A novel multiplex detection array revealed systemic complement activation in oral squamous cell carcinoma

Abstract

Oral squamous cell carcinoma (OSCC) is one of the most common tumors within the oral cavity. Early diagnosis and prognosis tools are urgently needed. This study aimed to investigate the activation of the complement system in OSCC patients as potential biomarker. Therefore, an innovative complement activation array was developed. Characterized antibodies detecting the complement activation specific epitopes C3a, C5a and sC5b-9 along with control antibodies were implemented into a suspension bead array. Human serum from a healthy (n = 46) and OSCC patient (n = 57) cohort were used to investigate the role of complement activation in oral tumor progression. The novel multiplex assay detected C3a, C5a and sC5b-9 from a minimal sample volume of human tears, aqueous humor and blood samples. Limits of detection were 0.04 ng/mL for C3a, 0.03 ng/mL for C5a and 18.9 ng/mL for sC5b-9, respectively. Biological cut-off levels guaranteed specific detections from serum. The mean serum concentration of a healthy control cohort was 680 ng/mL C3a, 70 ng/mL C5a and 2247 ng/mL sC5b-9, respectively. The assay showed an intra-assay precision of 2.9-6.4% and an inter-assay precision of 9.2-18.2%. Increased systemic C5a (p < 0.0001) and sC5b-9 (p = 0.01) concentrations in OSCC patients were determined using the validated multiplex complement assay. Higher C5a concentrations correlated with tumor differentiation and OSCC extension state. Systemic sC5b-9 determination provided a novel biomarker for infiltrating tumor growth and C3a levels were associated with local tumor spreading. Our study suggests that systemic complement activation levels in OSCC patients may be useful to assess disease progression.

Keywords: C3a; C5a; complement proteins; mulitplex assay; oral squamous cell carcinoma.

Figure 1. Principle for multiplex-complement activation assay

(A) The complement system is a part of the innate immunity and activated by three different pathways: the classical, the lectin, and the alternative pathway. Activation results in enzymatic cleavage of C3 to C3a and C3b. The latter triggers the terminal complement pathway by cleavage of C5 and the formation of the terminal complement complex sC5b-C9. The formation of sC5b-C9 and the anaphylatoxins C3a and C5a determines complement activation. (B, C) The detection of the complement activation products was performed in multiplex suspension bead array based on the x-MAP technology (Luminex^®). (B) Activation product specific antibodies were immobilized to distinct fluorescent, magnetic beads to capture either C3a, C5a or sC5b-C9 from solution, respectively. Antigen specific biotinylated antibodies and phycoerythrin conjugated to streptavidin detected the captured activation products simultaneously. (C) False positive signals by cross-reacting human anti-species antibodies and in vitro complement activation were excluded by utilizing an appropriate reaction buffer.

Figure 2. Complement activation markers were simultaneously and sensitively quantified in the multiplex system

(A) Serial dilutions of native, human C3a-desArg (black circle), recombinant, human C5a-desArg (black square) and native human sC5b-C9 (black triangle) depicted different assay performances for the simultaneous analysis of complement activation markers. The target antigens were only detected by respective coupled magnetic beads and not by control beads (grey). Limit of detection (orange) and assay cutoffs (red square) for each analyte are shown. (B) The concentration range of complement activation markers determined with the novel multiplex assay from a human serum pool (black square with dot) corresponded to previously reported ranges for C3a, C5a and sC5b-C9 single detection either from plasma (grey), serum (black) or unknown sample matrix (white), respectively [–62].

Figure 3. Bead-specific detection of purified antigens in singleplex and multiplex assays

(A, B, D, F) Specificity of C3a-beads, (C, B, D, F) C5a-beads and (E, B, D, F) sC5b-C9-beads were either analysed in a (A, C, E) one-bead assay with a single antigen dilution series or (B, D, F) all five bead regions and a singular antigen dilution series. (B, D, F) All beads showed a specific signal for the respective antigen and no cross reactivities. The detection of (A, B) C3a, (C, D) C5a and (E, F) sC5b-C9 was comparable in the (A, C, E) single and (B, D, F) multiplex assays, indicating a bead-specific detection.

Figure 4. The multiplex assay detected complement activation products specifically from complement component-depleted human sera

(A–C) C3a, C5a and sC5b-C9 were quantified either in normal human control serum (control) or C2-, C3-, C4-, C5-, C6-, C7-, C8-, C9-, properdin-, complement factor H-depleted serum. The lowest concentrations for (A) C3a and (B) C5a were observed in C3-depleted or C5-depleted serum, respectively. (C) The terminal complement complex was detected at a reduced level in C5-depleted serum. We did not observe a detection signal for any of the tested sera on the control beads (data not shown).

Figure 5. Simultaneous quantification of complement activation markers from aqueous humor, tears and sera of OSCC patients and controls

C3a and C5a were either measured in (A) aqueous humor of glaucoma and cataract patients, (B) in tears of healthy controls or (C–E) in sera of OSCC-patients (n = 57) and matched controls (n = 46) using the validated 5-plex immunoassay based on luminex technology. (A) The C3a levels of glaucoma and cataract patients did not show any significant difference, but a tendency for higher C3a levels in glaucoma patients. C5a concentrations in aqueous humor were below the lower detection limit. (B) Quantification of C3a and C5a in tears ranged between 4.4–26 ng/mL for C3a and 0.11–3.3 ng/mL for C5a, respectively. (D, E) C5a and sC5b-C9 concentrations were significantly increased in OSCC patients compared to the control group. Mean with standard deviations are depicted. (^**p < 0.01, ^****p < 0.0001 two-tailed, unpaired t-test).

Figure 6. Complement activation markers were associated with tumor differentiation (G), tumor extension (T) and lymph node metastases (N)

(A–C) The histologic grade (G) describes the cell differentiation in the patients’ tumors/ neoplasms. Patients with G2 tumor grading had significantly elevated (A) C3a, as well as (B) C5a and (C) sC5b-9 serum levels compared to the control group. (D–F) The T-classification system categorizes the extension of the patients′ primary tumors. (D) There was no relation between C3a serum levels and T-classification. (E) All tumor extension classifications (T-classifications) showed a significant increase in systemic C5a levels compared to the control group. (F) Invasive growth (T4) is associated with increased sC5b-9 serum levels compared to the control, T2 and T3 group. (G–J) The N-classification describes the degree of spread of tumor cells to regional lymph nodes. (G) C3a concentration was significantly correlated with tumor spread to lymph nodes. C3a concentration was highest in N2 classified lymph node metastases. (H, I) There was no significant correlation between complement markers C5a, sC5b-C9 and lymph node metastases. (J) Patients, that presented a N2-tumor, were significantly younger than patients with a N0 or N1 tumor. (K–M) Age was not associated with T-classification or correlated with complement activation marker concentration in serum. (^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001 two-tailed, unpaired t-test).