C5b-9 Membrane Attack Complex Formation andExtracellular Vesicle Shedding in Barrett's Esophagus and Esophageal Adenocarcinoma.

Cathryn M Kolka,Ian Brown,Wioleta M Zelek,B Paul Morgan,Sandra Brosda,Michelle M Hill,Matt Trau,Alain Wuethrich,Ailin Lepletier,Yilang Cao,Alok K Shah,Ramlah Khamis,Guy D Eslick,Renee S Richards,Karthik B Shanmugasundaram,Andrew Barbour,Julie Webster,Nicholas J Clemons,Clay Winterford

doi:10.3389/fimmu.2022.842023

Abstract

The early complement components have emerged as mediators of pro-oncogenic inflammation, classically inferred to cause terminal complement activation, but there are limited data on the activity of terminal complement in cancer. We previously reported elevated serum and tissue C9, the terminal complement component, in esophageal adenocarcinoma (EAC) compared to the precursor condition Barrett’s Esophagus (BE) and healthy controls. Here, we investigate the level and cellular fates of the terminal complement complex C5b-9, also known as the membrane attack complex. Punctate C5b-9 staining and diffuse C9 staining was detected in BE and EAC by multiplex immunohistofluorescence without corresponding increase of C9 mRNA transcript. Increased C9 and C5b-9 staining were observed in the sequence normal squamous epithelium, BE, low- and high-grade dysplasia, EAC. C5b-9 positive esophageal cells were morphologically intact, indicative of sublytic or complement-evasion mechanisms. To investigate this at a cellular level, we exposed non-dysplastic BE (BAR-T and CP-A), high-grade dysplastic BE (CP-B and CP-D) and EAC (FLO-1 and OE-33) cell lines to the same sublytic dose of immunopurified human C9 (3 µg/ml) in the presence of C9-depleted human serum. Cellular C5b-9 was visualized by immunofluorescence confocal microscopy. Shed C5b-9 in the form of extracellular vesicles (EV) was measured in collected conditioned medium using recently described microfluidic immunoassay with capture by a mixture of three tetraspanin antibodies (CD9/CD63/CD81) and detection by surface-enhanced Raman scattering (SERS) after EV labelling with C5b-9 or C9 antibody conjugated SERS nanotags. Following C9 exposure, all examined cell lines formed C5b-9, internalized C5b-9, and shed C5b-9+ and C9+ EVs, albeit at varying levels despite receiving the same C9 dose. In conclusion, these results confirm increased esophageal C5b-9 formation during EAC development and demonstrate capability and heterogeneity in C5b-9 formation and shedding in BE and EAC cell lines following sublytic C9 exposure. Future work may explore the molecular mechanisms and pathogenic implications of the shed C5b-9+ EV.

Highlights

Recent studies have generated strong evidence of tumorpromoting actions of early complement components in the tumor microenviroment (TME) [1]
tissue microarray (TMA) sections co-stained for C9 and C5b-9 were evaluated for staining intensity in regions of specific tissue phenotypes (NSE: normal squamous epithelium, Barrett’s esophagus (BE): Barrett’s Esophagus, LGD: Low grade dysplasia, HGD: high grade dysplasia, HGD+IEC: HGD with intraepithelial carcinoma, esophageal adenocarcinoma (EAC)) using a semiquantitative scoring system from 0 to 3 by a specialist gastrointestinal pathologist (Figure 1)
We developed and applied two novel techniques to show that the local activation of complement pathway leads to C9 deposition and C5b-9/membrane attack complex (MAC) formation on the epithelial component of BE and EAC, and Extracellular Vesicles (EV) shedding of C9 and C5b-9

Summary

Introduction

Recent studies have generated strong evidence of tumorpromoting actions of early complement components in the tumor microenviroment (TME) [1]. The complement system is a protease cascade of the innate immune system of plasma proteins that triggers inflammation and helps immune cells to fight infections following activation by immune complexes, apoptotic cells (classical pathway) or foreign sugar motifs (lectin pathway). There is a constitutive low sentinel activity via the alternative pathway. Each pathway leads to the cleavage of the central component C3 into bioactive fragments C3a and C3b, activating the terminal pathway via C5 cleavage into C5a and C5b [1]. The C5b fragment binds with C6, C7, C8 and multiple units of C9 in the membrane, forming C5b-9, known as the membrane attack complex (MAC), in the cell membranes of cells and microorganisms to cause cell lysis

Methods

Results

Conclusion