Immune Signaling Research Articles

Unraveling IFN-I response dynamics and TNF crosstalk in the pathophysiology of systemic lupus erythematosus.

The innate immune system serves the crucial first line of defense against a wide variety of potential threats, during which the production of pro-inflammatory cytokines IFN-I and TNFα are key. This astonishing power to fight invaders, however, comes at the cost of risking IFN-I-related pathologies, such as observed during autoimmune diseases, during which IFN-I and TNFα response dynamics are dysregulated. Therefore, these response dynamics must be tightly regulated, and precisely matched with the potential threat. This regulation is currently far from understood. Using droplet-based microfluidics and ODE modeling, we studied the fundamentals of single-cell decision-making upon TLR signaling in human primary immune cells (n = 23). Next, using biologicals used for treating autoimmune diseases [i.e., anti-TNFα, and JAK inhibitors], we unraveled the crosstalk between IFN-I and TNFα signaling dynamics. Finally, we studied primary immune cells isolated from SLE patients (n = 8) to provide insights into SLE pathophysiology. single-cell IFN-I and TNFα response dynamics display remarkable differences, yet both being highly heterogeneous. Blocking TNFα signaling increases the percentage of IFN-I-producing cells, while blocking IFN-I signaling decreases the percentage of TNFα-producing cells. Single-cell decision-making in SLE patients is dysregulated, pointing towards a dysregulated crosstalk between IFN-I and TNFα response dynamics. We provide a solid droplet-based microfluidic platform to study inherent immune secretory behaviors, substantiated by ODE modeling, which can challenge the conceptualization within and between different immune signaling systems. These insights will build towards an improved fundamental understanding on single-cell decision-making in health and disease.

Placental TLR recognition of salivary and subgingival microbiota is associated with pregnancy complications

BackgroundPre-term birth, the leading cause of neonatal mortality, has been associated with maternal periodontal disease and the presence of oral pathogens in the placenta. However, the mechanisms that underpin this link are not known. This investigation aimed to identify the origins of placental microbiota and to interrogate the association between parturition complications and immune recognition of placental microbial motifs.61FbVQciH1C2BbwKYgvy28Video MethodsSaliva, plaque, serum, and placenta were collected during 130 full-term (FT), pre-term (PT), or pre-term complicated by pre-eclampsia (PTPE) deliveries and subjected to whole-genome shotgun sequencing. Real-time quantitative PCR was used to measure toll-like receptors (TLR) 1–10 expression in placental samples. Source tracking was employed to trace the origins of the placental microbiota.ResultsWe discovered 10,007 functionally annotated genes representing 420 taxa in the placenta that could not be attributed to contamination. Placental microbial composition was the biggest discriminator of pregnancy complications, outweighing hypertension, BMI, smoking, and maternal age. A machine-learning algorithm trained on this microbial dataset predicted PTPE and PT with error rates of 4.05% and 8.6% (taxonomy) and 6.21% and 7.38% (function). Logistic regression revealed 32% higher odds of parturition complication (95% CI 2.8%, 81%) for every IQR increase in the Shannon diversity index after adjusting for maternal smoking status, maternal age, and gravida. We also discovered distinct expression patterns of TLRs that detect RNA- and DNA-containing antigens in the three groups, with significant upregulation of TLR9, and concomitant downregulation of TLR7 in PTPE and PT groups, and dense correlation networks between microbial genes and these TLRs. 70–82% of placental microbiota were traced to serum and thence to the salivary and subgingival microbiomes. The oral and serum microbiomes of PTPE and PT groups displayed significant enrichment of genes encoding iron transport, exosome, adhesion, quorum sensing, lipopolysaccharide, biofilm, and steroid degradation.ConclusionsWithin the limits of cross-sectional analysis, we find evidence to suggest that oral bacteria might translocate to the placenta via serum and trigger immune signaling pathways capable of inducing placental vascular pathology. This might explain, in part, the higher incidence of obstetric syndromes in women with periodontal disease.

Advances in the mechanism of inflammasomes activation in herpes virus infection.

Herpesviruses, prevalent DNA viruses with a double-stranded structure, establish enduring infections and play a part in various diseases. Despite their deployment of multiple tactics to evade the immune system, both localized and systemic inflammatory responses are triggered by the innate immune system's recognition of them. Recent progress has offered more profound understandings of the mechanisms behind the activation of the innate immune system by herpesviruses, specifically through inflammatory signaling. This process encompasses the initiation of an intracellular nucleoprotein complex, the inflammasome associated with inflammation.Following activation, proinflammatory cytokines such as IL-1β and IL-18 are released by the inflammasome, concurrently instigating a programmed pathway for cell death. Despite the structural resemblances between herpesviruses, the distinctive methods of inflammatory activation and the ensuing outcomes in diseases linked to the virus exhibit variations.The objective of this review is to emphasize both the similarities and differences in the mechanisms of inflammatory activation among herpesviruses, elucidating their significance in diseases resulting from these viral infections.Additionally, it identifies areas requiring further research to comprehensively grasp the impact of this crucial innate immune signaling pathway on the pathogenesis of these prevalent viruses.

Lipopolysaccharide Regulates the Macrophage RNA-Binding Proteome.

RNA-protein interactions within cellular signaling pathways have significant modulatory effects on RNA binding proteins' (RBPs') effector functions. During the innate immune response, specific RNA-protein interactions have been reported as a regulatory layer of post-transcriptional control. We investigated changes in the RNA-bound proteome of immortalized mouse macrophages (IMM) following treatment with lipopolysaccharide (LPS). Stable isotope labeling by amino acids in cell culture (SILAC) of cells followed by unbiased purification of RNP complexes at two time points after LPS stimulation and bottom-up proteomic analysis by LC-MS/MS resulted in a set of significantly affected RBPs. Global RNA sequencing and LFQ proteomics were used to characterize the correlation of transcript and protein abundance changes in response to LPS at different time points with changes in protein-RNA binding. Il1α, MARCKS, and ACOD1 were noted as RBP candidates involved in innate immune signaling. The binding sites of the RBP and RNA conjugates at amino acid resolution were investigated by digesting the cross-linked oligonucleotide from peptides remaining after elution using Nuclease P1. The combined data sets provide directions for further studies of innate immune signaling regulation by RBP interactions with different classes of RNA.

Abstract 6842: The effect of intra-tumoral Candida albicans on host gene expression

Abstract The gut and tumor microbiome have been shown to affect tumorigenesis, tumor immune infiltration, and response to traditional cancer therapeutics such as radiation therapy. Further, specific microbe-cancer relationships have been established. One such microbe is Candida albicans, which has been identified within several gastrointestinal cancers and is associated with an increased risk of metastases and decreased overall survival. By completing RNA-sequencing on murine rectal tumors colonized with C. albicans, we can better understand whether intratumoral changes in gene expression exist based on colonization of specific microbes. This study utilizes gut-to-tumor translocation and whole transcriptome analysis for a discovery-based approach to identify the effects of C. albicans on the host tumor. We established a rectal tumor model with C57BL/6 mice using MC38 rectal cancer cells subcutaneously injected on the right flank, local radiation to the tumor (standard-of-care for rectal cancer), and gavage with either C. albicans (Ca) or PBS. Fungal presence within tumors was validated by staining with calcofluor white and subsequently, tumors were sent for bulk RNA-sequencing to determine differences in gene expression using DeSeq2 and a gene set enrichment analysis (GSEA). In unsupervised analysis of gene expression differences between PBS-gavaged and Ca-gavaged with bulk RNA sequencing, tumors gavaged with C. albicans possessed unique gene expression and pathway analysis when compared to PBS-gavaged mice. Global differential gene expression showed 1,947 genes upregulated in Ca gavage and 593 genes upregulated in the PBS gavage (p < 0.05). C. albicans colonized tumors showed decreased expression in gene pathways relating to immune activation (interferon alpha response, IL-6 and STAT3 signaling), p53 expression, DNA repair, oxidative phosphorylation, NOTCH signaling, and apoptosis. We subsequently used a network-based approach to identify the most important genes driving the differences in the tumors, calculating the degree, closeness, and betweenness centralities. Several microbes have been regarded as potential contributors to decreased treatment response and survival in rectal cancer. In the case of tumors colonized with C. albicans, bulk RNA-sequencing indicates down regulation of gene pathways related to cell proliferation, immune activation, and DNA maintenance. Down regulation of gene pathways related to cell cycle control and arrest suggests that intratumoral colonization of C. albicans may causally affect tumorigenesis. Decreases in immune signaling in tumors suggest a potentially causal role of C. albicans immune suppression. These results highlight initial steps to understand how intratumoral C. albicans may be affecting rectal cancer progression and treatment response and serve as a technique to allow better insight to individual microbial impact on tumors. Citation Format: Alexander Loncar, Dennis Grencewicz, Rebecca Hoyd, Aaditya Pallerla, Nyelia Williams, Martin Benej, Yogita Mehra, Shiva Jahanbakhshi, Matthew Anderson, Nicholas Denko, Daniel Spakowicz. The effect of intra-tumoral Candida albicans on host gene expression [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 6842.

Abstract 5088: Evaluating TNF-receptor associated factor 2 (TRAF2) as a targetable driver of immune resistance in LKB1 deficient non-small cell lung cancer

Abstract STK11 is a serine-threonine kinase and tumor suppressor lost in about 25% of NSCLC. It potently regulates a network of downstream effector kinases in the AMPK family, thereby exerting major effects on cell signaling, metabolic activity, and epigenetic patterning. Clinically loss of STK11 in NSCLC is associated with poor response to immune checkpoint blockade. Several mechanisms of immune resistance have been identified involving both tumor intrinsic pathways as well as changes in the immune microenvironment. Dysregulation of cellular inhibitor of apoptosis proteins, specifically cIAP1 has been identified as a specific resistance mechanism, which was shown to also influence the STING pathway in the context of STK11 loss. However, there is a gap in understanding how loss of STK11 influences cIAP1 signaling.We evaluated cIAP1, its TRAF family member binding partners, and downstream NF-kB and immune signaling using a multiomic approach. Using CPTAC multiomic data of 110 lung adenocarcinomas, we observe discordance between protein vs mRNA expression for cIAP1, which shows no significant association with STK11 loss in mRNA, but is significantly over-expressed at the protein level (P<1e-4). Conversely, we identify TRAF2 as the only TRAF member whose expression is increased in STK11-deficient NSCLC, with concordant increase in both mRNA (P<1e-8) and protein (P<1e-10). Further, on the protein level TRAF2 and cIAP1 levels were highly correlated (P<1e-12), strongly suggesting that TRAF2 is the proximal factor driving dysregulation of cIAP1 signaling in these tumors. We also observe a correlated pattern of phosphorylation sites within cIAP1, TRAF2, and other NF-kB signaling members that we putatively link to STK11 loss. We confirm that TRAF2 mRNA is consistently over-expressed in STK11 deficient NSCLC across multiple additional transcriptomic datasets together comprising >2000 tumors. Across this large dataset we observe that among STK11-deficient tumors, TRAF2 is anti-correlated with NF-kB signaling and immune infiltration signatures, a relationship that is strikingly absent among the STK11-WT subset. Using GEMM cell lines (KRAS, TP53, STK11 GEMM; Winslow lab, Stanford), we derived STK11 mutant vs WT isogenic pairs and performed T-cell co-culture experiments using ovalbumin antigen and OT-I CD8 T-cells. This showed that inhibition of cIAP1 using birinapant was synergistic to CD8 cytotoxicity, as previously known, but the synergy was much more pronounced in STK11 mutant vs STK11-WT context (Bliss index 38, P<1e-10). Together, these data demonstrate that the link between STK11-loss and cIAP1 dysregulation is likely through over-expression of TRAF2, and support the strategy of targeting TRAF2/cIAP1 to overcome immune resistance STK11-deficient NSCLC. Citation Format: Bahareh Nourmohammadi, Joseph M. Amann, Rahul Shivahare, Qin Ma, Zihai Li, David P. Carbone, Jacob Kaufman. Evaluating TNF-receptor associated factor 2 (TRAF2) as a targetable driver of immune resistance in LKB1 deficient non-small cell lung 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 5088.

Abstract 965: Urine cell-free DNA multi-omics combining copy number analysis with fragmentomics to detect minimal residual disease in bladder cancer patients

Abstract Background: Standard-of-care for patients with localized muscle-invasive bladder cancer (BC) is neoadjuvant chemotherapy followed by radical cystectomy (RC). Bladder-sparing treatments are limited by our current inability to sensitively detect minimal residual disease (MRD). Here, we analyzed urine, a noninvasive and proximal biofluid, from patients with bladder cancer and utilized an approach that incorporates copy number-derived tumor fraction as well as fragmentomics to sensitively detect MRD and predict pathologic complete response (pCR). Methods: We acquired urine preoperatively from 51 BC patients (80% muscle-invasive) on the day of standard-of-care RC, and after neoadjuvant chemotherapy in 57% of patients. We performed whole genome sequencing (WGS) of urine cfDNA (median depth: 10X) from all 51 BC patients and 13 healthy adults. Tumor fraction level based on genome-wide copy number alterations was estimated using ichorCNA. We inferred fragment size and genomic coverage of urine cfDNA using Picard Tools. Then, we computed the short-to-long (S/L) fragment ratio for genomic bins in each sample. The S/L ratio per bin was derived by partitioning the genome into 100 kb bins and evaluating the ratio of GC-corrected short fragments (50-150 bp) to long fragments (151-250 bp) within each bin. A normalized genome-wide S/L score was calculated by averaging across all bins. Results: In our cohort of 51 BC patients, 45% of patients achieved pCR (n = 23) while 55% had residual disease detected in their surgical sample (no pCR; n = 28). Patients with no pCR had significantly higher copy number-derived tumor fractions in urine compared to patients with pCR (median 10.6% vs 1.9%, p = 0.0002) and healthy adults (n = 12) (median 10.6% vs 1.8%, p = 0.003). Tumor fraction achieved an AUC of 0.89 for classification of No pCR from pCR with sensitivity of 82% and specificity of 92%. Presumed cancer-free patients (healthy and pCR) had significantly longer urine cfDNA median fragment sizes than patients with no pCR (median 177 bp vs 156 bp, p = 0.002). Presumed cancer-free patients (healthy and pCR) also had significantly lower S/L ratio scores than no pCR patients (mean .98 vs 1.5, p = 0.007). The top 100 bins with maximally differential S/L ratio between these two patient groups were enriched for genes involved in KEGG pathways implicated in cancer and immune signaling (autophagy, transcriptional misregulation in cancer, Th1 differentiation) indicating biological relevance. Conclusion: Copy number alteration-derived tumor fraction inference combined with fragmentomic analysis of urine cell-free DNA can enable sensitive detection of MRD to predict pCR and reveal potentially onco-relevant pathways. Citation Format: Arpit Panda, Irfan Alahi, Pradeep S. Chauhan, John Sheng, Nathan Colon, Cayce Nawaf, Alexander Shiang, Peter K. Harris, Faridi Qaium, Eric H. Kim, Melissa A. Reimers, Woodson Smelser, Zachary L. Smith, Aadel A. Chaudhuri. Urine cell-free DNA multi-omics combining copy number analysis with fragmentomics to detect minimal residual disease in bladder cancer patients [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 965.

Abstract 7568: Molecular effects of neo-adjuvant hormone therapy for locally advanced prostate cancer: An ancillary study of the ARNEO phase 2 trial

Abstract Intensified hormone therapy in combination with radiotherapy improves outcome for very high-risk locally advanced prostate cancer. The impact of hormone therapy with surgery, however, is uncertain. In order to understand this better, we ran an ancillary translational study on ARNEO (NCT03080116), a Phase 2 clinical trial, which randomized 89 locally-advanced prostate cancer patients to 12 weeks LHRHa +/- apalutamide (APA) followed by prostatectomy. We performed clinical-grade transcriptome-wide expression array analysis (Veracyte, Inc. San Diego, CA) of FFPE diagnostic core biopsies (N=89) and tumors removed at prostatectomy (N=32 LHRHa, N=23 LHRHa + APA). We also performed RNASeq on fresh frozen biopsies of prostatectomy tumors (N=42 LHRHa, N=45 LHRHa + apalutamide) and LHRHa-naive control cases (N=61) matched for clinical and pathological risk factors. Pre-surgery serum PSA decreased by a median of 98% (IQR:90.2-99.3) after hormone treatment. This decrease was better associated with tumor AR signaling (r2=0.11) versus pre-treatment serum PSA (r2=0.0005), or between pre-treatment serum PSA with diagnostic biopsies (r2=0.008). We confirmed APA more potently suppressed AR signaling than LHRHa alone, with no detectable difference in direction of effect for differentially regulated genes/pathways. LHRHa + APA had less residual tumor (median Residual Cancer Burden, RCB=0.48cm3) than in LHRHa (median RCB=1.7cm3) but in cases with sufficient tumor, we observed residual active AR signaling pathways at prostatectomy in both treatment arms. To orthogonally confirm this, we selected 28 ERG positive cases (by IHC pre-treatment) and found retained ERG expression in 12/13 on LHRHa and 10/15 on LHRHa + APA. Using pathway analysis in the RNAseq data, we identified LHRHa +/- APA induced down-regulation of AR, metabolic pathways, as well as DNA repair, and up-regulation of hallmark NOTCH, Hedgehog, TGF-beta, WNT and immune signaling (adjusted p-value cutoff=0.001). Comparing differentially expressed genes on RNASeq between treatment-naïve versus LHRHa +/- APA treated tumors, we observed STING1 as significantly higher in LHRHa +/- APA vs control (Wilcoxon rank sum test p-value <0.001). We found that expression of STING1 was positively correlated with hallmark interferon alpha response (r=0.7, rs=0.6) that was correlated with apoptosis (r=0.5, rs=0.5). Whilst we show that adding APA to LHRHa increases suppression of AR activity and ERG expression, we find that 12 weeks is insufficient to abrogate AR signaling in most tumors, despite ~98% decrease in serum PSA. We identify novel processes modulated in humans by potent suppression of androgen activity, including upregulation of STING1-type 1 interferon response as a potential key modulator of treatment effect. Citation Format: Mazlina Ismail, Wout Devlies, Marina Parry, Gaetan Devos, Stefanie Friedrich, Charles Parker, Larissa Mendes, Elai Davicioni, Mark Linch, Frank Claessens, Steven Joniau, Gerhardt Attard. Molecular effects of neo-adjuvant hormone therapy for locally advanced prostate cancer: An ancillary study of the ARNEO phase 2 trial [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 7568.

Abstract 1385: Crosstalk between hepatocyte and myeloid-specific IL17 signaling in colorectal cancer metastasis

Abstract Advanced colorectal cancer (CRC) is resistant to current immunotherapies and prone to metastasis. Chronic inflammation mediated by cytokines such as interleukin (IL)17 activates the oncogenic pathways in cancer cells, promoting sporadic and inflammation-associated CRC. Increased levels of IL17A in early stages of CRC portends poor prognosis indicating its potential involvement into metastasis. We and others previously showed that IL17A signaling is required for early CRC tumorigenicity in a variety of models and that in early CRC, its ability to signal to epithelial cells is essential. What remains unknown is whether IL17 signaling to other cell types within the tumor microenvironment (TME) is essential to control tumor progression and metastasis. Using a model of colitis-associated cancer (CAC) we found that “late” IL17A neutralization decreases tumor burden, possibly acting through regulation of myeloid cells and chemokine network essential for the control of immunosuppressive TME. Next, using models of colorectal cancer liver metastasis (CRLM) including intraportal cell injection and cecum transplantation of organoids with distinct set of oncogenic mutation we found that IL17 neutralization reduces metastasis. FACS analysis revealed that IL17A promotes the recruitment of tumor-associated macrophages and other key myeloid subsets into the metastatic liver environment. Interestingly, ablation of IL17RC (receptor) in myeloid cells only (using Il17rcf/f -LysMCre mice) led to reduction in metastasis and reshaping of metastatic myeloid cell compartments. Meanwhile, IL17 signaling in hepatocytes was required for early metastasis and pre-metastatic niche formation, as the ablation of IL17RC in hepatocytes (using Il17rcf/f -AlbCre mice) led to a reduction of metastatic numbers but not growth. This coincided with reduced neutrophil recruitment and enhanced activation of cytotoxic innate lymphoid cells and CD8 T lymphocytes, indicating the role of hepatocyte specific IL17 signaling in blunting anti-tumor immunity. Our findings illuminate the role of inflammatory cytokine IL17 into CRC progression and metastasis and imply its distinct cell type specific role within the metastatic liver TME. Citation Format: Katarzyna Chojnacka, Dan Kamen, Katrina M. Reyes, Hana Tomizawa, Sergei I. Grivennikov. Crosstalk between hepatocyte and myeloid-specific IL17 signaling in colorectal cancer metastasis [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 1385.

Abstract 4993: CJRB-101 induces immune responses through the GUT-TME axis in immune cell-driven mechanism in lung cancer models

Abstract Background Live biotherapeutic products (LBPs) are known to enhance immune responses through the GUT-TME axis. Here, we investigate the GUT-TME-related immune cell profiling and signals associated with the anti-cancer effects of CJRB-101. Methods Tumors from NSCLC patients (anti-PD-1 refractory) were transplanted in Hu-CD34-NSG to establish humanized patient-derived xenograft (PDX) models. CJRB-101 was administered at 1x109 CFU (p.o., BID) or combination with anti-PD-1 (10 mpk, i.p., BIW). TME was analyzed using multiplex IHC, flow cytometry and scRNA sequencing. Samples were collected from C3PQ syngeneic mice at multiple timepoints for GUT-TME immune cell profiling. For depletion assay, immune cells were individually depleted during the combination treatment. TLR4-mediated mechanism was evaluated using ex vivo and in vivo assay treated with CJRB-101 or cell membrane of CJRB-101. Results CJRB-101 combined with pembrolizumab effectively suppressed tumor growth in anti-PD-1 resistant PDX models. Further analysis revealed a correlation between the activity of NK cells and angiogenesis inhibition. Abundance of NK/NKT was higher in the CJRB-101 treated group compared to the vehicle group in multiple PDX models. The expression of GZMB and IFNG in NK/NKT cells was significantly higher in the CJRB-101 treated group compared to the vehicle group in YHIM2014 (p<0.001). CJRB-101 treated group showed significantly higher expression of antiangiogenic IL1B (p<0.001) while the expression of pro-angiogenic VEGFA (p=0.002) was lower compared to the vehicle group in YHIM2014 cancer cells. Macrophages in the intestine were increased at Day 3 in the CJRB-101 group compared to anti-PD1, while NK cells, granulocytes, CD3+, CD4+, CD8+ T cells increased at Day 10 in the syngeneic model. Depletion assay confirmed that macrophages, CD8+ T cells and neutrophils were pivotal in the anti-cancer effects of CJRB-101. We observed that TAK242 and MD2 reduced the IL-6 secretion of Raw264.7 in a dose-dependent manner. The cell membrane played a key role in increasing BMDM M1 polarization and repolarization of M2 to M1, and that inhibition of TLR4 resulted in a decrease in BMDM repolarization. TLR inhibition also demonstrated that TGI decreased from 34% to 20% when treated with cell membrane + TAK242 compared to cell membrane monotherapy. Conclusions This study showed that macrophages were the dominant immune population in the early stages then T cells (CD3, CD4, CD8), NK cells and granulocytes became more active in the latter stages of the GUT-TME-axis immune response. In vivo results indicated that anti-cancer efficacy of CJRB-101 is immune-cell driven by TLR4-dependent stimulation of key immune cell populations (macrophages, CD8+ T cells, neutrophils) modulated by the cell membrane of CJRB-101. CJRB-101 is currently undergoing clinical investigation for treatment of patients with advanced NSCLC. Citation Format: Arim Min, Bo-eun Kwon, Seong-san Kang, Sujeong Baek, Junwon Yang, Hyunkyung Park, Jieun Im, Hyunjeong Kim, Jaemin Kim, Jieun Kwon, Dong Kwon Kim, Jii Bum Lee, Hyeonseok Oh, Seung Min Yang, Yu Jin Han, Mi hyun Kim, Heekyung Han, Kwangmin Na, Young Taek Kim, Mi Ran Yun, Jae Hwan Kim, Youngseon Byeon, Young Seob Kim, Min Hee Hong, Sun Min Lim, Kyoung-Ho Pyo, Byoung Chul Cho. CJRB-101 induces immune responses through the GUT-TME axis in immune cell-driven mechanism in lung cancer models [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 4993.

Abstract 3387: Spatial analysis revealed distinct molecular and immune landscape in genetic versus carcinogen-induced lung adenocarcinoma precancer mouse models

Abstract Introduction: Our understanding of early carcinogenesis of lung adenocarcinoma (LUAD) is still rudimentary. Single-cell technologies have unveiled the intricate interaction between tumor cells and tumor microenvironment (TME) with unprecedented precision. However, the spatial context of single-cell phenotypes within TME during LUAD early carcinogenesis remain poorly understood. Here, we sought to characterize human-relevant murine LUAD precancer models to elucidate the molecular and immune changes in the spatial context during early LUAD carcinogenesis to inform lung cancer interception strategies. Methods: We have developed genetically engineered mouse models (GEMMs) (129S4 KrasG12D) and carcinogen-induced LUAD precancer model (CITM) (129S4 Urethane). Lung tissues were collected at various time points after induction and analyzed using whole exome sequencing (WES), imaging mass cytometry (IMC), and spatial transcriptomics (10x Visium). Results: Pathological image analysis demonstrated a significantly higher tumor burden (p<0.01) in the 129S4 KrasG12D model compared to the 129S4 Urethane model across different time points. WES revealed a progressive increase in tumor mutation burden (TMB) from precancers to invasive LUADs in both models, with the CITM model having significantly higher TMB (p<0.05) at matched timepoints than GEMM model. IMC assessed 39 biomarkers in 284 regions of interest (ROIs) and identified 1.4 million individual cells with 17 major cell types. Macrophages were the most prevalent immune cell type in TME, representing 9.00% of total cells in GEMM and 8.29% in CITM. The percent immune cells of the total cells progressively decreased from early-stage hyperplasia to late-stage adenocarcinoma in both models. The CITM exhibited a significantly (p<0.05) higher immune proportion than the GEMM model at hyperplasia and adenoma stages, with no significant difference at the invasive adenocarcinoma tumor cores between these 2 models. On the other hand, CITM had a significantly higher overall immune proportion (p<0.05) and the CD8 T cell and B cell proportions than GEMM tumor adjacent lung tissues at the adenocarcinoma stage leading to a significantly higher immune cell gradient between tumor adjacent lung tissues and tumor cores in CITM models. Spatial transcriptomics (ST) data demonstrated an increased presence of immune response signaling at the tumor adjacent lung tissues compared to the tumor core in the latest stage. Conclusion: Spatial analysis revealed different molecular and immune landscapes in the genetic and carcinogen-induced LUAD precancer models, leading to distinct cellular spatial arrangements within the tumor microenvironment in each model. Overall, innate immunity, particularly macrophage, probably plays critical roles during early carcinogenesis of LUAD regardless of carcinogen exposure. Citation Format: Bo Zhu, Pingjun Chen, Jian-Rong Li, Muhammad Aminu, Chenyang Li, Hong Chen, Yanhua Tian, Frank R. Rojas, Parra Cuentas Edwin Roger, Ou Shi, Monique B. Nilsson, Alissa Poteete, Shawna M. Hubert, Khaja B. Khan, Wei Lu, Xingzhi Song, Jianhua Zhang, Don L. Gibbons, Luisa M. Solis Soto, Ignacio I. Wistuba, John V. Heymach, Chao Cheng, Jia Wu, Jianjun Zhang. Spatial analysis revealed distinct molecular and immune landscape in genetic versus carcinogen-induced lung adenocarcinoma precancer mouse models [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 3387.

Abstract 4939: Computational pathology approach for prognostic advancements and therapeutic benefits in gastrointestinal cancer: A multi-centric retrospective study

Abstract Background: The current cancer staging methods cannot accurately predict survival and therapeutic benefits in cancer patients. Digital pathomics is an emerging field with potential to revolutionize disease evaluation. The present study aims to develop and validate a deep learning pathomics signature from digital hematoxylin-eosin (H&E) staining slides to predict the survival of pan-gastrointestinal cancer, and further investigated its associations with chemotherapy and immunotherapy response. P atients and Methods: This international multicenter study included 2,463 patients with pan-gastrointestinal cancer from twelve cohorts of seven cancer centers, among which 1653 patients were diagnosed with GC. We proposed a deep learning pathomics signature (DLPS) by integrating information on three scales from the whole slide images (WSIs) of H&E, including pathomics nucleus, pathomics microenvironment, and single-cell spatial distribution features. Next, we evaluated the predictive accuracy of DLPS for prognosis, chemotherapy response, and immunotherapy response. To interpret the predictive ability of the DLPS, we analyzed multi-omics data and employed the Shapley value strategy to provide its biological insights and importance ranking. Results: The DLPS was significantly associated with overall survival in patients with GC (hazard ratio range: 1.203-2.717, all p < 0.001), which was further validated in other gastrointestinal cancers (all p < 0.02). The DLPS remained an independent predictor of prognosis in multivariable analysis (all p < 0.001). Furthermore, a nomogram incorporating the DLPS and TNM stage shows significantly improved accuracy in predicting cancer prognosis compared to that with TNM stage alone (all p < 0.05). Shapley value analysis highlighted DLPS as the strongest predictor for prognosis. Importantly, GC patients with a low-DLPS (but not those with a high-DLPS) exhibited substantial benefits from adjuvant chemotherapy (all p < 0.05). Furthermore, we found the objective responses of anti-PD-1 immunotherapy is significantly higher in the low-DLPS group (29.6%) than in the high-DLPS group (8.3%, p < 0.05). Upon analyzing multi-omics data, we found that a higher DLPS was positively correlated with tumor promoting, chemotherapy resistance, immune tumor microenvironment and metabolic signaling. Conclusion: The DLPS enabled improved assessment on prognosis, and has the potential to identify patients who will benefit from adjuvant chemotherapy and immunotherapy, which can further be extended to many gastrointestinal cancers or other solid tumors. Citation Format: Taojun Zhang, Zepang Sun, Zhe Li, M. Usman Ahmad, Md Tauhidul Islam, Fan Yang, Zhenhui Li, Yuming Jiang. Computational pathology approach for prognostic advancements and therapeutic benefits in gastrointestinal cancer: A multi-centric retrospective study [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 4939.

Fucosyltransferase 8 regulates adult neurogenesis and cognition of mice by modulating the Itga6-PI3K/Akt signaling pathway.

Fucosyltransferase 8 (Fut8) and core fucosylation play critical roles in regulating various biological processes, including immune response, signal transduction, proteasomal degradation, and energy metabolism. However, the function and underlying mechanism of Fut8 and core fucosylation in regulating adult neurogenesis remains unknown. We have shown that Fut8 and core fucosylation display dynamic features during the differentiation of adult neural stem/progenitor cells (aNSPCs) and postnatal brain development. Fut8 depletion reduces the proliferation of aNSPCs and inhibits neuronal differentiation of aNSPCs in vitro and in vivo, respectively. Additionally, Fut8 deficiency impairs learning and memory in mice. Mechanistically, Fut8 directly interacts with integrin α6 (Itga6), an upstream regulator of the PI3k-Akt signaling pathway, and catalyzes core fucosylation of Itga6. Deletion of Fut8 enhances the ubiquitination of Itga6 by promoting the binding of ubiquitin ligase Trim21 to Itga6. Low levels of Itga6 inhibit the activity of the PI3K/Akt signaling pathway. Moreover, the Akt agonist SC79 can rescue neurogenic and behavioral deficits caused by Fut8 deficiency. In summary, our study uncovers an essential function of Fut8 and core fucosylation in regulating adult neurogenesis and sheds light on the underlying mechanisms.

Abstract 2021: The RXR agonist MSU-42011 and the MEK inhibitor selumetinib reduce pERK levels in NF1-deficient cells and inhibit cytokine production in macrophages

Abstract Neurofibromatosis type 1 (NF1) is a common genetic disease that predisposes approximately 50% of affected individuals to develop plexiform neurofibromas (PNFs), which can progress to highly aggressive malignant peripheral nerve sheath tumors (MPNSTs) in approximately 10% of patients. NF1 is caused by mutations in the tumor suppressor gene NF1, which encodes for neurofibromin, a negative regulator of RAS activity. Selumetinib, a specific inhibitor of MEK1/2, is the only FDA-approved drug for NF1-associated PNFs. However, the anti-tumor effects of selumetinib are limited in MPNSTs and have dose-limiting side effects. Deficiency of the NF1 gene not only promotes tumorigenesis but also has broad effects on the immune cells and cytokine signaling driven by hyperactive RAS signaling. Because macrophages account for almost half of cells in NF1 lesions and their infiltration correlates with disease progression, we hypothesized that targeting tumor-promoting immune cells could be an alternative approach for treating NF1. The novel retinoid X receptor (RXR) agonist MSU-42011 reduces tumor growth in experimental Kras-driven cancers by decreasing pERK expression, reducing tumor-promoting immune cells like CD206+ macrophages and regulatory T cells, and increasing activated cytotoxic T cells. Here, we treated NF1-deficient cells and macrophages with MSU-42011 and selumetinib, either alone or in combination, using monoculture and conditioned media (CM) conditions. In human PNF cells and mouse MPNSTs, treatment with 200 nM MSU-40211 or 50 nM selumetinib for 3 hours reduced pERK protein levels compared with untreated controls, and the combination treatment enhanced this reduction in pERK protein levels. Additionally, there was a trend toward reduction in cell viability with increasing drug concentrations after 72 hours of the combination treatment. Moreover, CM from human and mouse PNF cells increased the mRNA expression of monocyte chemoattractant CCL2 (C-C motif chemokine ligand 2) and the secretion of IL-6 and TNFα in human THP1 monocytes/macrophages and bone marrow derived macrophages (BMDM). Notably, MSU-42011 and selumetinib alone inhibited CCL2 mRNA expression in THP1 macrophages and BMDM stimulated with CM from human and mouse PNF cells, respectively, and the inhibition of CCL2 mRNA expression was greatest with combination treatment. The combination of MSU42011 and selumetinib also significantly reduced tumor burden in a LL2 model of lung cancer driven by an activating Kras mutation. Based on the similarities in RAS activation and immune cell infiltration in NF1 and lung cancer, our next step is to confirm the immunomodulatory and anti-tumor effects of MSU-42011 and selumetinib in a syngeneic model of PNF and MPNST. Citation Format: Pei-Yu Hung, Jessica A. Moerland, Karen T. Liby. The RXR agonist MSU-42011 and the MEK inhibitor selumetinib reduce pERK levels in NF1-deficient cells and inhibit cytokine production in macrophages [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 2021.

Prolonged dialysis during ex vivo lung perfusion promotes inflammatory responses.

Ex-vivo lung perfusion (EVLP) has extended the number of transplantable lungs by reconditioning marginal organs. However, EVLP is performed at 37°C without homeostatic regulation leading to metabolic wastes' accumulation in the perfusate and, as a corrective measure, the costly perfusate is repeatedly replaced during the standard of care procedure. As an interesting alternative, a hemodialyzer could be placed on the EVLP circuit, which was previously shown to rebalance the perfusate composition and to maintain lung function and viability without appearing to impact the global gene expression in the lung. Here, we assessed the biological effects of a hemodialyzer during EVLP by performing biochemical and refined functional genomic analyses over a 12h procedure in a pig model. We found that dialysis stabilized electrolytic and metabolic parameters of the perfusate but enhanced the gene expression and protein accumulation of several inflammatory cytokines and promoted a genomic profile predicting higher endothelial activation already at 6h and higher immune cytokine signaling at 12h. Therefore, epuration of EVLP with a dialyzer, while correcting features of the perfusate composition and maintaining the respiratory function, promotes inflammatory responses in the tissue. This finding suggests that modifying the metabolite composition of the perfusate by dialysis during EVLP can have detrimental effects on the tissue response and that this strategy should not be transferred as such to the clinic.

Abstract 6428: Transcriptome analysis of tissues and peripheral blood revealed potential biomarkers for chemo/radio-immunotherapy in non-small cell lung cancer

Abstract Background: Combination of anti-PD-(L)1 antibody with chemotherapy has shown promising efficacy in advanced/metastatic non-small cell lung cancer (NSCLC). Dynamics of peripheral blood mononuclear cells (PBMC) might have impact on repertore of immune cells further resulting different immune infiltration in tumor microenvironment. Thus, monitoring changes in PBMC during therapy or measuring tumor microenvironment provides the possibility to predict the efficacy of tumor immunotherapy. Methods: Patients diagnosed with NSCLC (Stage III-IV)were enrolled in this study. Anti-PD-L1 and chemo/radio combination therapy was performed as adjuvant therapy. Tumor samples were obtained prior to adjuvant therapy PBMC samples were obtaind at multiple time points, including prior adjuvant therapy, 3 months after adjuvant therapy, and 3 months after adjuvant therapy. Tumor and PBMC mRNA sequencing were performed by Amoy Diagnostics Co Ltd. (Xiamen, China). Patients who achieved CR/PR/SD and PFS > 6 months were defined as well responders. Pathway enrichment and GSEA analyses were performed by clusterProfiler (version 4.0.5) on GO, KEGG, and from six different gene sets. Correlations between immune cellular signature ES scores and gene expression were estimated using Pearson correlation. Results: 28 patients were enrolled; mean age was 60.38±7.86 years, 27 (96.4%) patients were male. 21 (75.0%) patients were identified as responder and 7 (25.0%) were non-responder. In the baseline tumor samples, immune infiltration was more extensive in responders compared to non-responders. Cytotoxic lymphocytes, activated CD8+ T cell, and NK cells were significantly enriched in responders. High expression of CCL17 and down regulation of CTSG/PLK1/PRTN3, a signaling axis that inhibits histone H3 hydrolysis and promotes monocyte to macrophage differentiation, was significant in responders. Dynamic monitoring of PBMC before and after treatment showed significant upregulation of immune infiltration-related pathways in responders, including effector cells, NK cells and CD8+ T cells. In the non-responder group, the up-regulated pathways after treatment were mainly enriched in tumor growth rate, EMT, and matrix remodeling. Multi-time point analysis showed that CEACAM5/MKI67 level and tumor proliferation rate in non-responders were higher than those in responders at each time point after treatment. Conclusion: Baseline tumor transcripton signatures and dynamic changes in PBMC could be applied to develop novel biomarkers for immune-related therapy. Patients with stronger immune infiltration signals in baseline tumor samples and PBMC after treatment had better immune response. The upregulation of proliferation-related signals in PBMC predicts poor immune response. Citation Format: Jing Hu, Jianguo Zhang, Peng Cheng, Yanhua Chen, Changbin Zhu, Feilin Yi, Conghua Xie. Transcriptome analysis of tissues and peripheral blood revealed potential biomarkers for chemo/radio-immunotherapy in non-small cell lung 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 6428.

Comparative transcriptome analysis reveals the importance of phenylpropanoid biosynthesis for the induced resistance of 84K poplar to anthracnose

BackgroundPoplar anthracnose, which is one of the most important tree diseases, is primarily caused by Colletotrichum gloeosporioides, which has been detected in poplar plantations in China and is responsible for serious economic losses. The characteristics of 84K poplar that have made it one of the typical woody model plants used for investigating stress resistance include its rapid growth, simple reproduction, and adaptability.ResultsIn this study, we found that the resistance of 84K poplar to anthracnose varied considerably depending on how the samples were inoculated of the two seedlings in each tissue culture bottle, one (84K-Cg) was inoculated for 6 days, whereas the 84K-DCg samples were another seedling inoculated at the 6th day and incubated for another 6 days under the same conditions. It was showed that the average anthracnose spot diameter on 84K-Cg and 84K-DCg leaves was 1.23 ± 0.0577 cm and 0.67 ± 0.1154 cm, respectively. Based on the transcriptome sequencing analysis, it was indicated that the upregulated phenylpropanoid biosynthesis-related genes in 84K poplar infected with C. gloeosporioides, including genes encoding PAL, C4H, 4CL, HCT, CCR, COMT, F5H, and CAD, are also involved in other KEGG pathways (i.e., flavonoid biosynthesis and phenylalanine metabolism). The expression levels of these genes were lowest in 84K-Cg and highest in 84K-DCg.ConclusionsIt was found that PAL-related genes may be crucial for the induced resistance of 84K poplar to anthracnose, which enriched in the phenylpropanoid biosynthesis. These results will provide the basis for future research conducted to verify the contribution of phenylpropanoid biosynthesis to induced resistance and explore plant immune resistance-related signals that may regulate plant defense capabilities, which may provide valuable insights relevant to the development of effective and environmentally friendly methods for controlling poplar anthracnose.

Abstract 3181: Immune modulatory activity of radio-sensitizing gold nanoparticle

Abstract Radiation therapy (RT) is frequently used in the neoadjuvant setting in borderline resectable and some resectable pancreatic ductal adenocarcinoma (PDAC) and in the definitive setting in locally advanced PDACs which is third leading cause of cancer associated death. Higher doses of RT improve overall treatment outcomes but increasing the dose significantly above 50Gy in standard 2Gy fractions risks profound gastrointestinal (GI) toxicity. Additionally, genotoxic stress such as RT, can induce CXCL12 secretion by cancer associated fibroblast (CAFs) and overexpression of CXC motif chemokine receptor 4 (CXCR4) in cancer cells. CXCL12-CXCR4 downstream signaling results in immune suppression and consequent enhanced tumor growth and metastasis and poor prognosis in PDAC. To overcome these clinical challenges, we have developed multimodal gold nanoparticle constructs (GNPs) by functionalizing the CXCR4 antagonists to (i) bind to CXCR4 and mediate GNP internalization, (ii) abolish the feedback immune inhibitory signals mediated by CXCR4 overexpression, and (iii) enhance therapeutic benefit of radiation. To achieve these multimodal activities, we have covalently attached the CXCR4 antagonist, AMD070, to thiol-terminated polyethylene glycol (PEG) to synthesize AMD-PEG which was then conjugated with GNPs to produce AMD-PEG-GNP. In our experiment AMD-PEG-GNP has successfully inhibited CXCL12-CXCR4 downstream signalling such as reduction of ERK phosphorylation in PDAC cells. To analyze the radiosensitization role of GNP nanoconjugates, PDAC cells were incubated with AMD-PEG, PEG-GNP and AMD-PEG-GNPs for 4 hours and then treated with increasing doses of radiated, from 1-4Gy. AMD-PEG-GNP demonstrated a systematic enhancement on reactive oxygen species (ROS) production and downregulation of a colony formation. Collectively, the results from in vitro study with PDAC cells confirm that the nanoconjugate with AMD070 enhances radio-cytotoxicity and impair CXCL12-CXCR4 downstream signaling. Hence AMD-PEG-GNP has strong merit to enhance radiation dose locally and simultaneously restrict RT induced immunosuppression in PDAC. Citation Format: Tanmay Kulkarni, Shamit Dutta, Hazel Parent, Karington Perry, Yuri Mackeyev, Santanu Bhattacharya. Immune modulatory activity of radio-sensitizing gold nanoparticle [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 3181.

Abstract 4345: Evaluation of the prevalence and impact of molecular variation in the three-prime repair exonuclease 1 TREX1 across mammalian species and in human malignancies

Abstract The three-prime repair exonuclease 1, TREX1, degrades cytosolic DNA to prevent aberrant immune activation. Inactivation of TREX1 results in DNA accumulation in the cytosol and subsequent induction of the cGAS-STING DNA sensing pathway, downstream interferon-mediated innate immune signaling, and inflammation. Correspondingly, germline pathogenic mutational defects in the TREX1 gene lead to hereditary autoimmune and autoinflammatory disorders. While TREX1 mutations are also reported in some tumors, their consequences in cancer remain poorly understood. To assess the functional importance of amino acid residues in TREX1, we analyzed protein sequences of the functional TREX1 isoform, TREX1b, from 168 mammalian species. We also conducted a survey of genomic variant databases COSMIC, ClinVar, TCGA, LOVD, Decipher, and gnomAD for disease-associated TREX1 mutations. Finally, we modeled TREX1 mutations occurring in human tumors to evaluate predicted impact on protein stability and function using structure-based approaches FoldX and INPS-3D, and compared results to available predictions from AlphaMissense and COSMIC-3D. In the Cancer Genome Atlas (TCGA), 16 out of 32 cancer histologies have tumor samples bearing protein-altering TREX1 variants, with bladder cancer and melanoma having the highest frequency of TREX1 variants at 2.7% of tumor samples. Moreover, all 32 cancer types have some samples with loss of one or both copies of TREX1; frequencies of heterozygous loss range from 0.6% (thymoma) to 86% (kidney clear cell and lung squamous cell carcinomas). We also observed TREX1 copy gain with lower frequency than instances of copy loss, but also occurring in 31 out of 32 studies. Multiple tumors have a combination of a TREX1 protein sequence variant and copy number gain or loss, suggesting that their potential interplay may increase functional impact of TREX1 variants. Out of 15 cancer-associated TREX1 variants, 5 (33%) were predicted to be pathogenic by AlphaMissense. For FoldX and INPS-3D mutagenesis modeling, we used the published experimental 3D crystal structure of the wild type (WT) human TREX1 (PDB: 7TQQ). The changes in Gibbs free energy of folding ΔΔG between WT and mutant predicted by FoldX and INPS-3D both correlated strongly with the AlphaMissense pathogenic probability scores (Pearson r=0.69 and 0.73, respectively). We will discuss implications of TREX1 variation and conservation across mammalian species and potential effects of germline TREX1 variants in hereditary autoimmune and autoinflammatory disorders and on somatic and germline TREX1 variants in human cancer cells and tumor samples. Citation Format: Mariam M. Konate, Marwa Shekfeh, Julia Krushkal. Evaluation of the prevalence and impact of molecular variation in the three-prime repair exonuclease 1 TREX1 across mammalian species and in human malignancies [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 4345.

Abstract 6853: Spatial proteomic and transcriptomic characterization of the inflammatory landscape of formalin fixed paraffin embedded Barrett’s esophagus tissues

Abstract Esophageal adenocarcinoma (EAC), a significant cause of morbidity and mortality, develops from its metaplastic precursor lesion termed Barrett’s esophagus (BE). BE progresses to EAC within an inflammation rich environment, however a detailed understanding of the types of inflammatory cells present, how that population changes over time or diagnosis, and the related immunomodulatory pathways that may mediate BEs progression is still understudied. Traditional immunohistochemistry is often inadequate to provide detailed information about a broad number of cells, local cellular proportions, and cellular heterogeneity; while bulk or single cell RNA sequencing lose the spatial context and relationships between the stroma and epithelium. Utilizing modern multiplex profiling technologies such as Nanostring’s GeoMx digital Profiler (DSP) aid in identifying both cellular composition and distribution of cells within the epithelium and stroma. Therefore, our aim of this study was to determine the suitability of the DSP platform for analyzing formalin fixed paraffin embedded (FFPE) endoscopic samples of BE and determine any differences between non-dysplastic BE (NDBE) samples from patients who eventually progressed (progressors) compared to those who did not (non-progressors), with the goal to gain deeper insights about cellular composition and spatial distribution of immune cells that may dictate progression of BE. Our secondary goal was to investigate the relationship of these cells to the BE epithelial cells through understanding the immune signaling and regulatory pathways. Our pilot study utilizing a 49-plex antibody panel on 7 NDBE samples from progressors and 8 NDBE samples from non-progressors showed good quality metrics and deciphered an immune rich microenvironment in BE with several immune markers including CD66b (neutrophils), CD14 (monocytes), CD68 (macrophages) and CD56 (NK cells) being overexpressed in the stroma of progressors compared with non-progressors, corrected p value less than 0.05. The myeloid activation marker CD80 was overexpressed in the BE epithelial cells of progressors. To determine how spatial transcriptomic profiling performed in FFPE endoscopic samples, 1,812 transcripts within 48 regions of interest of four BE, HGD, and EAC samples was interrogated. Only 36 (2%) probes fell below the limit of detection quality threshold. Thus, our pilot results show that robust proteomic and transcriptomic profiling can be obtained from endoscopic BE biopsies using this system. Based on these results, we systematically interrogated the immune cell makeup and distribution as well as their related inflammatory and immunoregulatory pathways in 40 NDBE biopsies (20 progressors and 20 with stable non-progressing disease) within discrete compartments (stroma and epithelium) through spatial whole transcriptome profiling. Citation Format: Qurat ul Ain, Nicola Frei, Amir M. Khoshiwal, Pim Stougie, Robert Odze, Sophie Camilleri-Broet, Lorenzo Ferri, Lucas C. Duits, Jacques Bergman, Matthew D. Stachler. Spatial proteomic and transcriptomic characterization of the inflammatory landscape of formalin fixed paraffin embedded Barrett’s esophagus tissues [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 6853.