Spatial Profile Research Articles

On the Energy Cost for Nitrogen Fixation in DC Glow Discharges in Atmospheric‐Pressure Air

ABSTRACTModeling of the production of nitrogen oxide molecules in DC discharges in laminar longitudinal flows of atmospheric‐pressure air is performed. Two‐dimensional nonequilibrium discharge model includes the system of gas dynamic equations and balance equations for various plasma components. Simulations are performed for the discharge currents 50–600 mA and gas flow velocities 1–10 m/s. In considered conditions, the gas temperature in the discharge core varies in the range of 3000–6000 K. Spatial profiles of the densities and fluxes of produced species are obtained, both inside the discharge and in the afterglow region. The energy cost for the production of nitrogen oxides is calculated, depending on the discharge current, gap length, and gas flow velocity. The results of the simulation are compared with available experimental data.

ADP-heptose attenuates Helicobacter pylori-induced dendritic cell activation

ABSTRACT Sophisticated immune evasion strategies enable Helicobacter pylori (H. pylori) to colonize the gastric mucosa of approximately half of the world’s population. Persistent infection and the resulting chronic inflammation are a major cause of gastric cancer. To understand the intricate interplay between H. pylori and host immunity, spatial profiling was used to monitor immune cells in H. pylori infected gastric tissue. Dendritic cell (DC) and T cell phenotypes were further investigated in gastric organoid/immune cell co-cultures and mechanistic insights were acquired by proteomics of human DCs. Here, we show that ADP-heptose, a bacterial metabolite originally reported to act as a bona fide PAMP, reduces H. pylori-induced DC maturation and subsequent T cell responses. Mechanistically, we report that H. pylori uptake and subsequent DC activation by an ADP-heptose deficient H. pylori strain depends on TLR2. Moreover, ADP-heptose attenuates full-fledged activation of primary human DCs in the context of H. pylori infection by impairing type I IFN signaling. This study reveals that ADP-heptose mitigates host immunity during H. pylori infection.

Primordial Black Hole Compaction Function from Stochastic Fluctuations in Ultraslow-Roll Inflation.

We study the formation of primordial black holes (PBHs) with ultraslow-roll inflation when stochastic effects are important. We use the ΔN formalism and simplify the stochastic equations with an analytical constant-roll approximation. Considering a viable inflation model, we find the spatial profile of the PBH compaction function numerically for each stochastic patch, without assumptions about Gaussianity or the radial profile. The stochastic effects that lead to an exponential tail for the density distribution also make the compaction function very spiky, unlike as assumed in the literature. Naively using collapse thresholds found for smooth profiles, the PBH abundance is enhanced by up to a factor of 10^{9}, and the PBH mass distribution is spread over 3 orders of magnitude in mass. The results point to a need to redo numerical simulations of PBH formation with spiky profiles.

Magnetic field enhanced terahertz emission from metallic nanostructures with response to laser spatial profile distribution

This communication proposes an analytical model that exhibits terahertz (THz) radiation generation from spatially corrugated noble-metal spherical and cylindrical nanoparticles (NPs) placed under the influence of an externally applied static magnetic field. This scheme involves the resonant excitation of THz radiation through the beat-wave of two lasers in the presence of ponderomotive nonlinearities. Effect of magnetic field as well as laser intensity profiles, which include super, cosh, flat-top and ring-shaped Gaussian profiles, have been investigated. The incorporation of the magnetic field induces substantially more dynamic laser-metal coupling and influences the surface plasmon resonance condition associated with electrons of the NPs, thereby leading to stronger THz emission. Our results also demonstrate that the spatial profile of the laser affects the THz output, as it impacts the excitation and dynamics of the NPs. Additionally, we find that both the shape, size and interparticle-separation of NPs play crucial roles in enhancing THz generation. Furthermore, we explore the effects of varying other parameters like electric field strength and beam waist of incident lasers. These findings provide valuable insights for the design and optimization of THz sources based on NP systems, offering new avenues for applications in spectroscopy, imaging, communication and biomedical sciences.

Abstract B073: Organ-specific diversity of tumor immune microenvironment in metastatic pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) that is refractory to treatment frequently leads to extensive systemic and multi-organ metastases. PDAC is characterized as an immune “cold” cancer; however, the persistence of this “cold” tumor immune microenvironment (TIME) across distant metastases, and the mechanisms by which metastases influence their respective TIME, remain poorly understood. In this study, we conducted spatial protein expression profiling of the TIME using the Nanostring GeoMx Digital Spatial Profiling (DSP) platform. We employed a 43- plex panel on 54 tissue samples, including 14 primary tumors and 40 matched metastases from organs commonly affected by PDAC metastases (liver, lung, and peritoneum), obtained through a rapid (“warm”) autopsy program. We quantified protein expression in three cellular compartments: PanCK+ epithelial cells, α-SMA+ fibroblasts, and CD45+ immune cells, across 181 regions of interest (ROIs). Our analysis revealed a significant enrichment of CD45+ immune cells in lung metastases compared to other organ sites, with a notably lower presence of these cells in liver metastases. Focusing on the immune cell compartment, we analyzed the CD45 segments for further investigation. After normalization using housekeeping proteins, including Histone H3, GAPDH, and ribosomal protein S6, we found that the immune profiles were most distinct between liver and lung metastases. Specifically, liver metastases exhibited the greatest deviation in immune profile compared to primary PDAC, lung, and peritoneal metastases. We scored protein expression signatures for various immune cell subsets based on specific marker expressions. When comparing liver to lung metastases, scores for T cells, myeloid cells, checkpoint markers, macrophages, and interferon signaling were significantly lower in the liver, underscoring the immune “coldness” of the liver metastasis environment. Our cohort included six untreated patients and nine patients treated with chemotherapies. We compared protein expressions in each organ site between untreated and treated patients. Notably, treatment induced divergent expression patterns of multiple proteins between liver and lung metastases. While treatment significantly reduced the expression of several immune markers, including CD45 (total immune), CD3 (T cell), CD4 (T helper cell), CD45RO (memory T cell), CD163 (M2 macrophage), and B7-H3 (checkpoint marker), in lung metastases, these markers were significantly upregulated in liver metastases of treated patients. This finding indicates a pronounced divergence in TIME modulation in response to treatment between liver and lung metastases, potentially fostering a treatment-resistant environment in PDAC patients with multi- organ metastasis. Consequently, this urges the need to develop tailored therapeutic strategies for PDAC metastasis, taking into account the distinct immune landscapes of different metastasis sites. Citation Format: Jimin Min, Vittorio Branchi, Kimal I Rajapakshe, Nisha Narayanan, Guangsheng Pei, Linghua Wang, Jean L Grem, Thomas C Caffrey, Paul M Grandgenett, Michael A Hollingsworth, Paola A Guerrero, Anirban Maitra. Organ-specific diversity of tumor immune microenvironment in metastatic pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B073.

Abstract C073: FOLFIRINOX with Glycogen Synthase Kinase-3 Beta (GSK-3β) Inhibitor Elraglusib and Transforming Growth Factor- β (TGFβ) Inhibitor Losartan in Untreated Metastatic Pancreatic Ductal Adenocarcinoma(PDAC): Interim analysis of safety cohort

Abstract Introduction: Epithelial-mesenchymal transition (EMT) is one of the key drivers of aggressive biology and development of chemoresistance in PDAC. Inhibition of glycogen synthase kinase 3β (GSK3β) may offer a promising novel approach for modulating dynamic EMT plasticity to overcome resistance to FOLFIRINOX (5-Fluororuacil, Irinotecan, Oxaliplatin, FFX). Methods: Cell line panels comprising both epithelial (E) and quasi-mesenchymal (QM) subtypes were used and treated with IC50 (0.3125 mM) and IC100 (1.25 mM) doses of elraglusib (ELR). Gene expression patters in response to treatment were analyzed using bulk RNA sequencing. 6 patients in the safety run-in cohort of NCT05077800 treated with standard doses of FFX, ELR and losartan were included in this interim analysis. Biopsy specimens were processed for RNA-in situ hybridization (RNA-ISH), a clinically validated assay, and Nanostring (GeoMx) spatial transcriptomics. Results: In preclinical models, exposure to the higher IC100 (1.25 mM) doses of ELR in the QM cell lines caused a shift in transcriptional expression patterns similar to the E cell line, with strong downregulation of EMT. FFX exposure alone in QM cell lines was associated with significant resistance, but addition of ELR (even at IC50 dose) caused a synergistic effect with decreased cell viability. However, in the E cell lines, there was no benefit from addition of ELR. In the safety cohort, clinical results are encouraging with a long-term (>6 month) response in 3/6 patients. 2/6 achieved partial response, 1/6 had stable disease. Nanostring (GeoMx) spatial transcriptomic profiling on available biopsy samples showed significant global gene expression changes before and after treatment. There were concordant changes in 97 differentially expressed genes (DEGs) within the epithelial compartment with treatment, similar to ELR induced changes in PDAC cell lines, with a shift in transcriptional expression patterns from QM to E subtype. Gene set enrichment analysis demonstrated downregulation of the EMT pathway, as well as decreased TGF-β signaling. RNA-ISH analysis on baseline biopsy samples showed that the two partial (best) responders had higher degrees of QM marker expression (82.58% and 63.76%). The regimen was well tolerated with no dose limiting toxicity noted in the safety cohort. Conclusion: Our experiments demonstrate that the E subtype PDAC cell lines are inherently sensitive to FFX chemotherapy with no added advantage with GSK3β inhibition. Meanwhile, GSK3β inhibition in QM cell lines induces mesenchymal to epithelial transition, thereby rendering it sensitive to FFX. The data from our safety cohort provide a clinical proof of concept that GSK-3β inhibition with ELR, in combination with FFX, may be an effective therapeutic strategy in metastatic PDAC. NCT05077800 is currently enrolling. Citation Format: Priyadarshini Pathak, Eric Lin, Ildiko Phillips, David Ting, Colin Weekes. FOLFIRINOX with Glycogen Synthase Kinase-3 Beta (GSK-3β) Inhibitor Elraglusib and Transforming Growth Factor- β (TGFβ) Inhibitor Losartan in Untreated Metastatic Pancreatic Ductal Adenocarcinoma(PDAC): Interim analysis of safety cohort [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C073.

Abstract PR-14: Meta-analysis of spatial transcriptomics data from pancreatic cancer precursors proposes a common molecular framework for PanIN and IPMN

Abstract INTRODUCTION Pancreatic ductal adenocarcinoma (PDAC) arises from either pancreatic intraepithelial neoplasia (PanIN) or intraductal papillary mucinous neoplasm (IPMN). Transcriptomic studies of PanIN and IPMN conducted in isolation both aim to improve early detection, risk stratification, and therapeutic target selection. Here, we curated existing and new whole transcriptome digital spatial profiles (DSP-RNA) from normal to malignant pancreatic epithelium to map the molecular landscape of PDAC progenitors. METHODS New NanoString GeoMx DSP-RNA datasets were generated from 1) surgical IPMN specimens resected at Duke University and 2) deceased donor pancreas specimens collected by the Gift of Life Michigan Donor Care Center that could not be used for transplantation and thus allocated to the University of Michigan for approved research. Published studies were downloaded from GEO (GSE229752, GSE226829, GSE199102) and the NanoString Spatial Organ Atlas. Epithelial areas of interest (AOIs) were processed and analyzed with stgeomx (https://github.com/mkiyer/stgeomx) and R/Bioconductor packages. Quality control (QC) procedures removed low quality AOIs. Batch effects were mitigated by defining highly variable genes within each dataset and merging them into a common set for downstream analysis. Molecular subtype gene signatures were obtained from the pdacR package and quantified using Gene Set Variation Analysis. RESULTS We assembled a pancreatic epithelial DSP-RNA cohort consisting of 768 AOIs from 62 patients. AOIs were annotated as normal duct (n=193), acini (n=53), acinar-ductal metaplasia (ADM) (n=57), PanIN (n=108), IPMN (n=92), and PDAC (n=265). Principal component analysis (PCA) revealed compelling associations between DSP-RNA profiles and the established classical, basal-like, and exocrine molecular subtypes of PDAC. The first principal component (PC1) stratified the exocrine and basal-like PDAC subtypes on opposite ends of its axis (PC1 vs exocrine Pearson r=0.929, p < 0.001, PC1 vs basal-like Pearson r=-0.775, padj < 0.001). PC2 further distinguished classical from basal-like AOIs, and PC3 separated normal (CRISP3+) from neoplastic (MUC5AC+) ductal epithelium. Unsupervised clustering of AOIs proposed four epithelial subtypes: “acinar/ADM” (cAcinar), “normal duct” (cDuct), “low-risk neoplasm” (cLR), and “high-risk/malignant” (cHR). PanIN and IPMN lesions exhibited remarkably similar global gene expression patterns, suggesting that they arise from a common lineage. CONCLUSIONS Meta-analysis of pancreatic epithelial DSP-RNA datasets provided a unifying molecular context for PanIN, IPMN, and PDAC. The established classical, basal-like, and exocrine PDAC subtypes explained global patterns of gene expression variation. Intriguingly, PanIN and IPMN transcriptomes appeared similar, suggesting that early detection and therapeutic selection strategies may be broadly applicable to PDAC precursors. Citation Format: Matthew K Iyer, Ashley Fletcher, Elishama Kanu, Chanjuan Shi, Rosina Carr, Ahmed M. Elhossiny, Daniel P. Nussbaum, Eileen S. Carpenter, Marina Pasca di Magliano, Arul M. Chinnaiyan, Peter J. Allen, Timothy L. Frankel. Meta-analysis of spatial transcriptomics data from pancreatic cancer precursors proposes a common molecular framework for PanIN and IPMN [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr PR-14.

Abstract B091: Spatial Transcriptomics Unveils Intraductal Papillary Mucinous Neoplasm Heterogeneity: From Novel Clusters to Immune Dynamics

Abstract Intraductal papillary mucinous neoplasms (IPMNs), a common precursor to pancreatic ductal adenocarcinoma, present as a spectrum of benign indolent behavior and progression to invasive carcinoma, which highlights the importance of identifying biomarkers of IPMN progression. This study utilized whole transcriptome spatial transcriptomics to define the molecular characteristics of Epithelial, Immune, and Fibroblast cell populations of the three distinct histological sub-types of IPMN to understand the dynamics between the cell types and identify biomarkers of neoplastic progression. Fifty-two surgically resected IPMN cores from 21 patients with resected IPMNs comprising 24 Gastric, 14 Intestinal, and 14 Pancreaticobiliary with a distribution of low-grade dysplasia (LGD), high-grade dysplasia (HGD), and Invasive carcinoma (IC) were analyzed. Epithelial, Immune, and Fibroblast components were analyzed using the GeoMx spatial profiler. Unsupervised hierarchical clustering of epithelial areas of interest (AOI) identified 3 distinct clusters demonstrating a spectrum of molecular phenotypes in IPMN that were correlated with the three histological subtypes. Comparing the gene expression profile based on the shared genes expressed on all three subtypes, the Intestinal type tends to have an overexpression of genes whereas the Pancreaticobiliary represents a repressed phenotype relative to the Gastric phenotype. Supervised clustering using Moffitt’s classifier to identify the Epithelial-Mesenchymal transition demonstrates the Pancreaticobiliary subtype to be more Basal-like versus the Gastric and Intestinal inclined towards the classical phenotype. Supervised clustering based on the degree of dysplasia within the epithelial compartments of each of these subtypes, demonstrated a spectrum of molecular heterogeneity and identified biomarkers specific to High-Grade dysplasia in the Gastric and Intestinal subtype. Immune AOI deconvolution analysis demonstrated that Tregs were associated with Pancreaticobiliary, Macrophage enrichment associated with the Intestinal phenotype, while there is enrichment of CD8 T-cell and CD4 T-cell gene signatures associated with the Gastric subtype. Fibroblast AOI analyses demonstrated heterogenous myCAF- iCAF signatures between the subtypes. In summary, Spatial whole transcriptome analysis of IPMN identifies molecular signatures in epithelial and immune compartments that differentiate between the three histological subtypes of IPMN and helps understand their progression from Low-grade dysplasia to High-grade dysplasia. Citation Format: Amaya Pankaj, Michael J Raabe, Yuhui Song, Bidish K Patel, Katherine Xu, Joshua R Kocher, Nicholas J Caldwell, Maria L Ganci, Linda T Nieman, Mari Mino-Kenudson, Vikram Deshpande, Nabeel Bardeesy, Carlos Fernandez-Del Castillo, Martin J Aryee, David T Ting, Yasmin G Hernandez-Barco. Spatial Transcriptomics Unveils Intraductal Papillary Mucinous Neoplasm Heterogeneity: From Novel Clusters to Immune Dynamics [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B091.

Abstract A030: Modulation of lymphocyte adhesion and migration by iCAF and myCAF fibroblasts in PDAC

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) poses significant challenges in treatment due to its complex interaction with the tumour microenvironment (TME). Fibroblasts, a crucial component of the TME, influence tumour progression and therapeutic responses. This study explores the diversity of cancer-associated fibroblast (CAF) populations in PDAC tumours versus adjacent normal fibroblasts (ANFs), focussing on myofibroblasts (myCAFs) and inflammatory fibroblasts (iCAFs). These fibroblast subtypes exhibit distinct characteristics and localisation within the tumour. The research examines the impact of CAFs and ANFs on immune cell behaviour, particularly in relation to adhesion and migration. Methods: Tumour and juxta-tumoral tissue from patients were digested into single cell suspensions. Cells were stained with a 22-plex antibody panel by flow cytometry. Pseudo-emperipolesis assays were assessed by co-culture of lymphocytes with CAF and ANF fibroblasts. Furthermore, these were performed with or without stimulation of fibroblasts with by TGF-b or TNF-a/IFN-g, to promote myCAF or iCAF phenotypes respectively. Gene expression analysis of tumour-derived fibroblast subtypes was assessed by single-cell RNA sequencing using the 10X Genomics platform. The transcriptional profile of spatially resolved fibroblasts which were proximal or distant to tumour epithelium was determined by the NanoString GeoMx Digital Spatial Profiler (DSP) platform. Results: Flow cytometry analysis revealed that iCAFs predominated within the tumour stroma compared to myCAFs, although iCAF populations were less prevalent within ANF populations. Upon stimulation with inflammatory cytokines (TNF-α/IFN-γ), CAFs promoted lymphocyte migration in contrast to ANFs. However, TGF-β stimulation of CAF downregulated the migration of lymphocytes. As such, TGF-β or TNF-α/IFN-γ stimulation exhibited antagonistic effects on lymphocyte adhesion and migration in CAFs. Tumour-proximal fibroblasts exhibited myCAFs characteristics expressing α-SMA and PDPN, as well as ECM molecules such as collagen and integrins, suggesting a barrier to lymphocyte migration. Conversely, distal fibroblasts displayed an inflammatory profile, expressing cytokines and chemokines including CCL2, CXCL9, CXCL12, IL6, IL11 and LIF, thereby contributing to the inflammatory milieu within the tumour microenvironment. Additionally, iCAFs demonstrated higher transcription levels of hyaluronan synthase (Has1) and fibulin 2 (FBLN2) which are crucial for ECM formation and remodelling. This study identifies a differential role for iCAFs and myCAFs in the modulate of lymphocyte adhesion and migration within the PDAC microenvironment, highlighting the complex interplay between fibroblast subtypes and immune cell dynamics. Citation Format: Fouzia Zayou, Hayden Pearce, Wayne Croft, Samantha Nicol, Sandra Margielewska-Davies, Sarah Powell-Brett, Rachel Brown, Jianmin Zuo, Keith Roberts, Helen M. McGettrick, Paul Moss. Modulation of lymphocyte adhesion and migration by iCAF and myCAF fibroblasts in PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A030.

Abstract B023: Unravelling fibroinflammatory and immune signatures for pancreatic cancer early detection

Abstract Pancreatic cancer is associated with late-stage diagnosis and poor patient prognosis. The disease is characterised by chronic dysregulated immunity and inflammation. Moreover, chronic pancreatitis is linked with an increased risk of pancreatic cancer. Tissue remodelling during disease progression involves a complex network of cells and a crosstalk between cancer cells, stromal components and immune cells, but it remains poorly explored. Thus, to improve the outcome of patients, there is a need for better understanding of biological changes associated with the initiation and progression of pancreatic cancer at the molecular and spatial levels. Using samples obtained from our Accelerated Diagnosis of neuroEndocrine and Pancreatic TumourS (ADEPTS) biobank, we have evaluated blood samples for the discovery of non- invasive biomarkers to discriminate patients with pancreatic cancer from symptomatic patients without cancer. In this pilot study, the transcriptome of peripheral blood mononuclear cells (PBMCs), isolated using a ficoll gradient was explored to identify PDAC-associated mRNA signatures. Moreover, the Olink® Explore Inflammation and Oncology panels were performed in a cohort of 40 patients with early stage (I-II) PDAC, 40 chronic pancreatitis samples and 40 control patients with benign symptomatic upper GI diseases. To validate and enrich these results, a panel of inflammatory and immune markers including immunoglobulins, cytokines and chemokines was also run using multiplex techniques. Symptoms observed in our cohort were also analysed together with clinical (e.g diabetic status, biochemistry profile) and demographic data (age, sex, ethnicity, post code etc). We have identified mRNA transcriptome signatures and differentially expressed proteins associated with inflammatory and immunological processes in PDAC compared to benign symptomatic samples. Our results offer potential diagnostic utility for early-stage disease. Additionally, our unique cohort of patient samples comprising of those with confirmed PDAC and those presenting to clinic with benign disease and similar non-specific symptoms, allows for the development of non-invasive diagnostic tests with better translational applicability. Future work will involve the validation of these findings in a larger patient cohort and the use of NanoString GeoMx® Digital Spatial Profiler to perform whole transcriptome and proteome profiling in human chronic pancreatic and pancreatic cancer tissue samples. Citation Format: Kyra Fraser, Maria Rosado, Stephen P Pereira, Pilar Acedo. Unravelling fibroinflammatory and immune signatures for pancreatic cancer early detection [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B023.

A Comprehensive Proteogenomic and Spatial Analysis of Innate and Acquired Resistance of Metastatic Melanoma to Immune Checkpoint Blockade Therapies.

This study paves the way for the creation of innovative therapeutic strategies, aimed at subverting resistance to immune checkpoint blockade (ICB) therapies in metastatic melanoma patients. By unraveling the specific molecular mechanisms underlying resistance, scientists can design effective alternative treatments that target pathways such as pathways associated with cell cycle dysregulation and c-MYC signaling. Furthermore, through the application of advanced immune monitoring techniques such as NanoString Digital Spatial Profiling (DSP) and Cyclic Immunofluorescence (CyCIF), this study has significantly enriched our understanding of the tumor microenvironment. This enhanced characterization facilitates the discovery of potential biomarkers that may forecast a patient's response to ICB treatment. Ultimately, these advancements could potentially refine patient outcomes and foster the development of more personalized cancer treatments in the future.

Abstract C029: Window-of-opportunity trial of metastatic pancreatic cancer reveals potential mechanisms of response to targeting RAS/MEK signaling

Abstract KRAS is mutated in ∼85% of pancreatic ductal adenocarcinoma (PDAC) patient's tumors, suggesting that it is a key node of PDAC targeted therapy. RAF/MEK/ERK signaling is downstream of KRAS signaling. Preclinical studies targeting MEK showed promises but clinical trials failed to show the benefit compared to Standard-of-Care treatment chemotherapy. To elucidate the mechanism of action and resistance to several targeted therapies including MEK inhibition in human PDAC, we conducted a Window-Of-Opportunity trial for Metastatic pancreatic cancer (WOOM trial). In this trial, metastatic PDAC patients received 10 days of Cobimetinib, an FDA approved MEK inhibitor, therapy and specimens of pre- and post- treatment were obtained by biopsy to evaluate the response to targeted treatment using deep multi-omic analytics including DNA-seq, RNA-seq, multiplex IHC, cyclic IF and Digital Spatial Profiling (DSP). Four of 13 patient’s tumors were defined as Responsive tumors by using CA19- 9 and KI67 as a readout. Genomic sequence showed that KRAS amplifications were frequently seen in non-responsive tumors and KrasG12R mutations were seen with higher frequency in Responsive tumors. RNA-seq analysis showed that these Responsive tumors had lower KRAS activity and were more likely to be classical-like subtype compared to non-responsive tumors. DSP analysis confirmed the downregulation of ERK phosphorylation with paradoxical phospho- MEK activation after treatment in all examined cases which is consistent with effective target inhibition. Phospho-MYC protein expression and MYC transcriptomic activity were significantly decreased in responsive tumors, which suggest MYC downregulation could contribute to an active response to MEK inhibition. Single cell RNA-seq and spacial analysis showed tumor microenvironment changes after Cobimetinib treatment including the increase of CD8 T cell infiltration in responsive tumors. Furthermore, PDX from patient’s biopsy recapitulated patient's response to Cobimetinib and faithfully metastasized to the liver. Interestingly, Cobimetinib resistant PDX was also resistant to KRAS inhibitor. This model could be a great tool for testing new therapeutic strategies targeting the KRAS/MEK pathway. Here, we summarize the multi- omics data and discuss potential mechanisms of response and resistance to targeting KRAS signaling and related microenvironmental changes. Citation Format: Motoyuki Tsuda, Colin Daniel, Xiaoyan Wang, Carl Peltz, Hayley Zimmny, Alexander Smith, John Muschler, Xi Li, Tugba Yildiran Ozmen, Furkan Ozmen, Dove Keith, Christopher Corless, Koei Chin, Jonathan Brody, Charles Lopez, Gordon Mills, Rosalie Sears. Window-of-opportunity trial of metastatic pancreatic cancer reveals potential mechanisms of response to targeting RAS/MEK signaling [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C029.

Subcellular Level Spatial Transcriptomics with PHOTON.

The subcellular localization of RNA is closely linked to its function. Many RNA species are partitioned into organelles and other subcellular compartments for storage, processing, translation, or degradation. Thus, capturing the subcellular spatial distribution of RNA would directly contribute to the understanding of RNA functions and regulation. Here, we present PHOTON (Photoselection of Transcriptome over Nanoscale), a method which combines high resolution imaging with high throughput sequencing to achieve spatial transcriptome profiling at subcellular resolution. We demonstrate PHOTON as a versatile tool to accurately capture the transcriptome of target cell types in situ at the tissue level such as granulosa cells in the ovary, as well as RNA content within subcellular compartments such as the nucleolus and the stress granule. Using PHOTON, we also reveal the functional role of m6A modification on mRNA partitioning into stress granules. These results collectively demonstrate that PHOTON is a flexible and generalizable platform for understanding subcellular molecular dynamics through the transcriptomic lens.

Cylindrical compression of thin wires by irradiation with a Joule-class short-pulse laser

Equation of state measurements at Jovian or stellar conditions are currently conducted by dynamic shock compression driven by multi-kilojoule multi-beam nanosecond-duration lasers. These experiments require precise design of the target and specific tailoring of the spatial and temporal laser profiles to reach the highest pressures. At the same time, the studies are limited by the low repetition rate of the lasers. Here, we show that by the irradiation of a thin wire with single-beam Joule-class short-pulse laser, a converging cylindrical shock is generated compressing the wire material to conditions relevant to the above applications. The shockwave was observed using Phase Contrast Imaging employing a hard X-ray Free Electron Laser with unprecedented temporal and spatial sensitivity. The data collected for Cu wires is in agreement with hydrodynamic simulations of an ablative shock launched by highly impulsive and transient resistive heating of the wire surface. The subsequent cylindrical shockwave travels toward the wire axis and is predicted to reach a compression factor of 9 and pressures above 800 Mbar. Simulations for astrophysical relevant materials underline the potential of this compression technique as a new tool for high energy density studies at high repetition rates.

Dissipative spatiotemporal soliton in a driven waveguide laser

A distributed Kerr-lens mode locking regime can be realized in a waveguide laser by spatial profiling of the pump beam, thus creating a spatio-temporal soliton. Additional slow temporal modulation of the pump source stabilizes the spatio-temporal solution in a broad range of parameters defined by the dynamic gain saturation. We choose a Cr:ZnS waveguide laser as a practical example, but such a regime is feasible in various waveguide and fiber oscillators. A far-reaching analogy with Bose–Einstein condensates allows this approach to stabilize the weakly dissipative BECs.

Spatial resolved transcriptomics reveals distinct cross-talk between cancer cells and tumor-associated macrophages in intrahepatic cholangiocarcinoma

BackgroundMultiple studies have shown that tumor-associated macrophages (TAMs) promote cancer initiation and progression. However, the reprogramming of macrophages in the tumor microenvironment (TME) and the cross-talk between TAMs and malignant subclones in intrahepatic cholangiocarcinoma (iCCA) has not been fully characterized, especially in a spatially resolved manner. Deciphering the spatial architecture of variable tissue cellular components in iCCA could contribute to the positional context of gene expression containing information pathological changes and cellular variability.MethodsHere, we applied spatial transcriptomics (ST) and digital spatial profiler (DSP) technologies with tumor sections from patients with iCCA.ResultsThe results reveal that spatial inter- and intra-tumor heterogeneities feature iCCA malignancy, and tumor subclones are mainly driven by physical proximity. Tumor cells with TME components shaped the intra-sectional heterogenetic spatial architecture. Macrophages are the most infiltrated TME component in iCCA. The protein trefoil factor 3 (TFF3) secreted by the malignant subclone can induce macrophages to reprogram to a tumor-promoting state, which in turn contributes to an immune-suppressive environment and boosts tumor progression.ConclusionsIn conclusion, our description of the iCCA ecosystem in a spatially resolved manner provides novel insights into the spatial features and the immune suppressive landscapes of TME for iCCA.

Full-length target sequences of GeoMx digital spatial profiling probes reveal that gene-promiscuity predicts probe sensitivity to EDTA tissue decalcification

GeoMx Digital Spatial Profiling (Nanostring) is a commercial spatial transcriptomics method to selectively analyze regions of interest within intact tissue sections. We show that decalcification with ethylene-diamine-tetra-acetic (EDTA) variably attenuates probe counts, while probes that are more resistant to this effect consequently appear overexpressed after quantile normalization. By determining the undisclosed full-length target sequences of probes used in the human whole transcriptome panel, hereby updating target transcripts and genes, we find that the gene-promiscuity of probes is an important factor that determines sensitivity to EDTA incubation.

Tumor and α-SMA-expressing stromal cells in pancreatic neuroendocrine tumors have a distinct RNA profile depending on tumor grade.

Alpha-smooth muscle actin (α-SMA) expression in the stroma is linked to the presence of cancer-associated fibroblasts and is known to correlate with worse outcomes in various tumors. In this study, using a GeoMx digital spatial profiling approach, we characterized the gene expression of the tumor and α-SMA-expressing stromal cell compartments in pancreatic neuroendocrine tumors (PanNETs). The profiling was performed on tissues from eight retrospective cases (three grade 1, four grade 2, and one grade3). Selected regions of interest were segmented geometrically based on tissue morphology and fluorescent signals from synaptophysin and α-SMA markers. The α-SMA-expressing stromal-cell-associated genes were involved in pathways of extracellular matrix modification, whereas, in tumor cells, the gene expression profiles were associated with pathways involved in cell proliferation. The comparison of gene expression profiles across all three PanNET grades revealed that the differences between grades are not only present at the level of the tumor but also in the α-SMA-expressing stromal cells. Furthermore, the tumor cells from regions with a rich presence of adjacent α-SMA-expressing stromal cells revealed an upregulation of matrix metalloproteinase-9 (MMP9) expression in grade 3 tumors. This study provides an in-depth characterization of gene expression profiles in α-SMA-expressing stromal and tumor cells, and outlines potential crosstalk mechanisms.

Spatial Metabolomic Profiling of Pinelliae Rhizoma from Different Leaf Types Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging.

Pinelliae Rhizoma (PR), a highly esteemed traditional Chinese medicinal herb, is widely applied in clinical settings due to its diverse pharmacological effects, including antitussive, expectorant, antiemetic, sedative-hypnotic, and antitumor activities. Pinellia ternata exhibits morphological variation in its leaves, with types resembling peach, bamboo, and willow leaves. However, the chemical composition differences among the corresponding rhizomes of these leaf phenotypes remain unelucidated. This pioneering research employed Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI-MSI) to conduct the in situ identification and spatial profiling of 35 PR metabolites in PR, comprising 12 alkaloids, 4 organic acids, 12 amino acids, 5 flavonoids, 1 sterol, and 1 anthraquinone. Our findings revealed distinct spatial distribution patterns of secondary metabolites within the rhizome tissues of varying leaf types. Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) effectively differentiated between rhizomes associated with different leaf morphologies. Furthermore, this study identified five potential differential biomarkers-methylophiopogonanone B, inosine, cytidine, adenine, and leucine/isoleucine-that elucidate the biochemical distinctions among leaf types. The precise tissue-specific localization of these secondary metabolites offers compelling insights into the specialized accumulation of bioactive compounds in medicinal plants, thereby enhancing our comprehension of PR's therapeutic potential.

Spatiotemporal Exploration of Ridesharing Services Ridership through Geovisualization: A Case Study of the New York City Region

App-based ridesharing services (RSSs), exemplified by platforms like Uber, play a pivotal role in modern transportation by offering convenient and on-demand services. The exploration of RSSs necessitates a comprehensive consideration of the inherent spatiotemporal variability within the data. Prior research, however, has tended to analyze the spatial and temporal dimensions separately, with many studies omitting the temporal aspect. This study addresses the gap by using geovisualization techniques to illustrate emerging hot spot analysis in New York City in 2022, derived from space–time data mining. Overall, despite temporal variations in overall RSSs ridership, certain taxi zones maintain distinct ridership patterns. Across the five New York City boroughs (Manhattan, Bronx, Queens, Brooklyn, and Staten Island), Midtown Manhattan and the Brooklyn areas adjacent to Queens exhibit saturated intensifying hot spots, signaling a notable increase in RSSs ridership throughout 2022, surrounded by sporadic hot spots. Conversely, peripheral areas of New York City reveal diminishing cold spots, indicating a decrease in their intensity as cold spots. Furthermore, the study conducts separate spatial and temporal profiling. By presenting the spatiotemporal trends of RSSs, this research complements existing literature and provides valuable insights for more informed interventions. The study also highlights certain limitations that could be addressed in future endeavors.