Tyramide Signal Amplification Research Articles

Micromonas, a small pigmented flagellate, predominates the nanoflagellate and photosynthetic picoeukaryote communities in the northern South China Sea.

A small pigmented flagellate, Micromonas, is prevalently distributed in coastal and pelagic waters. However, there have been few studies conducted to quantify their abundance in the marginal seas of the Northwest Pacific Ocean. In this study, we used fluorescent in situ hybridization with tyramide signal amplification (TSA-FISH) to reveal the spatial distribution of Micromonas in the northern South China Sea (SCS). On average, the abundance of Micromonas was 317 cells mL-1, with the average proportions in the nanoflagellates (NF) and photosynthetic picoeukaryotes (PPE) communities being 10.94% and 15.39%, respectively. This indicates a wide distribution and dominance of this genus in the studied area. The relationships between Micromonas abundance and various environmental factors suggested that biotic correlations play more important roles than physicochemical filtering on Micromonas assemblage. This may indicate a broad environmental adaptation spectrum of this genus through its flexibility in terms of resource acquisition strategies. In summary, this study provides insight into the spatial distribution pattern of Micromonas and highlights its crucial contribution to the composition of NFs and PPE communities, which rely on biological interaction to respond to the changing environmental conditions in the northern SCS.

Abstract 5525: Tissue-resident cytotoxic T cell infiltration predicts metachronous colorectal cancer metastasis

Abstract Background: Identifying patients at high risk for metachronous colorectal cancer metastasis is essential for improving prognosis. The balance between host immune regulation and tumor immune evasion ultimately permits or prevents cancer progression. Lymphocytic infiltration in colorectal cancer can predict long-term clinical outcomes. However, these lymphocytes' origin, subtype and function have yet to be adequately explored. We investigated the association between the development of metastasis and CD8+T cells expressing the integrin protein CD103, a marker of tissue-resident memory T cells. Methods: In this retrospective, case-control matched study, we conducted multiplex immunofluorescence staining of tumor samples from 124 patients with colorectal cancer. Tissue microarrays containing representative cores of the central tumor, invasive margin, and adjacent normal tissue were immunostained for CD3, CD8, CD103, pSMAD3, and cytokeratin, using tyramide signal amplification. Cells were quantified using StrataQuest digital image analysis software, with intratumoral and stromal regions analyzed separately. Kruskal-Wallis one-way analysis of variance with subsequent Dunn’s test for pair-wise comparisons was performed using the Bonferroni method of multiple-comparison correction. Results: Central tumor and invasive margin CD3+ densities were associated with synchronous metastasis. Compared to non-metastatic patients, those who went on to develop metachronous metastasis (n=42) had a low proportion of intra-epithelial CD8+CD103+ T cells at the central tumor (non-metastatic: 32.8%, IQR 15.0-42.7%; metachronous metastasis: 12.9%, IQR 2.08-28.4%; P = 0.013). Similarly, patients with metachronous metastasis had a low proportion of stromal CD8+CD103+ T cells at the invasive margin (non-metastatic: 3.51%, IQR 1.83-6.55%; metachronous metastasis: 1.75%, IQR 0.62-3.72%; P = 0.019). On multivariate Cox regression of histological features, amongst patients with metastasis, significant overall survival prognostic markers included a low proportional CD8+CD103+ T cell infiltration (HR 2.41, 95%CI 1.16-5.01, P = 0.019), mucinous features (HR 4.28, 95%CI 1.98-9.24, P < 0.001), and the presence of perineural invasion (HR 2.75, 95%CI 1.28-5.89, P < 0.001). Conclusion: CD8+CD103+ lymphocytes may protect from the development of colorectal metastasis. Proportional CD8+CD103+ T cell infiltration is a promising prognostic marker in colorectal adenocarcinoma for the development of metachronous metastasis, and, amongst patients with metastasis, it is a prognostic marker of overall survival. Citation Format: Ryan Cohen, Tracey Lee-Pullen, Timothy Miller, Shadi Pirasteh, Cameron Platell, Katie Meehan, Kathy Fuller, Melanie McCoy. Tissue-resident cytotoxic T cell infiltration predicts metachronous colorectal cancer metastasis [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 5525.

Abstract 1156: A systematic comparison of common multiplex immunohistochemistry (mIHC) methodologies to the novel SignalStarTM assay in the characterization of the tumor microenvironment (TME)

Abstract The emergence of an increasing number of immunotherapy biomarkers and their importance within the spatial context of the TME has resulted in a concomitant need for reliable and accurate mIHC assays. Many of the currently available mIHC technologies do not offer amplification, resulting in the loss of detection of low expressing cells, while others require deposition and cycling of antibodies, which can lead to epitope masking and degradation, respectively. The SignalStar mIHC assay from Cell Signaling Technology is a novel methodology used to label, amplify and visualize up to 8 targets within the same formalin fixed, paraffin-embedded carcinoma (FFPE) tissue section without the need for fluorophore deposition or antibody cycling. In this study, the SignalStar mIHC assay was used to simultaneously label 8 targets including TIM-3, PD-1, PD-L1, LAG3, CD3, CD68, CD8 and Pan-Keratin in FFPE carcinoma tissue. A network of complementary, fluorescently-labeled oligonucleotides was used to amplify the signal of the first 4 antibody-oligo conjugates followed by tissue imaging. The fluorescent signal was then removed, and the process repeated.The resulting two 4-plex images were then computationally aligned with QuPath. This methodology was compared to the Tyramide Signal Amplification (TSA) deposition assay, as well as to indirect and direct immunofluorescent detection with respect to mean fluorescence intensity (MFI) per cell and percent positivity of cells in matched regions of interest in serial sections. All mIHC strategies were in turn compared to the canonical chromogenic IHC assay. Furthermore, the fluorescent signal from the 8-plex SignalStar assay was removed and the same tissue was stained with fluorescent direct conjugates to achieve even higher plex. Our data demonstrates that the SignalStar assay detected similar percentages of cells when compared to chromogenic and TSA staining. Alternatively, the SignalStar assay often outperformed indirect and direct immunofluorescent detection. SignalStar was also successfully combined with a panel of direct Alexa Fluor® dye conjugates, showing the complementarity of these assays in such a way that both plex and flexibility can be increased. Ultimately, we demonstrate that SignalStar mIHC is a useful tool in the characterization and analysis of the complex TME. SignalStar mIHC outshines existing technologies as it does not require deposition in order to amplify the signal of multiple colocalizing targets, including low abundance proteins, while also maintaining dynamic range. Citation Format: Jennifer Ziello, Derek Papalegis, Lily Vu, Kelsey Goldman, Jean Loebelenz, Shleby Sponaugle, Sasha Tkachev, Sarah Klein. A systematic comparison of common multiplex immunohistochemistry (mIHC) methodologies to the novel SignalStarTM assay in the characterization of the tumor microenvironment (TME) [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 1156.

Visualization of PD-L1-positive and PD-1-positive immune cell contact in the breast cancer microenvironment

Functioning of the Pd-1/Pd-L1 immune checkpoint in the microenvironment of breast cancer may lead to the tumor escape from the immune response. However, it is unknown how often Pd-L1 binds to Pd-1 in breast cancer patients, which Pd-L1-positive cells are predominantly involved in the interaction, and what prognostic significance it has. The objective of the study was to assess the frequency of co-location of Pd-1/Pd-L1-positive cells in the microenvironment of breast cancer as well as to determine the population of these cells. Material and Methods. The study included 25 patients with invasive breast carcinoma. Interaction between cells carrying the Pd-1 receptor and the Pd-L1 ligand in the tumor microenvironment were visualized using multiplex TSA (tyramide signal amplification)-modified immunohistochemistry. Participation of M1 macrophages (Cd68+Cd163-Cd3-CKAE1/3-), M2 macrophages (Cd68+/-Cd163+Cd3-CKAE1/3-), lymphocytes (Cd68-Cd163-Cd3+CKAE1/3-) and other immune cells in these interactions was assessed. Results. Half of the breast cancer patients included in the study had interactions of immune cells of the microenvironment, one of which carried Pd-1, and the other carried Pd-L1. The contact of cells carrying Pd-1 and Pd-L1 was associated with the level of TILs and the ratio of Pd-1+/ Pd-L1+ cells in the tumor microenvironment. The Pd-1/Pd-L1 interaction was found with similar frequency in Pd-L1 positive and negative patients. In the cell contacts, macrophages acted as Pd-L1+ cells in the vast majority of cases. Lymphocytes were Pd-1-positive cells rather than Pd-L1-carrying cells. In addition, it was found that metastasis-free survival was not associated with the presence or absence of co-localized cells carrying Pd-1 and Pd-L1 in the tumor microenvironment. Conclusion. Co-location of immune cells carrying Pd-1 and Pd-L1 occurs in breast cancer. M1 and M2 macrophages, Cd3+ lymphocytes and other immune cells are involved in these interactions. However, further studies are needed to establish the prognostic significance of these contacts.

Characterization of Vascular Niche in Systemic Sclerosis by Spatial Proteomics.

Systemic sclerosis (SSc) is a connective tissue disease that can serve as a model to study vascular changes in response to inflammation, autoimmunity, and fibrotic remodeling. Although microvascular changes are the earliest histopathologic manifestation of SSc, the vascular pathophysiology remains poorly understood. We applied spatial proteomic approaches to deconvolute the heterogeneity of vascular cells at the single-cell level in situ and characterize cellular alterations of the vascular niches of patients with SSc. Skin biopsies of patients with SSc and control individuals were analyzed by imaging mass cytometry, yielding a total of 90 755 cells including 2987 endothelial cells and 4096 immune cells. We identified 7 different subpopulations of blood vascular endothelial cells (VECs), 2 subpopulations of lymphatic endothelial cells, and 3 subpopulations of pericytes. A novel population of CD34+;αSMA+ (α-smooth muscle actin);CD31+ VECs was more common in SSc, whereas endothelial precursor cells were decreased. Co-detection by indexing and tyramide signal amplification confirmed these findings. The microenvironment of CD34+;αSMA+;CD31+ VECs was enriched for immune cells and myofibroblasts, and CD34+;αSMA+;CD31+ VECs expressed markers of endothelial-to-mesenchymal transition. The density of CD34+;αSMA+;CD31+ VECs was associated with clinical progression of fibrosis in SSc. Using spatial proteomics, we unraveled the heterogeneity of vascular cells in control individuals and patients with SSc. We identified CD34+;αSMA+;CD31+ VECs as a novel endothelial cell population that is increased in patients with SSc, expresses markers for endothelial-to-mesenchymal transition, and is located in close proximity to immune cells and myofibroblasts. CD34+;αSMA+;CD31+ VEC counts were associated with clinical outcomes of progressive fibrotic remodeling, thus providing a novel cellular correlate for the crosstalk of vasculopathy and fibrosis.

Automatic ultrasensitive lateral flow immunoassay based on a color-enhanced signal amplification strategy

Lateral flow immunoassays (LFIAs) are an essential and widely used point-of-care test for medical diagnoses. However, commercial LFIAs still have low sensitivity and specificity. Therefore, we developed an automatic ultrasensitive dual-color enhanced LFIA (DCE-LFIA) by applying an enzyme-induced tyramide signal amplification method to a double-antibody sandwich LFIA for antigen detection. The DCE-LFIA first specifically captured horseradish peroxidase (HRP)-labeled colored microspheres at the Test line, and then deposited a large amount of tyramide-modified signals under the catalytic action of HRP to achieve the color superposition. A limit of detection (LOD) of 3.9 pg/mL and a naked-eye cut-off limit of 7.8 pg/mL were achieved for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleoprotein. Additionally, in the inactivated virus detections, LOD equivalent to chemiluminescence (0.018 TCID50/mL) was obtained, and it had excellent specificity under the interference of other respiratory viruses. High sensitivity has also been achieved for detection of influenza A, influenza B, cardiac troponin I, and human chorionic gonadotrophin using this DCE-LFIA, suggesting the assay is universally applicable. To ensure the convenience and stability in practical applications, we created an automatic device. It provides a new practical option for point-of-care test immunoassays, especially ultra trace detection and at-home testing.

Isolation and characterization of rabbit limbal niche cells

Limbal niche cells (LNCs) are one of the most important supporting cells for corneal epithelial stem cells (CES), however, research on LNCs has been mostly limited to humans and rats previously. To expand the research work into the rabbit animal model, one of the most often used animals in stem cell study, this study was carried out for the in vitro isolation and identification of rabbit LNCs.Rabbit LNCs were isolated by collagenase A digestion method and single cells were obtained, the cells were then seeded on 5% Matrigel-coated plastic surface and cultured in modified embryonic stem cell medium (MESCM). Three biological replicates of the isolating and characterization were recorded from New Zealand White rabbits aged from 2.5 months to 5 months. LNC markers (VIM/CD90/CD105/SCF/PDGFRβ) were analyzed using tyramide signal amplification (TSA) staining, immunohistochemical staining (IHC), western blotting (WB), and real-time reverse transcription polymerase chain reaction (qPCR). TSA staining suggested that VIM was highly expressed in rabbit limbus stroma, which was confirmed by WB, and P63α was expressed in the basal limbus epithelium. Pan-CK and CK12 were highly expressed in the central corneal epithelium but lightly expressed in the limbal epithelium. The WB result indicated that PDGFRβ and VIM expressions in rabbit-LNCs P4 were higher than in P1 and P7. In addition, rabbit corneal epithelium highly expressed Paired Box 6 (PAX6) and Epidermal growth factor-like domain 6(EGFL6). For the three repeat experiments, the cell expansion activity of rabbit-LNC was highest at P4. Rabbit-LNCs were passaged from P0 to P7, and the number of cell doublings (NCD) of P4 for the three repeat experiments was 2.816, 2.737, and 2.849. qPCR showed that high mRNA expression levels of VIM, CD90, CD105, SCF, and PDGFRβ in rabbit-LNCs P4.In conclusion, rabbit-LNCs could be successfully isolated by the collagenase A digestion method as used in human tissue. There were similar characteristics between rabbit and human LNCs (VIM+/CD90+/CD105+/SCF+/PAX6+/PDGFRβ+).

Abstract C045: Multiplex fluorescent immunohistochemistry reveals immune microenvironment alterations in COMPASS-like complex gene mutated pancreatic cancer

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is highly resistant to chemoradiation and immune therapies. There is limited biomarker to identify subsets of PDAC that responds to immunotherapy or targeted therapy. We have identified a subset of PDACs (approximately 25%) carrying COMPASS-like complex (CLC) gene mutations. Analysis of the clinicopathologic and molecular features of this PDAC subset revealed that these tumors are an aggressive group of PDACs with poor or squamous histologic differentiation, increased rates of TP53 mutations, and poor patient survival compared to control PDACs. Our preliminary data showed that PDACs carrying mutations in two members of the CLC genes (KDM6A and KMT2D) have immunosuppressive tumor microenvironment (TME), suggesting that CLC genes are candidate biomarkers for immunotherapy. However, a comprehensive analysis of the immune composition, immune checkpoint expression, and the relationship between tumor cells and immune cells in CLC gene mutated PDACs compared to controls has not been performed. In this study, we characterized the immune composition in CLC gene mutated PDACs compared to paired control PDACs using tyramide signal amplification multiplex fluorescent immunohistochemistry (mfIHC). Methods: Human PDAC tissue slides were stained with 7 markers using mfIHC and allowed for phenotyping of 7 cell types: PD-L1- epithelial cells (ECs) (pancytokeratin+PD-L1-), PD-L1+ ECs (pancytokeratin+PD-L1+), cytotoxic T lymphocytes (CTLs) (CD3+CD8+), helper T cells (CD3+CD8-FoxP3-), Tregs (CD3+CD8-FoxP3+), PD-L1- antigen presenting cells (APCs) (CD163+PD-L1-), and PD-L1+ APCs (CD163+PD-L1+). Random image sections were selected in each sample (5~8 sections per sample) and divided into wild-type (WT) group (n=49) and mutant group (n=49) and analyzed using inForm Cell Analysis software. Tissue segmentation training was performed to separate stromal and epithelial elements. Cell percentage, cell density, and nearest neighbor distance of each cell type were calculated using R program. Results: There is an increased infiltration of T cells, Tregs, and helper T cells in the epithelial component of the mutant group compared with WT group. The epithelial cells are more frequent to express PD-L1. Interestingly, we also observed more Treg infiltration in the stromal component and total tissue in the mutant group compared with WT group. The CTLs are more engaged with the epithelial cells, regardless of their PD-L1 status, in the mutant group compared with WT group. Conclusions: Our results provide insight into the effects of CLC gene mutations on shaping the immune microenvironment of PDAC and suggest that CLC gene mutated PDACs have a different immune microenvironment than other PDACs and may respond differently to immunotherapy. Citation Format: Shungang Zhang, Jake McGue, Elaina Daniels, Hong Sun Kim, Dafydd Thomas, Jiaqi Shi. Multiplex fluorescent immunohistochemistry reveals immune microenvironment alterations in COMPASS-like complex gene mutated pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C045.

Loss of Human Epidermal Receptor 2 Expression in Formalin-Fixed Paraffin-Embedded Breast Cancer Samples and the Rescuing Effect of Enhanced Antigen Retrieval and Signal Amplification.

As an important therapeutic target in breast cancer, HER2 expression assessed by immunohistochemistry plays a critical role in breast cancer treatment. Recent advances in HER2 antibody-drug conjugate therapy have enabled patients with HER2-low expression breast cancer to benefit from the drugs. However, it is not known whether the HER2-low expression in breast cancer FFPE blocks would be lost as storage time increased. In this study, we aimed to assess the loss of HER2 antigenicity in stored FFPE blocks of breast cancer and the rescue effect of modifying the protocol of antigen staining. We selected archived HER2-low breast cancer FFPE blocks with stored time ranging from 1 year to over 15 years and re-detected the expression of HER2. Our study showed that HER2 antigenicity loss increased with storage time and could cause false negativity in HER2-low detection. Moreover, we showed that by either increasing the antigen retrieval time or applying the tyramide signal amplification (TSA) kit, the HER2 signal can be rescued and detected in about half of the cases with HER2-low loss without causing false positivity.

Cellular and subcellular localization of Rab10 and phospho-T73 Rab10 in the mouse and human brain

Autosomal dominant pathogenic mutations in Leucine-rich repeat kinase 2 (LRRK2) cause Parkinson’s disease (PD). The most common mutation, G2019S-LRRK2, increases the kinase activity of LRRK2 causing hyper-phosphorylation of its substrates. One of these substrates, Rab10, is phosphorylated at a conserved Thr73 residue (pRab10), and is one of the most abundant LRRK2 Rab GTPases expressed in various tissues. The involvement of Rab10 in neurodegenerative disease, including both PD and Alzheimer’s disease makes pinpointing the cellular and subcellular localization of Rab10 and pRab10 in the brain an important step in understanding its functional role, and how post-translational modifications could impact function. To establish the specificity of antibodies to the phosphorylated form of Rab10 (pRab10), Rab10 specific antisense oligonucleotides were intraventricularly injected into the brains of mice. Further, Rab10 knock out induced neurons, differentiated from human induced pluripotent stem cells were used to test the pRab10 antibody specificity. To amplify the weak immunofluorescence signal of pRab10, tyramide signal amplification was utilized. Rab10 and pRab10 were expressed in the cortex, striatum and the substantia nigra pars compacta. Immunofluorescence for pRab10 was increased in G2019S-LRRK2 knockin mice. Neurons, astrocytes, microglia and oligodendrocytes all showed Rab10 and pRab10 expression. While Rab10 colocalized with endoplasmic reticulum, lysosome and trans-Golgi network markers, pRab10 did not localize to these organelles. However, pRab10, did overlap with markers of the presynaptic terminal in both mouse and human cortex, including α-synuclein. Results from this study suggest Rab10 and pRab10 are expressed in all brain areas and cell types tested in this study, but pRab10 is enriched at the presynaptic terminal. As Rab10 is a LRRK2 kinase substrate, increased kinase activity of G2019S-LRRK2 in PD may affect Rab10 mediated membrane trafficking at the presynaptic terminal in neurons in disease.

Protein and lipid expansion microscopy with trypsin and tyramide signal amplification for 3D imaging

Expansion microscopy, whereby the relative positions of biomolecules are physically increased via hydrogel expansion, can be used to reveal ultrafine structures of cells under a conventional microscope. Despite its utility for achieving super-resolution imaging, expansion microscopy suffers a major drawback, namely reduced fluorescence signals caused by excessive proteolysis and swelling effects. This caveat results in a lower photon budget and disfavors fluorescence imaging over a large field of view that can cover an entire expanded cell, especially in 3D. In addition, the complex procedures and specialized reagents of expansion microscopy hinder its popularization. Here, we modify expansion microscopy by deploying trypsin digestion to reduce protein loss and tyramide signal amplification to enhance fluorescence signal for point-scanning-based imaging. We name our new methodology TT-ExM to indicate dual trypsin and tyramide treatments. TT-ExM may be applied for both antibody and lipid staining. TT-ExM displayed enhanced protein retention for endoplasmic reticulum and mitochondrial markers in COS-7 cell cultures. Importantly, TT-ExM-based lipid staining clearly revealed the complex 3D membrane structures in entire expanded cells. Through combined lipid and DNA staining, our TT-ExM methodology highlighted mitochondria by revealing their DNA and membrane structures in cytoplasm, as well as the lipid-rich structures formed via phase separation in nuclei at interphase. We also observed lipid-rich chromosome matrices in the mitotic cells. These high-quality 3D images demonstrate the practicality of TT-ExM. Thus, readily available reagents can be deployed in TT-ExM to significantly enhance fluorescence signals and generate high-quality and ultrafine-resolution images under confocal microscopy.

A Functional Hydrogel Bead-Based High-Throughput Screening System for Mammalian Cells with Enhanced Secretion of Therapeutic Antibodies.

Droplet-based high-throughput screening systems are an emerging technology that provides a quick test to screen millions of cells with distinctive characteristics. Biopharmaceuticals, specifically therapeutic proteins, are produced by culturing cells that secrete heterologous recombinant proteins with different populations and expression levels; therefore, a technology to discriminate cells that produce more target proteins is needed. Here, we present a droplet-based microfluidic strategy for encapsulating, screening, and selecting target cells with redox-responsive hydrogel beads (HBs). As a proof-of-concept study, we demonstrate the enrichment of hybridoma cells with enhanced capability of antibody secretion using horseradish peroxidase (HRP)-catalyzed hydrogelation of tetra-thiolate poly(ethylene glycol); hybridoma cells were encapsulated in disulfide-bonded HBs. Recombinant protein G or protein M with a C-terminal cysteine residue was installed in the HBs via disulfide bonding to capture antibodies secreted from the cells. HBs were fluorescently stained by adding the protein L-HRP conjugate using a tyramide signal amplification system. HBs were then separated by fluorescence-activated droplet sorting and degraded by reducing the disulfide bonds to recover the target cells. Finally, we succeeded in the selection of hybridoma cells with enhanced antibody secretion, indicating the potential of this system in the therapeutic protein production.

Abstract B048: Multiplex analysis of pancreatic, oesophageal and rectal adenocarcinoma: A cross-cancer approach on the impact of neoadjuvant therapy on the tumour microenvironment

Abstract Introduction: The prognosis of gastrointestinal malignancies has improved significantly with the introduction of neoadjuvant therapy (neoTx). For rectal (RCa) and esophageal adenocarcinoma (OCa), neoTx has become the standard of care, resulting in tumor-downsizing and significantly prolonged patient survival. However, the effects of neoTx on the topographical interactions between different populations of tumor-infiltrating immune cells, the degree of intratumoral immune infiltration, the distance to tumor cells, as well as their spatial interactions with other important features of the tumor microenvironment (TME), are still unknown. In this cross-cancer histopathological profiling of the microenvironment of rectal and esophageal tumors, we aim to investigate the local effects of cytotoxic agents and determine whether these correspond to the pathological changes observed in pancreatic cancer (PCa). Methods In this study, we employed multiplex immunohistochemistry (mIHC) based on tyramide signal amplification (TSA) using OPAL® dyes and a customized 6-plex approach to characterize tumor-associated stromal features (CD11c, CD34, NCAM, PGP9.5, aSMA, PanCK) as well as tumor-infiltrating lymphoid (CD3, CD4, CD8, CD20, FOXP3, PanCK) and myeloid cell subsets (CD11b, CD33, CD68, CD208, HLA-DR, PanCK). FFPE samples from 60 neoadjuvantly treated RCa and 40 OCa patients and a matched cohort of primary resected patients were included in the analysis. Results In PCa, neoTx alters the TME by depleting pro-tumorigenic immune cells, such as myeloid-derived suppressor cells (MDSC) and regulatory T cells, and suppressing tumor-associated stromal activation, neural invasion, and microangiogenesis. In our current study, we generated three standardized 6-plex panels for comprehensive histopathologic analysis of the immune architecture and associated stromal features of neoadjuvantly treated RCa and OCa patients compared with primary resected patients. Lymphoid subpopulations included Th-cells, cytotoxic T-cells, regulatory T-cells, and B-cells, while MDSC, dendritic cells, M1- and M2-macrophages were analyzed in the myeloid panel within the same FFPE slide. Conclusion: Our findings highlight new differential cell identities in neoTx-treated PCa patients and confirm their suitability for optimizing future clinical immunotherapy trials. Our current approach validates multiplex-IHC approaches for histological profiling of FFPE tumor samples in RCa and OCa and demonstrates the importance of a deep topographic characterization to understand the TME composition of gastrointestinal malignancies after neoTx. Citation Format: Leonard Richter. Multiplex analysis of pancreatic, oesophageal and rectal adenocarcinoma: A cross-cancer approach on the impact of neoadjuvant therapy on the tumour microenvironment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B048.

Downregulation of N4-acetylcytidine modification in myeloid cells attenuates immunotherapy and exacerbates hepatocellular carcinoma progression.

N4-acetylcytidine (ac4C) is a conserved and abundant mRNA modification that controls protein expression by affecting translation efficiency and mRNA stability. Whether the ac4C modification of mRNA regulates hepatocellular carcinoma (HCC) development or affects the immunotherapy of HCC is unknown. By constructing an orthotopic transplantation mouse HCC model and isolating tumour-infiltrated immunocytes, we evaluated the ac4C modification intensity using flow cytometry. Remodelin hydrobromide (REM), an ac4C modification inhibitor, was systematically used to understand the extensive role of ac4C modification in immunocyte phenotypes. Single-cell RNA-seq was performed to comprehensively evaluate the changes in the tumour-infiltrating immunocytes and identify targeted cell clusters. RNA-seq and RIP-seq analyses were performed to elucidate the underlying molecular mechanisms. Tyramide Signal Amplification (TSA) analysis on the HCC tissue microarray was performed to explore the clinical relatedness of our findings. Ac4C modification promoted M1 macrophage infiltration and reduced myeloid-derived suppressor cell MDSCs infiltration in HCC. The inhibition of ac4C modification induces PDL1 expression by stabilising mRNA in the myeloid cells, thereby attenuating the CTL-mediated tumour cell-killing ability. High infiltration of ac4C+CD11b+ cells is positively related to a better prognosis in patients with HCC. Ac4C modification of myeloid cells enhanced the HCC immunotherapy by suppressing PDL1 expression.

A novel method for the whole-cell detection of environmental microorganisms using hemin and tyramide signal amplification (Hemin-TSA) with a desired fluorescent dye

A method called hemin-tyramide signal amplification (Hemin-TSA) was developed for visualization of environmental microorganisms using hemin and tyramide signal amplification. In Hemin-TSA, hemin, which has peroxidase activity, is bound to microbial cells, and a desired fluorescent dye is deposited on the microbial cells by a hemin-catalyzed TSA reaction. The protocol was initially optimized in terms of hemin concentration, hemin binding time and repeated reaction times of TSA. Hemin-TSA showed a comparative or improved signal-to-noise ratio compared to DAPI staining. The shapes of fluorescent signals obtained from microbial cells were almost morphologically identical to those observed in phase contrast microscopy. Hemin-TSA staining provided more accurate cell counts than DAPI staining, especially for actively growing cells, for which two or three spotty DAPI signals were obtained from a single cell. In addition, microbial cells that were not detected by DAPI staining were detected by Hemin-TSA with fluorescein, which enabled us to avoid high non-specific fluorescence under UV excitation. The method developed in this study allows us to visually detect microbial cells in various environments with the characteristics of better cell morphological identification, improved enumeration accuracy and selectivity of fluorescent dyes.

Optimising multiplex immunofluorescence staining for characterising the tumour immune micro-environment.

Exploring the tumour microenvironment provides insight into the unique interaction between the host and tumour. Ultimately, its study improves understanding of how an individual mounts and achieves an anti-tumour immune response. In the context of colorectal cancer, immune biomarkers within the tumour microenvironment outperform traditional histopathological staging in predicting disease recurrence. Multiplex immunofluorescence enables simultaneous assessment of multiple markers to provide a highly accurate classification of immune cells and their spatial characterisation relative to tumour tissue. Further, automated slide staining provides staining consistency and reduces labour costs. Image acquisition using a non-spectral scanner allows more researchers to utilise multiplexed immunofluorescence for translational research. Herein we describe the optimisation process of conducting automated staining using a five-colour, tyramide signal amplification-based multiplex immunofluorescence panel. Using antibodies against CD3, CD8, CD103 and cytokeratin, the panel characterises T cell populations within human colorectal adenocarcinoma tissue. We provide an overview of primary antibody titration and the development of tyramide signal amplification immunofluorescence monoplex assays. We detail the processes of antibody stripping and the role of exogenous horseradish peroxidase inhibition to facilitate multiplexing. An account of determining the staining sequence and fluorophore assignment is provided. We describe image acquisition using a standard fluorescence microscope slide scanner and the management of spectral crosstalk using this system. Finally, we briefly document the digital image analysis required to characterise cells and determine their spatial distribution within the colorectal tumour microenvironment.

Double Digital Assay for Single Extracellular Vesicle and Single Molecule Detection.

Extracellular vesicles (EVs) have emerged as a promising source of biomarkers for disease diagnosis. However, current diagnostic methods for EVs present formidable challenges, given the low expression levels of biomarkers carried by EV samples, as well as their complex physical and biological properties. Herein, a highly sensitive double digital assay is developed that allows for the absolute quantification of individual molecules from a single EV. Because the relative abundance of proteins is low for a single EV, tyramide signal amplification (TSA) is integrated to increase the fluorescent signal readout for evaluation. With the integrative microfluidic technology, the technology's ability to compartmentalize single EVs is successfully demonstrated, proving the technology's digital partitioning capacity. Then the device is applied to detect single PD-L1 proteins from single EVs derived from a melanoma cell line and it is discovered that there are ≈2.7 molecules expressed per EV, demonstrating the applicability of the system for profiling important prognostic and diagnostic cancer biomarkers for therapy response, metastatic status, and tumor progression. The ability to accurately quantify protein molecules of rare abundance from individual EVs will shed light on the understanding of EV heterogeneity and discovery of EV subtypes as new biomarkers.

Molecular profiles of different PD-L1 expression in patients with esophageal squamous cell carcinoma

ABSTRACT Background PD-1/PD-L1 inhibitors are approved treatments for patients with esophageal squamous cell carcinoma (ESCC). The present investigation aspired to explore the interrelation between molecular phenotype and PD-L1 expression in ESCC. Methods PD-L1 testing and targeted next-generation sequencing (NGS) were performed on tumoral tissues from 139 ESCC patients. Tumor-infiltrating lymphocytes (TILs) were scrutinized using a tyramide signal amplification system combined with immunohistochemistry. Results Among enrolled patients, 36.7% displayed high PD-L1 expression (combined positive score [CPS] ≥10). BRCA1 and NF1 gene mutations were significantly associated with high PD-L1 expression (p < .05) while TGFβ pathway alterations were linked to low PD-L1 expression (p = .02). High copy number instability (CNI) and copy number alterations (CNA) were correlated with low PD-L1 expression. Patients with CDKN2A deletion exhibited higher PD-L1 expression. Varying types of TILs were observed across different PD-L1 expression groups. The ratio of CD8+PD-L1+ T cells and CD8+PD-1+ T cells to CD8+ T cells remained comparable in both tumoral and stromal regions, but the ratio of CD68+PD-L1+ macrophages to CD68+ macrophages was higher than the ratio of CD68+PD-1+ macrophages to CD68+ macrophages. CPS was significantly correlated with PD-L1+ lymphocytes and CD68+ macrophages in the tumoral region. CD8+ T cell infiltration was positively correlated with PD-1+ cells in both tumoral and stromal regions. Conclusion In this study, we presented the prevalence rates of PD-L1 expression in Chinese ESCC patients. The association of genetic profiles with PD-L1 expression levels also provide the clue that genomic phenotype may interact with the immunologic phenotype in ESCC.

RNA methylation-driven assembly of fluorescence-encoded nanostructures for sensitive detection of m6A modification writer METTL3/14 complex in human breast tissues

N6-methyladenosine (m6A) is an ubiquitous post-transcriptional modification catalyzed by METTL3/14 complex in eukaryotic mRNAs. The abnormal METTL3/14 complex activity affects multiple steps of RNA metabolism and may induce various diseases. Herein, we demonstrate the RNA methylation-driven assembly of fluorescence-encoded nanostructures for sensitive detection of m6A modification writer METTL3/14 complex in human breast tissues. METTL3/14 complex can catalyze the methylation of RNA probe to prevent it from being cleaved by MazF. The intact RNA probe is recognized by the magnetic bead (MB)-capture probe conjugates to induce duplex-specific nuclease (DSN)-assisted cyclic digestion, exposing numerous shorter ssDNAs with 3′-OH end. The shorter ssDNAs on the MB surface can act as the primers to initiate terminal deoxynucleotidyl transferase (TdT)-enhanced tyramide signal amplification (TSA), forming the Cy5 fluorescence-encoded nanostructures. After magnetic separation, the Cy5 fluorescence-encoded nanostructures are digested by DNase I to release abundant Cy5 fluorophores that can be simply quantified by fluorescence measurement. This assay achieves good specificity and high sensitivity with a detection limit of 58.8 aM, and it can screen METTL3/14 complex inhibitors and quantify METTL3/14 complex activity at the single-cell level. Furthermore, this assay can differentiate the METTL3/14 complex level in breast cancer patient tissues and healthy volunteer tissues.

BMI1-induced CD127+KLRG1+ memory T cells enhance the efficacy of liver cancer immunotherapy

The efficacy of HCC (hepatocellular carcinoma) immunotherapy is hindered by the limited reactivity and short duration of tumor-infiltrating T cells. These deficiencies may be ascribed to the proliferative ability of T cells. The primary objective of this study was to identify the key factor regulating tumor-infiltrating lymphocytes (TIL) proliferation within the HCC microenvironment. Through the utilization of tissue-infiltrated T cell proteomics and fraction proteomics, we analyzed the differential proteins in T cells among HCC, liver fibrosis, and hemangioma (serving as controls) groups. Additionally, we examined the differential regulatory TFs of T cells between the HCC and VH (volunteer healthy, as a control) groups. Using cyTOF and flow cytometry technologies, as well as generating CD8+ T-specific BMI1 knockout mice, we confirmed that BMI1 controls CD127+KLRG1+ memory cell differentiation. Through RNA-seq and MeRIP-seq, we verified that BMI1 regulates TCF1 expression independently of its classical function. Furthermore, by conducting Tyramide signal amplification (TSA) IHC analysis, employing a hydrodynamic mouse HCC model, and utilizing liver-specific nanoparticle targeting therapy, we demonstrated that BMI1 in HCC influences the proliferation of infiltrating CD8+T. BMI1 inhibition promotes effector T cell differentiation while suppressing memory T cell differentiation. Moreover, liver-specific BMI1 knockdown proves beneficial in ameliorating T cell dysfunction and decelerating HCC progression. Our research group has pioneered the exploration of the proteomics of HCC-infiltrated T cells, shedding light on the pivotal role of BMI1 in controlling CD127+KLRG1+ memory CD8+ T cell differentiation, which serves as the cornerstone for achieving immunotherapy efficacy in HCC.