Hsp70 in Liquid Biopsies-A Tumor-Specific Biomarker for Detection and Response Monitoring in Cancer

Abstract

In contrast to normal cells, tumor cells of multiple entities overexpress the Heat shock protein 70 (Hsp70) not only in the cytosol, but also present it on their plasma membrane in a tumor-specific manner. Furthermore, membrane Hsp70-positive tumor cells actively release Hsp70 in small extracellular vesicles with biophysical characteristics of exosomes. Due to conformational changes of Hsp70 in a lipid environment, most commercially available antibodies fail to detect membrane-bound and vesicular Hsp70. To fill this gap and to assess the role of vesicular Hsp70 in circulation as a potential tumor biomarker, we established the novel complete (comp)Hsp70 sandwich ELISA, using two monoclonal antibodies (mAbs), that is able to recognize both free and lipid-associated Hsp70 on the cell surface of viable tumor cells and on small extracellular vesicles. The epitopes of the mAbs cmHsp70.1 (aa 451-461) and cmHsp70.2 (aa 614-623) that are conserved among different species reside in the substrate-binding domain of Hsp70 with measured affinities of 0.42 nM and 0.44 nM, respectively. Validation of the compHsp70 ELISA revealed a high intra- and inter-assay precision, linearity in a concentration range of 1.56 to 25 ng/mL, high recovery rates of spiked liposomal Hsp70 (>84%), comparable values between human serum and plasma samples and no interference by food intake or age of the donors. Hsp70 concentrations in the circulation of patients with glioblastoma, squamous cell or adeno non-small cell lung carcinoma (NSCLC) at diagnosis were significantly higher than those of healthy donors. Hsp70 concentrations dropped concomitantly with a decrease in viable tumor mass upon irradiation of patients with approximately 20 Gy (range 18-22.5 Gy) and after completion of radiotherapy (60-70 Gy). In summary, the compHsp70 ELISA presented herein provides a sensitive and reliable tool for measuring free and vesicular Hsp70 in liquid biopsies of tumor patients, levels of which can be used as a tumor-specific biomarker, for risk assessment (i.e., differentiation of grade III vs. IV adeno NSCLC) and monitoring of therapeutic outcomes.

Keywords: Hsp70; glioblastoma; liquid biopsy; non-small cell lung carcinoma (NSCLC); prediction; response monitoring; sandwich ELISA; small extracellular vesicles; tumor biomarker.

Figure 1

Binding affinity of the cmHsp70.1 and cmHsp70.2 mAbs. The binding affinity of cmHsp70.1 (K_D 0.42 nM) (a) and cmHsp70.2 (K_D 0.44 nM) (b) to Hsp70 protein was measured using the Microscale Thermophoresis technique. The X-axis represents the concentration of the Hsp70 protein (M) and the Fnorm (‰) is displayed on the Y-axis. Arrows indicate the determined K_D values.

Figure 2

Western blot of recombinant Hsp70 protein and tumor cell lysates, flow cytometric analysis of membrane-bound Hsp70 on viable tumor cells, and dot blot analysis using BSA, recombinant Hsp27, Hsp60, Hsp70, Hsc70 as target proteins. (a) Hsp70 protein and lysates of tumor cells were subjected to an SDS gel and blots were stained with cmHsp70.1, cmHsp70.2, ctrl Hsp70A and ctrl Hsp70B antibodies. Representative immunoblots of recombinant Hsp70 (upper, 72 kDa) and tumor cell lysates (lower) representing Hsp70 (72 kDa), Hsc70 (73 kDa) and β-actin (40 kDa) as a loading control are shown. Molecular weight markers are indicating 70 kDa and 40 kDa. Detailed information about Western blot can be found at Supplementary Materials. (b) Detection of membrane-bound Hsp70 on A549, HCT116 cell lines by flow cytometry using the cmHsp70.1, cmHsp70.2, ctrl Hsp70A and ctrl Hsp70B antibodies (white histograms). Staining with the respective isotype-matched control antibodies is represented by grey histograms. As a control, PBL of healthy donors were stained with cmHsp70.1 and cmHsp70.2 mAb. The numbers in the histograms indicate the proportion of Hsp70 positively stained cells. (c) Dot blot analysis of BSA, recombinant Hsp27, Hsp60, Hsp70 and Hsc70 (100 ng, 10 ng, 1 ng, 0.1 ng) using cmHsp70.1, cmHsp70.2 and ctrl Hsp70B antibodies.

Figure 3

Representative eight-point calibration curve for measuring samples of healthy individuals. The X-axis and Y-axis represent the OD (a.u.) and the Hsp70 protein concentration (ng/mL), respectively.

Figure 4

Detection and recovery of liposomal Hsp70 in serum. Quantification (a) and recovery (b) of lipid-bound Hsp70 in artificial lipid vesicles (liposomal Hsp70: 500 ng/mL), as determined by the sandwich Hsp70 ELISA using the cmHsp70.2 mAb as a coating antibody, and cmHsp70.1 and ctrl Hsp70A mAbs as detection antibodies. The data represent mean values of three independent experiments, *** p < 0.001, a two-sided t-test was used.

Figure 5

Interference factors potentially influencing the Hsp70 concentrations measured by the compHsp70 ELISA. (a) Comparison of the Hsp70 concentrations in plasma and serum. Plasma (black bars) and serum (grey bars) from 13 healthy individuals were taken at the same time. (b) Hsp70 concentrations in the serum of 17 healthy individuals taken before (black bars) and 2 h after a high fat diet (grey bars). (c) Hsp70 concentration of 108 healthy volunteers in different age groups ranging from 21–77 years. Characteristics of the healthy donors are summarized in Table 3. A Pearson correlation test was performed.

Figure 6

Serum and exosomal Hsp70 concentrations in healthy donors and tumor patients. (a) Serum Hsp70 concentrations in healthy individuals (n = 108), patients with NSCLC (n = 166) and HGG (n = 34), as measured using the compHsp70 ELISA. Significantly higher Hsp70 levels were found in both tumor patient cohorts compared to healthy controls (* p < 0.05, *** p < 0.001, Mann-Whitney U Test). (b) ROC curve analysis using the data shown in (a). (c) Serum Hsp70 concentrations in squamous cell carcinoma patients stage III (234.4 ± 29.2 ng/mL; n = 59) and IV (321.2 ± 68.8 ng/mL; n = 12) as well as adeno carcinoma patients stage III (260.3 ± 75.3 ng/mL; n = 24) and IV (561.3 ± 173.4 ng/mL; n = 29) compared to Hsp70 levels of healthy individuals (35.1 ± 3.99 ng/mL; n = 108) (p < 0.001, two-sided t-test). Differences in serum Hsp70 concentrations in adeno carcinoma stage III and IV were significantly different (* p < 0.05; ANOVA Tukey test) (d) Serum Hsp70 concentrations in patients before radiotherapy (Before RT; 494.1 ± 72.2 ng/mL; n = 80), during radiotherapy (During RT; 310.5 ± 36.8 ng/mL; after approximately 20 Gy; n = 58) and after radiotherapy (After RT; 380.0 ± 51.8 ng/mL; n = 56), compared to healthy individuals (Healthy; 35.1 ± 4.0 ng/mL; n = 108), as measured by the compHsp70 ELISA (*** p < 0.001, t-test). Following a dose of approximately 20 Gy (range 18–22.5 Gy), the Hsp70 levels dropped significantly from 494.1 ± 72.2 to 310.5 ± 36.8 ng/mL (* p < 0.05, t-test). For pairwise comparisons, two-sided t-test was applied, for comparison of all groups, ANOVA Tukey test was used. (e) Hsp70 content in exosomes isolated from the plasma of a healthy control donor, a tumor patient, as determined by the compHsp70 ELISA and a control (ctrl) Hsp70 ELISA that only detects free Hsp70. Size and purity of the plasma-derived exosomes from the plasma was determined by dynamic light scattering. (f) Hsp70 content in exosomes isolated from the supernatant of tumor cells, as determined by the compHsp70 ELISA and a control (ctrl) Hsp70 ELISA and protein content in tumor cell lysates and exosomes, as determined by Western blot analysis. Detailed information about Western blot can be found at Supplementary Materials.

Figure 6

Serum and exosomal Hsp70 concentrations in healthy donors and tumor patients. (a) Serum Hsp70 concentrations in healthy individuals (n = 108), patients with NSCLC (n = 166) and HGG (n = 34), as measured using the compHsp70 ELISA. Significantly higher Hsp70 levels were found in both tumor patient cohorts compared to healthy controls (* p < 0.05, *** p < 0.001, Mann-Whitney U Test). (b) ROC curve analysis using the data shown in (a). (c) Serum Hsp70 concentrations in squamous cell carcinoma patients stage III (234.4 ± 29.2 ng/mL; n = 59) and IV (321.2 ± 68.8 ng/mL; n = 12) as well as adeno carcinoma patients stage III (260.3 ± 75.3 ng/mL; n = 24) and IV (561.3 ± 173.4 ng/mL; n = 29) compared to Hsp70 levels of healthy individuals (35.1 ± 3.99 ng/mL; n = 108) (p < 0.001, two-sided t-test). Differences in serum Hsp70 concentrations in adeno carcinoma stage III and IV were significantly different (* p < 0.05; ANOVA Tukey test) (d) Serum Hsp70 concentrations in patients before radiotherapy (Before RT; 494.1 ± 72.2 ng/mL; n = 80), during radiotherapy (During RT; 310.5 ± 36.8 ng/mL; after approximately 20 Gy; n = 58) and after radiotherapy (After RT; 380.0 ± 51.8 ng/mL; n = 56), compared to healthy individuals (Healthy; 35.1 ± 4.0 ng/mL; n = 108), as measured by the compHsp70 ELISA (*** p < 0.001, t-test). Following a dose of approximately 20 Gy (range 18–22.5 Gy), the Hsp70 levels dropped significantly from 494.1 ± 72.2 to 310.5 ± 36.8 ng/mL (* p < 0.05, t-test). For pairwise comparisons, two-sided t-test was applied, for comparison of all groups, ANOVA Tukey test was used. (e) Hsp70 content in exosomes isolated from the plasma of a healthy control donor, a tumor patient, as determined by the compHsp70 ELISA and a control (ctrl) Hsp70 ELISA that only detects free Hsp70. Size and purity of the plasma-derived exosomes from the plasma was determined by dynamic light scattering. (f) Hsp70 content in exosomes isolated from the supernatant of tumor cells, as determined by the compHsp70 ELISA and a control (ctrl) Hsp70 ELISA and protein content in tumor cell lysates and exosomes, as determined by Western blot analysis. Detailed information about Western blot can be found at Supplementary Materials.