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UBE2N is a K63-specific ubiquitin conjugase linked to various immune disorders and cancer. Here, we demonstrate that UBE2N and its partners UBE2V1 and UBE2V2 are highly expressed in malignant melanoma. Silencing of UBE2N and its partners significantly decreased melanoma cell proliferation and subcutaneous tumor growth. This was accompanied by increased expression of E-cadherin, p16, and MC1R and decreased expression of melanoma malignancy markers including SOX10, Nestin, and ABCB5. Mass spectrometry–based phosphoproteomic analysis revealed that UBE2N loss resulted in distinct alterations to the signaling landscape: MEK/ERK signaling was impaired, FRA1 and SOX10 gene regulators were downregulated, and p53 and p16 tumor suppressors were upregulated. Similar to inhibition of UBE2N and MEK, silencing FRA1 decreased SOX10 expression and cell proliferation. Conversely, exogenous expression of active FRA1 increased pMEK and SOX10 expression, and restored anchorage-independent cell growth of cells with UBE2N loss. Systemic delivery of NSC697923, a small-molecule inhibitor of UBE2N, significantly decreased melanoma xenograft growth. These data indicate that UBE2N is a novel regulator of the MEK/FRA1/SOX10 signaling cascade and is indispensable for malignant melanoma growth. Our findings establish the basis for targeting UBE2N as a potential treatment strategy for melanoma.Significance: These findings identify ubiquitin conjugase UBE2N and its variant partners as novel regulators of MAPK signaling and potential therapeutic targets in melanoma. Cancer Res; 78(22); 6462–72. ©2018 AACR.

Supplementary Table S5. UBE2N loss resulted in distinct alterations to the signaling landscape, as revealed by Mass spectrometry-based phosphoproteomic analysis

The Supplementary file contains supplementary methods, figures and tables that depict the importance of UBE2N, UBE2V1, and UBE2V2 in melanoma growth and progression.

Understanding tissue heterogeneity is critical for elucidating cell-cell interactions with important implications in immuno-oncology, inflammation, and neuroscience. Tissue heterogeneity poses immense challenges to understanding underlying molecular mechanisms using techniques such as qRT-PCR or bulk sequencing. While single-cell RNA sequencing can provide information about precise cellular composition of tissues, data analysis can be cumbersome and spatial context is lost. With single-cell spatial platforms such as RNAscope, target gene and protein expression can be visualized to characterize cell types and tissue neighborhoods. Here, we demonstrate a novel method for the simultaneous detection of RNA and protein using a modified co-detection assay. This novel co-detection assay enables visualization of a combination of up to 12 RNA and/or protein targets on the same sample. We used a set of antibodies targeting key immune and tumor cell markers- PD1, CD3, CD4, CD8, CD68, FOXP3 and KRT17, along with RNA biomarkers to interrogate the tumor microenvironment (TME) in human FFPE tumor samples. Using a combination of RNA and protein targets, we characterized different cell types such as T cells, macrophages, and tumor cells in the TME. We also developed a novel method for visualizing intercellular interactions between PD1 and PD-L1, offering insights into popular checkpoint mechanisms that are targeted for therapeutic intervention in cancer treatment. The assay offers a powerful technique for visualizing target RNA biomarkers in specific cell-types identified by cell-marker protein expression. This is a valuable tool for multi-omic analysis and accurate interrogation of complex tissues to obtain insights into novel biomarkers and therapeutic targets. Citation Format: Anushka Dikshit, Sayantani Basak, Vasudha Murlidhar, Ge-Ah Kim, Julia Yu, Sonali Deshpande, Li-Chong Wang, Maithreyan Srinivasan. Interrogating the tumor-immune landscape with a novel automated RNAscope™ assay for multiplexed detection of RNA and protein. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6771.

Purpose The next breakthroughs in immuno-oncology will be driven by high-plex tools that decipher the spatial arrangement of different cell types within the tumor microenvironment (TME). Imaging Mass Cytometry™ (IMC™) is a proven tool for the study of complex cellular interactions in the TME. It utilizes CyTOF® technology for simultaneous assessment of 40-plus protein markers at subcellular resolution without spectral overlap or background autofluorescence, thus providing unprecedented insight into the organization and function of the TME. Despite this, some protein targets are challenging to include in IMC as they have very few or no commercial antibodies available. Moreover, although cellular identity can easily be deciphered through detection of protein targets, knowledge of the cell’s transcriptome improves understanding of cellular function and activation state. Here, we present a robust and reliable workflow that combines the highly sensitive and specific RNAscope™ technology for RNA detection with the multiplexing capability of IMC to visualize key RNA and protein markers in the same tumor samples. Methods The RNAscope HiPlex v2 assay was combined with protein detection on the Hyperion+™ Imaging System to evaluate expression of both RNA and protein targets in formalin-fixed, paraffin-embedded (FFPE) tumor tissue microarray (TMA). The RNAscope assay was used with 12 target RNA marker probes and associated metal-labeled detection probes, suitable for use in IMC. The RNAscope HiPlex v2 assay workflow was followed as recommended up until applying the fluoros. Instead of using the fluoros, metal-conjugated probes were used for RNA detection. Metal-conjugated antibodies were used for protein detection in the same tissue. Results Target protein markers identified a range of immune cells, tumor cells, stromal cells, endothelial cells, and extracellular matrix. Co-detection of RNA and protein allowed visualization of several cytokines and chemokines, such as CXCL13, CXCL9, CXCL10, IFNγ, IL10, IL8, and IL1B, enabling the identification of the cellular source of these secreted factors. Additionally, immune cell subpopulations and their activation states were visualized using marker-specific antibodies. Immune cell hubs associated with anti-tumor immune responses were detected in tumor niches across the TMA. Conclusions Overall, combining RNAscope with protein co-detection on the IMC platform allowed simultaneous visualization of RNA and protein targets to interrogate the tumor microenvironment. The single-cell resolution offered by the RNAscope assay provides superior sensitivity for RNA detection in addition to the existing protein-detection capability of the IMC platform. This workflow can be applied to FFPE tissues to study therapeutic efficacy, stratify patients based on inflammatory signature, and study cell-cell interactions within the TME. Citation Format: James Pemberton, Smriti Kala, Anushka Dikshit, Clinton Hupple. High-plex co-detection of RNA and protein to explore tumor-immune interactions utilizing RNAscope with Imaging Mass Cytometry. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4636.

Regulatory RNA molecules such as microRNAs (miRNA) and long non-coding RNAs play critical roles in regulating translation of mRNA to protein. Several miRNAs have been implicated in disease initiation and progression, especially in cancer. The regulatory mechanisms of miRNAs and other small RNAs such as antisense oligos (ASOs) and silencing RNAs (siRNA) have been exploited to develop oligonucleotide therapies for 1. undruggable targets, 2. achieving longer-term effects 3. rapid production of therapeutics and 4. lower drug development costs. These therapeutics have the promise to treat debilitating neurodegenerative diseases, rare and inherited disorders. RNA therapies are typically delivered as a part of nanoparticles or a viral vector. Regardless of the delivery mechanism, methods to understand the biodistribution of the vector, transgene expression and cell type identification in a spatial context is crucial to study the safety and efficacy of these therapies. Built on the flagship RNAscope technology, the RNAscope Plus assay can detect 1 small RNA and 3 mRNA targets using TSA-based fluorescent readouts and is compatible with the new fluorescent Vivid™ dyes. Fixed, fresh frozen samples and formalin fixed paraffin embedded (FFPE) tissues are supported by manual and automated workflows using the Leica Bond Rx system. We have leveraged this technology to investigate spatial expression profile of miRNA and associated RNA targets across different tissue types and applications. We can also combine protein detection using a target antibody to visualize cell-type specific markers. This assay was used to demonstrate expression of miRNAs and target genes implicated in tumor initiation, progression, and angiogenesis. Expression of miR-205, associated tumor target genes such as PanCK, PTEN and tumor suppressor TP53 was visualized in head and neck cancer tumors. Downregulation of tumor-suppressor, TP53 resulted in upregulation of miR-205 which downregulates PTEN expression. Pan-CK stained the tumor region in the tissue. Similarly, miR-155 expression was observed in niche areas within breast cancer, head and neck cancer and cervical cancer tumors. Expression of miR-155 demonstrated correlation with high VEGF expression suggesting its role in angiogenesis. This novel platform will enable researchers to visualize regulatory RNA simultaneously with target RNAs, cell-type and morphology markers in intact cells/tissues with single cell resolution. This technology can provide meaningful insights into disease pathology driven by miRNAs as well as assess biodistribution and efficacy of oligonucleotide therapeutics. Citation Format: Anushka Dikshit, Sayantani Basak, Sonali Deshpande, Manvir Sambhi, Li-Chong Wang, Maithreyan Srinivasan. Multi-omic spatial analysis with simultaneous detection of small RNAs, mRNAs and proteins using the novel RNAscope™ Plus technology. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3775.

Background: It has been well established that the tumor microenvironment (TME), which comprises cancer cells, stromal cells, and surrounding extracellular matrix, plays a critical role in cancer development, progression, and control. The immunological components within tumors, known as the tumor immune microenvironment (TiME), have also been implicated in tumor development, recurrence, and metastasis. Effective strategies for cancer immunotherapies will require a deep understanding of the factors that shape both the TME and TiME. Here, we describe a spatial multiomics approach that utilizes RNAscope™ ISH technology paired with high-plex whole-slide spatial phenotyping with the PhenoCycler™-Fusion platform. This two-step approach is compatible with human FFPE tissues and enables researchers to characterize the spatial biology of the TiME more accurately by detecting RNA and protein markers on serial sections. The resulting multiomic data more accurately reveal the interplay between TME and TiME by giving insight into cell lineages, surrounding structures, as well as secreted chemokines and cytokines that exist within the TME ecosystem. Methods: We performed ultrahigh-plex spatial phenotyping on the PhenoCycler-Fusion on FFPE tumor tissue sections, using an antibody panel that is designed for immune cell phenotyping, evaluation of immune contexture and proliferation across the TME. Using serial sections from the same tissue blocks, we then ran the RNAscope HiPlex v2 assay automated on the PhenoCycler-Fusion system. This assay consisted of a 12-plex immuno-oncology panel of RNA target probes, which were selected to detect macrophages, chemokines, and cytokines within tumors. We used Phenoplex software to analyze the protein and RNA datasets and to compute cell phenotypes and spatial associations. Results and Conclusions: In this proof-of-concept study, we demonstrate the utility of multiomic spatial profiling on the PhenoCycler-Fusion platform. Analysis of the resulting multiplex imaging data not only revealed the structural organization of cells within the TME, but also activation states of immune cells. Together, this information provides a more complete functional map of immune cells within the TME and TiME and thereby enriches our understanding of tumor biology that may be deterministic of immunotherapy responsiveness. This work paves the way for future research that will rely on deep spatial phenotyping with protein biomarkers coupled with accurate quantification of the expression of regulatory cytokines, chemokines, growth factors, or non-coding RNAs that only RNA probes can detect. Citation Format: Niyati Jhaveri, HaYeun Ji, Anushka Dikshit, Jessica Yuan, Emerald Doolittle, Steve Zhou, Maithreyan Srinivasan, Bassem B. Cheikh, Fabian Schneider, James Mansfield, Julia Kennedy-Darling, Oliver Braubach. Multiomic spatial profiling of the tumor immune microenvironment at single cell resolution. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5626.

Interrogating complex tumor microenvironment requires a multi-omics approach that can provide high level of sensitivity and specificity. Identifying immune cell subsets within the tumor can be vital for predicting response and determining therapeutic efficacy. Detecting target immune cell markers using immunohistochemistry/Immunofluorescence (IHC/IF) and visualizing cytokine expression with in situ hybridization (ISH) can provide comprehensive information about the activation states of immune cells. Here, we demonstrate a newly developed integrated ISH and IHC/IF workflow compatible with manual and automated platforms that can substantially improve RNA-protein co-detection. We demonstrate the use of our RNA-Protein Co-detection assay in combination with the automated RNAscope Multiplex Fluorescent v2 assay, automated RNAscope Chromogenic Duplex assay and manual RNAscope Multiplex Fluorescent v2 assay to detect T cell markers, macrophage markers and checkpoint markers in the tumor microenvironment by using a microarray with different tumor samples. We identified CD4+ helper T cells and CD8+ cytotoxic T lymphocytes. Additionally, we determine the activation states of CD8+ T cells by visualizing IFNG, GZMB and IL-2 expression. We were also able to identify macrophages detected by CD68 protein expression and the M1 and M2 subsets were differentiated by using the M2-specific marker, CD163. We could also delineate tumor-stroma border in the samples by using the Pan-CK probe which distinctly marks the tumor cells and visualize the expression of immunoregulatory receptors PD-L1 and CTLA4 in the tumor cells. This assay is enabled for multiplexing by combining with our fluorescent assay platforms. Similarly, for researchers interested in detecting target expression while retaining morphological context, the codetection assay should be combined with our chromogenic assays. Overall, the new RNAscope-ISH-IHC co-detection workflow and reagents enable optimized simultaneous visualization of RNA and protein targets by enhancing the compatibility of antibodies with minimal optimization. Citation Format: Anushka Dikshit, Sayantani Basak, Emerald Doolittle, Ilya Kovalenko, Michaeline Bunting. Spatial analysis of tumor-infiltrated immune cells with highly specific RNAscope™ RNA-protein Co-detection assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1723.

The tumor microenvironment (TME) is highly complex, comprised of tumor cells, immune cells, stromal cells, and extracellular matrix. Understanding spatial interactions between various cell types and their activation states in the TME is crucial for implementing successful immunotherapy strategies against various types of cancer. This study demonstrates a highly sensitive and specific multiplexed technique, the RNAscope HiPlex v2 in situ hybridization (ISH) assay for spatial and transcriptomic profiling of target genes to assess immune regulation in human lung, breast, cervical and ovarian FFPE tumor tissues. We have expanded our current RNAscope HiPlex assay capability of iteratively multiplexing up to 12 targets in fixed and fresh frozen samples to include formalin fixed paraffin embedded (FFPE) tissues. The novel FFPE reagent effectively reduces background autofluorescence, improving the signal to noise ratio. We have leveraged this technology to investigate spatial expression of 12 oncology and immuno-oncology target genes, including tumor markers, immune checkpoint markers, immunosuppression markers, immune cell markers and secreted chemokine RNA expression profile within the TME. The targets were simultaneously registered using HiPlex image registration software v2 that enables background subtraction. We visualized T cell infiltration and identified T cell subsets within tumors using CD3 and CD8 expression and activated T cells by IFNG expression. We further identified subsets of pro- and anti-inflammatory macrophages by CD68 and CD163 expression as well effector cells which secrete chemokines and cytokine. We also detected the hypoxia markers HIF1A and VEGF to elucidate the immunosuppressive state of tumor cells. Preliminary analysis and quantification of the HIF1A expression using HALO® image analysis software showed higher copy numbers in the lung tumor as compared to the other tumors, demonstrating the sensitivity of the assay through differential expression. We additionally showed the differential expression of immune checkpoint markers PDCD1, and CD274 within the TME. Using a highly sensitive multiplexed RNAscope HiPlex v2 ISH assay, we have demonstrated the capability of this technique to spatially resolve 12 targets in four different tumor types. The FFPE reagent efficiently quenched background autofluorescence in the tissues and identified immune cell signatures within the TME. Quantification of immunosuppressive markers further depicted differential expression among various tumors. This technology is highly beneficial for investigating complex and spatial tumor-stroma interactions in basic science and translational research. The assay can also provide valuable understanding of the biological crosstalk among various cell types in complex and heterogeneous tissues. Citation Format: Anushka Dikshit, Sayantani Basak, Ching-Wei Chang, Michaeline Bunting, Kim Collins. Spatial multiplex profiling of immune cell markers in FFPE tumor tissues using the RNAscope™ HiPlex v2 in situ hybridization assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3865.

Background Spatially resolved gene expression has emerged as a crucial technique to understand complex multicellular interactions within the tumor and its microenvironment. Interrogation of complex cellular interactions within the tumor microenvironment (TME) requires a multi-omics approach where multiple RNA and protein targets can be visualized within the same tumor sample and be feasible in FFPE sample types. Simultaneous detection of RNA and protein can reveal cellular sources of secreted proteins, identify specific cell types, and visualize the spatial organization of cells within the tissue. Examination of RNA by in situ hybridization (ISH) and protein by immunohistochemistry (IHC) or immunofluorescence (IF) are widely used and accepted techniques for the detection of biomarkers in tumor samples. Given the similarities in workflow, co-detection of RNA and protein by combining ISH and IHC/IF in a single assay can be a powerful multi-omics solution for interrogating the complex tumor and its microenvironment. Methods In this report we combined the single cell, single molecule RNA ISH technology known as RNAscope with IHC/IF to simultaneously detect RNA and protein in the same FFPE tumor section using both chromogenic and fluorescence detection methods. Results We demonstrate co-localization of target mRNA and the corresponding protein in human cancer samples, visualize infiltration of immune cells into the TME, characterize the activation state of immune cells in the TME, identify single cell gene expression within cellular boundaries demarcated by IHC/IF, examine cell type-specific expression of multiple immune checkpoint markers, and distinguish endogenous T cells from activated CAR+ T cells. Overall, we show that co-detection of RNA by the RNAscope ISH assay and protein by the IHC/IF assay in the same FFPE section is a feasible methodology. The combined RNAscope ISH-IHC/IF workflow is a powerful technique that can be used to study gene expression signatures at the RNA and protein level with spatial and single cell resolution. Conclusions By leveraging the strength of the similar workflows of RNAscope ISH and IHC/IF assays, this methodology combines transcriptomics and proteomics in the same tissue section, providing a multi-omics approach for characterizing complex tissues and revealing cell type specific gene expression with spatial and single cell resolution.

Background The canonical WNT-β-catenin signaling pathway is vital for development and tissue homeostasis but becomes strongly tumorigenic when dysregulated. and alter the transcriptional signature of a cell to promote malignant transformation. However, thorough characterization of these transcriptomic signatures has been challenging because traditional methods lack either spatial information, multiplexing, or sensitivity/specificity. To overcome these challenges, we developed a novel workflow combining the single molecule and single cell visualization capabilities of the RNAscope in situ hybridization (ISH) assay with the highly multiplexed spatial profiling capabilities of the GeoMx™ Digital Spatial Profiler (DSP) RNA assays. Using these methods, we sought to spatially profile and compare gene expression signatures of tumor niches with high and low CTNNB1 expression. Methods After screening 120 tumor cores from multiple tumors for CTNNB1 expression by the RNAscope assay, we identified melanoma as the tumor type with the highest CTNNB1 expression while prostate tumors had the lowest expression. Using the RNAscope Multiplex Fluorescence assay we selected regions of high CTNNB1 expression within 3 melanoma tumors as well as regions with low CTNNB1 expression within 3 prostate tumors. These selected regions of interest (ROIs) were then transcriptionally profiled using the GeoMx DSP RNA assay for a set of 78 genes relevant in immuno-oncology. Target genes that were differentially expressed were further visualized and spatially assessed using the RNAscope Multiplex Fluorescence assay to confirm GeoMx DSP data with single cell resolution. Results The GeoMx DSP analysis comparing the melanoma and prostate tumors revealed that they had significantly different gene expression profiles and many of these genes showed concordance with CTNNB1 expression. Furthermore, immunoregulatory targets such as ICOSLG, CTLA4, PDCD1 and ARG1, also demonstrated significant correlation with CTNNB1 expression. On validating selected targets using the RNAscope assay, we could distinctly visualize that they were not only highly expressed in melanoma compared to the prostate tumor, but their expression levels changed proportionally to that of CTNNB1 within the same tumors suggesting that these differentially expressed genes may be regulated by the WNT-β-catenin pathway. Conclusions In summary, by combining the RNAscope ISH assay and the GeoMx DSP RNA assay into one joint workflow we transcriptionally profiled regions of high and low CTNNB1 expression within melanoma and prostate tumors and identified genes potentially regulated by the WNT- β-catenin pathway. This novel workflow can be fully automated and is well suited for interrogating the tumor and stroma and their interactions.GeoMx Assays are for RESEARCH ONLY, not for diagnostics.

Background The ability of T cells to mediate anti-tumor immunity has been harnessed to develop successful immunotherapies in recent years. Although direct presentation of tumor antigens by tumor cells plays an important role in the effector function of cytotoxic T lymphocytes (CTLs), cross-presentation by professional antigen presenting cells such as dendritic cells (DCs) is vital for priming naive CD8+ T cells and developing a sustainable cytotoxic response. Natural killer (NK) cells within the tumor microenvironment (TME) recruit a specific population of DCs called conventional type 1 DCs (cDC1s) into the TME by secreting chemokines such as CCL5 and XCL1. However, these cells are very low in abundance and are characterized by the expression of numerous markers, making their detection in the tissue context challenging. Methods Therefore, to interrogate the presence of cDC1 and NK cells in the TME and reveal their spatial relationship we utilized the highly sensitive and specific RNAscope Multiplex Fluorescence in situ hybridization (ISH) assay. Probes for XCR1, THBD, CLEC9A, and CCR5 were used to identify cDC1 cells within 4 cervical cancer tumors. These tumors were then assessed for the presence of NK cells by using specific marker probes such as CD56 and NCR1 and chemokines XCL1 and CCL5. Finally, CTLs were visualized to determine if there is a correlation between the presence of cDC1 and NK cells and CTL infiltration within the cervical cancer tumors. Results Our results revealed a strong correlation between the presence of NK cells, cDC1 cells, and CTLs within 3 out of 4 cervical cancer samples. The NK cells showed expression of the chemokines XCL1 and CCL5, which are the ligands for XCR1 and CCR5 respectively, suggesting that the XCR1+/CCR5+ cDC1 cells may have been potentially recruited by these NK cells. Regions high in cDC1 and NK cells also showed significantly higher levels of CTL recruitment, as indicated by the presence of CD8+/IFNG+ T cells. Conversely, 1 of the 4 cervical cancer samples demonstrated relatively lower levels of NK cells which correlated with lower cDC1 cells and in turn lower CTL infiltration. Conclusions In conclusion, by utilizing the RNAscope Multiplex ISH assay we were able to identify and visualize the spatial relationship between NK cells, CTLs, and cDC1 cells, a rare subset of DC cells that excel at presenting tumor antigens to T cells. Using this technology, it is possible to spatially interrogate the TME and detect specialized immune cells when assessing response to immunotherapies.

Gene expression analysis is critical to precisely characterize complex tissues and provide insight into a disease condition. Techniques like PCR, sequencing, and northern blotting are highly sensitive and specific but are unable to provide information about spatial positioning of target genes. Visualization of gene expression with a spatial context can be critical in identifying complex milieus in heterogenous tissues like tumors. The RNAscope in situ hybridization (ISH) technology detects target RNA expression with high sensitivity and specificity at single-cell resolution. To understand the cellular cross talk between different cell populations, it is essential to simultaneously study gene and protein expression within a complex tissue. This chapter details combining the RNAscope ISH assay with immunofluorescence (IF) in one protocol to simultaneously visualize gene expression and protein expression in human tumor tissue and mouse brain tissue.

The tumor microenvironment (TME) is highly complex, comprised of tumor cells, immune cells, stromal cells, and extracellular matrix. This study demonstrates a highly sensitive and specific multiplexed technique, the RNAscope HiPlex v2 in situ hybridization (ISH) assay for spatial and transcriptomic profiling of target genes to assess immune regulation in FFPE tumor tissues. The RNAscope HiPlex v2 assay iteratively multiplexes up to 12 targets in frozen and FFPE tissues. The novel FFPE reagent effectively reduces background autofluorescence, improving the signal to noise ratio. Here we investigate spatial expression of 12 immuno-oncology target genes, including tumor markers, immune checkpoint markers, immunosuppression markers, immune cell markers and secreted chemokines The targets were simultaneously registered using HiPlex image registration software v2 that enables background subtraction. We visualized T cell infiltration and activation within tumors using CD3, CD8 and IFNG expression. Subsets of pro- and anti-inflammatory macrophages were detected by CD68 and CD163 expression Hypoxia markers HIF1A and VEGF indicated immunosuppressive state of tumor cells. Preliminary analysis and quantification of the HIF1A expression using HALO® image analysis showed higher copy numbers in the lung tumor as compared to the other tumors. We additionally showed the differential expression of immune checkpoint markers PDCD1, and CD274 within the TME. Using a highly sensitive multiplexed RNAscope HiPlex v2 ISH assay, 12 key targets were spatially resolved in four different tumor types. The assay can provide valuable understanding of the biological crosstalk among various cell types in complex and heterogeneous tissues.

Interrogating complex tumor microenvironment requires a multi-omics approach. Detecting target immune cell markers using immunohistochemistry/Immunofluorescence (IHC/IF) and visualizing cytokine expression with in situ hybridization (ISH) can provide comprehensive information about the activation states of immune cells. Here, we demonstrate a newly developed integrated ISH and IHC/IF workflow compatible with manual and automated platforms with improved RNA-protein co-detection. We demonstrate the use of our RNA-Protein Co-detection assay in combination with the automated and manual RNAscope Multiplex Fluorescent assay as well as the automated RNAscope Chromogenic Duplex assay to detect T cell markers, macrophage markers and checkpoint markers in the tumor microenvironment by using a microarray with different tumor samples. We identified CD4+ helper T cells, CD8+ cytotoxic T cells and determined their activation states by visualizing IFNG, GZMB and IL-2 expression. We were also able to identify macrophages detected by CD68 protein expression and differentiate the M1 and M2 subtypes by using M2-specific marker, CD163. We could also delineate tumor-stroma border in the samples by using the Pan-CK probe which distinctly marks the tumor cells and visualize expression of immunoregulatory receptors PD-L1 and CTLA4 in the tumor. This assay is enables for multiplexing by combining with our fluorescent assays or enables target detection while retaining morphological context when combined with our chromogenic assays. Overall, the new RNAscope-ISH-IHC/IF co-detection workflow and reagents enable simultaneous visualization of RNA and protein targets by enhancing the compatibility of antibodies with minimal optimization.

UBE2N is a K63-specific ubiquitin conjugase linked to various immune disorders and cancer. Here, we demonstrate that UBE2N and its partners UBE2V1 and UBE2V2 are highly expressed in malignant melanoma. Silencing of UBE2N and its partners significantly decreased melanoma cell proliferation and subcutaneous tumor growth. This was accompanied by increased expression of E-cadherin, p16, and MC1R and decreased expression of melanoma malignancy markers including SOX10, Nestin, and ABCB5. Mass spectrometry–based phosphoproteomic analysis revealed that UBE2N loss resulted in distinct alterations to the signaling landscape: MEK/ERK signaling was impaired, FRA1 and SOX10 gene regulators were downregulated, and p53 and p16 tumor suppressors were upregulated. Similar to inhibition of UBE2N and MEK, silencing FRA1 decreased SOX10 expression and cell proliferation. Conversely, exogenous expression of active FRA1 increased pMEK and SOX10 expression, and restored anchorage-independent cell growth of cells with UBE2N loss. Systemic delivery of NSC697923, a small-molecule inhibitor of UBE2N, significantly decreased melanoma xenograft growth. These data indicate that UBE2N is a novel regulator of the MEK/FRA1/SOX10 signaling cascade and is indispensable for malignant melanoma growth. Our findings establish the basis for targeting UBE2N as a potential treatment strategy for melanoma. Significance: These findings identify ubiquitin conjugase UBE2N and its variant partners as novel regulators of MAPK signaling and potential therapeutic targets in melanoma. Cancer Res; 78(22); 6462–72. ©2018 AACR.

BackgroundThe tumor microenvironment (TME) is highly complex, comprised of tumor cells, immune cells, stromal cells, and extracellular matrix. Understanding spatial interactions between various cell types and their activation states in the TME is crucial for implementing successful immunotherapy strategies against various types of cancer. This study demonstrates a highly sensitive and specific multiplexed technique, the RNAscope HiPlex v2 in situ hybridization (ISH) assay for spatial and transcriptomic profiling of target genes to assess immune regulation in human lung, breast, cervical and ovarian FFPE tumor tissues.MethodsWe have expanded our current RNAscope HiPlex assay capability of iteratively multiplexing up to 12 targets in fixed and fresh frozen samples to include formalin fixed paraffin embedded (FFPE) tissues. The novel FFPE reagent effectively reduces background autofluorescence, improving the signal to noise ratio. We have leveraged this technology to investigate spatial expression of 12 oncology and immuno-oncology target genes, including tumor markers, immune checkpoint markers, immunosuppression markers, immune cell markers and secreted chemokine RNA expression profile within the TME. The targets were simultaneously registered using HiPlex image registration software v2 that enables background subtraction.ResultsWe visualized T cell infiltration and identified T cell subsets within tumors using CD3and CD8 expression and activated T cells by IFNG expression. We further identified subsets of pro- and anti-inflammatory macrophages by CD68 and CD163 expression as well effector cells which secrete chemokines and cytokine. We also detected the hypoxia markers HIF1A and VEGF to elucidate the immunosuppressive state of tumor cells. Preliminary analysis and quantification of the HIF1A expression using HALO® image analysis software showed higher copy numbers in the lung tumor as compared to the other tumors, demonstrating the sensitivity of the assay through differential expression. We additionally showed the differential expression of immune checkpoint markers PDCD1, and CD274 within the TME.ConclusionsUsing a highly sensitive multiplexed RNAscope HiPlex v2 ISH assay, we have demonstrated the capability of this technique to spatially resolve 12 targets in four different tumor types. The FFPE reagent efficiently quenched background autofluorescence in the tissues and identified immune cell signatures within the TME. Quantification of immunosuppressive markers further depicted a differential expression among various tumors. This technology is highly beneficial for investigating complex and spatial tumor-stroma interactions in basic science and translational research. The assay can also provide valuable understanding of the biological crosstalk among various cell types in complex and heterogeneous tissues.

The study reported here demonstrates that a flaxseed-supplemented diet causes ovarian tumors in the laying hen to undergo apoptosis, resulting in a reduction of tumor burden, reducing the frequency and severity of ovarian cancer. We have previously shown in normal ovaries that flaxseed and its components down-regulate ERalpha and alter the expression of enzymes that metabolize estrogen. In this study, we analyzed the effects of the two main components of whole flaxseed, ligan and omega 3 fatty acids on estrogen metabolism and the estrogen receptor in ovarian tumors. ER alpha expression was up-regulated in the ovarian tumors and was not affected by diet. Liver CYP1A1 expression was significantly increased by the whole flaxseed diet with a corresponding increase in 2-methoxyestradiol plasma levels. We also observed increased p38 and ERK 1/2 MAPK activation in the ovary as well as an increase in apoptosis in the tumor epithelium. SMAD 7, a factor involved in the 2-methoxyestradiol-mediated apoptosis pathway was also up-regulated in tumors from the whole flaxseed diet group. 2-methoxyestradiol-induced antitumor effects were further validated by in human ovarian cancer cells. This study details the effect of flaxseed diet on estrogen metabolism and demonstrates the antiovarian cancer effects of 2-methoxyestradiol.

Gene expression analysis is critical to precisely characterize complex tissues and provide insight into a disease condition. Techniques like PCR, sequencing, and northern blotting are highly sensitive and specific but are unable to provide information about spatial positioning of target genes. Visualization of gene expression with a spatial context can be critical in identifying complex milieus in heterogenous tissues like tumors. The RNAscope in situ hybridization (ISH) technology detects target RNA expression with high sensitivity and specificity at single-cell resolution. To understand the cellular cross talk between different cell populations, it is essential to simultaneously study gene and protein expression within a complex tissue. This chapter details combining the RNAscope ISH assay with immunofluorescence (IF) in one protocol to simultaneously visualize gene expression and protein expression in human tumor tissue and mouse brain tissue.

Complex tissues such as tumors are comprised of multiple cells types and extracellular matrix. These cells include heterogenous populations of immune cells that infiltrate the tumors. Understanding the composition of these immune infiltrates in the tumor microenvironment (TME) can provide key insights to guide therapeutic intervention and predict treatment response. Thorough understanding of complex tissue dynamics and immune cell characterization requires a multi-omics approach. Simultaneous detection of RNA and protein using in situ hybridization (ISH) and immunohistochemistry/immunofluorescence (IHC/IF) can reveal cellular sources of secreted proteins, identify specific cell types, and visualize the spatial organization of cells within the tissue. However, a sequential workflow of ISH followed by IHC/IF frequently yields suboptimal protein detection because the protease digestion step in the ISH protocol resulting in poor antibody signal. Here we demonstrate a newly developed integrated ISH/IHC workflow that can substantially improve RNA-protein co-detection, enabling the visualization and characterization of tumor immune infiltrates at single-cell resolution with spatial and morphological context. To characterize tumor-infiltrating immune cells in a tumor TMA (tumor microarray), we utilized the RNAscope Multiplex Fluorescence assay in combination with the RNA-Protein Co-detection Kit to detect multiple immune cell populations. Immune cells such as macrophages, T cells and NK cells were detected using specific antibodies against CD68, CD8, CD4 and CD56, respectively. Precise characterization of these immune cells was achieved by using probes against targets such as CCL5, IFNG, GNZB, IL-12, NCR1 etc. that not only help in identifying specific immune cells but also assist in determining their activation states. We identified subsets of T cells such as CD4+ regulatory T cells and CD8+ cytotoxic T lymphocytes. Additionally, we were able to determine the activation states of CD8+ T cells by visualizing the expression of IFNG and GZMB. Furthermore, infiltrating macrophages were identified by detecting the CD68 protein expression while the M1 and M2 subsets were differentiated by detecting the M2-specific target RNA for CD163. Similarly, NK cells were identified by detecting CD56 protein in combination with CCL5 and NCR1 RNA expression. Interestingly, the degree of infiltration of the different immune cell populations varied based on the tumor type. In conclusion, the new RNAscope-ISH-IHC co-detection workflow and reagents enable optimized simultaneous visualization of RNA and protein targets by enhancing the compatibility of antibodies - including many previously incompatible antibodies - with RNAscope. This new workflow provides a powerful new approach to identifying and characterizing tumor infiltrating populations of immune cells. Citation Format: Anushka Dikshit, Jyoti Phatak, Lydia Hernandez, Emerald Doolittle, Vasudha Murlidhar, Bingqing Zhang, Xiao-Jun Ma. Characterizing tumor-infiltrated immune cells with spatial context using an integrated RNAscope-immunohistochemistry co-detection workflow in FFPE tissues [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 LB235.

Characterizing heterogenous populations of tumor-infiltrating immune cells requires a multi-omics approach. Here we demonstrate a newly developed integrated in situ hybridization (ISH) and immunohistochemistry (IHC/IF) workflow that can substantially improve RNA-protein co-detection, enabling the visualization and characterization of tumor immune infiltrates at single-cell and spatial resolution. To characterize tumor-infiltrating immune cells in a tumor TMA (tumor microarray), we utilized the RNAscope Multiplex Fluorescence assay in combination with the RNA-Protein Co-detection Kit to detect multiple immune cell populations. Immune cells such as macrophages, T cells and NK cells were detected using antibodies against CD68, CD8, CD4 and CD56 in combination with probes targeting CCL5, IFNG, GNZB, IL-12, NCR1 etc. We identified CD4+ regulatory T cells and CD8+ cytotoxic T lymphocytes. Additionally, we determine the activation states of CD8+ T cells by visualizing IFNG and GZMB expression. Furthermore, infiltrating macrophages were detected by CD68 protein expression while the M1 and M2 subsets were differentiated by using the M2-specific marker, CD163. NK cells were identified by detecting CD56 protein in combination with CCL5 and NCR1 RNA expression. The degree of immune cell infiltration varied based on the tumor type. In conclusion, the new RNAscope-ISH-IHC co-detection workflow and reagents enable optimized simultaneous visualization of RNA and protein targets by enhancing the compatibility of antibodies, including many previously incompatible antibodies with RNAscope . This new workflow provides a powerful approach to identifying and characterizing tumor infiltrating immune cells.

Flaxseed has been studied for decades for its health benefits that include anti-cancer, cardio-protective, anti-diabetic, anti-inflammatory properties. The biologically active components that mediate these effects are the omega-3 fatty acids and the lignan, secoisolariciresinol diglucoside. We have previously shown that whole flaxseed supplemented diet decreases the severity and incidence of ovarian cancer while a 15% dose of flaxseed is most protective against inflammation and estrogen-induced chemical and genotoxicity. The objective of this study was to dissect the independent effects of the two flaxseed components on estrogen signaling and metabolism. Two and half year old hens were fed either a control diet, 15% whole flaxseed diet, defatted flax meal diet or 5% flax oil diet for 3 months after which the animals were sacrificed and blood and tissues were harvested. Whole flaxseed diet caused a decrease in expression of ERα. ERα target gene expression was assessed using RT(2) profiler PCR array. Some targets involved in the IGF/insulin signaling pathway (IRS1, IGFBP4, IGFBP5) were downregulated by flaxseed and its components. Flaxseed diet also downregulated AKT expression. A number of targets related to NF-kB signaling were altered by flaxseed diet including a series of targets implicated in cancer. Whole flaxseed diet also affected E2 metabolism by increasing CYP1A1 expression with a corresponding increase in the onco-protective E2 metabolite, 2-methoxyestradiol. The weak anti-estrogens, enterolactone, enterodiol and 2-methoxyestradiol, might be working synergistically to generate a protective effect on the ovaries from hens on whole flaxseed diet by altering the estrogen signaling and metabolism.

The canonical WNT-β-catenin signaling pathway is vital for development and tissue homeostasis but becomes strongly tumorigenic when dysregulated. This pathway can alter the transcriptional signature of a cell to promote malignant transformation, growth and metastasis. However, thorough characterization of these transcriptomic signatures has been challenging because traditional methods lack either spatial information, multiplexing, or sensitivity/specificity. To overcome these challenges, we developed a novel workflow combining the single molecule and single cell visualization capabilities of the RNAscope in situ hybridization (ISH) assay with the highly multiplexed spatial profiling capabilities of the GeoMx™ Digital Spatial Profiler (DSP) RNA assays. Using these methods, we sought to spatially profile and compare gene expression signatures of tumor niches with high and low CTNNB1 expression. After screening 120 tumor cores from multiple tumors for CTNNB1 expression by the RNAscope assay, we identified melanoma as the tumor type with the highest CTNNB1 expression while prostate tumors had the lowest expression. Using the RNAscope Multiplex Fluorescence assay we selected regions of high CTNNB1 expression within 3 melanoma tumors as well as regions with low CTNNB1 expression within 3 prostate tumors. These selected regions of interest (ROIs) were then transcriptionally profiled using the GeoMx DSP RNA assay for a set of 78 genes relevant in cancer. Target genes that were differentially expressed were further visualized and spatially assessed using the RNAscope Multiplex Fluorescence assay to confirm GeoMx DSP data with single cell resolution. The GeoMx DSP analysis comparing the melanoma and prostate tumors revealed that they had significantly different gene expression profiles and many of these genes showed concordance with CTNNB1 expression. Furthermore, immunoregulatory targets such as ICOSLG, CTLA4, PDCD1 and ARG1, also demonstrated significant correlation with CTNNB1 expression. On validating selected targets using the RNAscope assay, we could distinctly visualize that they were not only highly expressed in melanoma compared to the prostate tumor, but their expression levels changed proportionally to that of CTNNB1 within the same tumors suggesting that these differentially expressed genes may be regulated by the WNT-β-catenin pathway. In summary, by combining the RNAscope ISH assay and the GeoMx DSP RNA assay into one joint workflow we transcriptionally profiled regions of high and low CTNNB1 expression within melanoma and prostate tumors and identified genes potentially regulated by the WNT- β-catenin pathway. This novel workflow can be fully automated and is well suited for interrogating the tumor and stroma and their interactions. GeoMx Assays are for RESEARCH ONLY, not for diagnostics. Citation Format: Anushka Dikshit, Daniel Zollinger, Chris Merritt, Karen Nguyen, Jill McKay-Fleisch, Courtney Anderson, Xiao-Jun Ma. Molecularly guided highly multiplexed digital spatial analysis reveals differential gene expression profiles in the WNT-β-catenin pathway between melanoma and prostate tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2707.

Spatially resolved gene expression has emerged as a crucial technique to understand complex multicellular interactions within the tumor and its microenvironment. Interrogation of complex cellular interactions within the tumor microenvironment (TME) requires a multi-omics approach where multiple RNA and protein targets can be visualized within the same tumor sample and be feasible in FFPE sample types. Simultaneous detection of RNA and protein can reveal cellular sources of secreted proteins, identify specific cell types, and visualize the spatial organization of cells within the tissue. Examination of RNA by in situ hybridization (ISH) and protein by immunohistochemistry (IHC) or immunofluorescence (IF) are widely used and accepted techniques for the detection of biomarkers in tumor samples. Given the similarities in workflow, co-detection of RNA and protein by combining ISH and IHC/IF in a single assay can be a powerful multi-omics solution for interrogating the complex tumor and its microenvironment. In this report we combined the single cell, single molecule RNA ISH technology known as RNAscope with IHC/IF to simultaneously detect RNA and protein in the same FFPE tumor section using both chromogenic and fluorescence detection methods. We demonstrate co-localization of target mRNA and the corresponding protein in human cancer samples, visualize infiltration of immune cells into the TME, characterize the activation state of immune cells in the TME, identify single cell gene expression within cellular boundaries demarcated by IHC/IF, examine cell type-specific expression of multiple immune checkpoint markers, and distinguish endogenous T cells from activated CAR+ T cells. Overall, we show that co-detection of RNA by the RNAscope ISH assay and protein by the IHC/IF assay in the same FFPE section is a feasible methodology. The combined RNAscope ISH-IHC/IF workflow is a powerful technique that can be used to study gene expression signatures at the RNA and protein level with spatial and single cell resolution. By leveraging the strength of the similar workflows of RNAscope ISH and IHC/IF assays, this methodology combines transcriptomics and proteomics in the same tissue section, providing a multi-omics approach for characterizing complex tissues and revealing cell type specific gene expression with spatial and single cell resolution. Citation Format: Anushka Dikshit, Jyoti Phatak, Siobhan Kernag, Helly Pimental, Hailing Zong, Courtney Todorov, Lydia Hernandez, Jeffrey Kim, Bingqing Zhang, Courtney Anderson, Xiao-Jun Ma. Spatially resolve RNA and protein simultaneously in FFPE tumor samples by combining RNAscope in situ hybridization and immunohistochemistry assays [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2706.

About 25% of non-small cell lung cancer (NSCLC) patients bear one or more KRAS mutations in their tumors, which is correlated with poor prognosis. The precise identification of somatic mutations in tumors is becoming increasingly important for studying tumor progression and developing targeted therapies. While sequencing technologies allow for mutation-profiling, they do not permit direct visualization and association of genetic alterations with cellular morphology. In addition, DNA mutational status does not predict expression of the mutant allele which may provide information connecting genotype to phenotype. Therefore, a technology for mutation detection at the transcript level directly in the tumor context is desirable. To address this need we developed a specialized RNA in situ hybridization (ISH) method known as BaseScope. The BaseScope assay has a unique signal amplification system that allows for highly sensitive and specific detection of single nucleotide point mutations in tissues. BaseScope probes specific for KRAS G12C, G12A, G12V, G12S and wild type KRAS were designed and expression of each point mutation was assessed in a NSCLC tumor microarray with 48 tumor cores with known KRAS mutation status as determined by DNA sequencing. RNA quality and background signal threshold for each tumor core were determined using PPIB (positive) and dapB (negative) control probes. Using the sequencing data as the gold standard, the BaseScope assay demonstrated 83-100% sensitivity and 97-100% specificity for various KRAS mutations [Table 1]. For KRAS G12C, the assay correctly identified all 6 sequencing-positive cores and identified the rest as negatives. For KRAS G12V, the assay detected 5 of 6 mutated cores with 100% specificity. Interestingly, for KRAS G12S and KRAS G12A mutations, the BaseScope assay demonstrated 100% sensitivity and 97% specificity. Furthermore, it was observed that 100% of the KRAS-mutated tumors showed expression for both wild type and mutant KRAS alleles within these NSCLC tumors. In summary, we demonstrate the development of an RNA ISH assay for point mutations detection with morphological context in FFPE tissues. Unlike current sequencing methods that lack spatial information this assay has the unique ability to identify very small subclones whose frequency within the tumor might fall below the detection limit of sequencing. Performance characteristics of BaseScope KRAS assaysKRAS POINT MUTATIONSNo. of cores with specified mutationsNo. of cores without specified mutationsBaseScope sensitivityBaseScope specificityG12C633100% (6/6)100% (33/33)G12A336100% (3/3)97.2% (35/36)G12V63383% (5/6)100% (33/33)G12S138100% (1/1)97.3% (37/38) Citation Format: Anushka Dikshit, Helen Jarnagin, Emerald Doolittle, Courtney Anderson, Bingqing Zhang, Xiao-Jun Ma. Visualization of KRAS point mutations in non-small cell lung cancer tumors with morphological context using the BaseScope in situ hybridization assay [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2705.

The ability of T cells to mediate anti-tumor immunity has been harnessed to develop some of the most successful immunotherapies in recent years. Although direct presentation of tumor antigens by tumor cells plays an important role in the effector function of cytotoxic T lymphocytes (CTLs), cross-presentation by professional antigen presenting cells such as dendritic cells (DCs) is vital for priming naive CD8+ T cells and developing a sustainable cytotoxic response. Natural killer (NK) cells within the tumor microenvironment (TME) recruit a specific population of DCs called conventional type 1 DCs (cDC1s) into the TME by secreting chemokines such as CCL5 and XCL1. However, these cells are very low in abundance and are characterized by the expression of numerous markers, making their detection in the tissue context challenging. Therefore, to interrogate the presence of cDC1 and NK cells in the TME and reveal their spatial relationship to each other we utilized the highly sensitive and specific RNAscope Multiplex Fluorescence in situ hybridization (ISH) assay. Probes for XCR1, THBD, CLEC9A, and CCR5 were used to identify cDC1 cells within 4 cervical cancer tumors. These tumors were then assessed for the presence of NK cells by using NK cell-specific marker probes such as CD56 and NCR1 as well as the chemokines XCL1 and CCL5. Finally, CTLs were visualized to determine if there is a correlation between the presence of cDC1 and NK cells and infiltration of CTLs within the cervical cancer tumors. Our results revealed a strong correlation between the presence of NK cells, cDC1 cells, and CTLs within 3 out of 4 cervical cancer samples. The NK cells showed expression of the chemokines XCL1 and CCL5, which are the ligands for XCR1 and CCR5 respectively, suggesting that the XCR1+/CCR5+ cDC1 cells may have been potentially recruited by these NK cells. Regions high in cDC1 and NK cells also showed significantly higher levels of CTL recruitment, as indicated by the presence of CD8+/IFNG+ T cells. Conversely, 1 of the 4 cervical cancer samples demonstrated relatively lower levels of NK cells which correlated with lower cDC1 cells and in turn lower CTL infiltration. In conclusion, by utilizing the RNAscope Multiplex ISH assay we were able to identify and visualize the spatial relationship between NK cells, CTLs, and cDC1 cells, a rare subset of DC cells that excel at presenting tumor antigens to T cells. Using this technology, it is possible to spatially interrogate the TME and detect specialized immune cells when assessing response to immunotherapies. Citation Format: Anushka Dikshit, Courtney Anderson, Binqing Zhang, Xiao-Jun Ma. Conventional type 1 dendritic cells and natural killer cells demonstrate strong correlation to cytotoxic T lymphocyte infiltration in cervical cancer tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3306.

Professional antigen presenting cells such as dendritic cells (DCs) are vital for priming naive CD8+ T cells and developing a sustainable anti-tumor immune response. Natural killer (NK) cells within the tumor microenvironment (TME) recruit a specific population of DCs called conventional type 1 DCs (cDC1s). However, these cells are low in abundance making their detection in the tissue context challenging. To interrogate the presence of cDC1 and NK cells in the TME and reveal their spatial relationship to each other we utilized the highly sensitive and specific RNAscope Multiplex Fluorescence in situ hybridization (ISH) assay. NK cells and cDC1 cells were identified by using cell specific marker probes in 4 cervical cancer samples. Similarly, CTLs were visualized to determine if there is a correlation between the presence of cDC1 and NK cells and infiltration of CTLs within the cervical cancer tumors. Our results revealed a strong correlation between the presence of NK cells, cDC1 cells, and CTLs within 3 out of 4 cervical cancer samples. The NK cells showed expression of the chemokines XCL1 and CCL5, suggesting that the XCR1+/CCR5+ cDC1 cells may have been potentially recruited by these NK cells in the TME. Regions high in cDC1 and NK cells also showed significantly higher levels of CTL recruitment, indicated by the presence of CD8+/IFNG+ T cells. Conversely, 1 of the 4 cervical cancer samples demonstrated relatively lower levels of NK cells which correlated with lower cDC1 cells and in turn lower CTL infiltration. In conclusion, by utilizing the RNAscope Multiplex ISH assay we identified and visualized the spatial relationship between NK cells, CTLs, and cDC1 cells, highlighting the strength of this technology to spatially interrogate the TME.

Due to its exposure to a harsh luminal environment, the intestinal epithelium has a remarkably fast turnover rate that is facilitated by a resident intestinal stem cell (ISC) population present at the base of the intestinal crypt. These ISCs, marked by the GPCR Lgr5, allow the intestinal epithelium to adapt to different types of damage, such as inflammation. Chronic intestinal inflammation is a hallmark of the inflammatory bowel diseases (IBD) yet the interplay between inflammatory immune cells and ISCs remains to be elucidated. Therefore, we utilized the single-molecule RNA in situ hybridization (ISH) technology RNAscope to visualize the expression of multiple immune and ISC markers within the inflamed intestinal tissue environment. To interrogate the expression pattern of inflammatory immune cell and ISC markers within the intestinal crypt, we performed the assay on colons from either control or TNBS-treated mice. We visualized the location of each intestinal cell population, including the resident Lgr5+ ISC population, within the crypt. The impact of inflammation on the Lgr5+ ISC population, as well as the Wnt/β-catenin pathway, was also examined. Using a multiplex assay, we assessed infiltration of regulatory T cells and Th17 cells in the inflamed region. Lastly, we examined the expression of several receptor-ligand pairs for cytokines and ISC markers. Taken together, these results demonstrate the ability of the RNAscope assay to visualize the ISCs within the morphological context of the intestinal crypt and in relationship to inflammatory immune cells. This work may help understand mechanisms behind the pathogenesis of IBD and other inflammatory diseases and also aid in the development of potential therapies.

The objective of the present study was to find the optimum dose of flaxseed that would decrease PG and alter oestrogen pathway endpoints implicated in ovarian cancer. In the study, four groups of fifty 1·5-year-old chickens were fed different amounts of flaxseed (0, 5, 10 or 15 % of their total diet) for 4 months and were then killed to collect blood and tissues. Levels of flaxseed lignan metabolites, Enterolactone (EL) and Enterodiol (ED) were measured in the serum, liver and ovaries by liquid chromatography–MS/MS, and n-3 and n-6 fatty acid (FA) levels were measured by GC. The effects of the varied flaxseed doses were assessed by measuring levels of PGE2 and oestrogen metabolites (16-hydroxyestrone (16-OHE1) and 2-hydroxyestrone (2-OHE1)) as well as by analysing the expression of the oestradiol metabolising enzymes CYP3A4 (cytochrome p450, family 3, subfamily A, polypeptide 4), CYP1B1 (cytochrome p450, family 1, subfamily B, polypeptide 1) and CYP1A1 (cytochrome p450, family 1, subfamily A, polypeptide 1) and that of oestrogen receptor α (ERα) in the ovaries. The ratio of n-3:n-FA increased with an increase in flaxseed supplementation and corresponded to a dose-dependent decrease in cyclo-oxygenase-2 protein and PGE2 levels. EL and ED increased in the serum, liver and ovaries with increased concentrations of flaxseed. Flaxseed decreased the expression of ERα in the ovaries. The ratio of 2-OHE1:16-OHE1 in the serum increased significantly in the 15 % flaxseed diet, and there was a corresponding increase in CYP1A1 in the liver and decrease in CYP3A4 in the ovaries. CYP1B1 mRNA also decreased with flaxseed diet in the ovaries. The 15 % flaxseed-supplemented diet significantly decreased inflammatory PGE2, ERα, CYP3A4, CYP1B1 and 16-OHE1, but it increased CYP1A1 and 2-OHE1, which thus reduced the inflammatory and pro-carcinogenic micro-environment of the ovaries.