Protein Expression Profiles Research Articles

Integration of omics technologies for the identification of predictive biomarkers in type 2 diabetes: a comprehensive analysis of recent literature

Background. Omics technologies, such as genomics, proteomics, metabolomics, and Transcriptomics are being used for identifying biomarkers. These biomarkers are unraveling the molecular mechanisms underlying type 2 diabetes mellitus (T2DM), which can help to promote more personalized treatment strategies and advance our understanding of disease pathogenesis. Omics approaches enable the examination of genetic, protein, metabolic, and gene expression profiles more comprehensively while offering insights into T2DM risk, progression, and potential therapeutic targets. Methods: This review follows a systematic methodology, aimed at evaluating omics technology’s role in diabetes research. Utilizing literature searches, we got an initial pool of 257 studies with a rigorous selection process and narrowed the selection to 10 high-quality studies. Our methodology approach ensured the inclusion of relevant, peer-reviewed articles that contribute significantly to understanding the application of omics technologies in predicting biomarkers for type 2 diabetes. Results: The systematic review identifies ten high-quality studies illuminating substantial omics technology’s role in advancing our understanding of type 2 diabetes (T2D). Collectively, these studies demonstrate how genomics, proteomics, metagenomics, metabolomics, and Transcriptomics have uncovered novel biomarkers and molecular pathways for T2D. Our findings underscore all the omics potentials specifically for developing predictive biomarkers, enhancing diagnostics, and tailoring personalized treatment strategies. Genetic variations, metabolic alterations, and protein and RNA expression profiles were highlighted as key areas where omics technologies offer insights into the pathophysiology and management of T2D.

Progressive changes in the protein expression profile of alveolar septa in early-stage lung adenocarcinoma.

Adenocarcinomas show a stepwise progression from atypical adenomatous hyperplasia (AAH) through adenocarcinoma in situ (AIS) to invasive adenocarcinoma (IA). Immunoglobulin superfamily containing leucine-rich repeat (ISLR) is a marker of tumor-restraining cancer-associated fibroblasts (CAFs), which are distinct from conventional, strongly α-smooth muscle actin (αSMA)-positive CAFs. Fibroblastactivation protein (FAP) has been focused on as a potential therapeutic and diagnostic target of CAFs. We investigated the changes in protein expression during adenocarcinoma progression in the pre-existing alveolar septa by assessing ISLR, αSMA, and FAP expression in normallung, AAH, AIS, and IA. Fourteen AAH, seventeen AIS, and twenty IA lesions were identified and randomly sampled. Immunohistochemical analysis was performed to evaluate cancer-associated changes and FAP expression in the pre-existing alveolar structures. Normal alveolar septa expressed ISLR. The ISLR level in the alveolar septa decreased in AAH and AIS tissues when compared with that in normal lung tissue. The αSMA-positive area gradually increased from the adjacent lung tissue (13.3% ± 15%) to AIS (87.7% ± 14%), through AAH (70.2% ± 21%). Moreover, the FAP-positive area gradually increased from AAH (1.69% ± 1.4%) to IA (11.8% ± 7.1%), through AIS (6.11% ± 5.3%). Protein expression changes are a feature of CAFs in the pre-existing alveolar septa that begin in AAH. These changes gradually progressed from AAH to IA through AIS. FAP-positive fibroblasts may contribute to tumor stroma formation in early-stage lung adenocarcinoma, and this could influence the development of therapeutic strategies targeting FAP-positive CAFs for disrupting extracellular matrix formation.

The Contribution of Proteomics in Understanding Endometrial Protein Expression in Women with Recurrent Implantation Failure.

Recurrent implantation failure (RIF) poses a significant challenge in assisted reproductive technology (ART) outcomes. The endometrium plays a crucial role in embryo implantation, and its protein expression profile is integral in determining receptivity. Proteomics has emerged as a valuable tool in unraveling the molecular intricacies underlying endometrial receptivity and RIF. The aim of the present review is to analyze the contribution of proteomics to the understanding of endometrial protein expression in women with RIF, based on the results of significant proteomic studies. Medline/Pubmed databases were searched using keywords pertaining to proteomics combined with terms related to RIF. 15 studies were included in the present review. Several proteins have been found to exbibit differential expression in endometrial biopsies and fluid samples between fertile women and women with RIF during the receptive endometrial phase. The profile of endometrial proteins varied significantly among the studies. Nevertheless, similar changes in the expression levels of annexin-6, progesterone receptor, MMP-2, and MMP-9 in the endometrium of women with RIF, were found in more than one study indicating that certain proteins could potentially be effective biomarkers of endometrial receptivity. Proteomics contributes significantly to the understanding of protein expression in the endometrium of women with RIF and the analysis of proteins in endometrial fluid are promising for improving the clinical management of RIF.

Proteomic analysis of DEN and CCl4-induced hepatocellular carcinoma mouse model.

Hepatocellular carcinoma (HCC) seriously threatens human health, mostly developed from liver fibrosis or cirrhosis. Since diethylnitrosamine (DEN) and carbon tetrachloride (CCl4)-induced HCC mouse model almost recapitulates the characteristic of HCC with fibrosis and inflammation, it is taken as an essential tool to investigate the pathogenesis of HCC. However, a comprehensive understanding of the protein expression profile of this model is little. In this study, we performed proteomic analysis of this model to elucidate its proteomic characteristics. Compared with normal liver tissues, 432 differentially expressed proteins (DEPs) were identified in tumor tissues, among which 365 were up-regulated and 67 were down-regulated. Through Gene Ontology (GO) analysis, Ingenuity Pathway Analysis (IPA), protein-protein interaction networks (PPI) analysis and Gene-set enrichment analysis (GSEA) analysis of DEPs, we identified two distinguishing features of DEN and CCl4-induced HCC mouse model in protein expression, the upregulation of actin cytoskeleton and branched-chain amino acids metabolic reprogramming. In addition, matching DEPs from the mouse model to homologous proteins in the human HCC cohort revealed that the DEN and CCl4-induced HCC mouse model was relatively similar to the subtype of HCC with poor prognosis. Finally, combining clinical information from the HCC cohort, we screened seven proteins with prognostic significance, SMAD2, PTPN1, PCNA, MTHFD1L, MBOAT7, FABP5, and AGRN. Overall, we provided proteomic data of the DEN and CCl4-induced HCC mouse model and highlighted the important proteins and pathways in it, contributing to the rational application of this model in HCC research.

Administration of Gas6 attenuates lung fibrosis via inhibition of the epithelial-mesenchymal transition and fibroblast activation

The epithelial-mesenchymal transition (EMT) and fibroblast activation are major events in idiopathic pulmonary fibrosis pathogenesis. Here, we investigated whether growth arrest-specific protein 6 (Gas6) plays a protective role in lung fibrosis via suppression of the EMT and fibroblast activation. rGas6 administration inhibited the EMT in isolated mouse ATII cells 14 days post-BLM treatment based on morphologic cellular alterations, changes in mRNA and protein expression profiles of EMT markers, and induction of EMT-activating transcription factors. BLM-induced increases in gene expression of fibroblast activation-related markers and the invasive capacity of primary lung fibroblasts in primary lung fibroblasts were reversed by rGas6 administration. Furthermore, the hydroxyproline content and collagen accumulation in interstitial areas with damaged alveolar structures in lung tissue were reduced by rGas6 administration. Targeting Gas6/Axl signaling events with specific inhibitors of Axl (BGB324), COX-2 (NS-398), EP1/EP2 receptor (AH-6809), or PGD2 DP2 receptor (BAY-u3405) reversed the inhibitory effects of rGas6 on EMT and fibroblast activation. Finally, we confirmed the antifibrotic effects of Gas6 using Gas6−/− mice. Therefore, Gas6/Axl signaling events play a potential role in inhibition of EMT process and fibroblast activation via COX-2-derived PGE2 and PGD2 production, ultimately preventing the development of pulmonary fibrosis.

Application of phytochemicals in photodynamic therapy of cancer

SignificanceHerbal medicine is an important part of the health care system in many countries as they exhibit chemopreventive and chemotherapeutic effects. The combination of phytochemicals with Photodynamic therapy is a novel approach to enhancing therapeutic efficacy. ApproachWe studied the effect of various plant extracts, phytochemicals, photosensitizers and their combinations to induce cell death in the lung, breast, colon, cervical and skin cancer cells. The efficacy of the drug moieties was evaluated using viability, proliferation, and cytotoxicity studies. Detailed cell death mechanism was studied using protein expression profiling using immunofluorescence, flow cytometry, ELISA and western blotting. ResultsThe results revealed that PDT and combination therapy groups showed significant cell death in breast and lung cancer cells. Conjugation of photosensitizers with nanoparticles and phytochemicals enhanced the drug delivery and treatment outcome. ConclusionsCombination of PDT with other existing treatments or replacing the synthetic PSs with natural ones is a promising approach in the fight against cancer with improved specific cancer cell targeting.

Spatial Profiles in Triple-negative Breast Cancer: Unraveling the Tumor Microenvironment and Biomarkers for Immune Checkpoint Inhibitors

Abstract Objective: Immune checkpoint inhibitors (ICIs) have become an important treatment option for cancer. However, the predictive power of current biomarkers is limited for treatment response, especially in triple-negative breast cancer (TNBC). Investigation of the tumor microenvironment (TME) may provide biological insights into the response to ICIs by uncovering the interactions among tumor and immune cells. Emerging technologies of spatial transcriptomics (ST) and proteomics allow clinical researchers to better understand the TME. Data Sources and Study Selection: We reviewed the results of articles published in the past 10 years worldwide. Results: Emerging spatial profiling technologies can be classified into image-based and sequencing-based methods, both of which preserve information on tissue architecture with gene expression and/or protein abundance profiles. Here, we reviewed articles studying TNBC using spatial profiling techniques. By integrating spatial profiles, recent studies showed the relevance of gene and protein expression profiles in the TME of different subgroups. These ST and proteomic characteristics were shown to be associated with patients’ survival. Conclusion: The application of spatial profiling techniques to cancer research has significantly advanced our understanding of breast cancer biology, particularly in the context of TNBC. We are confident that the technology has the potential to revolutionize the prediction of treatment outcomes in the near future. By elucidating the nuances within the TME, spatial profiling opens up new possibilities for personalized strategies for immunotherapy.

Expression profiles of heat shock protein 70 and high-mobility groups box-1 protein in gnotobiotic brine shrimp challenged with different virulence levels of isogenic Vibrio harveyi strains.

Expression profiles of heat shock protein 70 and high-mobility groups box-1 protein in gnotobiotic brine shrimp challenged with different virulence levels of isogenic Vibrio harveyi strains.

Licensing effects of inflammatory factors and TLR ligands on the regenerative capacity of adipose-derived mesenchymal stem cells.

Introduction: Adipose tissue-derived mesenchymal stem cells are promising contributors to regenerative medicine, exhibiting the ability to regenerate tissues and modulate the immune system, which is particularly beneficial for addressing chronic inflammatory ulcers and wounds. Despite their inherent capabilities, research suggests that pretreatment amplifies therapeutic effectiveness. Methods: Our experimental design exposed adipose-derived mesenchymal stem cells to six inflammatory factors for 24h. We subsequently evaluated gene expression and proteome profile alterations and observed the wound closure rate post-treatment. Results: Specific pretreatments, such as IL-1β, notably demonstrated an accelerated wound-healing process. Analysis of gene and protein expression profiles revealed alterations in pathways associated with tissue regeneration. Discussion: This suggests that licensed cells exhibit potentially higher therapeutic efficiency than untreated cells, shedding light on optimizing regenerative strategies using adipose tissue-derived stem cells.

Enhanced starch accumulation in Chlorella sorokiniana as sugar platform and the expression profiling of key regulatory proteins

Microalgae can accumulate considerable starch as a renewable sugar platform for biofuel production. In this study, Chlorella sorokiniana exhibited a significant increase in starch content from 3.0% to 49.5% DM under sulfur limitation (SL) conditions, and the lower the concentration of sulfur was, the more effective it was. Direct enzymatic hydrolysis of wet biomass resulted in a glucose yield of 97.1%, indicating its potential for fermentable sugar production. However, C. sorokiniana grew slowly, and cell division and biomass accumulation decreased under SL conditions, which could be explained by the decreased photosynthetic efficiency and protein content. The sulfur replenishment (SR) confirmed the rapid response of C. sorokiniana in starch accumulation to sulfur availability. Thus, we further investigated the expression profiling of key regulatory proteins under Control-SL-SR stages. The expression of AGPase A was strongly induced in the SL stage and subsequently downregulated during SR stage, suggesting its role as a key regulatory protein for enhanced starch accumulation. Additionally, downregulation of SBE and GBE expression likely contributed to structural modifications of the starch molecules. These proteins could serve as potential targets for metabolic engineering in further improving the starch production capacity in C. sorokiniana.

Age-related changes after intracerebral hemorrhage: a comparative proteomics analysis of perihematomal tissue.

The risk factors and causes of intracerebral hemorrhage (ICH) and the degree of functional recovery after ICH are distinct between young and elderly patients. The increasing incidence of ICH in young adults has become a concern; however, research on the molecules and pathways involved ICH in subjects of different ages is lacking. In this study, tandem mass tag (TMT)-based proteomics was utilized to examine the protein expression profiles of perihematomal tissue from young and aged mice 24h after collagenase-induced ICH. Among the 5,129 quantified proteins, ICH induced 108 and 143 differentially expressed proteins (DEPs) in young and aged mice, respectively; specifically, there were 54 common DEPs, 54 unique DEPs in young mice and 89 unique DEPs in aged mice. In contrast, aging altered the expression of 58 proteins in the brain, resulting in 39 upregulated DEPs and 19 downregulated DEPs. Bioinformatics analysis indicated that ICH activated different proteins in complement pathways, coagulation cascades, the acute phase response, and the iron homeostasis signaling pathway in mice of both age groups. Protein-protein interaction (PPI) analysis and ingenuity pathway analysis (IPA) demonstrated that the unique DEPs in the young and aged mice were related to lipid metabolism and carbohydrate metabolism, respectively. Deeper paired-comparison analysis demonstrated that apolipoprotein M exhibited the most significant change in expression as a result of both aging and ICH. These results help illustrate age-related protein expression changes in the acute phase of ICH.

Abstract 6915: Integrated proteogenomic analysis for the NCI patient-derived cancer model repository

Abstract Background: Studying protein expression involved in cancer from patient-derived xenografts (PDX) enhances understanding of tumorigenesis and may shed light on patient therapeutic decisions. Recently, mass spectrometry-based proteomics sequencing techniques have enabled the acquisition of high-throughput PDX protein/phosphoprotein expression profiles and paved a way for investigating oncogenic signaling pathways and their interactions in different tumor histologies. Integrated analysis of these proteomic data with existing biomarkers derived from whole exome and RNAseq data will provide the community with a rich resource for translational research. Materials and methods: iTRAQ-based proteomics/phospho-proteomics data, with 11 Tandem Mass Tag (TMT) channels, were processed and median-normalized at gene level. The gene expression data were derived from tximport and DESeq2 from of RNA-Seq. In addition, whole exome sequencing (WES) data were used to compute mutations and copy number profiles. For data visualization, the R Bioconductor package ComplexHeatmap was used extensively. Results: PDX proteomics/phospho-proteomics data were analyzed alongside with gene expression, copy number and mutational profiles for several cancer types, revealing vivid molecular regulation for many cancer-related pathways. A moderate yet significant Spearman correlation coefficient of over 0.45 and 0.5 was observed between proteomics and RNA-Seq data at gene and pathway level, respectively. The PDX protein and gene expression pattern from common cancer regulatory pathways of bladder (BLCA, n=32 models), colon (COAD, n=82), lung (NSCLC, n=32), pancreatic (PAAD, n=52), metastatic breast cancer (CSNOS) (n=16) and other histologies were aggregated and discussed at the model level. EGFR, MET and cell-cycle genes CDKN2A and CDKN2B exhibited significant positive correlation among copy number, gene expression and protein expression profiles. Protein and gene expression profiles of PAM50 genes were shown for CSNOS samples, to further sub-classify or correct misclassification. Further analysis using proteomics/phospho-proteomics data with CNA identified several trans-regulatory events. Lastly, we identified RTN1 as a potential biomarker, exhibiting lower expression in sensitive rare-tumor PDX models prior to Axitinib and Vandetanib treatment. Conclusions: With the multi-omics datasets comprising proteomics/phospho-proteomics, RNA-Seq and WES datasets from the NCI Patient Derived Models Repository cohort, we were able to query some important cancer biological processes at a higher resolution. In addition, we revealed gene- and pathway-level regulatory differences from various histologies. Overall, the multi-omics data from PDX models showed promising recapitulation of original tumor activity and should continue to serve as an amenable and scalable drug screening platform for pre-clinical trials. Citation Format: Peter I. Wu, Li Chen, Yuri Kotliarov, Jianwen Fang, Yingdong Zhao, Yvonne Evrard, Lijun Chen, Shahanawaz Jiwani, Biswajit Das, Chris A. Karlovich, Hui Zhang, Lisa McShane, Melinda G. Hollingshead, Mickey Williams, James H. Doroshow. Integrated proteogenomic analysis for the NCI patient-derived cancer model repository [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 6915.

Abstract 2032: Osteoblasts alter their protein expression profile in the presence of prostate cancer cells to facilitate their invasion

Abstract Bone metastasis is the leading cause of death in prostate cancer patients, for which there is currently no effective treatment. Since the bone microenvironment plays an important role in this process, attention has been directed to the interactions between cancer cells and the bone microenvironment, including osteoblasts. Here, we investigated the mechanism of interactions between prostate cancer cells (DU-145 and PC-3) and osteoblast cell line hFOB.1.19. We are investigating the broader effects of four different nucleoside reverse transcriptase inhibitor (NRTI) analogs 5-fluoro-1-((1R,4R)-4-hydroxycyclopent-2-en-1-yl)pyrimidine-2,4(1H,3H)-dione (KBMD-E), 3-benzoyl-5-fluoro-1-((1S,4S)-4-((tetrahydro-2H-pyran-2-yl)oxy)cyclopent-2-en-1-yl)pyrimidine-2,4(1H,3H)-dione (KBMD-G), 1-((1R,4R)-4-hydroxycyclopent-2-en-1-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (KBMD-H) and 1-(5-hydroxymethyl)-2,5-dihydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (KBMD-S) along with aPKC specific inhibitors ICA-1S and ζ-Stat to develop as potential anti-cancer drugs. Our previous publications suggested that aPKC-ι and ζ are involved in prostate cancer progression and metastasis. Current data suggested that aPKC specific inhibitors along with KBMD compounds induced apoptosis and pyroptosis of DU-145 and PC-3 while having lesser effects on hFOB.1.19 cell line. Our data demonstrate that osteoblasts assist prostate cells to establish in the bone microenvironment by producing metastatic stimulating chemokines while upregulating responsible transcription factors such as c-Jun. In addition, our preliminary data suggested that PKC-ι and ζ are crucial for driving critical steps of the metastatic cascades of prostate cancer cells. Preliminary data suggested that all 6 compounds upregulated interleukin (IL)-18 and IL-1β while downregulating IL-8, IL-6 and CXCL-1. Upregulation of Caspase 8 with cleaved gasdermin D in prostate cells indicates an increase in pyroptosis. Our preliminary results suggest that all 6 compounds can be used to disrupt the main steps of prostate cancer bone metastasis which can be targeted to develop customized, tailored therapies for bone metastatic prostate cancer which merit further research. Citation Format: Aaron J. Todman, Wishrawana S. Ratnayake, Luke Lajmi, Sloan Breedy, Abiral H. Shourav, Kirpal Bisht, Mildred Acevedo-Duncan. Osteoblasts alter their protein expression profile in the presence of prostate cancer cells to facilitate their invasion [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 2032.

Abstract 585: Regorafenib antitumor immune response is enhanced by a novel drug combination in CRC syngeneic model

Abstract Purpose: In the United States, colorectal cancer (CRC) is the second leading cause of cancer mortality. Metastatic CRC (mCRC) refractory to traditional chemotherapy is managed with regorafenib, a multiple-kinase inhibitor. However, patients show only a modest improvement in overall survival but experience high drug toxicity, adverse side effects, and poor tolerability. Thus, to reduce regorafenib-induced toxicity and to enhance antitumor activity, we combined it with a hybrid/dual JAK/HDAC small-molecule inhibitor (JAK/HDACi) to leverage the advantages of both JAK and HDAC inhibition in a single agent. With syngeneic mice, the efficacy and impact on immunomodulation of this drug combination was assessed. Methods: To assess effects of the agents, C57BL/6 immunocompetent mice were injected with MC38 murine CRC cells and treated with regorafenib (6mg/kg body wt) or JAK/HDACi (30mg/kg body wt), and their combination every third day for 21 days. Upon completion of the experiment, the tumors were harvested and processed for RNA and protein expression profiles. The effect of drug treatment on immune response was analyzed by nCounter Gene Expression assays (NanoString Technologies). Cytokines were measured in serum using the MesoScale Discovery mouse V-Plex Proinflammatory Panel kit. For pharmacokinetic studies, plasma samples were assayed from C57BL/6 treated with drugs after 1, 7, 21, and 48 hrs of treatment. Results: The combination treatment significantly reduced tumor growth (volume and weight),relative to the vehicle control or single treatments. Gene expression showed higher CD45 abundance score in the combination treatment. This is an exciting observation, as CD45 has phosphatase activity and dephosphorylates key targets of the drugs used in the study. Additionally, after treatment with the combination, there was higher expression of Gzmb and Gzme and lower expression of Cx3cr1, indicating enhanced immuno modulation relative to regorafenib alone. We confirmed these findings by immunoprofiling of tumors. Higher CD45 staining, noted in tumors of mice treated with the combination, corroborated the gene expression results. Pronounced CD8 T lymphocytes infiltration was observed in combination, compared to regorafenib alone. Reduction of proinflammatory cytokine, TNF-α was noted in the combination group. This is an interesting observation as CD45 negatively regulates TNF, and combination treated mice had highest number of CD45 positive cells. Pharmacokinetic studies showed that the bioavailability of regorafenib was elevated after combination treatment relative to single-agent treatment. Conclusions: Relative to the single agents, the combination of regorafenib with JAK/HDACi was more effective, with an elevated antitumor immune response and with sustained inhibition of tumor growth. A clinical trial to evaluate this combination for the treatment of mCRCs is warranted. Citation Format: Prachi Bajpai, Farrukh Afaq, Sameer Al Diffalha, Sumit Agarwal, Hyung Gyoon Kim, Dennis Otali, Sooryanarayana Varambally, Ashish Manne, Ravi Paluri, Moh’d Khushman, Upender Manne. Regorafenib antitumor immune response is enhanced by a novel drug combination in CRC syngeneic model [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 585.

Abstract 5481: Spatial multiplex proteins profiling of tumor microenvironment to decipher de novo metastasis in nasopharyngeal carcinoma

Abstract Background: Nasopharyngeal carcinoma (NPC) has a high incidence rate in Southeast Asia, where more than 90% of NPC cases are associated with Epstein-Barr virus (EBV). Although NPC patients are generally sensitive to radiotherapy and chemotherapy, about 20-30% of patients have treatment failure due to the development of distant metastasis, i.e. de novo metastasis. In this study, we utilized GeoMx Digital Spatial Profiler (DSP), a multiplex and high-throughput sequencing platform designed for spatially- and morphologically resolved protein expression profiling to investigate the intra-tumoral and inter-tumoral heterogeneity of tumor microenvironment (TME) and identify the mechanisms underlying distant failure in NPC. Methods: We utilized GeoMx Protein Assays consisting of 147 proteins from 15 functional modules to compare the difference between NPC cases developing distant failure (DF) (n=21) and stage-, age-, gender- and treatment-matched NPC cases without developing distant failure (noDF) (n=15) within five years of treatment. Two fluorescent morphological markers including PanCK and CD45, were used for delineating the structural architecture of the tissues, identifying cell types, and selecting regions of interest (ROIs), followed by sequencing for protein expression quantification. NanoString GeoMx DSP Analysis Suite was used for quality control and data normalization. The NPC TME heterogeneity was further investigated by the 10X Visum platform for spatial transcriptome. The public single-cell RNA sequencing (scRNA-Seq) and bulk RNA sequencing datasets were obtained for the downstream analyses. Results: By analyzing protein expression profiles, the study revealed the intratumoral and intertumoral heterogeneity associated with EBV latent infection in the TME of NPC. Regions with high EBV latent gene expression exhibited a noticeable reduction in T cell infiltration, indicating an "immune cold" phenotype, whereas regions with low EBV latent gene expression displayed heavy T cell infiltration, indicating an "immune hot" phenotype. Moreover, the study identified a panel of autophagic and anti-apoptotic proteins that were significantly upregulated in the cancer cells of NPC cases developing distant failure after treatment. This observation, supported by the single-cell RNA sequencing datasets, further implicated EBV latent infection for the upregulation of autophagic and anti-apoptotic genes. Notably, the study also demonstrated that the genes upregulated in the immune cells in NPC cases with distant failure were associated with poor progression-free survival in an independent NPC cohort. Conclusions: These findings shed light on the crucial role of EBV latent infection in TME heterogeneity and highlight the potential involvement of autophagy and anti-apoptosis in de novo metastasis in NPC. Citation Format: Larry Ka-Yue Chow, Ying Wang, Lanqi Gong, Siqi Xu, Zhaozheng Hou, Xinyuan Guan, Dora Lai-Wan Kwong, Victor Ho-Fun Lee, Wai To ng, Anne Wing-Mui Lee, Zhonghua Liu, Yunfei Xia, Wei Dai. Spatial multiplex proteins profiling of tumor microenvironment to decipher de novo metastasis in nasopharyngeal carcinoma [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 5481.

Abstract 4682: Membrane protein expression and activity following inhibition of B-cell receptor-initiated signaling by WBMP-4, a therapeutic antibody for leukemia/lymphoma

Abstract The B-cell Receptor (BCR), comprising membrane Ig (mIg) and CD79a/b, controls normal signaling in B-cells, but dysregulation of this signaling is the dominant driver of leukemia/lymphoma. The class-specific mIg subunit of the BCR is a rational drug target for B-cell malignancies. It plays a critical role in the functioning of these cells, and allows for restricted targeting of only B-cells expressing an Ig class, offering a more tumor-specific approach than current pan-B-cell treatments. We developed a first-in-class antibody, WBMP-4, to the mIgM-BCR, which inhibits cell growth and induces apoptosis via widespread down-regulation of tyrosine kinase activity. Given the potent inhibition of intracellular signaling induced upon WBMP-4 binding to the mIgM-BCR, we expect that translation of membrane proteins will be impacted. Here we investigate the expression and phosphorylation profile of cell surface proteins following treatment of malignant B-cells with WBMP-4. Using western blot and flow cytometry, we measure levels of CD79a/b, CD19, CD20, CD21, CD22, CD32a, CD81, and CXCR4 at 6, 24, and 48 hours after low (5ug/mL) and high (20ug/mL) dose WBMP-4 treatment, as compared to control, untreated Burkitt lymphoma (CA46) cells. We analyze phosphorylation levels of those surface proteins that serve as kinases in this system (CD79a/b, CD19, CXCR4, etc.). Using these same methods, we also examine the expression and phosphorylation of key B-cell transcription factors (e.g. NF-kB, c-MYC, STAT5, PAX5, EBF1, FOXO, and BCL-2). Preliminary analyses show a complete absence of CD79a protein (western blot), and decreased phospho-kinase activity of CD79b (Pamgene phosphokinase array) following treatment of Burkitt lymphoma cells with WBMP-4, as compared to untreated cells. While the effect of an antibody binding to mIg may be most pronounced for CD79a/b relative to other proteins, as mIg and CD79a/b form a complex, we expect that the absence of CD79a/b is a result of WBMP-4-induced mIgM inhibition modulating downstream signaling and transcription factor activity. By examining cell surface markers, we may gain insight into how WBMP-4 impacts a cell’s interaction with the tumor microenvironment, and this information is valuable for diagnostic and patient monitoring purposes, and to determine drug combination strategies. This work will be considered in the context of our ongoing research on WBMP-4’s inhibition of intracellular signaling pathways, and the resulting biologic effects, and will contribute to the continued development of WBMP-4 by providing an understanding of the effects achieved (cell growth inhibition, cell differentiation, apoptosis) at different dosages and if these effects are maintained over time. As a novel, efficacious, and low toxicity treatment strategy for B-cell leukemia and lymphoma, WBMP-4 has significant clinical potential. Citation Format: Rachel S. Welt, David Kostyal, Virginia Raymond, Jose M. Lobo, Sydney Welt. Membrane protein expression and activity following inhibition of B-cell receptor-initiated signaling by WBMP-4, a therapeutic antibody for leukemia/lymphoma [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 4682.

Abstract 3704: Whole genome and targeted proteome analysis of single cancer cells

Abstract The simultaneous genome and proteome analysis of very rare single cells would enable addressing important biological and diagnostic questions particularly in cancer. Here, we introduce a novel genome and proteome (G&P) analytic workflow, a method allowing comprehensive analysis of single-cell DNA and targeted profiling of protein expression in single cells. As a proof of concept, we demonstrate the capability of the method by quantifying copy number of the ERBB2 gene alongside with protein expression levels of HER2 and phospho-HER2 in single cells. The G&P workflow utilizes antibodies conjugated with DNA oligonucleotides designed to enable their amplification alongside the single-cell genome facilitating parallel genome and proteome profiling by either quantitative PCR (qPCR) or sequencing. We utilized antibodies targeting cytokeratin 8/18/19, HER2 and phospho-HER (Tyr1248). Performance of each DNA-conjugated antibody was assessed by immunofluorescence staining in a variety of breast cancer cell lines (MDA-MB231, MDA-MB453, BT474 and MCF7) and sample types. Stained cells were isolated by manual micromanipulation and subjected to whole genome amplification (WGA). Protein expression levels, derived from quantification of the amplified DNA tags, were measured by qPCR. Shallow whole genome sequencing was employed to facilitate genome-wide profiling of copy number variations (CNVs), including the ERBB2 locus. We successfully developed and tested a G&P workflow allowing parallel genome-wide CNV profiling and quantification of three target proteins. G&P measurement yielded concordant results to immunofluorescence quantification for all tested markers. Furthermore, the workflow allowed direct correlation of copy numbers of the ERBB2 gene with HER2 expression in single cancer cells. Notably, the method enabled measurements of cellular and molecular responses at the single-cell level to lapatinib treatment, i.e. monitoring of the HER2 pathway activation level. Our novel multiplex G&P technique is capable of generating multiparametric measurements linking DNA copy numbers and expression levels of selected genes of interest. The method is optimized for analysis of single cells from rare cell populations and is readily translatable to study circulating tumor cells (CTCs) and disseminated cancer cells (DCCs), enabling for the first time pathway analysis via phosphoprotein analyses of these cells. Citation Format: Atieh Alyasin, Kathrin Weidele, Catherine Botteron, Christoph A. Klein, Zbigniew T. Czyż. Whole genome and targeted proteome analysis of single cancer cells [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 3704.

Abstract 1488: Spatial characterization of the tumor microenvironment of metastatic triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer associated with high rates of recurrence and metastasis, which contribute to poor overall survival. The challenges in treating TNBC stem from its high intratumor heterogeneity and lack of expression of the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 druggable targets. The tumor microenvironment (TME) plays a crucial role in disease progression. Within the TME, complex spatial interactions among tumor cells, immune cells, stroma, angiogenesis- and hypoxia-inducing factors collectively contribute to the advancement and spread of TNBC. In-depth understanding of the TME and interactions of the various components within can elucidate newer therapeutic targets, enhancing the prognosis of TNBC patients. Imaging mass cytometry (IMC) enables detailed analysis of cell-cell and cell-matrix interactions within the TME at the single-cell level. In this study, we aim to characterize the spatial relationships among various cell types within the TNBC TME using IMC, while also investigating differences in the TME of TNBC patients who developed distant metastasis (DM) compared to those who remained non-metastatic (NM). IMC was performed on formalin-fixed paraffin-embedded TNBC primary resection tissues (n= 16; DM= 7, NM= 9) from Stavanger University Hospital, Norway, using a panel of 25 metal-conjugated antibodies. Regions of interest were marked by the pathologist and the slides were laser ablated using Hyperion Imaging System. DeepCell was used to segment single cells based on surface markers. Mean intensity values of all markers for each cell were extracted and consolidated in R scripts for unsupervised clustering. Cell clusters were annotated based on phenotypic markers. Normalized expression of markers was compared for each cell type across DM and NM patients, including markers for cell proliferation, angiogenesis, hypoxia, and cell migration. Neighborhood analysis examined spatial associations between different cell types, highlighting differences in DM and NM patients. The IMC panel successfully provided spatial insights into the TME, including the characterization of tumor cells, immune cells (B cells, T cells, macrophages), endothelial cells, and proliferating cells. We identified 18 unique phenotypic cell clusters that were present at different frequencies in DM and NM patients. These clusters demonstrated different expression profiles of functional markers. t-SNE plots revealed distinct patterns of cell type distribution and unique protein expression profiles, highlighting significant differences between the two groups. Our data suggest a distinct spatial organization of the TME in TNBC patients who develop metastasis. This warrants further investigations to determine the role and interplay of TME features in predicting the risk of distant metastasis in TNBC. Citation Format: Mahak Bhargava, Jaspreet Kaur, Geetanjali Saini, Junjun Zheng, Yitian Xu, Shu-Hsia Chen, Ping-Ying Pan, Emma Rewcastle, Umay Kiraz, Emiel Adrianus Janssen, Ritu Aneja. Spatial characterization of the tumor microenvironment of metastatic triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1488.

Optimizing the Delivery of mRNA to Mesenchymal Stem Cells for Tissue Engineering Applications.

Messenger RNA (mRNA) represents a promising therapeutic tool in the field of tissue engineering for the fast and transient production of growth factors to support new tissue regeneration. However, one of the main challenges to optimizing its use is achieving efficient uptake and delivery to mesenchymal stem cells (MSCs), which have been long reported as difficult-to-transfect. The aim of this study was to systematically screen a range of nonviral vectors to identify optimal transfection conditions for mRNA delivery to MSCs. Furthermore, for the first time, we wanted to directly compare the protein expression profile from three different types of mRNA, namely, unmodified mRNA (uRNA), base-modified mRNA (modRNA), and self-amplifying mRNA (saRNA) in MSCs. A range of polymer- and lipid-based vectors were used to encapsulate mRNA and directly compared in terms of physicochemical properties as well as transfection efficiency and cytotoxicity in MSCs. We found that both lipid- and polymer-based materials were able to successfully condense and encapsulate mRNA into nanosized particles (<200 nm). The overall charge and encapsulation efficiency of the nanoparticles was dependent on the vector type as well as the vector:mRNA ratio. When screened in vitro, lipid-based vectors proved to be superior in terms of mRNA delivery to MSCs cultured in a 2D monolayer and from a 3D collagen-based scaffold with minimal effects on cell viability, thus opening the potential for scaffold-based mRNA delivery. Modified mRNA consistently showed the highest levels of protein expression in MSCs, demonstrating 1.2-fold and 5.6-fold increases versus uRNA and saRNA, respectively. In summary, we have fully optimized the nonviral delivery of mRNA to MSCs, determined the importance of careful selection of the mRNA type used, and highlighted the strong potential of mRNA for tissue engineering applications.

Validation and Evaluation of Reference Genes for Quantitative Real-Time PCR Analysis in Mythimna loreyi (Lepidoptera: Noctuidae).

Quantitative real-time PCR (qRT-PCR) is a widely applied technique for accurately assessing the expression of target genes. In practice, the evaluation of gene expression requires appropriate reference genes. To screen reliable reference genes for evaluating gene expression via qRT-PCR in Mythimna loreyi, a notorious migratory pest across Asia, Africa, Europe, and Australia, we assessed the expression stability of 13 candidate reference genes in M. loreyi using the ΔCt method, BestKeeper, Normfinder, GeNorm, and the web-based comprehensive platform RefFinder. These reference genes include RPL10, RPL27, RPL32, RPS3, TATA-box, GAPDH, AK, Actin, EF, α-tubulin, SOD, 18S rRNA, and FTZ-F1, which is frequently employed in Lepidoptera insects. Our findings revealed that the performance of the candidate reference gene depended on experimental conditions. Specifically, RPL27 and RPL10 were the most suitable for evaluating expression changes across developmental stages, tissues, and adult ages. The optimal reference genes were recommended in specific experiment conditions, for instance, EF and RPS3 were recommended for mating status, AK and RPL10 were recommended for temperature treatments, RPL27 and FTZ-F1 were recommended for larva diet, and EF and RPL27 were recommended for adult diet treatments. Additionally, expression profiles of pheromone-binding protein 2 (MlorPBP2) and glutathione S-transferase (MlorGST1) were used to validate the reference genes. This study provides reference genes for the accurate normalization of qRT-PCR data, laying the groundwork for studying the expression of target genes in M. loreyi.