Nuclear Counterstain Research Articles

7850 Developmental ZNHIT3 expression in mice embryos in Mayer-Rokitansky-Küster-Hauser (MRKH) Syndrome related organs

Abstract Disclosure: D. Kira: None. B. Eroglu, MS, PhD: None. Z. Hawkins: None. L.P. Chorich: None. S. Brakta: None. L.C. Layman: None. Introduction: MRKH is a severe form of the Mullerian duct (MD) anomaly where the upper 2/3 of the vagina and the uterus fail to develop. Individuals with MRKH still have normally functioning ovaries and normal chromosomes. MRKH may be isolated Mullerian aplasia (type 1) or associated with renal, skeletal, cardiac, and/or auditory dysfunction (type 2). The etiology of MRKH syndrome remains unknown for most probands, although a genetic contribution is likely. Most evidence suggests autosomal dominant inheritance with incomplete penetrance. WNT4 and HNF1B pathogenic variants have been identified in some MRKH individuals. Recently, our lab identified heterozygous (HTZ) ZNHIT3 loss of function variants in persons with MRKH, Both variants predict functional changes in the ZNHIT3 C-terminus and impair protein expression in vitro. Previously, other investigators found biallelic variants in the N-terminus of ZNHIT3 in individuals with PEHO syndrome, a severe autosomal recessive neurodevelopmental disorder characterized by extreme cerebellar atrophy. We are currently characterizing a novel Znhit3 knockout (KO) mouse model, in which homozygous KO mice exhibit embryonic lethality. We have demonstrated ZNHIT3 RNA expression in the human uterus, heart, kidney, and skeleton, but developmental expression of ZNHIT3 in wild type (WT) animals has not been studied. In the WT mouse embryo, the kidney forms between E8.0 and E11, while the MDs form through invagination of epithelium ∼E11.5 and extend along the Wolffian duct until reaching the cloaca at ∼E13.5. Hypothesis: We hypothesize that ZNHIT3 is involved in MD development and expressed in the urogenital system and MRKH-related organs during embryonic development of WT mice. Method: Timed mating of WT mice produced embryos for analysis. Embryos were dissected out at E11.5, 12.5 and 13.5, fixed in 4% PFA, embedded in paraffin, and sectioned sagittally at 5mm thickness. H&E staining was done to identify sections showing kidney, genital tubercle, skeleton and cerebellum. Immunohistochemistry was also done using a ZNHIT3 antirabbit antibody and DAPI as nuclear counter stain. Imaging was performed using the Keyence fluorescence microscope (BZ-X800) at 10x and 40x magnification. Results: At all 3 time points, ZNHIT3 protein expression was observed in the developing urogenital organs including kidneys and bipotential gonads, as well as in vertebral bones and cerebellum. Heart protein expression studies are ongoing. Conclusion: ZNHIT3 protein expression is seen as early as E11.5 and is present in MRKH-related organs, suggesting it may play a role in the development of urogenital and MRKH-related organs. These findings provide the basis to compare expression in the Znhit3 KO mouse, a mouse model for MRKH. Presentation: 6/2/2024

Abstract 5176: TIGIT expression increases with advancing clinical stages and does not differ across racial groups in resected pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) has a dismal 12% 5-year survival rate (SEER) due to a lack of early detection biomarkers and resistance to standard therapeutic options (surgery, chemotherapy, radiation). Black African Americans (BAA) have 20% increased incidence of PDAC compared to those with European Ancestry (EA). TIGIT, an immune checkpoint receptor, is a marker of T cell exhaustion and plays a key role in the inhibition of anti-tumor immune responses. Recent studies have demonstrated that immune checkpoint receptor expression (PD-1 and TIGIT) on specific T cell populations correlates to worse overall survival (OS). TIGIT inhibitors are being explored in clinical trials in pancreatic cancer due to implications of its ligand (CD155) promoting immune evasion. We hypothesize that targeted anti-TIGIT therapy, in conjunction with other therapies targeting the tumor microenvironment, could reverse the immune suppression that is characteristic of PDAC. We performed RNAscope in situ hybridization (ISH) with a probe specific for human TIGIT mRNA (combined with a nuclear counterstain) on 79 tissue samples. The cohort of tissue samples included 8 biopsies (endoscopic-guided fine needle biopsies at time of diagnosis), 66 primary (from surgical resection), and 5 metastatic (liver core biopsies from patients with a primary PDAC diagnosis) formalin-fixed paraffin-embedded (FFPE) tissue sections from patients with histologically confirmed PDAC. After cells were segmented based on nuclear recognition, the presence of TIGIT probe within the cytoplasm of each cell was determined and quantified. Percent positive cell values were then exported for statistical analysis in R. De-identified clinical metadata was obtained from REDCap, a cloud-based HIPAA-compliant database used to compile patient data for research purposes. We tested for associations between %TIGIT present and clinical covariates, using linear regression for continuous outcomes, and logistic regression for binary outcomes. ScRNAseq revealed that TIGIT mRNA is enriched in, but not exclusive to the T/NK cellular compartments in PDAC. Staining analysis showed that TIGIT expression did not differ significantly between racial groups (comparing BAA to non-BAA). The mean percentage of TIGIT positive cells was 64.0%. High expression of TIGIT was associated with clinical stage, where an increase in stage was associated with increasing %TIGIT (p < 0.05).The TIGIT biomarker assay can be conducted at time of diagnosis, time of surgical resection, and time of metastatic biopsy. If patients’ samples contain high levels of TIGIT expression, these patients may be candidates for anti-TIGIT drug therapy. Considering that TIGIT expression correlates with advancing clinical stage, patients with more advanced staging at diagnosis (stage IIB, III, IV) especially may benefit from anti-TIGIT therapy. Citation Format: Madison George, Julie Clark, Kendyll Gartrelle, Georges Nassif, Kailee Hartway, Daniel Long, Daniel Salas-Escabillas, Allison Wombwell, Thais Pichardo, Hui-Ju Wen, Simone Benitz, Samuel Zwernik, Rupen Shah, Hakmin Park, Philip, Gazala Khan, Howard Crawford, David Kwon, Brian Theisen, Nina Steele. TIGIT expression increases with advancing clinical stages and does not differ across racial groups in resected pancreatic 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 5176.

Abstract C012: TIGIT expression correlates to worse overall survival in primary and metastatic pancreatic ductal adenocarcinoma

Abstract Pancreatic Cancer has a 12% 5-year survival rate due to late-stage detection and lack of many chemotherapy or targeted therapy options. TIGIT is an immune checkpoint inhibitor being explored in clinical trials in pancreatic cancer due to the implications of its ligand (CD155) promoting immune evasion. TIGIT is a marker of T cell exhaustion and plays a key role in the inhibition of anti-tumor immune responses. We hypothesize that targeted anti-TIGIT therapy, in conjunction with other therapies targeting the tumor microenvironment, could reverse immune suppression that is characteristic of pancreatic ductal adenocarcinoma (PDAC). As such, here we aim to quantify TIGIT expression using automated RNAscope technology and to correlate it with overall patient survival. We performed RNAscope in situ hybridization and immunohistochemistry on 25 primary and 4 metastatic (liver) formalin-fixed paraffin-embedded (FFPE) tissue sections from patients with histologically confirmed PDAC. A nuclear counterstain combined with an RNAscope probe specific for the human TIGIT mRNA was utilized. Slides were scanned at 40X magnification with an automated slide scanner and quantified using the ISH-IHC module of the HALO-v3.5 software. After cells were segmented based on nuclear recognition, the presence of TIGIT probe within the cytoplasm of each cell was determined and quantified. Percent positive cell values were then exported for statistical analysis in SPSS. De-identified clinical metadata was obtained from REDCap, a cloud-based HIPAA-compliant database used to compile patient data for research purposes. Gender, survival months, ethnicity, race, tumor histology, stage, cancer history, treatment status, and comorbidities were evaluated. We analyzed a cohort of 29 histologically-confirmed surgically resected PDACs, comprised of 25 primary pancreatic tumor samples and 4 metastatic liver core biopsies. The mean percentage of TIGIT positive cells was 63%. From this, the TIGIT high versus low threshold was determined to be 70%. High TIGIT was associated with significantly lower overall survival (p=.013), suggesting that expression of TIGIT and T cell exhaustion may predict overall worse survival. The median overall survival months for the TIGIT low group was 321 months versus 106 months for the TIGIT high group. This data leads us to hypothesize that expression of TIGIT and T cell exhaustion may predict worse overall survival. Anti-TIGIT medications are currently in clinical trials for metastatic PDAC. Here we demonstrate that high TIGIT expression is associated with an overall worse prognosis and present a novel, automated TIGIT detection protocol that may be used to determine if patients are candidates for anti-TIGIT therapy. In a clinical setting, this novel, automated biomarker TIGIT detection protocol could be used to select candidates for anti-TIGIT therapy. This may lead to improved overall patient survival. Citation Format: Madison George, Julie Clark, Kendyll Gartrelle, Georges Nassif, Donald Rempinski, Daniel Long, Hui-Ju Wen, Simone Benitz, Samuel Zwernik, Rupen Shah, Hakmin Park, David Kwon, Philip Philip, Gazala Khan, Howard Crawford, Brian Theisen, Nina Steele. TIGIT expression correlates to worse overall survival in primary and metastatic pancreatic ductal adenocarcinoma [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 C012.

Segmental Unconventional Outflow in Mouse Eyes.

To investigate the flow pattern in unconventional outflow and its correlation with conventional outflow in mouse eyes. Fluorescent microspheres were injected into the anterior chamber of one eye of anesthetized C57BL/6J mice (n = 4), followed by perfused fixation with 4% paraformaldehyde in situ after 45minutes. Post-euthanasia, the injected eyes were enucleated, further immersion fixed, and dissected into 12 equal radial segments. Both sides of each segment were imaged using a confocal microscope after nuclear counterstaining. Both unconventional and conventional outflow patterns of each eye were analyzed by ImageJ and ZEN 2.3 imaging software. Segmental outflow patterns were observed in both the ciliary body (CB) and the supraciliary space and suprachoroidal space (SCS). In the CB, the tracer intensity was the lowest at 12 o'clock and highest at 9 o'clock, whereas in the SCS it was the lowest at 2 o'clock and the highest at 10 o'clock. Consequently, a segmental unconventional outflow was observed, with the lowest and highest flow regions in the superior and temporal quadrants, respectively. The overall segmental uveoscleral outflow has no correlation with trabecular outflow (P > 0.05). Four different outflow patterns were observed: (1) low-flow regions in both outflows, (2) primarily a high-flow region in conventional outflow, (3) primarily a high-flow region in unconventional outflow, and (4) high-flow regions in both outflows. Uveoscleral outflow is segmental and unrelated to the trabecular segmental outflow. These findings will lead to future studies to identify the best location for the placement of drainage devices and drug delivery.

Abstract B147: Preclinical evaluation of XPO1 inhibition with topoisomerase-I (topo-I) inhibition in colorectal cancer (CRC) cell lines and patient-derived xenograft (PDX) models

Abstract Background: Resistance to topo-I inhibitors is of significant importance in treating patients with metastatic CRC (mCRC). Selinexor and eltanexor are selective inhibitors of nuclear export, by binding to exportin 1 (XPO1, the primary nucleic export receptor, and is overexpressed in CRC). Eltanexor is an investigational XPO1 inhibitor, with similar mechanism and potency but improved tolerability in animal models. XPO1 inhibition results in nuclear localization of tumor suppressor proteins, oncoprotein mRNAs, and topoisomerases, and decreases DNA damage repair (DDR) response. Irinotecan is a topo-I inhibitor which induces DNA damage and is commonly used in mCRC. We hypothesized that XPO1 inhibition may improve response to topo-I inhibitor in mCRC by impairing the DDR response. Methods: The synergism of XPO1 inhibition (selinexor or eltanexor) with SN38 was assessed in CRC cell lines by CellTiter-Glo 2.0 and quantitated using Bliss Additivity Model using Synergy Finder 2.0. Each condition was plated either concurrent (both drugs for 72 hours) or sequential (SN38 for 24 hours then removed, followed by XPO1 inhibitor for 48 hours). Nude mice were implanted with one microsatellite stable CRC PDX model in the flanks followed by treatment with vehicle, XPO1 inhibitor, irinotecan (15 mg/kg intraperitoneal weekly), or combination. Tumor growth was measured by specific growth rate (SGR), within an IACUC-approved protocol. Tumor tissue was collected at day 7 for molecular analysis. Tissues were lysed and protein was run on 4%-12% SDS-PAGE Gel. p21 Waf/Cip and P-Histone H2A.X were used to determine DNA damage by western blot. P-Histone H2AX (pH2AX) was evaluated by immunofluorescence using primary conjugated P-Histone H2A.X antibody and DAPI for nuclear counter stain. Slides were imaged using Olympus iX83 microscope and analysis was performed using Olympus CellSens software. Results: In CRC cell lines, concurrent combination eltanexor and SN38 did not synergistically decrease viability but increased pH2AX compared to SN38 alone (~30-fold). However, sequential SN38 followed by eltanexor synergistically decreased viability at multiple dose levels; evaluation of pH2AX is ongoing. In PDX model CRC254, eltanexor (10 mg/kg and reduced to 7.5 mg/kg on day 8 to improve tolerability, orally 5 days per week) with irinotecan decreased SGR compared to irinotecan alone (SGR=0.007 vs 0.026, respectively, p=0.0003) and compared to eltanexor alone (SGR=0.029, p <0.0001). Combination treatment increased pH2AX and increased p21 expression compared to either single-agent treatment. Similar SGR results were observed with selinexor (5 mg/kg orally twice weekly). Conclusions: XPO1 inhibition and topo-I inhibition decreased CRC tumor growth in PDX models, presumably by increasing DNA double strand breaks. Cell line data suggests sequential dosing of topo-I inhibition followed by XPO1 inhibition may be effective. This work supports continued development of this combination in mCRC. Citation Format: Robert W Lentz, Adrian Dominguez, Stacey Bagby, Cameron Binns, Alexis Leal, Sunnie Kim, Sarah Lindsey Davis, Christopher H Lieu, Wells A Messersmith, Todd M Pitts. Preclinical evaluation of XPO1 inhibition with topoisomerase-I (topo-I) inhibition in colorectal cancer (CRC) cell lines and patient-derived xenograft (PDX) models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B147.

Abstract B008: Machine-learning enabled quantification of colocalized multiplex IHC signals with spectral overlap

Abstract Background: Immunohistochemistry (IHC) is the gold standard for detecting protein biomarkers in cancer. In breast cancer, several nuclear biomarkers are relevant for guiding treatment decisions, including estrogen receptor (ER) and progesterone receptor (PR), indicative of response to endocrine therapy, and the proliferation marker Ki67, indicative of response to CDK inhibition1. However, these nuclear markers are detected using chromogens that colocalize and spectrally overlap with hematoxylin (HTX), the standard nuclear counterstain for IHC. Thus, the accurate detection and quantification of nuclear antigens is challenging. To overcome this limitation of IHC-based biomarker assays, we developed a method to identify colocalized signals after staining with chromophores with spectral overlap. Methods: Multiplex IHC (mIHC) using primary antibodies against Ki67, PR, and ER was performed on a breast cancer tissue microarray (N=65 cores). Antigens were detected by dabsyl-, tamra-, and Cy5-conjugated secondary antibodies, respectively, and slides were counterstained with HTX. The cores were scanned with brightfield imaging (Aperio AT2 and 3DHistech microscopes). The same cores were imaged on a custom brightfield microscope using an LED light source and 10 nm wide band-pass filters at 405, 450, 520, 550, 589, 632 and 660 nm. Images were spectrally unmixed using absorption spectra of the different chromophores and the background estimated based on the images themselves and aligned with the brightfield images. Pathologist-trained individuals collected exhaustive manual annotations in regions of interest for each marker and HTX using unmixed channels. A convolutional neural network (CNN) was trained using the annotations for each marker and HTX and applied to the WSI. Predicted cell locations were aggregated across the four channels to obtain an exhaustive “super-annotation” co-expression map of the WSI. Super-annotations were then used to train a CNN to detect ER/PR/Ki67/HTX colocalization in the aligned RBG image of the same slide. Predictions from the super-annotation model were compared to ER/PR/Ki67/HTX detection done via HALO (Indica Labs). Results: Our multi-spectral imaging pipeline allowed for comprehensive annotations of all nuclei positive for at least one of the three markers or HTX. Using our super-annotation model, the count error of nuclei that are positive for HTX and one, two, or three markers was reduced by 7.8%, 6.4%, and 0.03%, respectively, compared to HALO. Conclusions: We have developed a method to identify colocalized antigens after mIHC using chromogens with spectral overlap. Related work to extend this approach to membranous biomarkers is ongoing and will enable additional novel analyses (e.g., describing the precise spatial organization of PD-L1+ tumor cells relative to CD8+ tumor-infiltrating lymphocytes). The approach that we describe here has great potential to facilitate the utilization of mIHC for the clinical detection of biomarkers in oncology.

Pattern of CD8+ tissue resident memory T cells between local recurrence and distant metastasis in nasopharyngeal carcinoma.

e15161 Background: CD8+ T lymphocytes are believed to be the major anti-tumor immune subsets. Consequently, most cancer immunotherapeutic approaches seek to amplify cytotoxic T lymphocytes specific to malignant cells. A recently identified subpopulation of memory CD8+ T cells, named tissue-resident memory T (Trm) cells, persists in non-lymphoid tissues, including solid tumors. This T-cell subset is considered an independent memory T-cell lineage with cytotoxic features. However, their function, spatial location and influence on disease-related outcomes (local recurrence [LR], distant metastasis [DM] in nasopharyngeal carcinoma (NPC), a disease entity characterized by substantial immune infiltrate, remain poorly understood. We aim to close this gap through investigation on the abundance and spatial localization of CD8+ Trm in relation to tumor site (primary or recurrence [LR and DM]). Methods: This retrospective cohort analysis based on 20 NPC patient samples: primary (initial diagnosis; [I; n = 19], recurrence ([LR; n = 14] and [DM; n = 10]). We accessed the spatial localization in tumor tissues within tissue-segmented classifications of either cancer islands (pan-cytokeratin–positive [CK+] areas) or stromal areas (CK– areas). Tumor infiltrating lymphocytes (TILs) among patients from I, LR and DM group was quantified through multiplexed 5-colour opal staining using biomarkers against CD8, CD103, IL17, CK plus DAPI (nuclear cell counterstain). Results: While total CD8+ TILs (total tumor) as well as their spatially localized cell densities (tumor islet [TI] vs. tumor stroma [TS]) did not show any significant changes among I, LR and DM patient groups, a higher proportion of CD8+ TILs in the TI co-express the CD103 molecule, an indication of CD8+ Trm (CD103+CD8+) infiltration (mean CD103+CD8+/CD8+ in TI: 32.1% in I group, 44.8% in LR group, 46.0% in DM group; mean CD103+CD8+/CD8+ in TS: 19.0% in I group, 26.2% in LR group 26.2% in DM group). Notably, we observed that such augmentation only reached statistically significant within TI from patient in LR group (p = 0.0451). Furthermore, we found that CD8+ Trm in the LR group expressed higher level of cytokine IL-17, which was ~2.1-fold (p = 0.0788) and 3.6-fold upregulated (p = 0.0285) as compared to I and DM, respectively. Conclusions: Cancer islet–localized CD8+ TILs are composed of CD103+ Trms, which make up nearly 45% the total CD8+ T cell population within LR tumors of NPC patients, highlighting spatial localization of CD103+CD8+ Trms as a key phenotype of CD8+ TIL subset. The CD8+ Trm expressed with increased IL17 suggests that enrichment of this T-cell subset might exerts cytotoxic functions. Our results demonstrate that CD8+ Trm had a greater likelihood as an indicator of LR risk for NPC patients and help identify patient cohorts likely to benefit from immunotherapy.

Cover Image

Coronal section of a day 15.5 mouse embryo. NetrinG1-positive thalamocortical axons (red) project from the thalamus, through the internal capsule, into the cortex of a mouse with constitutive deletion of Jnk2 and Jnk3. Dlx5/6-CIE positive cells are labeled with EGFP, Hoechst nuclear counterstain is in blue. Image acquired on Olympus VS120 Slide Scanner at 10x. From: Early construction of the thalamocortical axon pathway requires c-Jun N-terminal kinase signaling within the ventral forebrain; Jessica G. Cunningham, James D. Scripter, Stephany A. Nti, Eric S. Tucker; https://doi.org/10.1002/dvdy.416; DevDyn 251:3, pp. 459-480.

Niche formed by bone morphogenetic protein antagonists gremlin 1 and gremlin 2 in human hair follicles.

Niche formed by bone morphogenetic protein antagonists gremlin 1 and gremlin 2 in human hair follicles.

Reduction in trabecular meshwork stem cell content in donor eyes with primary open angle glaucoma

We previously identified and characterized human trabecular meshwork stem cells (TMSCs) based on high expression of ABCG2/p75 positivity and high nucleus to cytoplasmic ratio. These TMSCs expressing high ABCG2 and p75 were located to the insert region of the human TM. Additionally, we demonstrated an age-related reduction in the TMSC content which was significantly associated with TM cell loss. In continuation, this study was aimed to determine the TMSC content in glaucomatous donor eyes wherein a drastic reduction in TM cellularity has already been reported. Anterior segments from known glaucomatous (n = 6) and age-matched normal (n = 8) donors were dissected into four quadrants. A minimum of three sections from each quadrant were used for histopathological analysis as well as immunostaining. Analysis of hematoxylin and eosin-stained sections from glaucomatous tissues revealed a decrease in total TM cellularity, thickening of trabecular beams, fusion of trabeculae, absence of patent Schlemm’s canal compared to age-matched controls. In addition, the TM thickness at various positions of the meshwork and the coronal as well as the meridional diameters of the Schlemm’s canal were observed to be significantly reduced in glaucomatous eyes. Further, sections from both the groups were immunostained for universal stem cell marker ABCG2 and neural crest derived stem cell marker p75. The images were acquired using Leica SP8 confocal microscope. Quantification of total TM cellularity based on nuclear counterstain (mean ± SD) using ImageJ identified 69.33 ± 12.77 cells/section in control eyes. In glaucomatous donors, the TM cellularity was found to be reduced significantly to 41.83 ± 9.0 (p = 0.0007). In addition, a reduction in the percentage of TMSCs (cells with high ABCG2 expression and p75 positivity) was evident in glaucomatous donors (0.14 ± 0.17%) compared to age-matched controls (4.73 ± 5.46%) (p = 0.064). Thus, the present study confirmed the significant decline in TM cellularity and a reducing trend in the TMSC content, though this reduction was non-significant in glaucomatous donor eyes. Further studies are essential to elucidate the role of TMSCs in the pathogenesis of primary open angle glaucoma.

Cover Image

The tardigrade brain is key to understanding the evolution of the central nervous systems in Panarthropoda. In this issue of the Journal of Morphology, Gross and coauthors (pp. 1298–1312) reinvestigated the central nervous system of the heterotardigrade Echiniscus testudo using immunohistochemistry to visualize the number and position of tracts, commissures, and neuropils. They document five major synapsinimmunoreactive domains along the body: a large unitary, horseshoe‐shaped neuropil in the head and four neuropils in the trunk ganglia, supporting the hypothesis that the dorsal brain is serially homologous with the ventral trunk ganglia. The cover image shows the dorsal part of the saddle shaped brain [anti‐synapsin immunolabeling (red) and nuclear counterstain (cyan)]. The centrally located synapsin‐rich region, corresponding to the central brain neuropil.

H2A.B is a cancer/testis factor involved in the activation of ribosome biogenesis in Hodgkin lymphoma.

Testis-specific regulators of chromatin function are commonly ectopically expressed in human cancers, but their roles are poorly understood. Examination of 81 primary Hodgkin lymphoma (HL) samples showed that the ectopic expression of the eutherian testis-specific histone variant H2A.B is an inherent feature of HL. In experiments using two HL cell lines derived from different subtypes of HL, H2A.B knockdown inhibited cell proliferation. H2A.B was enriched in both nucleoli of these HL cell lines and primary HL samples. We found that H2A.B enhanced ribosomal DNA (rDNA) transcription, was enriched at the rDNA promoter and transcribed regions, and interacted with RNA Pol I. Depletion of H2A.B caused the loss of RNA Pol I from rDNA chromatin. Remarkably, H2A.B was also required for high levels of ribosomal protein gene expression being located at the transcriptional start site and within the gene body. H2A.B knockdown reduced gene body chromatin accessibility of active RNA Pol II genes concurrent with a decrease in transcription. Taken together, our data show that in HL H2A.B has acquired a new function, the ability to increase ribosome biogenesis.

Abstract 239: Integrated computational image analysis of cellular and acellular tissue components as a method for detailed tumor tissue mapping and structural patterns recognition

Abstract Tumor microenvironment (TME) represents an integrated system that affects cancer cell behavior and contributes directly to disease outcome. Systemic approach to analysis of TME should uncover its complexity and facilitate discovery of mechanisms orchestrating tumor development and metastasis. Multiplex fluorescence tissue staining followed by spatial analysis of tumor tissue architecture can provide insights to pivotal interactions of cellular and acellular components of TME. Extracellular matrix (ECM is represented mainly by collagen deposition. Number of reports indicates that ECM contribution to TME state not only depends upon amount of accumulated collagen but its geometrical features and spatial orientation of fibers. These characteristics of collagen fibers contribute directly to physical and mechanical properties of tissue and can change tumor growth and metastasis. Current methods of computational image analysis of tissue implement assessment of cellular or acellular components separately. The goal of current work was to develop a new computational tool to perform integrated analysis of fibrous and cellular components of tumor tissue in spatial dependent manner to achieve detailed tumor tissue mapping and structural patterns recognition. To pursue this goal, we generated images of human lung adenocarcinoma tissue characterized by indolent and aggressive behavior. We performed multiplex immunofluorescence staining for following markers: CD3 - marker of T-lymphocytes, PanCytokeratin - marker of epithelial/tumor cells, collagen hybridizing peptide (3Helix) - marker of collagen, DAPI - nuclear counterstain. To develop image analysis pipeline, we utilized an open source graphical interface analytical platform KNIME, where we generated modular workflow. For ECM analysis, we integrated Python written code into KNIME node. Segmentation of collagen fibers was performed using skeletonization with subsequent calculation of geometrical properties (length, alignment, widths) and orientation of each fiber. Data, collected from single cell analysis and ECM architecture assessment, were combined and forwarded to downstream spatial analysis, where distances from cell to cell or cell to ECM were computed and neighborhood analysis was performed. We demonstrated that tumor cells in aggressive adenocarcinoma samples were co-localized with a smaller number of collagen fibers. In addition, length of that fibers was less in comparison to indolent group. Correlation analysis revealed positive correlation between length of collagen fibers and number of tumor cells in indolent group, but we did not observe this phenomenon in indolent group. Developed computational method provides additional dimensionality to tissue image analysis and can reveal underrecognized structural patterns of the tumor microenvironment. Citation Format: Georgii Vasiukov, Tatiana Novitskaya, Maria-Fernanda Senosain, Anna Menshikh, Andries Zijlstra, Sergey Novitskiy, Pierre Massion. Integrated computational image analysis of cellular and acellular tissue components as a method for detailed tumor tissue mapping and structural patterns recognition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 239.

Abstract 2778: Comparative multiplex digital phenotyping of the tumor microenvironment across multiple tumor types using a well characterized multi-tumor tissue microarray

Abstract The tumor microenvironment (TME) represents a complex interaction of immune and host cells and can be described broadly as ‘inflamed', ‘immune excluded' and ‘desert'; categories that may serve as predictive parameters for response to different immunotherapies. Multiplexed fluorescence and associated digital imaging platforms are important tools for defining the spatial components of the TME and can accelerate development of novel immunotherapy strategies. To investigate the TME across multiple tumor types simultaneously we have utilized a multi-tumor tissue microarray (TMA) covering 11 different tumor types comprising a total of 144 cases, each in duplicate with cores (1mm) taken from invasive margin (IM) and tumor center (TC). The TMA was stained using the UltiMapper I/O Immuno8 panel, which includes markers for CD3, CD4, CD8, FOXP3, CD68, PD-1, PD-L1, and a pan-CK/SOX10 cocktail as a tumor indicator. Stained TMAs were scanned on a fluorescence whole slide scanner (20X magnification) and marker images aligned using the UltiStacker software using the nuclear counterstain images as references from two rounds of imaging, to provide accurate marker colocalization data. Digital image analysis was performed using Visiopharm software to generate cell phenotype data, including localization to tumor and stroma, for each of the 288 cores. Comparative analysis of all tumor types regardless of core location (IM and TC combined) revealed gastric cancer, CRC, TNBC and NSCLC to be most highly infiltrated with CD3+ T cells, and ER+ and Her2+ breast cancers least infiltrated. The contribution of cytotoxic T cells (CD3+ CD8+) to the TME was greater for gastric cancer (approx. 50%) compared with NSCLC (approx. 20%) and the proportion of cytotoxic T cells activated/exhausted as defined by expression of PD-1 ranged from approx. 25% (eg. TNBC, CRC) to 1% (eg. ER+ BC). Spatial localization analyses of cytotoxic T cells revealed pancreatic and hepatic cancers as the most ‘excluded' as evidenced by low CD3+/CD8+ T cell numbers in tumor (CK+) compared with stromal regions (CK-). Interestingly, cytotoxic T cells with an exhausted phenotype (CD3+ CD8+ PD-1+) were preferentially infiltrated within CK+ tumor areas compared with stroma (CK-) for inflamed tumors such as gastric, TNBC and CRC. These data highlight the benefits of combining a fast, multiplexed staining protocol, with a detailed digital analysis of immune phenotypes spatially within well characterized tumor samples to better understand the TME (eg. inflamed, excluded) and identify tumor types more likely to benefit from different immunotherapeutic strategies. Citation Format: Marie Cumberbatch, Douglas Wood, Christopher Womack, Milan Bhagat, Mael Manesse, Alison Bigley, Lorcan Sherry. Comparative multiplex digital phenotyping of the tumor microenvironment across multiple tumor types using a well characterized multi-tumor tissue microarray [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2778.

Abstract 1665: QuPath versus InForm: Digital image analysis of multiplex immunofluorescence (mIF) tumor-infiltrating lymphocyte (TIL) outcomes in an immunotherapy clinical trial

Abstract Background: mIF has emerged as a biomarker platform for characterizing TILs in breast cancer. One pitfall limiting wider application is the complex, proprietary nature of image analysis software. QuPath is an alternative, open-source platform recognized for its processing speed and ease of use. Here, we compare TIL analysis using QuPath version 0.2.3 versus InForm version 2.4, using specimens from a completed pre-operative phase Ib trial of locoregional cytokine therapy (IRX-2 regimen, Brooklyn ImmunoTherapeutics). Methods: 15 paired pre-/post-immunotherapy stage I-III early stage breast cancer specimens were analyzed using the PerkinElmer Vectra platform with a 6-plex panel: cytokeratin (CK, tumor marker), CD3 (T cells), CD8 (cytotoxic T cells), CD163 (macrophages), FoxP3 (regulatory T cells, Treg), programmed death ligand 1 (PD-L1), and DAPI nuclear counterstain. Four high-power fields (HPF) were randomly selected to represent each specimen. Images were analyzed per manufacturer instructions with InForm, and corresponding data were compared with QuPath analysis. Unmixed images were imported into QuPath and cells were segmented using a confidence threshold of DAPI stain. Cells were phenotyped using basic thresholding and random trees machine learning. Tissue was segmented using machine learning. We hypothesized that QuPath and InForm would generate correlating TIL counts data and similar cohort-level estimates of immunotherapy treatment effect. Results: We found a strong correlation in total cell count between QuPath v. InForm (R2 = 0.73, p < 0.001) and a moderate correlation in stromal/tumor area per HPF (R2 = 0.47, p < 0.001). QuPath cell phenotyping using thresholding v. random trees did not appreciably differ. However, QuPath v. InForm cell count correlations varied by cell type, with CD3+ CD8+ and CD3+ CD8- T cells strongly to moderately correlated (R2 = 0.65, p < 0.001 and R2 = 0.32, p < 0.001, respectively) and Treg and macrophage cell counts weakly correlated (R2 = 0.13, p < 0.001 and R2 = 0.08, p = 0.005). Continuous PD-L1 marker expression based on intensity was strongly correlated between softwares (μ: R2 = 0.97, p < 0.001 and σ: R2 = 0.93, p < 0.001). Estimated treatment effect on cell density varied by cell type (median fold change, QuPath v. Inform, CD8+: 3.1 v. 2.5; CD8-: 3.6 v. 1.5; Treg: 1.3 v. 1.1; macrophage: 3.2 v. 1.1). Outlier values were more common with QuPath in this analysis, resulting in unstable estimates of mean fold change. Conclusion: Exploratory mIF biomarker outcomes in clinical trials may depend on the method of image analysis. Further work is ongoing to evaluate and improve upon discordant results using QuPath v. InForm. These data also highlight the importance of collaborative efforts via the NIH Cancer Immune Monitoring and Analysis Centers to standardize and validate mIF methodologies across institutions. Citation Format: Benjamin M. Nikitin, Brie Chun, Isaac Kim, William L. Redmond, Katherine Sanchez, Zhaoyu Sun, Yaping Wu, David B. Page. QuPath versus InForm: Digital image analysis of multiplex immunofluorescence (mIF) tumor-infiltrating lymphocyte (TIL) outcomes in an immunotherapy clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1665.

Neoplastic Monocyte-Derived Osteoclasts Are Highly Proliferative, but Sub-Functional in MPN-Associated Myelofibrosis

Background: Myeloproliferative neoplasms (MPN) are clonal disorders characterized by the proliferation of myeloid progenitors. A defining hallmark of MPN-associated myelofibrosis (MF) is the deposition of fibrous tissue inside the bone marrow (BM) stroma, arising either as a primary process (PMF), or secondary to the essential thromocythemia (post-ET MF) or polycythemia vera. In at least 1/3 of cases, BM fibrosis is accompanied by the osteosclerotic thickening of the bone trabeculae. It was generally held that these changes are a reaction of mesenchymal cells to the stimuli from the malignant clone. However, the recent discovery of neoplastic monocyte-derived fibrocytes capable of directly inducing BM fibrosis in situ and in vivo has led us to hypothesize that other tissue remodeling cells of the myeloid lineage may play similar roles in the pathogenesis of MPN-associated MF. Osteoclasts (OC) are giant multinuclear cells formed by the fusion of multiple monocytic progenitors. Apart from being exclusively bone-resorbing cells, OCs have a critical role in regulating bone formation by the mesenchymal cell-derived osteoblasts (OB) and maintaining the hematopoietic BM microenvironment. The aim of this study was to examine the status of these cells in MF patients and explore the effects that the neoplastic OC progenitors exert on the development of MPN-associated osteosclerosis.Methods: In order to quantitate bone remodeling cells, trephine biopsies were obtained from patients with PMF (n = 32) and post-ET MF (n = 27), as well as healthy controls (n = 3). A tyramide signal amplification-based fluorescence IHC approach was employed using TRAP and cathepsin K as OC-specific and CD56and osterix as OB-specific markers. In addition, CD14+ low-density peripheral blood cells from the representative MF patients and healthy controls were cultured in the presence of M-CSF and RANKL. After 3 weeks mature OCs were visualized using fluorescently labeled F-actin and TRAP with DAPI as the nuclear counterstain, and the JAK2V617F allele frequency was assessed via RT-PCR. For the OC functional assay, cells were plated on a mineralized surface, which was subsequently stained using a modified von Kossa method. To assess the OC fusion capacity, two populations of progenitors labeled with different lipophilic probes were mixed in a 1:1 ratio and the colocalized signal was quantified for each mature cell. All imaging was conducted using an automated multispectral or a dual spinning disk confocal system.Results: Based on the multiplex BM imaging analysis, we observed a significant increase in the number of OCs in MF patients compared to healthy controls (median: 21.8 vs. 7.0 per 100 mm of bone perimeter; p < 0.0001) that was followed by a substantial increase in the total number of OBs and the density of trabecular bone. After inducing differentiation of JAK2+ monocytes from MF patients into mature TRAP+ OCs in vitro, these cells consistently showed a high mutant allele frequency. Compared to OCs cultured from healthy monocytes, MF patients' OCs exhibited a 40.5% decrease in the median number of nuclei per cell (p= 0.024) and a tendency to incompletely develop F-actin rings. Simultaneously, there was no change in the mean TRAP positivity between the two groups. Finally, MF OCs were significantly less apt at resorbing mineral-coated areas in contrast to healthy cells (median: 21.5% vs. 7.3%; p= 0.016), and there was a 3-fold decrease in the proportion of fused mature OCs from MF monocytes compared to healthy ones (p= 0.008).Conclusions: The development of OCs is highly skewed towards proliferation in both primary and secondary MPN-associated MF. We were able to demonstrate that neoplastic monocytic progenitors retain their aberrant genetic constitution even after differentiating into mature OCs. However, fusion of such progenitors seems to be profoundly impaired, and MF OCs are unable to fully acquire the phenotypical features associated with efficient bone resorption, particularly multinucleation and the development of actin-rich structures. At least to a certain extent, this process happens independently of the non-malignant stroma, although it seems to be impacting the OBs as well. Taken together, we show evidence that even though MF OCs are hyperproliferative, their function is intrinsically supressed due to the inherited neoplastic burden, which in turn contributes to the osteosclerotic dysplasia of the MPN-affected BM. DisclosuresVerstovsek:Blueprint Medicines Corp: Research Funding; Seattle Genetics: Research Funding; Genentech: Research Funding; Incyte: Research Funding; Galena BioPharma: Research Funding; Bristol Myers Squibb: Research Funding; Bristol Myers Squibb: Research Funding; Celgene: Research Funding; Roche: Research Funding; Pfizer: Research Funding; Lilly Oncology: Research Funding; Astrazeneca: Research Funding; Incyte: Research Funding; Promedior: Research Funding; Genentech: Research Funding; Galena BioPharma: Research Funding; Pfizer: Research Funding; Gilead: Research Funding; NS Pharma: Research Funding; Seattle Genetics: Research Funding; Astrazeneca: Research Funding; Lilly Oncology: Research Funding; Roche: Research Funding; CTI BioPharma Corp: Research Funding; NS Pharma: Research Funding; Blueprint Medicines Corp: Research Funding; Gilead: Research Funding; CTI BioPharma Corp: Research Funding; Celgene: Research Funding; Promedior: Research Funding.

Highly specific nuclear labeling via in situ formation of fluorescent copper nanoparticles.

When imaging cells, nuclear counterstaining is imperative; however, many commercial nuclear-staining dyes based on nucleic acid intercalation result in nonspecific signals in the cytoplasm. Here, we propose a new strategy that stains the nucleus with high specificity by in situ formation of DNA-templated copper nanoparticles (CuNPs). We demonstrated that genomic DNA in the nucleus enabled rapid formation of highly fluorescent CuNPs immediately following addition of a copper ion source and ascorbate as a reducing agent. Moreover, we found that RNA and mitochondrial DNA, largely responsible for nonspecific cytoplasmic signals from commercial nuclear-staining dyes, did not mediate the formation of the highly fluorescent CuNPs, resulting in highly specific nuclear staining at a reduced cost relative to commercially available methods. Furthermore, we verified the compatibility of the proposed method with other fluorescence-labeling techniques. These results demonstrated the efficacy of this method and its promise as a powerful tool for cell imaging.

Characterizing the Tumor Immune Microenvironment with Tyramide-Based Multiplex Immunofluorescence

Multiplex immunofluorescence (mIF) allows simultaneous antibody-based detection of multiple markers with a nuclear counterstain on a single tissue section. Recent studies have demonstrated that mIF is becoming an important tool for immune profiling the tumor microenvironment, further advancing our understanding of the interplay between cancer and the immune system, and identifying predictive biomarkers of response to immunotherapy. Expediting mIF discoveries is leading to improved diagnostic panels, whereas it is important that mIF protocols be standardized to facilitate their transition into clinical use. Manual processing of sections for mIF is time consuming and a potential source of variability across numerous samples. To increase reproducibility and throughput we demonstrate the use of an automated slide stainer for mIF incorporating tyramide signal amplification (TSA). We describe two panels aimed at characterizing the tumor immune microenvironment. Panel 1 included CD3, CD20, CD117, FOXP3, Ki67, pancytokeratins (CK), and DAPI, and Panel 2 included CD3, CD8, CD68, PD-1, PD-L1, CK, and DAPI. Primary antibodies were first tested by standard immunohistochemistry and single-plex IF, then multiplex panels were developed and images were obtained using a Vectra 3.0 multispectral imaging system. Various methods for image analysis (identifying cell types, determining cell densities, characterizing cell-cell associations) are outlined. These mIF protocols will be invaluable tools for immune profiling the tumor microenvironment.

Processing and Sectioning Undecalcified Murine Bone Specimens.

Preparation of mineralized tissue specimens for bone-specific staining encompasses a critical sequence of histological techniques that provides visualization of tissue and cellular morphology. Bone specimens are fixed in 10% neutral buffered formalin (NBF), dehydrated in graded ethanol (EtOH) solutions (and optionally cleared in xylene), infiltrated and embedded in polymethyl methacrylate (methyl methacrylate or MMA), classically sliced into 4-10 micrometer (μm) sections, and stained with bone-specific histological stains such as von Kossa (with either nuclear fast red solution counterstain or MacNeal's tetrachrome counterstain), modified Goldner's trichrome, Alizarin Red S, Safranin O, and tartrate-resistant acid phosphatase (TRAP) stain. Here, we describe the tissue processing of mineralized mouse bones from dissection to staining for histological analysis by light microscopy.

Activated adaptive immune system in dissected bicuspid aortic valve aortas: trigger for dissection?

Abstract Background/Introduction Bicuspid aortic valve (BAV) is associated with ascending aorta aneurysms and dissections. Presently, genetic factors and pathological flow patterns are considered responsible for aneurysm formation in BAV. Despite, indication for preventive surgery is presently only defined by vessel diameter which is poor marker for dissection, as it does not take other processes responsible for the vulnerability of the aorta into account. Purpose Inflammation is not considered a player in BAV aortopathy. We introduce a quantitative immunohistochemistry (IHC) approach to sensitively look at the potential role of both the innate and adaptive immune system in BAV aortopathy. Methods Dilated (n=8), non-dilated (n=14) and dissected (n=4) BAV ascending aortas were collected during surgery or from post-mortem donors. Median time from symptoms to surgery for dissections was just over 4 hours. Tissue was stained with a novel 8-colour IHC technique allowing for simultaneous visualization of 6 markers per slide, completed with DAPI nuclear counter-stain and elastin fiber autofluorescence. One panel focused on the adaptive immune system (identifying B cells and classic dendritic cells type 2 (cDC2s) and phenotyping T cells), and the other on the innate immune system (assessing macrophage polarization and neutrophil extravasation). All cells were identified and comprehensively phenotyped using automated quantitative analysis. Results Aneurysm formation was associated with an organized and consistent increase of lymphocytes in the adventitia. B cell follicles and helper T cell expansion were identified, suggestive of a targeted adaptive immune response (Fig. 1a). Only dissected aortas showed a statistically significant increase of helper T (p=0.3) and cDC2s (p=0.3) in the media, when compared to non-dilated and dilated samples (Fig. 1b). The short time between dissection symptoms and surgery suggests these cells were present before the dissection occurred. Furthermore, aneurysms and dissections are associated with a shift in macrophage phenotype to the more aggressive M1-like subset. In summary, we found that a progression of aggressive immune cells in the adventitia and media was correlated to a progression in disease state; from normal to dilated to dissected. Conclusions Aorta dilatation in patients with BAV is associated with an expansion of B and helper T cells in the adventitial compartment without changes in the media. This result might indicate an antigen-driven adaptive immune response. Only dissections show an increase in helper T cells and cDC2s in the media, together with polarization of macrophages to a more M1-like phenotype. We hypothesize that antigen-specific helper T cells expand in the adventitia, migrate to the media, and then potentiate macrophages which can eventually lead to tissue degeneration. These associations could shine light on the final step in the deterioration of the aorta towards a dissection. Figure 1. Microscopy results Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): TTW-NWO open technology grant (STW-14716), ERC-2014-StG-336454-CoNQUeST