Primary Antibody Research Articles

Confinement-enhanced electrochemiluminescence by Ru(dcbpy)32+-functionalized γ-CD-MOF@COF-LZU1 porous hybrid material as micro-reactor for CYFRA 21-1 detection

The improvement of electrochemiluminescence (ECL) performance relies on the electron transfer efficiency between luminophore and coreactant. An ultrasensitive ECL micro-reactor with confinement-enhanced performance was prepared by using the covalent organic framework-LZU1-functionalized metal-organic framework (MOF@COF-LZU1) as a platform to assemble enormous N,N-dibutyl-2-hydroxyethylamine (DBAE) and tris(4,4′-dicarboxylic acid-2,2′-bipyridyl) ruthenium(II) [Ru(dcbpy)32+] into its pore channels. Compared to individual substances of γ-CD-MOF and COF-LZU1, the synergistic effects can conduce to the enhancement of the intensity, durability and sensitivity of the micro-reactor. Besides, COF-LZU1 can provide a mild environment to accommodate a certain amount of DBAE by concentrating them from the aqueous solution into its hydrophobic cavities and boost the oxidation efficiency of DBAE to generate more DBAE●+ and profited the survival of DBAE●, leading to an improved reaction efficiency with the Ru(dcbpy)32+ intermediate. Thanks to the confinement-enhanced strategy, engineered as high-functioning luminescent materials, Ru@γ-CD-MOF@COF-LZU1 micro-reactors decorated with Au NPs can facilitate electron transfer and capture primary antibodies (Ab1). Moreover, Au–Pd–Pt noble metal aerogels (NMAs) functionalized MoS2 NFs (Au–Pd–Pt NMAs@MoS2 NFs) were chosen as base material due to its large specific surface areas, high porosity, and excellent electrical conductivity. Based on above merits, the sensor demonstrated a sensitive response to CYFRA 21-1 detection in a linear concentration gradient from 10 fg/mL to 50 ng/mL with a detection limit of 0.0055 pg/mL (S/N = 3). The COF-LZU1 decorated ECL micro-reactors were constructed based on the signal amplification strategies to realize accurate CYFRA 21-1 detection.

A novel ratiometric electrochemical immunosensor for the detection of cancer antigen 125 based on three-dimensional carbon nanomaterial and MOFs

Accurate and sensitive detection of cancer antigen 125 (CA125) is important in early ovarian cancer diagnosis and monitoring. In the present work, a novel electrochemical ratiometric immunosensor for the determination of CA125 based on three-dimensional reduced graphene oxide/multi-walled carbon nanotube carboxylic acid- thionine (3DrGO/MWCNTs-Thi) and UIO-66-NH2/ferrocenecarboxylic acid (UiO-66-Fc) was constructed. 3DrGO/MWCNTs-Thi of immobilized primary antibody (Ab1) and UiO-66-Fc of labeled second antibody (Ab2) were used as capture probes and signaling probes for CA125, respectively. As the concentration of CA125 increases, the oxidation peak currents (IThi and IFc) generated by the two materials were changed in opposite directions, and therefore the ratio of the currents (IThi/IFc) was negatively correlated with the concentration of CA125. Thanks to the amplification of high loading of signaling molecules by the framework nanocomposites and the ratiometric strategy, the constructed electrochemical immunosensor exhibits outstanding accuracy, stability, and sensitivity. Under optimal conditions, the detection range spanned from 0.01 U mL−1 to 80 U mL−1, with a calculated limit of detection (LOD) of 0.0089 U mL−1. The immunosensor was successfully applied to real serum sample tests, indicating its potential for clinical detection applications.

Abstract 6709: P329G-Engager: A novel universal antibody-based adaptor platform for cancer immunotherapy

Abstract Bispecific immune cell engagers are showing promise in cancer immunotherapy. To achieve optimal efficacy, there is a growing need for patient personalization, tackling intra-tumor heterogeneity, and simplified combination therapies. Here, we present a novel modular anti-P329G adaptor platform aiming to address these issues, which is based on the recognition of the P329G mutation in the Fc portion of a targeting adaptor antibody. This P329G mutation can serve to recruit different anti-P329G effector cell engagers, including innate cell engagers, T cell engagers, costimulators or immunocytokines. Specifically, upon identifying patient-specific tumor surface targets, the primary antigen-binding IgG1 antibodies bearing Fc-silencing P329G LALA mutations are applied. Subsequently, based on the tumor’s immune profile, the best-suited anti-tumoral effector cell type is chosen for recruitment and activation. Based on this information, the secondary antibody recognizing the P329G mutation is chosen from an array of effector cell engagers with different modes of action: P329G-T cell bispecifics (P329G-TCB), ADCC-competent P329G innate cell engagers (P329G-ICE), P329G costimulatory molecules (P329G-CD28/4-1BBL) or P329G immunocytokines (P329G-IL2v). In vitro assays showed all P329G modalities inducing anti-tumoral and/or immunomodulatory cell activity. Anti-tumoral efficacy of secondary antibodies was observed only in presence of tumor-targeted P329G adaptors, while no effect was observed in their absence. The readouts consisted of tumor killing quantification, CD4+ and CD8+ T cell activation, cytokine release and cytokine receptor signaling. As a proof of concept study in vivo, an experiment in MKN-45 (CEACAM5+)-bearing humanized mice was performed, using an anti-CEACAM5 P329G-IgG adaptor and a P329G-TCB. Both tumor volume shrinkage and T cell infiltration into the tumor confirmed anti-tumoral efficacy of the platform, as compared to a conventional CEACAM5-targeted TCB. Neither the individual P329G IgG nor the individual P329G-TCB induced anti-tumoral efficacy, validating the requirement for primary and secondary antibody binding for T cell engaging activity. These results provide in vitro and in vivo evidence that the universal P329G engager platform can be used as an efficacious cancer treatment. Ultimately, this modular approach may enable off-the-shelf personalization via combination of patient-specific antibodies and universal effector cell engagers based on the patient’s tumor target and immune profile. Citation Format: Marlena Surowka, Idil Hutter-Karakoc, Diana Darowski, Christina Claus, Claudia Ferrara Koller, Anne Freimoser-Grundschober, Thomas Hofer, Andrzej Sobieniecki, Denis Assisi, Stephane Leclair, Ekkehard Moessner, Pablo Umaña, Maria Amann, Christian Klein. P329G-Engager: A novel universal antibody-based adaptor platform for cancer immunotherapy [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 6709.

Abstract 6876: Cyclic immunofluorescent analysis of the tumor microenvironment across human brain cancer subtypes

Abstract Introduction: The tumor microenvironment (TME) has been the focus of a myriad of studies over the last decade and advancements in the field of cancer research have grown as a result. Improvements in the number of biomarkers that can be screened has increased across a variety of methods and the field of spatial profiling has largely adapted to keep pace with this. However, many challenges persist: 1) tissue microarrays can be limited by tumor heterogeneity and 2) newer technology can be cost-prohibitive. Brain tumors are of significant importance, especially those of glial origin, as they swiftly progress and result in a high fatality rate. As such, the need for more diverse therapeutic approaches is growing with many already pioneering new IHC-based strategies. The aim of this study was to address both challenges addressed above in the neurobiology space. Methods: Biomarkers of importance in brain cancer tissues were identified and three distinct panels were designed. The first panel consisted of CD68, CTLA-4, p16ink4A, p14ARF, and p53. The second included PDPN, Calretinin, MAGE-D2, PTEN, and Vimentin. The third panel included IDH2, BRAF, GFAP, VEGF, and BOP1. Staining was achieved using Bethyl Laboratories IHC-validated primary and secondary antibodies and Akoya Opal™ Polaris 7-color IHC kit fluorophores (Akoya Biosciences [NEL861001KT]). Optimization was achieved using FFPE tissue microarrays containing normal neural tissue and brain cancer subtypes. The final optimized order was then tested on whole serial sections of meningioma, astrocytoma, and glioblastoma in six-color mIF. Images were generated using the PhenoImager HT® and subsequently merged and analyzed by Pathomics. Results: Phenotyping and total cell counts for each marker were identified for each serially stained tissue set. Expression levels of MAGE-D2 and BRAF were high across malignant tissues of variable gradations. Conversely, in tissues with high expression of MAGE-D2, p53 levels in these samples were lower. This is consistent with findings that suggested that MAGE-D2 is a potential dissociator of p53 by impairing transcriptional activity. Macrophage expression appeared to correlate with CTLA4 and overall tumor infiltration. Conclusions: Utilizing several unique biomarkers to understand the spatial relationships and phenotypes within the tumor microenvironment is becoming a more common practice among researchers. In using this method of serially staining tissue sections and merging the resulting images, more researchers will have access to spatial information which may have been cost-prohibitive otherwise. Additionally, in utilizing whole sections of difficult-to-treat cancerous tissue rather than microarrays, phenotypic information is available across the entire tumor microenvironment enabling a more comprehensive assessment of the TME. Citation Format: Danielle Fails, Michael Spencer. Cyclic immunofluorescent analysis of the tumor microenvironment across human brain cancer subtypes [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 6876.

Abstract 3755: Impact of antigen retrieval and other parameters on detection of FGFR2b in various solid tumors on a BenchMark ULTRA automated staining platform: Development of a new IHC assay for FGFR2b

Abstract Background: Fibroblast growth factor receptor 2b (FGFR2b) is part of the FGFR transmembrane tyrosine kinase receptor family. This protein has gained more attention in recent years as a tumor progression biomarker in numerous solid tumors. The detection of FGFR2b by immunohistochemistry (IHC) has been used recently in immunotherapy clinical trials with multiple tumor types. During IHC assay development on formalin-fixed paraffin embedded tissue, a thorough study of antigen retrieval through cell conditioning or enzyme incubation, primary antibody incubation, and detection parameters is critical to evaluate staining performance. Design: A study of an investigational anti-FGFR2b (FPR2-D) assay developed on a BenchMark ULTRA automated staining platform explored the effect of antigen retrieval and detection parameters on staining performance. VENTANA System Software was used to modify steps in the IHC staining protocol resulting in 42 permutations. Ten different solid tumors were stained with these modifications to the assay and subsequently evaluated for FGFR2b expression. Result:s The results demonstrate that the staining clarity, intensity, and dynamic range within tissue cases of FGFR2b staining can vary widely between different solid tumor types. While most permutations of the initial assay parameters had minimal impact on the staining performance and readability by a pathologist, the order of the enzyme treatment and the cell conditioning steps (in addition to increased incubation time of primary antibody) had a significant impact when comparing across different solid tumors. Detection of FGFR2b seemed to have a greater susceptibility by tumor type to the antigen retrieval process than has traditionally been observed in other biomarkers. Conclusion: The overall results of this investigation show the importance of an extensive parameter screening approach when evaluating new antibody targets and indication combinations in IHC to ensure optimal staining performance. This is critical to downstream testing and the potential use of new assays in the context of in vitro diagnostics. Citation Format: Autumn Sky Watson, Catherine Le, Anne M. Wertheimer, Spencer Escobedo, Lisa So, Bhavani Bagevalu Siddegowda, John D. Palting, Susan Marion, Steven P. Stratton, Birte Aggeler. Impact of antigen retrieval and other parameters on detection of FGFR2b in various solid tumors on a BenchMark ULTRA automated staining platform: Development of a new IHC assay for FGFR2b [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 3755.

Abstract 5354: High-resolution analysis of immune checkpoint activation utilizing a combined PD1/PD-L1 in situ proximity ligation assay (isPLA) and multiplex immunofluorescence (mIF) imaging approach

Abstract The immune checkpoint involving PD-1 and PD-L1 generates suppressive signals in T cells, which help to prevent autoimmunity by inducing a state of immune exhaustion. In the context of the tumor microenvironment (TME), however, cancer cells can manipulate these pathways to camouflage themselves from an immune attack. Blocking these checkpoints has thus emerged as a key immunotherapeutic tactic. Nonetheless, the success rate of immune checkpoint inhibitors (ICIs) has been mixed, even for patients who test positive for relevant diagnostic biomarkers. Patient stratification currently depends on immunohistochemical staining for checkpoint proteins like PD-L1, but these tests do not provide adequate stratification. A more comprehensive stratification, one that includes immune profiling of the TME plus an evaluation of immune checkpoint interactions, might provide better patient stratification and ICI responsiveness. Here we describe mIF that is augmented with a PD-1 and PD-L1 protein-protein interaction assay. Human formalin-fixed, paraffin-embedded (FFPE) tissue sections were subjected to standard histological processing and then incubated with primary antibodies against PD1 and PD-L1. Sections were then treated with oligonucleotide-modified secondary probes suitable for in situ Proximity Ligation Assay (isPLA), enabling the detection of PD1 and PD-L1 interactions. The PLA protocol utilized a fluorescent probe complementary to amplified nucleic acid to enhance the visibility of the protein interactions. The sample was then mounted in a closed microfluidic chamber and imaged on the CellScape™ platform for Precise Spatial Multiplexing. After imaging of the isPLA signal, the CellScape cyclic multiplex mIF staining approach was used to iteratively stain immune and structural markers on the tissue sample utilizing the VistaPlex™ Spatial Immune Profiling Assay Kit. This proof-of-concept study demonstrates that high-plex mIF can be augmented with an isPLA on human FFPE samples. The combined application of both methods allows visualization of PD1/PD-L1 protein-protein interactions and integrates this interaction within the spatial context of the surrounding cell populations. This approach allows for a more comprehensive insight into the interplay of different immune cell and non-immune cell populations during checkpoint activation processes in normal and neoplastic tissues. Citation Format: Arne Christians, Jannik Boog, Charles E. Jackson, Matthew H. Ingalls, J Spencer Schwarz, Sara Bodbin, Agata Zieba Wicher, Subham Basu, Oliver Braubach. High-resolution analysis of immune checkpoint activation utilizing a combined PD1/PD-L1 in situ proximity ligation assay (isPLA) and multiplex immunofluorescence (mIF) imaging approach [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 5354.

Abstract 5166: A multiplexed immunofluorescence assay to assess Rb and phospho-Rb as predictive biomarkers for CDK4/6 inhibitors in HR+/HER2- breast cancer

Abstract Introduction: Cyclin-dependent kinases 4/6 inhibitors (CDK4/6i) are used as the first-line treatment of advanced/metastatic or high-risk HR+/HER2- breast cancer (BC) that make up about 60-70% of all cases. However, nearly 50% of patients exhibit drug resistance. Therefore, biomarkers that predict CDK4/6i response are needed to spare patients from toxicities and high costs. Resistance to CDK4/6i [palbociclib, ribociclib, and abemaciclib] is associated with the aberrant or loss of Retinoblastoma (Rb). A decline in phospho-Rb (pRb) levels on therapy is also associated with CDK4/6i response. We aimed to develop a multiplex immunofluorescence (IF) assay to assess Rb and pRb levels that can be used on circulating tumor cells (CTCs) to predict tumor response to CDK4/6i. Here, we report the selection of internal controls for Rb and pRb, assay repeatability and reproducibility, as well as compatibility of the CTC isolation protocol with the assay. Methods: Rb and pRb expression was evaluated by western blotting (WB) and IF in parental (P), estrogen deprivation-resistant (EDR), palbociclib-resistant (PalboR), and EDR/PalboR derivatives of two HR+/HER2- cell lines (MCF7 and T47D), Rb1 KO cells of MCF7 and T47D, as well as triple-negative cell lines, BT549 and MDA-MB-468. IF-stained cells were imaged using Leica STED SP8 confocal microscope and Zeiss Axioscan 7. The concordance between IF and WB results was assessed using a Bland-Altman plot. Selectivity and specificity of the assay as well as repeatability and reproducibility between investigators were also assessed. Blood from healthy subjects was used to test the compatibility of the ScreenCell® filtration system to isolate CTC with the IF assay. Results: Our initial approach utilized indirect IF staining for Rb and pRb using the same primary antibodies as WB. On the Bland-Altman plot, most data points were relatively close to the bias line, suggesting that the difference between the two methods was acceptable. The analytical range for mean Rb and pRb staining intensity was 0.744 - 46.779 and 1.001 - 52.919, respectively. Based on WB and IF data, we nominated T47D PalboR and MDA-MB-468 cells as negative controls for Rb and pRb expression. The selectivity and specificity were 100% and 70%, respectively, for the Rb expression and 99% and 61% for pRb, respectively. The IF assay was repeatable and reproducible between two investigators. The ScreenCell® CTC isolation method was not compatible with the assay, as Rb and pRb expression was either not detected or limited when processed using FC2 or PBTFC buffer, irrespective of the antigen retrieval. Conclusion: We have selected internal cell line controls for Rb and pRb and are in the process of optimizing signal-to-noise ratio and specificity using conjugated antibodies. We are also testing the compatibility of other CTC isolation methods with the IF assay. Citation Format: Mantasha Tabassum, Shivaani Suresh Kanna, Wangjia Cao, Mothaffar Rimawi, George E Miles, Meghana V Trivedi. A multiplexed immunofluorescence assay to assess Rb and phospho-Rb as predictive biomarkers for CDK4/6 inhibitors in HR+/HER2- breast cancer [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 5166.

Abstract 2588: Bioengineering of site-specific antibody labeling from basic research to spatial omics platforms

Abstract Over the last few years, spatial multi-omics platforms have emerged as a powerful tool for characterizing cancer biology in multiple dimensions. Notably, these systems typically rely on multiplex immunolabeling. Conventionally, multiplexing is accomplished through carefully selecting primary antibodies from different animal species, and in combination with their corresponding polyclonal secondary antibodies. However, given the practical complication related to the species availability of primary antibodies, the multiplexing labeling is still under constraint due to the necessity for matched secondary species labels and the availability. For decades, non-specific labeling of primary antibodies through amine- or thiol-reactive chemistries was widely performed. Evolving research and commercial products have sought to overcome this antibody-based multiplexing limitations by developing enzymatic reaction-directed chemical reaction or antibody targeting proteins that could contain affinity to the Fc or Fab regions of antibodies. However, these methods mentioned above have substantive limitations and side-effects on affinity, conjugation number, buffer incompatibilities, storage incompatibilities, insufficient conjugation, and uncontrolled stoichiometric labeling. Herein, we introduce an efficient, sustainable, reproducible site-specific antibody covalent labeling strategy through engineering anti-IgG secondary nanobodies. These infer the stability, high-affinity of species specific and easy-to-produce IgG binders with the permanence of controlled activation for proximity-driven intermolecular covalent conjugation, with a promise for efficient multiplexity. This labeling technology addresses antibody labeling challenges through currently available approaches, and would substantially enhance multiplexity in a wide variety of research and clinical settings. Citation Format: Kai Tao, Matthew J. Rames, MingChong Dai, Gulsu Sener, Samuel Olson, Srivathsan Ranganathan, Sila Ozdemir, Adam Oken, Michael Brasino, Randy Armstrong, Yu-Jui Chiu, Ece Eksi, Bruce Branchaud, Sadik Esener. Bioengineering of site-specific antibody labeling from basic research to spatial omics platforms [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 2588.

Abstract 2908: EDB+FN is an attractive therapeutic target in oncology: Insights from protein expression analysis of solid tumors

Abstract Stroma plays a major role in the initiation, growth, survival, and drug-resistance of solid tumors, yet few therapeutics specifically target tumor-associated stroma. Extra-domain-B fibronectin (EDB+FN) is an oncofetal splice variant of fibronectin upregulated in solid tumor stroma, which is associated with tumor growth, angiogenesis, and metastases. Here we developed an immunohistochemistry (IHC) assay to assess EDB+FN protein expression in the stroma of tumor and normal tissues to characterize the potential of EDB+FN as a therapeutic target for solid tumors with high unmet need. We validated a primary detection antibody and titrated assay conditions to achieve specificity and a broad dynamic range of EDB+FN expression levels across indications, including thyroid cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, soft tissue sarcoma, pancreatic cancer, non-small cell lung cancer, ovarian cancer, and renal cancer. Considering the stroma-specific expression pattern of EDB+FN, we developed a scoring algorithm weighting both the intensity and distribution of EDB+FN expression specifically in the stroma compartment and separately in tumor cells. Using this assay and scoring approach, we screened more than 200 tumor samples from 10 solid tumor indications and found EDB+FN to be highly differentially expressed in tumor-associated stroma with little to no expression on tumor cells and trace to undetectable levels in healthy tissue. EDB+FN was expressed across the indications evaluated with a wide range of expression both within and between indications. Stromal EDB+FN expression was not correlated with stromal density, which also varied widely within and across indications. In parallel, we developed an algorithm using digital pathology to approximate pathologist scoring of stromal density and EDB+FN expression, allowing streamlined screening of preclinical samples for in vivo studies and indication selection. The disease-specific expression pattern of EDB+FN and high incidence of expression across indications make EDB+FN an ideal target for high-unmet-need solid tumors. The specificity and dynamic range of our IHC assay for detection of EDB+FN protein expression position it as a robust potential biomarker assay for EDB+FN-targeting therapeutics. Citation Format: Sara Lewandowski, Liyang Diao, Alyssa Quigley, Marsha Crochiere, Jan Pinkas. EDB+FN is an attractive therapeutic target in oncology: Insights from protein expression analysis of solid tumors [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 2908.

An Unusual Presentation of Primary Anti-phospholipid Antibody Syndrome as Acute Coronary Syndrome in Young – A Case Report

A case report of a 32-year-old female patient without any traditional risk factors presented with angina to our hospital within 6 h. Her electrocardiogram (ECG) showed extensive anterior wall -ST elevation myocardial infarction (MI). She was treated with thrombolytic therapy in the form of tenecteplase and was given standard treatment for acute coronary syndrome (ACS). Her history revealed three recurrent pregnancy losses and a history of preeclampsia. Echocardiography showed regional wall motion abnormalities of the mid, distal, apex, and anterior wall with severe left ventricular systolic dysfunction. Routine blood investigations showed elevated total leukocyte count with neutrophilic predominance. With a history of recurrent pregnancy loss, she was investigated for anti-phospholipid anti-phospholipid antibody syndrome (APS) and was found to have positive lupus anticoagulant 1. She underwent coronary angiography (CAG) which showed a left main shaft 40% stenosis with an ulcerative lesion containing thrombus in the ostioproximal to mid-left anterior descending artery (LAD), other coronary arteries were normal. She was managed conservatively with glycoprotein IIb/IIIa inhibitor (Tirofiban) and switched to oral anticoagulation with Vitamin K antagonist (VKA-Acenocoumarin) along with dual-antiplatelet therapy.

Abstract 2912: SFRP2 and CD38 colocalize in triple negative breast cancer microenvironment

Abstract Introduction: A promising target for breast cancer is secreted frizzled-related protein 2 (SFRP2), and a humanized monoclonal antibody to SFRP2 (hSFRP2 mAb) inhibits primary triple negative breast cancer (TNBC) in vivo. CD38 is a known cause of PD-1 inhibitor resistance in some tumors, and in metastatic osteosarcoma hSFRP2 mAb overcomes PD-1 mAb resistance by reducing CD38 in tumor infiltrating lymphocytes (TILs). Could this phenomenon be replicated in TNBC? This is unknown as the expression of CD38 in TNBC has not been studied. This study will establish the presence of SFRP2 and CD38 in the TNBC microenvironment. Methods: Immunohistochemistry (IHC) was performed on an 88 core human TNBC tissue microarray (TMA) with antibodies to SFRP2 and CD38. Spatial analysis software was used to determine percent positivity. Each core was categorized using the scoring system for the estrogen receptor in breast cancer (<1% negative, 1-10% low positive, and >10% positive). Multiplex IHC was performed on 4 human triple TNBCs sections. Tissues were stained with antibodies to CD38, CD68 (macrophage), CD3 (T-cell), CD19 (B-cell), CytoKeratin (tumor), and SFRP2. Percent positivity of each marker was analyzed with a software package. For flow cytometry of tumor associated macrophages (TAMs), 1 million EO771 cells were injected into the mammary fat pad of female C57/BL6 mice. After 4 weeks tumors were extracted and a cell suspension was incubated in a mastermix with antibodies to SFRP2, CD38, PD1, CD11B and F480. Cells were fixed and conjugated to a fluorochrome secondary antibody. TAM gated cells were analyzed for the presence of SFRP2 and CD38. Results: For the TNBC TMA, 83 cores were positive and 4 cores were low positive for SFRP2, 1 not evaluable; 87 cores were positive and 1 low positive for CD38. For multiplex IHC there was a total of 1,753,544 individual cells in all 4 human TNBC tumors that were stained. 94% of cells stained positive for SFRP2, and 65% stained positive for CD38. Of the cells that were positive for SFRP2, 70% were also positive for CD38, 15% were positive for CD68, 1% were positive for CD19, 77% were positive for CytoKeratin, and 10% were positive for CD3. Of the cells that were CD38 positive, 99% were also positive for SFRP2, 15% were positive for CD68, 1% were positive for CD19, 81% were positive for CytoKeratin, and 9% were positive for CD3. Flow cytometry analysis of TAMs confirmed the presence of SFRP2, CD38 on TAMs. TAMs that were negative for PD1 and CD38 (pro-inflammatory) had a 56% reduction in SFRP2 compared to TAMs that were double positive for PD1 and CD38 (anti-inflammatory). Conclusion: SFRP2 and CD38 are prevalent in human TNBC and colocalize in the TNBC microenvironment in tumor cells, TAMs and TILs, but not B-cells. The presence and co-localization of SFRP2 and CD38 in TAMs was confirmed with flow cytometry. This confirms that SFRP2 is a therapeutic target in TNBC and provides a mechanistic rational for combination therapy with PD-1 inhibitor. Citation Format: Lillian Hsu, Patrick Nasarre, Rupak Mukherjee, Eleanor Hilliard, Martin Romeo, Elizabeth O'Quinn, Whitney Christians, Nancy Klauber DeMore. SFRP2 and CD38 colocalize in triple negative breast cancer 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 2912.

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

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

Abstract 84: StarBright Dyes: Fluorophores with superior performance for multiplexing in flow cytometry and Western blotting

Abstract New StarBrightTM Dyes from Bio-Rad are bright fluorescent fluorophores ideal for many antibody-based applications, for example, western blotting and flow cytometry. Traditional fluorophores can be dim, have high cross laser excitation, or don’t work with all protocols, leading to poor data. StarBright Dyes have been designed to address these pain points. Western blotting secondary antibodies are available conjugated to StarBright Blue 700 and StarBright Blue 520 Dyes. Their brightness, the short exposure time required, and low background make them ideal for detection of a single target, or as shown here for multiplex detection of several proteins in combination with other common fluorophores. For flow cytometry, we have a full range of 32 StarBright Dyes excited by the most common laser lines: 355 nm, 405 nm, 488 nm, 561 nm, or 640 nm. They are available conjugated to highly cited clones against common immunophenotyping targets. Data shown here on the ZE5 Cell Analyser demonstrates how the features of StarBright Dyes can improve multiplexing immunophenotyping data. They have narrow excitation and emission spectral profiles, which reduce compensation and spread. This, combined with their exceptional brightness, enables high resolution of cell populations. They work in all buffers and fixatives, so including them in existing panels does not require a change in protocol. They can also be premixed in a master mix for over six months with no loss in performance. Premixes are ideal when using the same panel over an extended period, such as in clinical and longitudinal studies, to ensure reproducible results, also saving time and money. StarBright Dyes are ideal for multiplexing assays in both western blotting and flow cytometry. BIO-RAD and STARBRIGHT are trademarks of Bio-Rad Laboratories, Inc. in certain jurisdictions. All trademarks used herein are the property of their respective owner. Citation Format: Sharon Sanderson, Oliver Rosenwasser, Michael Blundell. StarBright Dyes: Fluorophores with superior performance for multiplexing in flow cytometry and Western blotting [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 84.

Abstract 7259: Effectiveness and comparison of CAR-T cell therapies for synovial sarcoma; HER2-targeted CAR-T and NKG2D-based CAR-T

Abstract Introduction: In Japan since 2019, CAR-T cell therapy has been used to treat patients with relapsed or refractory B-cell acute lymphoblastic leukemia and diffuse large B-cell lymphoma and has shown excellent clinical results. Clinical studies of CAR-T cell therapies targeting various antigens, such as HER2, have been conducted, but the therapeutic outcome in sarcoma is still limited. We have previously reported that HER2-targeted CAR-T cells show antitumor effects for synovial sarcoma in vitro. The purpose of this study is to compare HER2-targeted CAR-T cells and NKG2D-based CAR-T cells in antitumor effect and specific responses for synovial sarcoma in vitro. Materials and Methods: Surface expressions of HER2 and NKG2D ligands on three synovial sarcoma cell lines were detected by using recombinant human HER2 Fc chimeric protein and that of NKG2D, followed by staining with PE-conjugated anti-human IgG secondary antibody. We generated 4-1BB-type second generation CAR-T cells targeting NKG2D ligands and examined their antitumor effects and specific responses against synovial sarcoma cells. In addition, we compared the differences between HER2-targeted CAR-T cells and NKG2D-based CAR-T cells by co-culturing with synovial sarcoma cells using short-term assay (WST-8 assay), long-term assay (Real-time Cell Analysis) and CD107a assay. Results: HER2 and NKG2D ligands were found to be expressed by flow cytometry in three synovial sarcoma cell lines. In vitro, NKG2D-based CAR-T cells showed antitumor effects against synovial sarcoma cells, including degranulation and cytokine production. Furthermore, NKG2D-based CAR-T cells showed stronger antitumor effects than HER2-targeted CAR-T cells in both short- and long-term assays and expressed higher levels of CD107a. Conclusions: In this study, NKG2D-based CAR-T cells showed more antitumor effects than HER2-targeted CAR-T cells in vitro. Although the reason for this is not clear, we hypothesize that NKG2D ligands may be induced in synovial sarcoma cells by co-culture with CAR-T cells because HER2 is a cancer-specific antigen, whereas NKG2D ligands are ligands for activating receptors of natural killer cells. Further studies, including in vivo studies, will be conducted to verify the difference in anti-tumor effects between these CAR-T cells. Citation Format: Tomohiro Miyazaki, Chihaya Imai, Naoki Oike, Yudai Murayama, Takashi Ariizumi, Minori Baba, Yasushi Kasahara, Hiroyuki Kawashima. Effectiveness and comparison of CAR-T cell therapies for synovial sarcoma; HER2-targeted CAR-T and NKG2D-based CAR-T [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 7259.

Comparative development of the serotonin- and FMRFamide-immunoreactive components of the nervous system in two distantly related ribbon worm species (Nemertea, Spiralia).

Neurodevelopment in larval stages of non-model organisms, with a focus on the serotonin- and FMRFamide-immunoreactive components, has been in the focus of research in the recent past. However, some taxonomic groups remain understudied. Nemertea (ribbon worms) represent such an understudied clade with only few reports on nervous system development mostly from phylogenetically or developmentally derived species. It would be insightful to explore neurodevelopment in additional species to be able to document the diversity and deduce common patterns to trace the evolution of nervous system development. Fluorescent immunohistochemical labeling with polyclonal primary antibodies against serotonin and FMRF-amide and a monoclonal antibody against synapsin performed on series of fixed larval stages of two nemertean species Cephalothrix rufifrons (Archinemertea, Palaeonemertea) and Emplectonema gracile (Monostilifera, Hoplonemertea) were analyzed with confocal laser scanning microscopy. This contribution gives detailed accounts on the development of the serotonin- and FMRFamide-immunoreactive subsets of the nervous system in two nemertean species from the first appearance of the respective signals. Additionally, data on synapsin-like immunoreactivity illustrates the general structure of neuropil components. Events common to both investigated species are the appearance of serotonin-like immunoreactive signals before the appearance of FMRF-like immunoreactive signals and the strict progression of the development of the lateral nerve cords from the anteriorly located, ring-shaped brain toward the posterior pole of the larva. Notable differences are (1) the proboscis nervous system that is developing much earlier in investigated larval stages of E. gracile and (2) distinct early, but apparently transient, serotonergic neurons on the frontal and caudal pole of the larva in E. gracile that seem to be absent in C. rufifrons. According to the results from this investigation and in line with previously published accounts on nervous system development, the hypothetical last common ancestor of Nemertea had a ring-shaped brain arranged around the proboscis opening, from which a pair of ventro-lateral nerve cords develops in anterior to posterior progression. Early frontal and caudal serotonergic neurons that later degenerate or cease to express serotonin are an ancestral character of Nemertea that they share with several other spiralian clades.

Abstract 3108: Antibody labeling reagents to rapidly screen for the binding and internalization of therapeutic antibodies

Abstract Introduction: There is a growing need in the immunotherapy research field for tools to rapidly screen novel therapeutic antibodies, whether looking for cells expressing IgG, target binding, or tracking internalization. Thermo Fisher Scientific has developed the next generation of Invitrogen™ Zenon™ reagents to enable screening of potential therapeutic antibodies, working across a variety of sample types including primary B cells and hybridomas, while being compatible with a wide range of platforms including flow cytometry, high content imaging, and in-incubator imaging systems. Methods: The Invitrogen™ Zenon™ Alexa Fluor™ Plus IgG Labeling Reagents to screen for antibody binding provide a fast, versatile, and reliable method to evaluate antibody binding to cell surface antigens. Zenon screening reagents are labeled with Invitrogen Alexa Fluor Plus 488, Alexa Fluor Plus 594, or Alexa Fluor Plus 647 dyes (i.e., labeling reagent). The labeling reagent is directed against the Fc portion of an intact mouse or human IgG primary antibody. The formation of the zenon-antibody complex is rapid and requires no purification steps. The Invitrogen™ Zenon™ pHrodo™ Deep Red Labeling Reagents for screening antibody internalization provides a highly sensitive method for monitoring antibody internalization. Zenon pHrodo Deep Red utilizes the same screening reagent as Zenon Alexa Fluor Plus reagents, replacing Alexa fluor dyes with the pH-sensitive dye, pHrodo Deep Red. The pHrodo Deep Red sensors have minimal fluorescence at neutral pH and only upon entry into the late endosome and lysosome do they brightly fluoresce. Zenon pHrodo Deep Red Labeling Reagents are perfectly suited for screening the endocytosis and degradation of potentially therapeutic antibodies including antibody-drug conjugates. Results: Live cells stained with Zenon-labeled antibodies show remarkable signal to background in both flow and imaging applications, while retaining their signal following fixation. Therapeutic antibodies such as anti-Her2 labeled with Zenon pHrodo Deep Red show as bright fluorescent puncta upon lysosomal degradation in the Alexa Fluor 647 channel and co-localize with lysosomal markers such as Invitrogen™ LysoTracker Red dye. Both Zenon Alexa Fluor Plus and Zenon phrodo Deep Red are ideal reagents for screening antibody panels of up to 96 different antibodies. Conclusions: Thermo Fisher Scientific has developed sensitive and rapid Zenon reagents to allow for high throughput therapeutic antibody screening. In addition to Zenon reagents, Thermo Fisher Scientific has developed additional tools that allow for specific monitoring of antibody and protein uptake into the lysosome for degradation. These tools not only greatly improve the workflow from our existing portfolio, but allow for increased sensitivity allowing the field to make decisive decisions when selecting candidate immunotherapies. Acknowledgments: I would like to thank the whole team that worked to develop these new labeling reagents including: Maria Olson and Chris Langsdorf. Citation Format: Ryan W. Holly. Antibody labeling reagents to rapidly screen for the binding and internalization of therapeutic antibodies [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 3108.

Changes of rat’s brain vesseles after air shock wave exposure

Mild blast-induced traumatic brain injury is common among the military, resulting in cognitive impairment, reduced socialization, which leads to disability and, as a result, a deterioration in the quality of life. It is considered that blood-brain barrier disruption and microvascular dysfunction are the key to this type of injury. The purpose of study was to study changes in brain vessels after air shock wave exposure. The study was carried out on 48 mature male Wistar rats, which were randomly divided into 2 groups: an experimental group, in which animals were subjected to inhalation anesthesia using halothane and exposed to a shock wave with an overpressure of 26.4±3.6 kPa, and a Sham group. After simulation of injury on days 1st, 3rd, 7th, 14th, and 21st, the rats were euthanized and the brain was removed and after all subjected to standard histological procedures and stained with hematoxylin and eosin. For immunohistochemical studies, as primary antibodies were used eNOS. The finished preparations were examined by light microscopy and photographed. Disorders of the cerebral vessels in experimental rats were detected from day 1st of the posttraumatic period. It was found that the blast wave led to vascular rupture, as well as increased vascular permeability with diapedesis of red blood cells and cerebral edema for up to 21st days. Focal violations of the vascular wall integrity in cortical and hippocampal hemocapillaries, venular link of the submembrane vessels; changes in the morphology of the metabolic vessels endothelium; uneven blood filling of the brain vessels were of major importance. These changes indicate that increased eNOS expression leads to dilation of cerebral vessels, which is a compensatory mechanism in response to injury to improve cerebral blood circulation. However, eNOS is not involved in vasodilation, which we observed up to 21st day post-trauma.

The role of pores in structure of polyethylenglycol diacrylate based‐inverse opal photonic crystal in binding protein applicable to optical biosensor

AbstractHere, the role of pores in the structure of inverse opal photonic crystals (IOPC) in binding proteins in comparison with their parent photonic crystal (PC) templates has been investigated. For this purpose, polyethyleneglycol diacrylate (PEGDA) coated with SiO2‐based PC (PEGDA/SiO2‐based PC) and PEGDA‐based IOPC were attached with fluorophore Alexa 488, which is a polyclonal secondary antibody, to investigate their fluorescence emission. Scanning electron microscopy (SEM) images showed face centered cubic (fcc) packing of the PEGDA/SiO2‐based PC. And it remained after the formation of PEGDA‐based IOPC. The presence of 3‐aminopropyl triethoxysilane (APTES) and Alexa 488 that immobilized the PEGDA‐based PC and PEGDA‐based IOPC was recognized by the appearance of bands at 850, 1175, and a dominant increase in the band at 1750 cm−1. Those were attributed to (N─H) wagging, (C─N) stretching, and (C═O) stretching vibrations, respectively. Reflectance spectra showed a blue shift of the Bragg photonic band gap of the PEGDA‐based IOPC in comparison with that of the PEGDA/SiO2‐based PC. The fluorescence images showed a significant increase in the fluorescence intensity of PEGDA‐based IOPC owing to the resonance effect. These obtained results indicated the role of pores in structure of PEGDA‐based IOPC in improvement of the attachment of Alexa 488 protein.

Target-induced photoelectrochemistry and colorimetric dual-mode platform for HIgG based on Ag2S/SnO2 composites and CoOOH nanoflakes

Human immunoglobulin G (HIgG) plays a significant role in diagnosing infections and monitoring autoimmune diseases. However, the development of sensitive and reliable sensing platforms remains a challenge. Based on the Ag2S/SnO2 composites, a dual-mode sensing platform was developed using multifunctional CoOOH nanoflakes for the sensitive detection of HIgG through photoelectrochemistry (PEC) and colorimetry. In the PEC mode, Ag2S QDs boost the PEC activity of SnO2 NFs and facilitate the subsequent antibody immobilization. Ascorbic acid (AA) captures holes in Ag2S/SnO2 to enhance the photocurrent. CoOOH, acting as a secondary antibody label, forms a sandwich structure with the electrode. The loading of HIgG and CoOOH-Ab2 due to specific recognition of antigen-antibody, leads to the quenching of PEC signal. Various amounts of HIgG will bind with different amounts of CoOOH-Ab2, displaying a linear response within the range of 100 pg/mL∼10 μg/mL, with the detection limit of 2.2 pg/mL. In the colorimetric mode, CoOOH efficiently catalyzes the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB), and various amount of the sandwich biocomplexes formed with different HIgG levels lead to distinct color changes for visual detection. The linear response range is 500 pg/mL∼9.375 μg/mL with a lower detection limit of 0.29 ng/mL. The two independent modes with different mechanisms, based on the same PEC electrode, can validate each other and provide more scientific and reliable results in response to the targets. Thus, this dual-mode sensing platform for targets-induced signaling changes presents a novel strategy for bioanalysis and detection.

Investigation of HEG1, VISTA (VSIR) and PD-L1 expression levels in malignant mesothelioma by immunohistochemical methods and their relationship with histological subtypes

Malignant Mesothelioma is a primary malignant tumor of the mesothelium lining the pleura, pericardium, peritoneum and tunica vaginalis of the testis with a poor prognosis. The epithelioid subtype is graded as high and low grade in the 2021 WHO classification of thoracic tumors. Recently, promising results from clinical trials of treatment with immune checkpoint inhibitors are reported. The aim of our study was to shed light on clinical studies on potential immune checkpoint inhibitors by examining VISTA and PD-L1 expression levels as well as HEG1 in malignant mesothelioma subtypes. Our study included 69 cases diagnosed with ‘malignant mesothelioma, well-differentiated papillary mesothelial tumor, atypical mesothelial hyperplasia’ at Dicle University Faculty of Medicine Department of Pathology between 2015 and 2021. Primary antibodies against HEG1, VISTA, and PD-L1 were used in the immunohistochemical study. The results showed a significant relationship between PD-L1 expression and sarcomatoid and high-grade epithelioid malignant mesotheliomas. VISTA was detected at a high rate in epithelioid malignant mesotheliomas and was negative in non-mesothelial tumors. HEG1 was positively monitored in mesothelial-derived tumors and negatively monitored in non-mesothelial tumors. The obtained results suggest that HEG1 could be further explored as a useful marker in determining mesothelial origin and that the expression levels of VISTA and PD-L1 markers may vary in histological subtypes in the selection of drugs for the treatment of malignant mesothelioma.