Development of EndoScreen Chip, a Microfluidic Pre-Endoscopy Triage Test for Esophageal Adenocarcinoma

Abstract

The current endoscopy and biopsy diagnosis of esophageal adenocarcinoma (EAC) and its premalignant condition Barrett’s esophagus (BE) is not cost-effective. To enable EAC screening and patient triaging for endoscopy, we developed a microfluidic lectin immunoassay, the EndoScreen Chip, which allows sensitive multiplex serum biomarker measurements. Here, we report the proof-of-concept deployment for the EAC biomarker Jacalin lectin binding complement C9 (JAC-C9), which we previously discovered and validated by mass spectrometry. A monoclonal C9 antibody (m26 3C9) was generated and validated in microplate ELISA, and then deployed for JAC-C9 measurement on EndoScreen Chip. Cohort evaluation (n = 46) confirmed the expected elevation of serum JAC-C9 in EAC, along with elevated total serum C9 level. Next, we asked if the small panel of serum biomarkers improves detection of EAC in this cohort when used in conjunction with patient risk factors (age, body mass index and heartburn history). Using logistic regression modeling, we found that serum C9 and JAC-C9 significantly improved EAC prediction from AUROC of 0.838 to 0.931, with JAC-C9 strongly predictive of EAC (vs. BE OR = 4.6, 95% CI: 1.6–15.6, p = 0.014; vs. Healthy OR = 4.1, 95% CI: 1.2–13.7, p = 0.024). This proof-of-concept study confirms the microfluidic EndoScreen Chip technology and supports the potential utility of blood biomarkers in improving triaging for diagnostic endoscopy. Future work will expand the number of markers on EndoScreen Chip from our list of validated EAC biomarkers.

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

“…Some co-localization with LAMP-1 (lysosome marker, Figures 5C , D ) could be observed, in agreement with previous data for other cells. We were intrigued that shedding of MAC-decorated EVs from BE and EAC cells could be a potential mechanism of our initial observation of increased serum C9 ( 7 , 21 ). Therefore, we next aimed to quantify MAC-decorated EVs from cell culture supernatant.…”

“…For all 3 cell lines, no C9 + EV was detected in C9-depleted serum alone at 0 min ( Figure 6E ), confirming that lack of C9 + EV within the depleted serum. Incubation with C9-depleted serum alone for 30 minutes led to significant shedding of C9 + EV ( Figure 6E ), presumably due to the trace residual C9 in the C9-depleted human serum ( 21 ). Exposure to 3 μg/ml purified C9 without incubation ( Figure 5E , 0 min +C9) did not induce shedding of C9 + EV but lead to strong C9 + EV release after incubation at 37°C ( Figure 6E ).…”

“…Immunopurification of C9 from human serum, generation of C9-depleted human serum and their detailed characterization were previously reported ( 21 ). Cells were seeded on to coverslips.…”

“…C9 treatment was conducted at 37°C, 5% CO 2 in medium containing 10% C9-depleted human serum, with/without addition of 3µg/ml immunopurified C9. This dose was chosen to be lower than a previously published lytic dose of 20µg/ml in K562 cells ( 16 ), as well as the reported serum concentration of C9 in BE and EAC patients (10-15 µg/ml) ( 21 ). 3µg/ml of immunopurified C9 did not cause substantial cell lysis in our experimental set up, determined by lactate dehydrogenase assay in 3 cell types.…”

“…C9+ and C5b-9+ EV analysis was performed using an immunoassay integrated on a microfluidic device with asymmetric gold electrodes and surface-enhanced Raman scattering (SERS) read-out, as previously reported ( 21 ). The electrodes of this SERS assay were functionalized with anti-CD9/CD63/CD81 antibodies (CD9: clone 5G6; CD63: clone H5C6; CD8: clone 1D6; Novus Biologicals) to capture EVs in the sample.…”