeks after treatment (p &amp;lt;0.001). Conclusion: It has been shown that ASCT2 expression is a marker of poor prognosis in RCC and that suppression of ASCT2 has an antitumor effect by affecting glutathione metabolism. On the other hand, the administration of V9302 raised the issue that a compensatory mechanism increases intracellular glutamine levels. Our results suggest that a novel combination strategy using CB839 in addition to V9302 could overcome the tolerance for the monotherapy and effectively target glutamine addiction in RCC. Citation Format: Yoshinari Muto, Akihito Takeuchi, Kenji Zennami, Eiji Sugihara, Ryoichi Shiroki, Hideyuki Saya, Makoto Sumitomo. Novel renal cell carcinoma therapy targeting glutaminolysis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3972.

Abstract 5556: Lemur tyrosine kinase 3 serves as a predictor of patient outcome and a target for the treatment of ovarian cancer

Abstract The objective of these in vitro and in vivo studies was to validate Lemur Tyrosine Kinase 3 (LMTK3) as a specific target and predictor of clinical outcome in ovarian cancer. LMTK3 belongs to a family of regulated tyrosine kinases with three structurally related isoforms, LMTK1, LMTK2, and LMTK3. Both nuclear and cytoplasmic LMTK3 expressions correlated with tumor grade and patient survival in cancers such as breast and colorectal cancer. We tested the clinical significance of the LMTK3 gene by immunohistochemistry (IHC) using LMTK3 monoclonal antibody on formalin-fixed paraffin-embedded sections (FFPE) collected from 204 early-stage (stage I-II) ovarian cancer patients. Results from this IHC LMTK3 study revealed a higher cytoplasmic to nuclear localization of LMTK3 correlated with worse overall survival (P&amp;lt;0.01). Further investigation of LMTK3’s prognostic value by screening LMTK3 signaling in 270 stage III-IV ovarian cancer patients is ongoing. We utilized the MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay for testing the killing efficacy of targeting LMTK3 by a monoclonal antibody, siRNA, and specific LMTK3 binding peptides (LMTK3BP) in ovarian cancer cell lines SKOV3, MDAH-2774, A2780, and TOV-21G. Treatment with LMTK3 specific monoclonal antibody, siRNA, and LMTK3BP significantly induced killing of both chemosensitive and chemoresistant ovarian cancer cells without affecting normal cells in vitro. Moreover, we observed this killing as synergistic with both Cisplatin and Taxotere treatment in vitro. Lastly, we used an A2780 cell line derived orthotopic xenograft mouse model of ovarian cancer to test the efficacy of specific LMTK3BP in vivo. Strikingly, LMTK3BP 20 mg/kg IV dose given three times a week for three weeks showed a 35% tumor reduction. Furthermore, in vivo safety studies showed no signs of toxicity of the LMTK3BP, even at a very high dose of 40 mg/kg. Thus, with dose optimization in ongoing experiments, we could expect a higher efficacy. In conclusion, this study highlighted the importance of LMTK3 as a predictor of patient clinical outcome and potential target for treatment in ovarian cancer. Citation Format: Thea K. Kirsch-Mangu, Axel S. Tullberg, Anna Portela, Khalil Helou, Ghassan M. Saed. Lemur tyrosine kinase 3 serves as a predictor of patient outcome and a target for the treatment of ovarian cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5556.