Fig 2: Schematic of sandwich ELISA, the sensitivity and specificity to detect ofCSPGs.a Schematic of sandwich ELISA workflow. The figure was created with Biorender.com. b Colocalization of ofCS with core proteins detected by rVAR2-3 and anti-CD44, CSPG4, and SDC1 antibodies (n = 3, biologically independent experiments). P = 0.0039 and P < 0.0001 for CD44 vs. CSPG4 and CD44 vs. SDC1 in A549, P = 0.0001 and P = 0.0025 for CD44 vs. CSPG4 and CD44 vs. SDC1 in SW480, P = 0.0549 and P = 0.0002 for CD44 vs. CSPG4 and CD44 vs. SDC1 in SW620. R = 1 represents perfect correlation. Scale bar = 10 μm. c The sensitivity of ELISA for ofCS-CD44, -CSPG4, and -SDC1 biomarkers. The cancer cell line (SW480) spiked samples were tested. d The ELISA specificity was detected by the competitive assay with gradient dilution of CSA (3.75 × 10−5–7.5 mg/mL), decorin, and chondroitinase-treated cancer cell line or plasma samples (n = 3, 2, and 2, respectively, biologically independent experiments). The chondroitinase treatment of the cancer cell line and patient plasma sample significantly reduced the rVAR2 binding (P = 0.0062 and P < 0.0001 respectively). No significant rVAR2 binding was detected in the decorin spiking sample indicating specific capture of the anti-CD44 antibody. Student t-test, all the tests were two-sided, data were shown as mean ± SD, ns. no significant, *p value <0.05, **p value <0.01, ***p value <0.001, ****p value <0.0001).

Fig 3: CD44 translocation to the nucleus and delayed ‘targeted nuclear degranulation’ mediated by CD44/ERM/F‐actin. (A) Neutrophils were fluorescently stained for nuclear membrane, cell membrane and CD63, respectively. After stimulation of neutrophils with PMA (100 nM), CD63 co‐localization with the cell membrane (15 min) was earlier than CD63 co‐localization with the nuclear membrane (20 min) (scale bar: 5 μm). (B) PMA (100 nM) stimulates neutrophils for 0–20 min. Immunofluorescence staining of nuclear membrane (LaminB1,blue), skeleton (F‐actin, red) and CD44 (green), respectively. (C) PMA (100 nM) stimulates neutrophils for 0–20 min. WB for changes in nuclear membrane CD44, ERM and CD43 content changes. ERM phosphorylation inhibitor (P‐ERMi, 10 µM, 30 min) pre‐incubation of neutrophils for 30 min. WB detection of intracellular MPO, histone (H3) changes. (D) WB and ELISA for changes in intranuclear, cytoplasmic and extracellular CD44 content, flow cytometry for changes in cytosolic CD44 content. (E and F) P‐ERMi (10 µM, 30 min) pre‐incubation of neutrophils. (E) PMA (100 nM) stimulates neutrophils for 30 min, immunofluorescence staining for nuclear (DAPI,blue), P‐ERM (green) and CD44 (red). (F) PMA (100 nM) stimulates neutrophils for 120 min, SYTOX fluorescence staining. (G) Importini pre‐incubation of neutrophils, immunofluorescence staining of nuclear membrane (LaminB1,blue), skeleton (F‐actin, red) and CD44 (green). (H) Importini (30 µM) pre‐incubation of neutrophils, WB detection of nuclear membrane CD44 and changes in nuclear MPO content

Fig 4: Validation of plasma ofCS modified CD44 in different types of the malignant tumor.a–c ROC curves analysis for the diagnosis of all stages of the pan-cancer set in validation study 1. d ROC curves analysis for the diagnosis of all stages (blue) of the pan-cancer set consisting of nine solid malignant tumor types except for hepatocellular carcinoma and early-stage patients (red) using the plasma ofCS-CD44. The dotted diagonal line denotes an AUC of 0.50. e–m ROC curves of all-stage (blue) and early-stage (red) bladder, esophageal squamous cell carcinoma, gastric, nasopharyngeal, lung, pancreatic, breast, and cervical cancer, respectively. n, o The OR and 95% CIs of the plasma ofCS-CD44 and cancer risk by decile at validation study for all stage patients of pan-cancer (n = 13,735) (n) and early-stage patients (n = 891) (o). Multiple logistic regression analysis was used to calculate the ORs adjusted for sex and age. All the tests were two-sided. The solid dots in the center for the error bars are the OR values, and the error bars are the corresponding 95% confidence intervals of the ORs. The dashed lines represent the OR values for samples with ofCS-CD44 ≥90% (upper line) and ofCS-CD44 <20% (lower line).

Fig 5: rVAR2 modified sandwich ELISA to detect plasma ofCS/ofCSPG in cancer patients at discovery study.a The study designed for the development and validation of a novel plasma oncofetal chondroitin sulfated proteoglycans for pan-cancer detection. b–e Plasma ofCS-CD44, -SDC1, -CSPG4 and total ofCS levels (median with inter-quartile range) from healthy individuals (n = 302) versus cancer patients with tongue cancer (n = 29), esophageal squamous cell carcinoma (ESCC, n = 27), nasopharyngeal carcinoma (NPC, n = 27), clear cell renal cell carcinoma (ccRCC, n = 27), colorectal cancer (CRC, n = 26), and bladder cancer (BLCA, n = 29), respectively. P for Mann–Whitney U-test. The middle line in the boxplot displays the median, and the box indicates the first and third quartiles. Value in each figure was compared with the first group. All the tests were two-sided. f–i ROC curves analysis for the diagnosis of malignant tumor patients (n = 165) from healthy individuals (n = 302) using the plasma ofCS, ofCS-CD44, -SDC1, and -CSPG4. The dotted diagonal line denotes an AUC of 0.50.