Multiple recognition assay reveals prostasomes as promising plasma biomarkers for prostate cancer

Abstract

Prostasomes are microvesicles (mean diameter, 150 nm) that are produced and secreted by normal and malignant prostate acinar cells. It has been hypothesized that invasive growth of malignant prostate cells may cause these microvesicles, normally released into seminal fluid, to appear in interstitial space and therewith into peripheral circulation. The suitability of prostasomes as blood biomarkers in patients with prostate cancer was tested by using an expanded variant of the proximity ligation assay (PLA). We developed an extremely sensitive and specific assay (4PLA) for detection of complex target structures such as microvesicles in which the target is first captured via an immobilized antibody and subsequently detected by using four other antibodies with attached DNA strands. The requirement for coincident binding by five antibodies to generate an amplifiable reporter results in both increased specificity and sensitivity. The assay successfully detected significantly elevated levels of prostasomes in blood samples from patients with prostate cancer before radical prostatectomy, compared with controls and men with benign biopsy results. The medians for prostasome levels in blood plasma of patients with prostate cancer were 2.5 to sevenfold higher compared with control samples in two independent studies, and the assay also distinguished patients with high and medium prostatectomy Gleason scores (8/9 and 7, respectively) from those with low score (≤ 6), thus reflecting disease aggressiveness. This approach that enables detection of prostasomes in peripheral blood may be useful for early diagnosis and assessment of prognosis in organ-confined prostate cancer.

Fig. 1.

Mechanism of 4PLA. Target molecules are captured by antibodies immobilized on the walls of a reaction vessel (A), the four PLA probes are added (B), and the probes are allowed to bind different epitopes on the target structure. The four oligonucleotides attached to the antibodies hybridize to each other (C) and guide hybridization of a further oligonucleotide (D). This oligonucleotide that is added together with ligation/amplification mix is joined by enzymatic DNA ligation to oligonucleotides attached to two of the antibodies, templated by oligonucleotides on the two other antibodies. Finally, the newly formed DNA template is amplified and quantified by qPCR.

Fig. 2.

Detection of prostasomes by using 4PLA and PLA. (A) Comparison of 4PLA (circles) and solid-phase PLA (squares) for measuring purified prostasomes. For 4PLA the SD of 0.021 and for solid-phase PLA the SD of 0.056 for negative controls were used to calculate the LOD. (B) 4PLA was used to detect serial dilutions of purified prostasomes, spiked in 4PLA buffer (squares) and in 10% human plasma (circles). (C) The 4PLA mechanism was investigated by omitting each of the four antibodies used in the probe mix in separate reactions while still adding the corresponding oligonucleotide. The omission of any antibody resulted in reduction of the detection signals to background levels. The y axes show the C_T average and the x axes indicate the concentration of prostasomes. Error bars indicate SDs from the mean for triplicate reactions.

Fig. 3.

4PLA was used to measure levels of prostasomes in plasma samples from patients and control subjects. (A) The analysis of samples from prostate cancer patients (n = 20) revealed, on average, significantly higher concentrations of prostasomes than those observed in samples from age-matched control subjects (n = 20; P < 0.001). (B) The higher concentrations of prostasomes in samples from patients were confirmed in a blind-test validation experiment examining the subgroup of 13 patients and 11 age-matched control samples (P < 0.001). The results are shown as box plots in which the dashed lines extend between the minimum and maximum values, boxes extend between the lower and higher quartiles, and the horizontal black bars indicate the medians for the patients and controls, respectively. (C) Plasma samples from five patients with prostate cancer were pooled, and the level of prostasomes in the supernatant after ultracentrifugation (open bar) was compared with the level in pooled plasma that had not been ultracentrifuged (gray bar). Error bars indicate SDs from the mean for triplicate reactions.

Fig. 4.

Plasma levels of prostasomes in samples from patients with prostate cancer classified in three groups according to histological Gleason scores. Each patient group was analyzed in a separate experiment together with the same control group. The levels of prostasomes in plasma samples from patients with Gleason score 7 (medium) and 8/9 (high) were both significantly elevated compared with those with Gleason score of 6 or lower and those of controls (P = 0.001). The levels of prostasomes in samples from patients with Gleason scores of 6 or lower were similar to those in samples from controls (P = 0.94).

Fig. 5.

(A) Box plots showing differences in levels of prostasomes and PSA, respectively, in patient groups with different Gleason scores. The P values were calculated by using a two-sample Wilcoxon rank-sum test. (B) Scatter plots illustrating the correlation between plasma prostasome and PSA levels for patients divided in three groups with Gleason scores of 6 or lower (n = 20), 7 (n = 19) or 8/9 (n = 20). ρ values are Spearman rank correlation coefficients, measuring the statistical dependence of PSA and prostasome levels as a function of Gleason scores.