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Staining of CD4 and CD8 T cells in spleen. Slides were stained with CD3 (A), CD4 (B), and CD8 (C) in that order with Ag stripping in between. (D) Merge of CD3 and CD4. (E) Merge of CD3 and CD8. (F) Merge of CD3, CD4, and CD8. (G) Pseudo-H&E image of CD4 staining with DAPI. (H) Pseudo-H&E image of CD3 (red), CD4 (brown), and CD8 (magenta) on cells adjacent to a central artery (CA). (I) H&E image of spleen sections showing lymphocyte distribution around a CA. Original magnification 320.
Source publication
Recent advances in multiplex immunohistochemistry techniques allow for quantitative, spatial identification of multiple immune parameters for enhanced diagnostic and prognostic insight. However, applying such techniques to murine fixed tissues, particularly sensitive epitopes, such as CD4, CD8α, and CD19, has been difficult. We compared different f...
Contexts in source publication
Context 1
... for subsequent staining with no cross-epitope staining (Fig. 4C- F). This demonstrates that the basic stripping buffer is suitable for multiplex fluorescent IHC detection of sensitive T and B cell markers in murine tissues. We were able to use the basic strip- ping buffer to successfully identify CD3, CD4, and CD8 pop- ulations in the spleen (Fig. ...
Context 2
... presence of these CD3 2 CD4 + cells in the spleen outlines the importance of multiplex staining to reliably study various immune cell populations. We were able to apply the technique in a multiplex platform to examine multiple parameters simulta- neously in fixed specimens of murine spleens (Fig. 5), as well as two orthotopically implanted tumor cell lines (Fig. 7). To study these sensitive epitopes, such as CD4 and CD8a, in a multiplex platform, a mild Ag-stripping solution, such as glycine, is superior for proper preservation of signal compared with heat-mediated Ag-stripping solution (Fig. 4). We conclude that the pH-10 ...
Citations
... Additionally, common proteomic assays for assessing biomarkers (e.g., conventional immunohistochemistry [IHC] or immunofluorescence [IF]) can provide in situ spatial context to therapeutic effect. Historically, these assays were limited to measuring 2-5 antigens per sample due to spectral quantification constraints; however, an evolution of highly multiplexed immunostaining techniques using two distinct methods has emerged: (1) conventional antibody staining (i.e., IF or IHC) in a cyclic fashion [59,60] or (2) mass spectrometry imaging (MSI) using rare earth metal labeled antibodies [23,[61][62][63][64][65][66][67][68][69][70][71]. While both of these approaches provide high-dimensional spatial proteomic tissue maps, they are inherently limited due to their destructive signal removal methods in cyclic imaging workflows and limited sensitivity to low abundance antigens using MSI. ...
Purpose: Small molecule drugs such as tyrosine kinase inhibitors (TKIs) targeting tumoral molecular dependencies have become standard of care for numerous cancer types. Notably, epidermal growth factor receptor (EGFR) TKIs (e.g., erlotinib, afatinib, osimertinib) are the current first-line treatment for non-small cell lung cancer (NSCLC) due to their improved therapeutic outcomes for EGFR mutated and overexpressing disease over traditional platinum-based chemotherapy. However, many NSCLC tumors develop resistance to EGFR TKI therapy causing disease progression. Currently, the relationship between in situ drug target availability (DTA), local protein expression and therapeutic response cannot be accurately assessed using existing analytical tools despite being crucial to understanding the mechanism of therapeutic efficacy.
Procedure: We have previously reported development of our fluorescence imaging platform termed TRIPODD (Therapeutic Response Imaging through Proteomic and Optical Drug Distribution) that is capable of simultaneous quantification of single-cell DTA and protein expression with preserved spatial context within a tumor. TRIPODD combines two complementary fluorescence imaging techniques: intracellular paired agent imaging (iPAI) to measure DTA and cyclic immunofluorescence (cyCIF), which utilizes oligonucleotide conjugated antibodies (Ab-oligos) for spatial proteomic expression profiling on tissue samples. Herein, TRIPODD was modified and optimized to provide a downstream analysis of therapeutic response through single-cell DTA and proteomic response imaging.
Results: We successfully performed sequential imaging of iPAI and cyCIF resulting in high dimensional imaging and biomarker assessment to quantify single-cell DTA and local protein expression on erlotinib treated NSCLC models. Pharmacodynamic and pharmacokinetic studies of the erlotinib iPAI probes revealed that administration of 2.5 mg/kg each of the targeted and untargeted probe 4 h prior to tumor collection enabled calculation of DTA values with high Pearson correlation to EGFR, the erlotinib molecular target, expression in the tumors. Analysis of single-cell biomarker expression revealed that a single erlotinib dose was insufficient to enact a measurable decrease in the EGFR signaling cascade protein expression, where only the DTA metric detected the presence of bound erlotinib.
Conclusion: We demonstrated the capability of TRIPODD to evaluate therapeutic response imaging to erlotinib treatment as it relates to signaling inhibition, DTA, proliferation, and apoptosis with preserved spatial context.
... Vectra has gained widespread adoption in the past half-decade, with several notable publications globally. It holds the potential for refining diagnostic criteria and assessing predictive biomarker values, particularly in lymphoid pathology, offering reproducibility and reliability [44]. Some institutions and hospitals use the Vectra system in clinical laboratory tests to assist clinicians in decision-making and treatment plans. ...
Cancer stands as the leading global cause of mortality, with rare cancer comprising 230 distinct subtypes characterized by infrequent incidence. Despite the inherent challenges in addressing the diagnosis and treatment of rare cancers due to their low occurrence rates, several biomedical breakthroughs have led to significant advancement in both areas. This review provides a comprehensive overview of state-of-the-art diagnostic techniques that encompass new-generation sequencing and multi-omics, coupled with the integration of artificial intelligence and machine learning, that have revolutionized rare cancer diagnosis. In addition, this review highlights the latest innovations in rare cancer therapeutic options, comprising immunotherapy, targeted therapy, transplantation, and drug combination therapy, that have undergone clinical trials and significantly contribute to the tumor remission and overall survival of rare cancer patients. In this review, we summarize recent breakthroughs and insights in the understanding of rare cancer pathophysiology, diagnosis, and therapeutic modalities, as well as the challenges faced in the development of rare cancer diagnosis data interpretation and drug development.
... B cells were primarily localized within the white pulp region, specifically within the splenic nodules or B cell follicles (labeled follicles in Fig. 5B). This observation aligns with existing knowledge regarding the compartmentalization of B cells in the spleen, where they play a crucial role in adaptive immunity [64]. The white pulp region, which houses the splenic nodules, is enriched with lymphoid cells and is a significant site for adaptive immune responses. ...
The communication between the nervous and immune systems plays a crucial role in regulating immune cell function and inflammatory responses. Sympathetic neurons, which innervate the spleen, have been implicated in modulating immune cell activity. The neurotransmitter norepinephrine (NE), released by sympathetic neurons, influences immune cell responses by binding to adrenergic receptors on their surface. The alpha-2 adrenergic receptor (α2AR), expressed predominantly on sympathetic neurons, has received attention due to its autoreceptor function and ability to modulate NE release. In this study, we used fast-scan cyclic voltammetry (FSCV) to provide the first subsecond measurements of NE released in the white pulp region of the spleen and validated it with yohimbine, a known antagonist of α2AR. For further application of FSCV in neuroimmunology, we investigated the extent to which subsecond NE from sympathetic neurons is important for immune cell physiology and cytokine production, focusing on tumor necrosis factor-alpha (TNF-α), interleukin-10 (IL-10), and interleukin-6 (IL-6). Our findings provide insights into the regulatory mechanisms underlying sympathetic-immune interactions and show the significance of using FSCV, a traditional neurochemistry technique, to study these neuroimmune mechanisms.
Graphical abstract
... There are currently two main modalities for highly multiplexed imaging that use either (1) antibody staining (i.e., immunohistochemistry [IHC] or immunofluorescence [IF]) or (2) mass spectrometry imaging (MSI) with rare earth metal-labeled antibodies [6][7][8][9][10][11][12][13][14][15][16][17]. Cyclic antibody-based approaches are broadly performed by repeated staining, imaging, and signal removal through fluorophore bleaching [9,14,15] or antibody stripping [17][18][19]. ...
... A variety of antibody staining methods and cyclic immunostaining methods have been proposed to facilitate highly multiplexed spatial proteomics [6][7][8][9][10][11][12][13][14][15][16][17][18][19]. However, it is often challenging to apply a single antibody labeling strategy across all desired biomarker targets, as some antibodies are sensitive to changes from conjugation and may lose specificity. ...
Simple Summary
Advances in our understanding of the complex spatial interactions between tumor epithelia and tumor microenvironmental cells have been driven by highly multiplexed imaging technologies. These techniques are capable of labeling many more biomarkers than conventional immunostaining methods. However, multiplexed imaging techniques suffer from low detection sensitivity, cell loss—particularly in fragile samples—and challenges with antibody labeling. Herein, we developed and optimized a DNA antibody barcoding strategy for cyclic immunofluorescence (cyCIF) that can be amplified to increase the detection efficiency of low-abundance antigens. Stained fluorescence signals can be readily removed using ultraviolet light treatment, preserving tissue and fragile cell sample integrity.
Abstract
Advances in our understanding of the complex, multifaceted interactions between tumor epithelia, immune infiltrate, and tumor microenvironmental cells have been driven by highly multiplexed imaging technologies. These techniques are capable of labeling many more biomarkers than conventional immunostaining methods. However, multiplexed imaging techniques suffer from low detection sensitivity, cell loss—particularly in fragile samples—, and challenges with antibody labeling. Herein, we developed and optimized an oligonucleotide antibody barcoding strategy for cyclic immunofluorescence (cyCIF) that can be amplified to increase the detection efficiency of low-abundance antigens. Stained fluorescence signals can be readily removed using ultraviolet light treatment, preserving tissue and fragile cell sample integrity. We also extended the oligonucleotide barcoding strategy to secondary antibodies to enable the inclusion of difficult-to-label primary antibodies in a cyCIF panel. Using both the amplification oligonucleotides to label DNA barcoded antibodies and in situ hybridization of multiple fluorescently labeled oligonucleotides resulted in signal amplification and increased signal-to-background ratios. This procedure was optimized through the examination of staining parameters including staining oligonucleotide concentration, staining temperature, and oligonucleotide sequence design, resulting in a robust amplification technique. As a proof-of-concept, we demonstrate the flexibility of our cyCIF strategy by simultaneously imaging with the original oligonucleotide conjugated antibody (Ab-oligo) cyCIF strategy, the novel Ab-oligo cyCIF amplification strategy, as well as direct and indirect immunofluorescence to generate highly multiplexed images.
... Then, sections were counterstained using haematoxylin and dehydrated through increasing concentrations of ethanol and xylene. For immunofluorescence staining of BrdU incorporation [44], the liver sections were incubated with 5 lgÁmL À1 anti-BrdU antibody (ab6326; Abcam) at 4°C overnight and then incubated with secondary antibodies for 1 h at room temperature. 4 0 ,6-diamidino-2-phenylindole (C1002; Beyotime, Shanghai, China) was used to stain the cell nuclei for 1 h at room temperature. ...
The strong regenerative ability of the liver safeguards the crucial hepatic functions. The balance between hepatocyte proliferation and death is critical for restoring liver size and physiology. Tumour necrosis factor (TNF) alpha‐induced protein 8‐like 1 (TIPE1) is highly expressed in liver and has been identified as a candidate regulator for cell proliferation and death, being involved in a variety of biological processes and diseases. However, the role of TIPE1 in liver regeneration remains unexplored. In the present study, we found that TIPE1 expression was elevated in the regenerating liver induced by either partial hepatectomy or 10% carbon tetrachloride administration. Mice with hepatocyte conditional Tipe1 knockout presented significantly impaired liver regeneration. Mechanistically, hepatic Tipe1 deficiency decreased the level of reactive oxygen species in hepatocytes, which in turn led to the inhibition of Forkhead box O1 acetylation and microtubule‐associated protein 1 light chain 3 I to microtubule‐associated protein 1 light chain 3 II conversion, and the accumulation of sequestosome 1. By contrast, forced expression of TIPE1 in hepatocyte significantly promoted liver regeneration following 70% partial hepatectomy and enhanced hepatocyte reactive oxygen species/acetylated‐Forkhead box O1 level and autophagy. These findings indicate that TIPE1 plays a crucial role in liver regeneration by finely regulating the oxidative stress and autophagy and is a potential target for medical intervention of liver regeneration.
... AI-powered scoring can also be applied to algorithms that include ICs, such as combined positive score 35 . In these algorithms, PD-L1 IC expression can be difficult to reliably assess visually, and thus pathologist concordance tends to be lower 13,14,36,37 . AIpowered scoring methods may thereby offer more precise and consistent results when defining PD-L1 expression on both TCs and ICs across multiple tumor types and cutoffs. ...
Assessment of programmed death ligand 1 (PD-L1) expression by immunohistochemistry (IHC) has emerged as an important predictive biomarker across multiple tumor types. However, manual quantitation of PD-L1 positivity can be difficult and leads to substantial inter-observer variability. Although the development of artificial intelligence (AI) algorithms may mitigate some of the challenges associated with manual assessment and improve the accuracy of PD-L1 expression scoring, use of AI-based approaches to oncology biomarker scoring and drug development has been sparse, primarily due to the lack of large-scale clinical validation studies across multiple cohorts and tumor types. We developed AI-powered algorithms to evaluate PD-L1 expression on tumor cells by IHC and compared it with manual IHC scoring in urothelial carcinoma, non-small cell lung cancer, melanoma, and squamous cell carcinoma of the head and neck (prospectively determined during the phase II and III CheckMate clinical trials). 1,746 slides were retrospectively analyzed, the largest investigation of digital pathology algorithms on clinical trial datasets performed to date. AI-powered quantification of PD-L1 expression on tumor cells identified more PD-L1–positive samples compared with manual scoring at cutoffs of ≥1% and ≥5% in most tumor types. Additionally, similar improvements in response and survival were observed in patients identified as PD-L1–positive compared with PD-L1–negative using both AI-powered and manual methods, while improved associations with survival were observed in patients with certain tumor types identified as PD-L1–positive using AI-powered scoring only. Our study demonstrates the potential for implementation of digital pathology-based methods in future clinical practice to identify more patients who would benefit from treatment with immuno-oncology therapy compared with current guidelines using manual assessment.
... Two main methods of high dimensional immunostaining employ (1) conventional antibody staining (i.e., immunohistochemistry [IHC] or immunofluorescence [IF]) or (2) mass spectrometry imaging (MSI) using rare earth metal-labeled antibodies [10][11][12][13][14][15][16][17][18][19][20][21] . Cyclic immunostaining produces precise spatial proteome maps by repeated staining, imaging, and signal removal (e.g., fluorophore bleaching 13,18,19 or antibody stripping [21][22][23] ) of a single sample. ...
A number of highly multiplexed immunostaining and imaging methods have advanced spatial proteomics of cancer for improved treatment strategies. While a variety of methods have been developed, the most widely used methods are limited by harmful signal removal techniques, difficulties with reagent production and antigen sensitivity. Multiplexed immunostaining employing oligonucleotide (oligos)-barcoded antibodies is an alternative approach that is growing in popularity. However, challenges remain in consistent conjugation of oligos to antibodies with maintained antigenicity as well as non-destructive, robust and cost-effective signal removal methods. Herein, a variety of oligo conjugation and signal removal methods were evaluated in the development of a robust oligo conjugated antibody cyclic immunofluorescence (Ab-oligo cyCIF) methodology. Both non- and site-specific conjugation strategies were assessed to label antibodies, where site-specific conjugation resulted in higher retained binding affinity and antigen-specific staining. A variety of fluorescence signal removal methods were also evaluated, where incorporation of a photocleavable link (PCL) resulted in full fluorescence signal removal with minimal tissue disruption. In summary, this work resulted in an optimized Ab-oligo cyCIF platform capable of generating high dimensional images to characterize the spatial proteomics of the hallmarks of cancer.
... The Vectra Polaris™ platform used in this study allows for up to eight targets to be simultaneously visualised on a single specimen, facilitating quantitative spatial analysis and immunophenotype identification 66,67 . It is the foremost used mIF system and has been successfully applied to various solid tumours for research purposes 25,26,66,68,69 . Newer systems, such as Akoya CODEX™, can accommodate up to 40 markers and studies have begun to use this system 67,70 . ...
Triple negative breast cancer (TNBC) comprises 10–15% of all breast cancers and has a poor prognosis with a high risk of recurrence within 5 years. PD-L1 is an important biomarker for patient selection for immunotherapy but its cellular expression and co-localization within the tumour immune microenvironment and associated prognostic value is not well defined. We aimed to characterise the phenotypes of immune cells expressing PD-L1 and determine their association with overall survival (OS) and breast cancer-specific survival (BCSS). Using tissue microarrays from a retrospective cohort of TNBC patients from St George Hospital, Sydney (n = 244), multiplexed immunofluorescence (mIF) was used to assess staining for CD3, CD8, CD20, CD68, PD-1, PD-L1, FOXP3 and pan-cytokeratin on the Vectra Polaris™ platform and analysed using QuPath. Cox multivariate analyses showed high CD68⁺PD-L1⁺ stromal cell counts were associated with improved prognosis for OS (HR 0.56, 95% CI 0.33–0.95, p = 0.030) and BCSS (HR 0.47, 95% CI 0.25–0.88, p = 0.018) in the whole cohort and in patients receiving chemotherapy, improving incrementally upon the predictive value of PD-L1⁺ alone for BCSS. These data suggest that CD68⁺PD-L1⁺ status can provide clinically useful prognostic information to identify sub-groups of patients with good or poor prognosis and guide treatment decisions in TNBC.
... This highlights the need to establish a standardised technique to quantitatively analyse the immune microenvironment in mouse models of IBD and colon cancer. Nonetheless, several studies suggest that certain murine immune markers such as CD4, CD8α are difficult to detect as these epitopes are highly sensitive to many fixation methods and/or heat-mediated antigen retrieval [2,16,17]. Repeated rounds of microwave treatment (MWT), for instance, are detrimental to tissue integrity, slide adhesion and damages antigens, thereby precluding further rounds of staining [18,19]. ...
... As repeated rounds of MWT can profoundly affect tissue integrity, adhesion to the slide and antigenicity, we sought to identify an alternative antibody stripping method that was efficient in the removal of the bound antibody complex. As outlined in Table 1, several antibody stripping methods have been proposed in the literature [16,28,29]. These methods include incubating slides in glycine-based buffers with high pH (pH 10.0), low pH (pH 2.2), and denaturing conditions using sodium dodecyl sulphate (SDS). ...
... MWT in citrate buffer was effective in stripping antibodies ( Figure 2F) as predicted. Some studies have reported effective stripping by incubating slides in a high pH glycine-based stripping buffer (pH 10.0) at 50 • C or room temperature [16,28]. However, we found that incubation in a high pH buffer at 50 • C resulted in residual signal ( Figure 2G) and failed to remove the antibody complex at room temperature in our laboratory settings ( Figure 2H). ...
Multiplex immunohistochemistry (mIHC) enables simultaneous staining of multiple immune markers on a single tissue section. Mounting studies have demonstrated the versatility of mIHC in evaluating immune infiltrates in different diseases and the tumour microenvironment (TME). However, the majority of published studies are limited to the analysis of human patient samples. Performing mIHC on formalin-fixed paraffin-embedded (FFPE) mouse tissues, particularly with sensitive antigens, remain challenging. The aim of our study was to develop a robust and reproducible protocol to uncover the immune landscape in mouse FFPE tissues. Effective antibody stripping while maintaining sensitivity to antigens and tissue adhesion to the glass slide is critical in developing an mIHC panel to allow successive rounds of staining. Thus, we identified a highly efficient stripping method that preserves signal intensity and antigenicity to allow multiple rounds of staining. We subsequently optimised an mIHC workflow with antibodies specific against CD4, CD8ɑ, FOXP3 and B220 to identify distinct T and B cell populations on mouse FFPE tissues. Lastly, the application of this mIHC panel was validated in a mouse model of inflammatory bowel cancer, two allograft mouse models of spontaneous colon adenocarcinoma and a sporadic mouse model of colon cancer. Together, these demonstrate the utility of the aforementioned protocol in establishing the quantity and spatial localisation of immune cells in different pathological tissues.
... We investigated the presence of the SARS-COV2 NP protein (Shirazi et al., 2021;Siripanthong et al., 2020;Le Bert et al., 2020;Grifoni et al., 2020) and the associated immune microenvironment by using multiplex IHC/IF technique (Ni et al., 2020;Thieme et al., 2020;Stack et al., 2014;Abel et al., 2014;Lovisa et al., 2015;Garnelo et al., 2015;Yeong et al., 2017;Garnelo et al., 2017;Esbona et al., 2016;Mlecnik et al., 2016;Nghiem et al., 2016;Feng et al., 2016;Lim et al., 2018). The SARS-COV2 NP protein was found predominantly near the perivascular regions colocalizing with receptors of SARS-COV2, ie ACE2 and TMPRSS2 (Yeong et al., 2019;Lam et al., 2019) (Figure 3). ...
... Particularly for this panel, we followed the detailed protocol that our group previous reported as protocol manuscript (Nghiem et al., 2016) and hereby briefly described. FFPE tissue sections were cut onto Bond Plus slides (Leica Biosystems Richmond) and heated at 60°C for 20 min (Feng et al., 2016). Tissue slides were then subjected to deparaffinisation, rehydration and heat-induced epitope retrieval (HIER) using a Leica Bond Max autostainer (Leica Biosystems Melbourne), prior to endogenous peroxidase blocking (Leica Biosystems Newcastle). ...
Coronavirus disease-19 (COVID-19) is caused by the newly discovered coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While the lung remains the primary target site of COVID-19 injury, damage to myocardium, and other organs also contribute to the morbidity and mortality of this disease. There is also increasing demand to visualize viral components within tissue specimens. Here we discuss the cardiac autopsy findings of 12 intensive care unit (ICU) naïve and PCR-positive COVID-19 cases using a combination of histological, Immunohistochemical/immunofluorescent and molecular techniques. We performed SARS-CoV-2 qRT-PCR on fresh tissue from all cases; RNA-ISH and IHC for SARS-CoV-2 were performed on selected cases using FFPE tissue from heart. Eight of these patients also had positive post-mortem serology for SARS-CoV-2. Histopathologic changes in the coronary vessels and inflammation of the myocardium as well as in the endocardium were documented which support the reports of a cardiac component to the viral infection. As in the pulmonary reports, widespread platelet and fibrin thrombi were also identified in the cardiac tissue. In keeping with vaccine-induced activation of virus-specific CD4+ and CD8+ T cells, and release of cytokines such as interferon-gamma (IFNγ), we observed similar immune cellular distribution and cytokines in these patients. Immunohistochemical and immunofluorescent localisation for the viral Spike (S-protein) protein and the nucleocapsid protein (NP) were performed; presence of these aggregates may possibly contribute to cardiac ischemia and even remodelling.
