Wilfrido Mojica

Supplementary Figure S4. Repeatability and reproducibility of SPR assay in detecting exosomal TF-Ag-α. The levels of exosomal TF-Ag-α in serum samples from one normal control, one lung cancer patient and one breast cancer patient were measured by operator 1 (a) and operator 2 (b) on 3 different days, 3 replicates per day. The mean, standard deviation, and coefficient of variation (CV) were reported. (c) Pearson correlation of results from operator 1 and operator 2.

Supplementary Figure S1. SPR assay detects exosomal TF-Ag-α for cancer diagnosis. Representative SPR curves for the detection of exosomal TF-Ag-α in serum samples from (a) a Stage II lung cancer patient using a biochip modified with IgG3 negative control antibodies; (b) a Stage 0 breast cancer patient using a biochip modified with IgG3 negative control antibodies, (c) a male normal control using a biochip modified with JAA-F11 antibodies, (d) a male normal control using a biochip modified with IgG3 negative control antibodies, (e) a female normal control using a biochip modified with JAA-F11 antibodies, and (f) a female normal control using a biochip modified with IgG3 negative control antibodies.

Supplementary Figure S2. SPR assay demonstrated exosomes carry TF-Ag-α. Exosomes were isolated from cancer patient serum samples using both total exosome isolation kit (from serum) (ThermoFisher) and exoRNeasy midi kit (Qiagen). Significantly higher levels of TF-Ag-α were observed in exosome samples isolated using both kits than the exosome-depleted serum samples. The exosome concentration was 3.29x10^11 exosomes/mL.

Supplementary Figure S3. Sensing performance of SPR assay in detecting exosomal TF-Ag-α. Exosomes from A549 NSCLC cells (a) and MDA-MB-231 breast cancer cells (b) were spiked in the serum of a normal control at concentrations of 0 to 10^11 exosomes/mL. The expression of exosomal TF-Ag-α was measured using the SPR assay. For A549 cell-derived exosomes, the LOD was 5×10^9 exosomes/mL and the linear range was from 5×10^9 to 10^11 exosomes/mL. For MDA-MB-231 cell-derived exosomes, the LOD was 10^9 exosomes/mL and the linear range was from 10^9 to 10^11 exosomes/mL.

Supplementary Table S2. Characteristics of Breast Cancer Patients and Controls.

Supplementary Table S1. Characteristics of Lung Cancer Patients and Controls.

Exosomes are nanosized extracellular vesicles released by cells to transport biomolecules such as proteins and RNAs for intercellular communication. Exosomes play important roles in cancer development and metastasis; therefore, they have emerged as potential liquid biopsy biomarkers for cancer screening, diagnosis, and management. Many exosome cargos, including proteins, RNAs, and lipids, have been extensively investigated as biomarkers for cancer liquid biopsy. However, carbohydrates, an important type of biomolecule, have not yet been explored for this purpose. In this study, we reported a new exosomal carbohydrate biomarker, α-linked Thomsen–Friedenreich glycoantigen (TF-Ag-α; Galβ1-3GalNAc-α). To translate our discovery into clinical settings, we developed a surface plasmon resonance–based assay which utilized a unique mAb, JAA-F11, with high specificity to measure the levels of exosomal TF-Ag-α in blood. To the best of our knowledge, we are the first to demonstrate that exosomes carry TF-Ag-α. We detected exosomal TF-Ag-α in as low as 10 μL serum samples from patients with cancer, but in contrast, levels were negligible in those from normal controls. With a total of 233 patients with cancer and normal controls, we showed that exosomal TF-Ag-α detected lung cancer (n = 60) and breast cancer (n = 95) from normal controls (n = 78) with ≥95% and ≥97% accuracy, respectively. These results demonstrated that exosomal TF-Ag-α is a potential liquid biopsy biomarker for cancer diagnosis.Significance:Exosomes or small extracellular vesicles have emerged as potent biomarkers of cancer liquid biopsy. We discovered a new exosomal carbohydrate marker, TF-Ag-α (Galβ1-3GalNAc-α), and showed that exosomal TF-Ag-α detected both lung and breast cancers with >95% accuracy. Our findings demonstrated that exosomal TF-Ag-α is a promising liquid biopsy biomarker for cancer screening and early detection.

ROC analysis to evaluate diagnostic performance of exosomal TF-Ag-α in breast cancer. ROC curves of comparisons between all patients with breast cancer (n = 70) and normal controls (n = 29; A); nonmetastatic breast cancer (stages 0–III, n = 55) and normal controls (n = 29; B); metastatic breast cancer (stage IV, n = 15) and normal controls (n = 29; C); luminal A/B breast cancer (n = 44) and normal controls (n = 29; D); triple-negative breast cancer (n = 9) and normal controls (n = 29; E); and HER2-positive breast cancer (n = 17) and normal controls (n = 29; F). The sensitivity (SEN), specificity (SPE), AUC, and cutoff values of exosomal TF-Ag-α for each comparison are summarized in G.

Supplementary Figure S4. Repeatability and reproducibility of SPR assay in detecting exosomal TF-Ag-α. The levels of exosomal TF-Ag-α in serum samples from one normal control, one lung cancer patient and one breast cancer patient were measured by operator 1 (a) and operator 2 (b) on 3 different days, 3 replicates per day. The mean, standard deviation, and coefficient of variation (CV) were reported. (c) Pearson correlation of results from operator 1 and operator 2.

Supplementary Table S1. Characteristics of Lung Cancer Patients and Controls.