Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2023.11.02.565001

Feasibility Study Utilizing NanoString Digital Spatial Profiling (DSP) Technology for Characterizing the Immune Microenvironment in Barrett's Esophagus FFPE Tissues

Ain, Q. u.·Frei, N.·Khoshiwal, A. M.·Stougie, P.·Odze, R.·Ferri, L.·Duits, L. C.·Bergman, J.·Stachler, M. D.
View original source ↗Export metadata ↓

Abstract

To date, characterization of the Barretts esophagus (BE) immune microenvironment in patients with known progression status to determine how the microenvironment may influence BE progression to esophageal adenocarcinoma (EAC) has been understudied, hindering both the biological understanding of progression and the development of novel diagnostics and therapies. Therefore, this studys aim was to determine if highly multiplex interrogation of the immune microenvironment can be performed on endoscopic formalin-fixed, paraffin-embedded (FFPE) samples utilizing the Nanostring GeoMx digital spatial profiling (GeoMx DSP) platform. We performed spatial proteomic analysis of 49 proteins expressed in the microenvironment and epithelial cells of histologically identical FFPE endoscopic biopsies from patients with non-dysplastic BE (NDBE) who later progressed to high-grade dysplasia (HGD) or EAC (N=7) or from patients who after at least 5 years follow up did not (N=8). In addition, we performed RNA analysis of 1,812 cancer related transcripts on a series of three endoscopic mucosal resections containing regions of normal tissue, BE, dysplasia (DYS), and EAC. Our primary goal was to determine feasibility of this approach and begin to identify the types of specific immune cell populations that may mediate the progression of pre-neoplastic BE to EAC. Spatial proteomic and transcriptomic profiling with GeoMx DSP showed reasonable quality metrics and detected expected differences between epithelium and stroma. Several proteins were found to have increased expression within non-dysplastic BE biopsies from progressors compared to non-progressors, suggesting further studies on the BE microenvironment are warranted. SummaryNew biological insights into the stepwise development and progression of esophageal adenocarcinoma (EAC) from Barretts esophagus (BE) are imperative to develop tailored approaches for early detection and optimal clinical management of the disease. This study aimed to determine the feasibility to spatially profile stromal and immunologic properties that accompany malignant transformation of BE to EAC in formalin-fixed, paraffin-embedded (FFPE) tissues. NanoStrings Digital Spatial Profiling (DSP) technology can detect and quantify protein and RNA transcripts in a highly multiplexed manner with spatial resolution, within specific regions of interest on FFPE tissue. Here, we performed a pilot study using the Nanostring GeoMx DSP, for measurement of protein and ribonucleic acid (RNA) expression on a series of FFPE slides from endoscopic biopsies and endoscopic mucosal resections (EMR) of BE. We compare a small series of biopsies of non-dysplastic BE (NDBE) from patients who progressed to more advanced disease to patients with NBDE who did not progress and then perform RNA profiling on EMRs with a range of histologic diagnoses.

Explore related subjects

cancer biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Ain, Q. u., Frei, N., Khoshiwal, A. M., Stougie, P., Odze, R., Ferri, L., Duits, L. C., Bergman, J., Stachler, M. D.. 2023-11-02. Feasibility Study Utilizing NanoString Digital Spatial Profiling (DSP) Technology for Characterizing the Immune Microenvironment in Barrett's Esophagus FFPE Tissues. https://doi.org/10.1101/2023.11.02.565001

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Tet2-Clonal Hematopoiesis Promotes Whereas Dnmt3a-CH Inhibits Breast Cancer Progression During Aging

Clonal hematopoiesis of indeterminate potential (CHIP) driven by somatic mutations in blood cells increases with age and is associated with adverse outcomes in cancer patients. How CHIP mutations influence solid tumor progression remains unclear. Using inducible murine models of Dnmt3a- or Tet2-mutant CHIP combined with orthotopic syngeneic breast cancer models, we demonstrate that upon aging Dnmt3a-mutant CHIP prevents whereas Tet2-mutant CHIP drives breast cancer progression. Dnmt3a-mutant immune infiltrates established interferon-mediated MHCIIhigh tumor-associated macrophages (TAMs) that supported cytotoxic CD8+ T cell-mediated tumor rejection. In contrast, Tet2-mutant TAMs exhibited an inflammatory, immunosuppressive phenotype characterized by increased IL-1 and CXCL2 production, reduced MHCII expression, and impaired T cell recruitment and activation. Therapeutic blockade of IL-1R1 or CXCR2 reduced tumor progression in aged Tet2-mutant CHIP mice and partially restored anti-tumor immunity. These findings establish that the effects of CHIP on cancer are mutation- and age-dependent and identify IL-1/CXCL2-driven inflammation as a therapeutic vulnerability of tumor-promoting Tet2-mutant CHIP.

cancer biology↗
bioRxiv2026

Single Nucleus Profiling Reveals Rewired Tumor-Immune-Stromal Cell Communication in Plasmacytoid Urothelial Carcinoma

Plasmacytoid urothelial carcinoma (PUC) is an aggressive histologic subtype of bladder cancer associated with worse patient outcomes and therapeutic response compared to conventional urothelial carcinoma (CUC). While recent work has demonstrated metastatic lymph nodes from PUC tumors have an inflamed phenotype, how the tumor microenvironment (TME) and crosstalk between tumor, stromal, and immune cells in primary PUC bladder tumors differs from those in CUC remains poorly understood. The impact of these differences on response to immunotherapy is also unknown. To understand TME differences between CUC and PUC and their impact on cell signaling, we performed single nucleus RNA sequencing on PUC and CUC tumors, as well as Xenium spatial transcriptomics on a mixed CUC / PUC tumor. We identified increased signaling between PUC-enriched, CDH1-low urothelial cells, immunosuppressive lipid-associated macrophages, and SFRP2+/CTHRC1+ fibroblasts in PUC tumors compared to CUC. Spatial transcriptomics identified increased macrophage infiltration in PUC histology and colocalization of PUC tumor cells, immunosuppressive macrophages, and SFRP2+ fibroblasts. Taken together, this study delineates PUC architecture and demonstrates interplay CDH1-low tumor cells, EMT, and immunosuppressive macrophages, identifying possible therapeutic vulnerabilities in PUC.

cancer biology↗
bioRxiv2026

The Phospholipase A2 Activity of Peroxiredoxin 6 Protects Hepatocarcinoma Cells Against Ferroptosis and Mitophagy Impairment

Peroxiredoxin 6 (PRDX6) is a moonlighting protein that plays a key role in regulating redox homeostasis and lipid metabolism through its unique dual peroxidase and calcium-independent phospholipase A2 (iPLA2) activities. Here, we generated a stable HepG2 cell line harboring a D140A mutation to selectively ablate iPLA2 activity, and we compared its phenotype with that of wild-type (WT) and PRDX6-knockout (KO) cells. D140A exhibited low ROS leves, similar to WT cells, confirming that oxidative stress is due to peroxidase deficiency. In contrast, D140A cells displayed higher lipid peroxidation than WT cells, though significantly lower than that of KO cells. Furthermore, the D140A mutant mirrored the KO phenotype regarding intracellular ferrous iron accumulation, impaired lipid droplet biogenesis, and the downregulation of the key ferroptosis regulator GPX4. Mechanistically, integrated proteomic, lipidomic, and enzymatic analyses revealed that a deficiency in iPLA2 activity of PRDX6 disrupted the release of key lipid species necessary for cell structure and signaling. Consequently, organelles involved in lipid synthesis and trafficking, such as peroxisomes, endoplasmic reticulum, mitochondria and lipid droplet, were affected. Specifically, we found that mitochondria exhibited a reduction in cristae and a diminished capacity for mitophagy and autophagy, which ultimately makes cells much more susceptible to erastin-induced ferroptotic cell death. Taken together, our findings demonstrate that PRDX6 iPLA2 activity uniquely coordinates organelle lipid homeostasis and mitophagy to protect cancer cells from ferroptotic cell death. Consequently, the use of iPLA2 inhibitors such as MJ33, which decreased PRDX6 iPLA2 activity to a level comparable to the mutant D140A, could represent a promising therapeutic strategy for inducing ferroptotic cell death in hepatocellular carcinoma.

cancer biology↗