C5b-9 Membrane Attack Complex Formation and Extracellular Vesicle Shedding in Barrett’s Esophagus and Esophageal Adenocarcinoma

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.

Discovery and validation of serum glycoprotein biomarkers for high grade serous ovarian cancer

“…Interestingly, AAL‐C9 glycoform levels have been reported to be elevated in lung and stomach cancers, intermediate in hepatocellular carcinoma and low in breast cancer [ 39 ]. Recently, we reported the release of C9 + EVs by esophageal adenocarcinoma cells as a potential mechanism of the elevated serum C9 glycoform in esophageal cancer [ 42 ]. However, the specific glycosylation differences in cancer serum and EVs, as well as the molecular mechanisms underpinning the altered C9 glycosylation in different cancer types remains to be determined.…”

The neoepitope of the complement C5b-9 Membrane Attack Complex is formed by proximity of adjacent ancillary regions of C9

“…Historically, aE11 has been used to detect C5b-9 deposition in a number of diseases, both experimentally and clinically. For example, aE11 has been used to detect MAC deposition associated with cancer, bowel, neurological, and kidney diseases [32][33][34][35][36][37][38] , as well as MAC in macrophage inflammasome activation 39 . Finally, in the development of the total complement activity ELISA, aE11 was compared with several other anti-C9 neoepitope antibodies, and found to be the most specific and the one with highest affinity 40 .…”

Birth and Death in Terminal Complement Pathway

“…Being one of the most abundant proteins in the drusen (Crabb et al, 2002; Hollborn et al, 2018), complement C9 is also referred to as ARMD15 (Age-Related Macular Degeneration 15) and has long been associated with Age-Related macular degeneration (Kremlitzka et al, 2018; Zipfel et al, 2006) and multiple sclerosis (Morgan et al, 1984). The MAC has a complex interaction with cancer cells at different stages (Fishelson and Kirschfink, 2019), and elevated levels of C9 have also been associated with esophageal adenocarcinoma (Joshi et al, 2017; Kolka et al, 2022) and colorectal cancer (Chantaraamporn et al, 2020). The complement system has considerable crosstalk with integrins to regulate single-cell metabolism (Merle et al, 2021).…”