Tonsil Tissue Research Articles

The first proteomics analysis of tonsils in patients with periodic fever, aphthous stomatitis, pharyngitis, and adenitis syndrome (PFAPA).

Periodic fever, aphthous stomatitis, pharyngitis, and adenitis (PFAPA) syndrome is a recurrent fever syndrome. The exact etiopathogenesis of PFAPA syndrome remains unknown. Biological fluids or tissues may provide disease-specific biomarkers that may help clinicians to find new pathogenic pathways. Tonsil tissues of seven patients with PFAPA were collected during the tonsillectomy. Seven patients who underwent tonsillectomy for reasons other than chronic tonsillitis were enrolled as a control group. The nHPLC LC-MS/MS system was used for protein identification and label-free quantification. Bioinformatics analysis was carried out using the UniProt accession numbers of the identified proteins. Proteomics analysis revealed to identity of proteins of which at least 23 were up and 57 were downregulated. Bioinformatics analysis of differentially regulated proteins by STRING indicated that protein folding and clearance machinery were interrupted in PFAPA patients compared to the controls. The affected pathways underlined the importance of the mitochondrial electron transport chain and ATP biosynthesis process. Although it is not clear that changes in tonsil protein expression whether directly related to pathogenesis or simply result of chronic inflammation, the identification of tonsil biomarkers for PFAPA may provide clinicians an opportunity to understand disease pathogenesis or develop new molecular targets for treatments. Proteomics analyses of tonsils revealed the identity of 80 proteins of which at least 23 were up and 57 were downregulated. Bioinformatics analysis underlined the importance of mitochondrial ETC and regulation of ATP biosynthetic process. This is the first study evaluating the proteomics of the tonsils of PFAPA patients. The identification of tonsil biomarkers for PFAPA may provide clinicians an opportunity to understand disease pathogenesis or develop new molecular targets for treatments.

Overexpression of YKL40,IL-6, IL-8, TNF-α in tonsils and the role of YKL40 in childhood with obstructive sleep apnea syndrome

To evaluate the levels of YKL40, IL-6(interleukin-6), IL-8(interleukin-8), IL-10(interleukin-10), TNF-α (tumor necrosis factor-α) in OSAS (obstructive sleep apnea syndrome)children and explore the mechanism of YKL40 promoting inflammatory factors overexpression in tonsils. qPCR and ELISA were used to identify the expression of YKL40, IL-6, IL-8, IL-10, and TNF-α in the tonsils of OSAS children. Primary tonsil lymphocytes (PTLCs) were cultured and recombinant human YKL40(rhYKL40)was used to stimulate PTLCs in different concentrations and at different time points. The activation of NF-κB in PTLCs was screened by western blotting. Relative mRNA of YKL40, IL-6, IL-8, TNF-α was over expressed in OSAS-derived tonsil tissue and the levels of YKL40, IL-6, IL-8, and TNF-α was increased in OSAS-derived protein supernatant of tonsil tissue.The relative mRNA expression of IL-6, IL-8 and TNF-α increased under the treatment of YKL40 (100 ng/mmol for 24 h). The phosphorylation of p65 in NF-κB pathway was stimulated in the process. The levels of YKL40, IL-6, IL-8, and TNF-α increases in OSAS children, and YKL40 promotes the overexpression of IL-6, IL-8 and TNF-α in PTLCs via NF-κB pathway. The result implements that inflammation may play an important role in the pathogenesis of OSAS in children.

Genetic Manipulation and Extended Culture of Human Germinal Center B Cells to Model Lymphomagenesis.

The germinal center (GC) is the stage of B cell differentiation that gives rise to a majority of B cell lymphomas. Here, we present an experimental coculture system for the ex vivo expansion and genetic manipulation of human GC B cells purified from discarded tonsil tissue. This system can be used to investigate the impact of defined genetic alterations, either individually or in combination, upon the growth and survival of human GC B cells in vitro. We provide examples of genetic combinations that lead to the immortalized growth of GC B cells in vitro, and others that result in malignant transformation in immunodeficient mice, allowing the creation of genetically bespoke, synthetic, human lymphoma models.

Tonsil explants as a human in vitro model to study vaccine responses.

Vaccination is one of the most effective infection prevention strategies. Viruses with high mutation rates -such as influenza- escape vaccine-induced immunity and represent significant challenges to vaccine design. Influenza vaccine strain selection is based on circulating strains and immunogenicity testing in animal models with limited predictive outcomes for vaccine effectiveness in humans. We developed a human in vitro vaccination model using human tonsil tissue explants cultured in 3D perfusion bioreactors to be utilized as a platform to test and improve vaccines. Tonsils cultured in bioreactors showed higher viability, metabolic activity, and more robust immune responses than those in static cultures. The in vitro vaccination system responded to various premanufactured vaccines, protein antigens, and antigen combinations. In particular, a multivalent in vitro immunization with three phylogenetically distant H3N2 influenza strains showed evidence for broader B cell activation and induced higher antibody cross-reactivity than combinations with more related strains. Moreover, we demonstrate the capacity of our in vitro model to generate de novo humoral immune responses to a model antigen. Perfusion-cultured tonsil tissue may be a valuable human in vitro model for immunology research with potential application in vaccine candidate selection.

Long-term effect observation after 10 years of tonsillotomy （Histological analysis of one case of secondary operation）

Objective:To investigate the long-term effect of partial tonsillectomy in children with tonsil hypertrophy. Methods:A total of 146 children with obstructive sleep apnea（OSA） who received surgical treatment for tonsil hyperplasia from January 2010 to January 2013 were selected and divided into the observation group（n=69） and the control group（n=77）. The observation group was received tonsillotomy（TT）, and the control group was received total tonsillectomy（TE）. Parental satisfaction and OSA quality of life questionnaire for children（OSA-18） were surveyed. Residual tonsil size was measured, and polysomnography（PSG） was monitored after 10 years. HE and immunohistochemical analysis were performed on tonsil tissues of one patient who performed a second operation after TT in 2017 year. Results:The results of questionnaire survey showed that the symptoms of respiratory obstruction were significantly improved in both groups, and the satisfaction of TT group was higher than that in the TE group. No increase in the number of respiratory tract infections was observed in all patients. In the TT group, nine cases（13.04%） had tonsil hyperplasia toⅡ°, and the remaining patients had tonsil hyperplasia to Ⅰ°. In addition, one case hadtonsil suppurative infection at the 14th month after surgery, and no recurrence or reoperation was found after treatment. There were seven cases in the TT group and eight cases in the TE group with occasional snoring and mouth breathing after surgery, but the PSG examination of the patients did not meet the diagnosis of OSA. The main causes were obesity and allergic rhinitis. Compared with the first operation, the cicatricial obstruction and infection of tonsil tissue in the second operation were not significantly changed, and the immunohistochemical results also demonstrated that the expression of CD20 was not changed, and the expression of CD3 was decreased. Conclusion:Both TT and TE can effectively improve the symptoms of OSA obstruction in children. TT has less trauma, less postoperative pain, faster recovery and lower rate of hyperplasia, which can be used as one of the main methods for the treatment of tonsil hypertrophy in children.

Testing a Nanoparticle Reagent for Imaging Mass Cytometry.

Mass cytometry (MC), a powerful single-cell analysis technique, has limitations in detecting low-abundance biomarkers. Nanoparticle (NP) reagents offer the potential for enhancing sensitivity by carrying large numbers of heavy metal isotopes. Here, we report NP reporters for imaging mass cytometry (IMC) based on NaYF4:Yb3+/Er3+ NPs. A two-step ligand exchange was used to coat NP surfaces with either methoxy-PEG2K-neridronate (PEG-Ner) and/or poly(sulfobetaine methacrylate)-neridronate (PSBMA-Ner). Both modifications provided long-term colloidal stability in PBS buffer. IMC measurements on tonsil tissue showed that PSBMA-Ner or a 1:1 mixture of PSBMA-Ner + PEG-Ner effectively suppressed nonspecific binding (NSB) at 2 × 1010 NPs/mL, unlike PEG-Ner alone. However, breast cancer tissue samples showed increased NSB at titers above 2 × 1010 NPs/mL. Reduced NSB with mixed PEG-Ner and PSBMA-Ner coatings opens the door for using heterobifunctional PEGs for the development of NP conjugates with bioaffinity agents, enabling more sensitive and specific MC analyses.

Deciphering the interplay of HPV infection, MHC-II expression, and CXCL13+ CD4+ T cell activation in oropharyngeal cancer: implications for immunotherapy

BackgroundHuman papillomavirus (HPV) infection has become an important etiological driver of oropharyngeal squamous cell carcinoma (OPSCC), leading to unique tumor characteristics. However, the interplay between HPV-associated tumor cells and tumor microenvironment (TME) remains an enigma.MethodsWe performed a single-cell RNA-sequencing (scRNA-seq) on HPV-positive (HPV+) and HPV-negative (HPV‒) OPSCC tumors, each for three samples, and one normal tonsil tissue. Ex vivo validation assays including immunofluorescence staining, cell line co-culture, and flow cytometry analysis were used to test specific subtypes of HPV+ tumor cells and their communications with T cells.ResultsThrough a comprehensive single-cell transcriptome analysis, we uncover the distinct transcriptional signatures between HPV+ and HPV‒ OPSCC. Specifically, HPV+ OPSCC tumor cells manifest an enhanced interferon response and elevated expression of the major histocompatibility complex II (MHC-II), potentially bolstering tumor recognition and immune response. Furthermore, we identify a CXCL13+CD4+ T cell subset that exhibits dual features of both follicular and pro-inflammatory helper T cells. Noteworthily, HPV+ OPSCC tumor cells embrace extensive intercellular communications with CXCL13+CD4+ T cells. Interaction with HPV+ OPSCC tumor cells amplifies CXCL13 and IFNγ release in CD4+T cells, fostering a pro-inflammatory TME. Additionally, HPV+ tumor cells expressing high MHC-II and CXCL13+CD4+ T cell prevalence are indicative of favorable overall survival rates in OPSCC patients.ConclusionsTogether, our study underscores a synergistic inflammatory immune response orchestrated by highly immunogenic tumor cells and CXCL13+CD4+ T cells in HPV+ OPSCC, offering useful insights into strategy development for patient stratification and effective immunotherapy in OPSCC.

TRPC6 is ubiquitously present in lymphatic tissues: A study using samples from body donors.

Transient receptor potential canonical channel 6 (TRPC6) is a non-selective cation channel that is activated by diacylglycerol. It belongs to the TRP superfamily, is expressed in numerous tissues and has been shown to be associated with diseases, such as focal segmental glomerulosclerosis, idiopathic pulmonary arterial hypertension and cardiac hypertrophy. The investigation of the channel in human lymphoid tissues has thus far been limited to mRNA analysis or the western blotting of isolated lymphoid cell lines. The present study aimed to detect the channel in human lymphoid tissue using immunohistochemistry. For this purpose, lymphatic tissues were obtained from body donors. The lymphatic organs analyzed included the lymph nodes, spleen, palatine tonsil, gut-associated lymphoid tissues (ileum and vermiform appendix) and thymus. A total of 102 samples were obtained and processed for hematoxylin and eosin (H&E) staining. The H&E staining method was employed to identify five samples with good morphology. In total, three samples of the palatine tonsil of patients were included. Immunostaining was carried out using a knockout-validated anti-TRPC6 antibody. As shown by the results, using immunohistochemical staining, the presence of TRPC6 was confirmed in all the analyzed lymphatic tissue samples. Lymphocytes in lymph nodes, spleen, palatine tonsil, thymus, and gut-associated lymphatic tissues in ileum and vermiform appendix exhibited a positive staining signal. The follicle-associated epithelium of the palatine tonsil, ileum and appendix also demonstrated staining. Vessels of the lymphatic organs, particularly the trabecular arteries of the spleen, the submucosal vessels of the appendix and ileum, as well as the high endothelial venules in the palatine tonsils and lymphatic vessels of the lymph nodes expressed TRPC6 protein. TRPC6 in follicles may be involved in the immune response. TRPC6 in high endothelial venules suggests a role in leukocyte migration. The role of TRPC6 and other channels of the TRP family in lymphatic organs warrant further investigations to elucidate whether TRP channels are a pharmacological target.

Chromogenic LMO2 mRNA ISH Expression Correlates with LMO2 Protein and Gene Expression and Captures Their Survival Impact in Diffuse Large B-Cell Lymphoma, NOS.

LMO2 is a relevant gene involved in B-cell ontogeny and a survival predictor of aggressive large B-cell lymphomas (aLBCL). Most studies assessing LMO2 mRNA expression have relied on microarray platforms or qRT-PCR methods, overlooking tissue morphology. In this study, we evaluate LMO2 RNA expression by chromogenic in situ hybridization (CISH) in normal tissue and in a series of 82 aLBCL. LMO2 CISH was performed in formalin-fixed paraffin-embedded tissues, scored by three different methods, and correlated with a transcriptome panel. We obtained statistically significant results correlating the methods of evaluation with LMO2 protein expression and gene expression results. Normal tonsil tissue showed high levels of LMO2, particularly within the light zone of the germinal center. Conversely, in aLBCL, a notable reduction in LMO2 expression was noted, remarkably in cases carrying MYC rearrangements. Furthermore, significant results were obtained through overall survival and Cox regression survival analysis, incorporating International Prognostic Index data alongside LMO2 expression levels. We show a reliable method to identify LMO2 mRNA expression by CISH, effectively capturing many of the reported biologic features of LMO2.

Podoplanin depletion in tonsil-derived mesenchymal stem cells induces cellular senescence via regulation of the p16Ink4a/Rb pathway

BackgroundMesenchymal stem cells (MSCs) are widely used in the development of therapeutic tools in regenerative medicine. However, their quality decreases during in vitro expansion because of heterogeneity and acquired cellular senescence. We investigated the potential role of podoplanin (PDPN) in minimizing cellular senescence and maintaining the stemness of tonsil-derived MSCs (TMSCs).MethodsTMSCs were isolated from human tonsil tissues using an enzymatic method, expanded, and divided into two groups: early-passaged TMSCs, which were cultured for 3–7 passages, and late-passaged TMSCs, which were passaged more than 15 times. The TMSCs were evaluated for cellular senescence and MSC characteristics, and PDPN-positive and -negative cells were identified by fluorescence-activated cell sorting. In addition, MSC features were assessed in siRNA-mediated PDPN-depleted TMSCs.ResultsTMSCs, when passaged more than 15 times and becoming senescent, exhibited reduced proliferative rates, telomere length, pluripotency marker (NANOG, OCT4, and SOX2) expression, and tri-lineage differentiation potential (adipogenesis, chondrogenesis, or osteogenesis) compared to cells passaged less than five times. Furthermore, PDPN protein levels significantly decreased in a passage-dependent manner. PDPN-positive cells maintained their stemness characteristics, such as MSC-specific surface antigen (CD14, CD34, CD45, CD73, CD90, and CD105) and pluripotency marker expression, and exhibited higher tri-lineage differentiation potential than PDPN-negative cells. SiRNA-mediated silencing of PDPN led to decreased cell-cycle progression, proliferation, and migration, indicating the significance of PDPN as a preliminary senescence-related factor. These reductions directly contributed to the induction of cellular senescence via p16Ink4a/Rb pathway activation.ConclusionPDPN may serve as a novel biomarker to mitigate cellular senescence in the clinical application of MSCs.Graphical

Abstract PO5-06-06: Tumor/stromal expression of CD3/CD8/PD-1/PD-L1 and overall survival (OS) in patients (pts) with metastatic (met) HER2+ breast cancer (BC)

Abstract Background: The “Thousand Patient HER-2 database” project at Saint Vincent’s University Hospital (SVUH) Dublin contains a cohort of met HER2+ BC pts treated with trastuzumab, with OS ranging from short term to durable complete response (never relapsed). While the majority of pts progress on treatment, higher stromal tumor immune infiltrate has been associated with longer OS in met HER2+ BC. Using the SVUH database, we have identified a cohort of met HER2+ BC pts that received trastuzumab and have a broad OS range (0.3 months (mos) to 200.9 mos). This study examines the tumor and stromal levels of pan T cell marker CD3, cytotoxic T cell marker CD8, and immune checkpoints PD-L1 and PD-1 by immunohistochemistry (IHC) in primary and met samples from this cohort. Methods: Clinico-pathological data was available for formalin-fixed, paraffin-embedded biopsy specimens (n=45 primary, n=25 matched met biopsies). IHC staining was conducted for CD3 (Agilent IR50361-2), CD8 (Agilent IR62361-2), PD-L1, (Agilent M365329-2), and PD-1 (Roche 07099029001). A DAKO Link 48 Autostainer was utilised for staining, incorporating positive (tonsil tissue) and negative controls (isotype controls). Slide processing used an Aperio AT2 Digital Slide Scanner (Leica Biosystems), Aperio ImageScope 12.4 software (Leica Biosystems) and QuPath analysis software (University of Edinburgh). Following annotation of tumor areas, an algorithm was trained to identify immune cells and designate them as tumor or stromal. Data was expressed as the number of positively stained cells/mm2 tissue. A median cut-off was applied to denote “high” and “low” expression for CD3 and CD8. For PD-1 and PD-L1, samples with ≥ 1 stained cell/mm2 were designated as positive (pos) and samples with zero stained cells as negative (neg). The Kaplan Meier method was utilised for survival studies. A paired Student’s t test was utilised for primary vs metastatic site comparisons. Results: 51.1% of primary samples displayed high CD3 (23/45) and CD8 (23/45) expression in both the stromal and tumor compartments. Within the tumor compartment, 18.6% (8/43) of samples were pos for PD-L1 expression, and 57.8% (26/45) were pos for PD-1 expression. In the stromal compartment, 30.2% (13/43) of samples were pos for PD-L1 expression, and 68.9% (31/45) were pos for PD-1 expression. High tumor levels of CD3 (median OS CD3 high 113.4 mos vs CD3 low 7.9 mos, HR 0.2938 (95% CI 0.130-0.618), p=0.0012) and CD8 (median OS CD8 high 73.9 mos vs CD8 low 10.8 mos, HR 0.4764 (95% CI 0.238-0.954), p=0.0365), but not stromal levels of these markers, were associated with improved OS. There were no significant differences in CD3, CD8, PD-1 and PD-L1 levels between matched primary and met samples (p >0.05). Tumor, but not stromal, PD-L1 expression was associated with longer OS (HR 0.3120 (95% CI 0.146-0.667), p=0.0027). Interestingly, stromal PD-1 expression, but not tumor PD-1 expression, was also associated with longer OS (median OS PD-1 pos 64.7 mos vs. PD-1 neg 5.6 mos, HR 0.158 (95% CI 0.060-0.411), p=0.0002). Conclusion: Our results report a link between OS and tumor (CD3+, CD8+ and PD-L1+)/stromal (PD-1+) immune cell infiltrate in met HER2+ BC patients treated with trastuzumab. Further expansion of this preliminary dataset is warranted. Citation Format: Denis Collins, Janet McCormack, Laura Ivers, Jose Javier Berenguer-Pina, Jo Ballot, Cecily Quinn, Darko Skrobo, Alex Eustace, Naomi Walsh, Aurelie Fabre, John Crown. Tumor/stromal expression of CD3/CD8/PD-1/PD-L1 and overall survival (OS) in patients (pts) with metastatic (met) HER2+ breast cancer (BC) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-06-06.

A comprehensive analytical validation framework for immunofluorescence assay design using CellScape precise spatial multiplexing

Abstract Multiplex immunofluorescence (mIF) is a powerful immunology research tool that enables deep phenotyping of cells within the tissue microenvironment. Careful optimization of multiplex antibody panels for specificity, sensitivity, and reproducibility, is crucial to achieving robust data and is a requirement for assay repeatability and standardization. Here, we describe our validation procedure for multiplex assay panels. Experiments were performed using the CellScape™ platform for precise spatial multiplexing. To design a ready-to-use panel for immune cell phenotyping, individual antibodies were first screened for specificity and then optimized for labeling sensitivity. Signal-to-noise, inter-assay reproducibility, and intra-assay precision were evaluated in a multi-stage testing matrix to validate each antibody in isolation and assembled into a panel. Measurements of both signal and quantitative immune phenotyping across technical replicates showed a high degree of intra-assay precision and inter-assay reproducibility. Pairing the CellScape platform with comprehensively validated antibody panels streamlined deep immune profiling. The 15-plex, ready-to-use VistaPlex™ immune profiling panel for human FFPE tissues was validated on human tonsil, lung, breast, and colon tissues demonstrating the ability to positively identify epithelial cells and 22 cellular immune phenotypes most critical to understanding tumor immune responses.

Ultrasensitive and Multiplexed Protein Imaging with Clickable and Cleavable Fluorophores.

Single-cell spatial proteomic analysis holds great promise to advance our understanding of the composition, organization, interaction, and function of the various cell types in complex biological systems. However, the current multiplexed protein imaging technologies suffer from low detection sensitivity, limited multiplexing capacity, or are technically demanding. To tackle these issues, here, we report the development of a highly sensitive and multiplexed in situ protein profiling method using off-the-shelf antibodies. In this approach, the protein targets are stained with horseradish peroxidase (HRP) conjugated antibodies and cleavable fluorophores via click chemistry. Through repeated cycles of target staining, fluorescence imaging, and fluorophore cleavage, many proteins can be profiled in single cells in situ. Applying this approach, we successfully quantified 28 different proteins in human formalin-fixed paraffin-embedded (FFPE) tonsil tissue, which represents the highest multiplexing capacity among the tyramide signal amplification (TSA) methods. Based on their unique protein expression patterns and their microenvironment, ∼820,000 cells in the tissue are classified into distinct cell clusters. We also explored the cell-cell interactions between these varied cell clusters and observed that different subregions of the tissue are composed of cells from specific clusters.

Abstract 1044: Low-cost, high-sensitivity InSituPlex® mIF assays on the Parhelia Omni-Stainer™

Abstract Background: Multiplex immunofluorescence (mIF) is an important tool for extracting complex phenotypic information from normal or diseased tissues, and provides accurate and predictive insights on the tissue microenvironment faster. For patient samples in translational and clinical research, high sensitivity, specificity, and reproducibility are critical; however, throughput and cost considerations are also important in designing clinical studies. Enabling an easy-to-use high-performance assay such as InSituPlex® on robust and affordable auto-stainers like the Parhelia Omni-Stainer™ can allow researchers to gain a deeper understanding of the tissue micro-environment through high-quality multiplexed biomarker detection in a fraction of the time and cost. Methods: We performed Ultivue InSituPlex® assays with OmniVUE™ panels using the Parhelia Omni-Stainer™, a low-cost, high-throughput tissue auto-stainer with reduced requirements for reagents with a minimal dead volume. We compared the whole-slide staining performance across replicates using human tonsil and tumor tissue arrays across multiple independent runs over multiple days. The stained slides were scanned using the Zeiss AxioScan.Z1 high-throughput imaging system, and image batches were processed using Ultivue’s UltiStacker.AI and UltiAnalyzer.AI software. Results: Tighter temperature control of the slide in the Omni-Stainer supports optimal InSituPlex® signal amplification. Intra-run and inter-day analysis on serial sections of human tonsil and tumor tissue arrays demonstrated a high degree of consistency in InSituPlex® assay performance, specifically with marker-specific signal intensities and cell densities. The Omni-Stainer facilitates the use of low reagent volumes at each step of the assay due to its efficient reagent use, thus lowering the cost of reagents per slide. Conclusions: This study demonstrated a fast, simple and robust workflow to perform high-performance InSituPlex® mIF assays on the low-cost, high-throughput Parhelia Omni-Stainer™. Ultivue’s configurable OmniVUE™ mIF panels can be run on the Omni-Stainer with reduced associated reagent costs per slide. This workflow can support larger clinical and translational studies in a more cost-effective fashion, leading to in-depth analysis of tissue microenvironment through highly precise multiplexed biomarker detection. Citation Format: Kevin Hwang, Lauren Duro, Alex Veith, Nelson Wedin, Nikolay Samusik, Je H. Lee. Low-cost, high-sensitivity InSituPlex® mIF assays on the Parhelia Omni-Stainer™ [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 1044.

Abstract 7517: Integrating in situ proximity ligation assay for PD1-PDL1 interaction with multiplexed immunofluorescence imaging

Abstract Background: Programmed cell death protein 1 (PD1) and its ligand PD-L1 are pivotal components of the immune checkpoint machinery, playing a crucial role in the regulation of immune responses in cancer. The interaction between PD1 and PD-L1 is a significant therapeutic target in oncology, as its inhibitors have shown improved outcomes across a variety of malignancies. However, the treatment response to immunotherapies such as pembrolizumab has shown a modest response (~30%) in previous studies. This could be explained by the lack of molecular insight pertaining to the interaction of PD1 on T-cells and PD-L1 in the tumor cells in malignant tissues at the time of clinical diagnosis. The recent development of in situ Proximity Ligation Assay (isPLA), marks a significant advancement in the field. This method allows for the visualization of direct protein interactions within tissues, for example between PD1 and PD-L1. This adds an important layer of information compared to multiplexed immunofluorescence, which is useful for deep phenotyping of cell types in a tissue but lacks the ability to detect direct interactions between specific cells. Methods: PD-L1-PD1 interactions in human FFPE tonsil and bladder cancer tissue were identified using the NaveniFlex Tissue Atto647N kit, following the guidelines provided by the manufacturer. This method is capable of detecting proteins within a proximity of less than 40 nm, utilizing oligonucleotide-antibody conjugates that generate an enhanced fluorescent signal. After completing the Naveni® detection process, we performed multiplexed immunofluorescence (IF) using the Phenocycler platform and a panel of 14 barcoded antibodies, to generate isPLA and multiplexed IF data on the same tissue section. Results & Discussion: We have successfully established an automated workflow for the isPLA protocol. In this work, we aim to continue this effort and combine the isPLA, with downstream highly multiplexed immunofluorescence using the Phenocycler platform. We have generated proof of concept data for the successful integration of the two methods, using tonsil and bladder cancer tissue to detect protein interactions between PD1 and PD-L1, as well as phenotypic profiling of the tumor microenvironment with a 14 plex panel. This panel allows us to phenotype the relevant cell types (B cells, dendritic cells, T cells, macrophages, tumor cells and their proliferation status) and subsets of T-cells like cytotoxic CD8+ cells. The integration of the isPLA and multiplexed IF is poised to significantly advance our understanding of the TME and hopefully aid in patient stratification for immune checkpoint inhibition therapy. The clinical significance of this method integration will later be explored in a retrospective cohort of muscle invasive bladder cancer, receiving immune checkpoint inhibition therapy with pembrolizumab as a second line treatment. Citation Format: Tony Ullman, David Krantz, Hampus Elofsson, Carolina Oses Sepulveda, Agata Zieba-Wicher, Päivi Östling, Anders Ullén, Subham Basu, Charlotte Stadler. Integrating in situ proximity ligation assay for PD1-PDL1 interaction with multiplexed immunofluorescence imaging [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 7517.

Abstract 7622: ASPYRE-Lung: Validation of a simple, fast, robust and novel method for multi-variant genomic analysis of actionable NSCLC variants in tissue

Abstract Genomic variant testing of tumors is critical for patients to access the full potential of personalized oncology therapeutics. Current methods such as NGS are costly and challenging to interpret, while PCR assays are limited in the number of variants they can cover. We developed ASPYRE® (Allele-Specific PYrophosphorolysis REaction) technology to address the urgent need for rapid, accessible and affordable diagnostics informing actionable genomic target variants of a given cancer. The targeted ASPYRE-Lung® panel for NSCLC covers 114 variants in 11 genes (ALK, BRAF, EGFR, ERBB2, KRAS, RET, ROS1, MET & NTRK1/2/3) to robustly inform clinical management, based on practice guidelines. The assay detects single nucleotide variants, insertions, deletions, and gene fusions from tissue-derived DNA and RNA simultaneously. We tested the limit of detection, specificity, analytical accuracy and analytical precision of ASPYRE-Lung using FFPE lung tissue samples from patients with NSCLC, variant-negative FFPE tissue from tonsil tissue, and FFPE-based contrived samples with controllable variant allele fractions. The sensitivity of ASPYRE-Lung was determined to be ≤ 3% variant allele fraction for single nucleotide variants and insertions or deletions, ≤ 100 copies for fusions, and ≤ 200 copies for MET exon 14 skipping. The specificity was 100% with no false positive results. The analytical accuracy test yielded no discordant variants between ASPYRE-Lung and orthogonal testing (targeted enrichment NGS) or contrived samples, and results were replicable across operators, reagent lots, runs, and real-time PCR instruments with a high degree of precision. The technology is simple and fast, requiring only four reagent transfer steps using standard laboratory equipment (PCR and qPCR instruments) with analysis via a cloud-based analysis algorithm. ASPYRE-Lung has the potential to be transformative in facilitating access to rapid, actionable molecular profiling of tissue for patients with NSCLC. Citation Format: Ryan T. Evans, Elizabeth Gillon-Zhang, Julia Brown, Katherine Knudsen, Candace King, Amanda S. Green, Ana-Luisa Silva, Justyna M. Mordaka, Rebecca N. Palmer, Alessandro Tomassini, Alejandra Collazos, Christina Xyrafaki, Iyelola Turner, Chau Ha Ho, Dilyara Nugent, Jinsy Jose, Simonetta Andreazza, Kristine von Bargen, Eleanor R. Gray, Magdalena Stolarek-Januszkiewicz, AIshling Cooke, Wendy J. Levin, Honey V. Reddi, Barnaby W. Balmforth, Robert J. Osborne. ASPYRE-Lung: Validation of a simple, fast, robust and novel method for multi-variant genomic analysis of actionable NSCLC variants in tissue [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 7622.

Abstract 1539: A comprehensive analytical validation framework for antibodies used in the CellScape multiplex immunofluorescence assay

Abstract Background: Multiplex immunofluorescence (mIF) is a powerful tool that enables deep phenotyping of cells within the tumor microenvironment (TME). Careful optimization of multiplex antibody panels for specificity, sensitivity and reproducibility is crucial to achieving robust data and is a precondition for assay repeatability and standardization. We describe our validation procedure for VistaPlex™ multiplex assay panels. Methods: Experiments were performed using the CellScape™ platform for precise spatial multiplexing. The platform enables automated cyclic mIF for readily available fluorophore-conjugated antibodies compatible with fresh frozen or FFPE tissues. The use of high dynamic range imaging further facilitates quantitative phenotyping, while excellent imaging resolution provides single-cell discrimination with high accuracy. To design a ready-to-use panel for immune cell phenotyping, individual antibodies were first screened for specificity and then optimized for labeling sensitivity. Signal-to-noise, inter-assay reproducibility, and intra-assay precision were evaluated across serial sections in a multi-stage testing matrix to validate each antibody in isolation and assembled into a panel. Resulting from this effort, we introduce a 15-plex ready-to-use VistaPlex immune profiling panel for human FFPE tissues. Results: Image analysis of signal-to-noise ratios demonstrated robust performance of multiplexed antibodies across multiple tissue sections and tissue types. Measurements of both signal and quantitative immune phenotyping across technical replicates showed a high degree of intra-assay precision and inter-assay reproducibility. The 15-plex panel was validated on human tonsil, lung, breast, and colon tissues. Conclusions: The CellScape platform enables simultaneous detection of multiple protein biomarkers on a single tissue section for deep immune cell profiling in the TME. Rigorously validated VistaPlex panels further streamline this spatial biology workflow and will aid in the generation of consistent and reliable results for end users. Citation Format: Charles E. Jackson, Arne Christians, Jannik Boog, Matthew H. Ingalls, Xenia Meshik, Adam Northcutt, Kayla E. Cashion, J Spencer Schwarz, Oliver Braubach. A comprehensive analytical validation framework for antibodies used in the CellScape multiplex immunofluorescence assay [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 1539.

Abstract 5187: VistaPlex immunofluorescence assay panels provide reliable deep spatial phenotyping for diverse immuno-oncology applications

Abstract Multiplex immunofluorescence (mIF) is a powerful tool that enables deep phenotyping of immune and cancer cells across a wide variety of tissue types. The initial effort and cost required to develop high-plex mIF panels is significant and poses a considerable hurdle for adoption of spatial biology. To streamline high-plex assay development, we developed a process for creating and validating multiplex antibody panels for use in immuno-oncology applications that leverage CellScape™, an automated end-to-end platform for Precise Spatial Multiplexing. Our multiplex assay development was a rigorous, multi-stage, iterative process to evaluate antibodies for suitability, specificity, and reproducibility. These assay panels are formulated as ready-to-use VistaPlex™ assay kits and are intended to enable rapid, reliable spatial phenotyping of key immune populations. To confirm the utility of VistaPlex, we deployed the Human FFPE Spatial Immune Profiling Panel to stain and image various FFPE tissue samples on the CellScape platform. With CellScape, tissues are mounted in enclosed fluidic chambers while on-instrument staining, imaging and signal erasure are conducted in iterative rounds. The panel presented here contains antibodies against 16 common biomarkers used in immune-oncology applications. Among these are CD3, CD4, CD8, CD20, and CD45 to detect cell lineages, PD-1 and PD-L1, to interrogate immune checkpoints, and markers that inform about cell state and activation. Spatial immune profiling data were obtained from human tonsil, colon, breast, and lung tissue samples. Deep immune phenotyping was performed using unsupervised clustering analysis and AI-assisted cluster labelling, and biomarker distributions were studied in the context of cellular neighborhoods and other proximity analyses. Overall, we identified more than 19 cell phenotypes with varying frequencies and spatial arrangements across tissue samples. Our data are robust and demonstrate that VistaPlex Assay kits for spatial profiling present an efficient and cost-effective solution for users that want to break into spatial biology. Citation Format: Arne Christians, Charles E. Jackson, Jannik Boog, Matthew H. Ingalls, Adam Northcutt, Kayla E. Cashion, Jonah Rauer, Karen Kwarta, J Spencer Schwarz, Oliver Braubach. VistaPlex immunofluorescence assay panels provide reliable deep spatial phenotyping for diverse immuno-oncology applications [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 5187.

Abstract 6879: Development of a rapid multiplex immunohistochemistry for characterizing tumor-immune microenvironment

Abstract Intratumoral immune profiles have been related to prognosis and therapeutic efficacy; this could result in personalized treatment based on biomarkers. Although techniques for multifactorial evaluation of different tumor-immune microenvironments have been established, their practical implementation in clinical settings still requires further improvement, where the time required for testing needs to be reduced and the technology needs to be clinically validated. To develop a multiplex, quantitative, and rapid tissue evaluation method based on clinically established standard immunohistochemistry (IHC), we attempted to develop 6-marker rapid multiplex IHC based on our previously reported 14-marker multiplex IHC via the reduction of the number of labels and speeding up the staining procedure (Tsujikawa et al. Cell Reports, 2017). First, we confirmed whether immunological features that were found to be associated with prognosis in 14-marker multiplex IHC analyses could be identified with fewer labels. Quantitative results by selected six markers exhibited significant correlation with those by 14 markers in terms of immune classifications based on the balance of lymphoid and myeloid cells. Next, we developed a rapid staining protocol by optimizing the reaction temperature, chromogen, and washing time, allowing the completion of 6-marker analysis in 5 hours and 49 minutes as opposed to several days required for the conventional multiplex IHC. By using six markers, validation in benign tonsil tissue and head and neck cancer tissue exhibited significant correlation between rapid and standard multiplex IHC in terms of staining intensities, densities of T cells, macrophages, lymphoid/myeloid immune cells ratios, and spatial profiles of intratumoral immune infiltrates. This method may enable a quantitative assessment of tumor-immune microenvironment on a clinically feasible time scale, which promote the development of tissue biomarker-guided therapeutic strategies. Citation Format: Alisa Kimura, Takahiro Tsujikawa, Hiroki Morimoto, Junichi Mitsuda, Sumiyo Saburi, Shigeyuki Mukudai, Hikaru Nagao, Nana Sakurai, Nanako Murakami, Aya Miyagawa-Hayashino, Hiroshi Ogi, Saya Shibata, Eiichi Konishi, Kyoko Itoh, Shigeru Hirano. Development of a rapid multiplex immunohistochemistry for characterizing tumor-immune microenvironment [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 6879.

Abstract 1532: Improved reproducibility in multiplex immunofluorescence analysis of the tumor microenvironment using a novel quenching solution

Abstract Formalin-fixed, paraffin-embedded (FFPE) tissues are commonly used in histopathology because the fixed tissues are stable and easy to store. In the context of immunofluorescence detection, however, endogenous autofluorescence of FFPE tissues can significantly reduce or mask immunofluorescent signals. The autofluorescence of FFPE tissues is caused by a variety of factors, including the presence of endogenous fluorophores, tissue fixation, and processing. While there are several methods that can be used to quench autofluorescence in FFPE tissues, chemical quenchers, which absorb or scatter light, are most frequently used for multiplex IF. Examples of chemical quenchers commonly used in manual multiplex staining include Sudan Black B and TrueBlack; however, these are not chemically compatible for use on automated platforms such as the Leica Biosystems BOND RX. Due to the need for increased throughput and decreased hands-on time, automation has risen in popularity versus manual staining, leading to a need for a versatile quenching solution compatible with automation. Here we demonstrate that NovaPlex Quenching Solution (NPQS), a novel proprietary quenching agent, reduces the background caused by autofluorescence and enhances the signal of the Cell IDx UltraPlex multiplex immunofluorescent (mxIF) assay across tissue types. Qualitative studies showed a decrease in autofluorescent signal in FFPE mouse spleen stained with antibodies against B220/Foxp3/CD3e, suggesting the utility of NPQS in mouse models of cancer and immunity. Similarly, nonspecific background was decreased across FFPE human tissue types, including lung adenocarcinoma stained for CD8/Foxp3/CD3e/CD4 and tonsil stained for CD8/Foxp3/PD-1/CD4. Further statistical studies in FFPE human tonsil tissue stained with CD8/Foxp3/PD-1/CD4 showed there was no significant decrease in dye intensity across all four markers and channels, resulting in a significant increase in signal-to-noise ratio for both CD8 at 490nm and PD-1 at 650nm (p<0.05). These experiments using Cell IDx/LBS UltraPlex mxIF technology demonstrate an effective method to improve the signal-to-noise ratios of fluorophores without reducing signal intensity from any of the target proteins in these parallel multiplex immunofluorescent assays. Citation Format: Matt Levin, Jack Heath, April Schrank-Hacker, Shelby Dunn, Minh-Chau Vu, Imane Hamza, David Schwartz. Improved reproducibility in multiplex immunofluorescence analysis of the tumor microenvironment using a novel quenching solution [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 1532.