Circulating peptidome to indicate the tumor-resident proteolysis

Abstract

Tumor-resident proteases (TRPs) are regarded as informative biomarkers for staging cancer progression and evaluating therapeutic efficacy. Currently in the clinic, measurement of TRP is dependent on invasive biopsies, limiting their usefulness as monitoring tools. Here we identified circulating peptides naturally produced by TRPs, and evaluated their potential to monitor the efficacy of anti-tumor treatments. We established a mouse model for ovarian cancer development and treatment by orthotopic implantation of the human drug-resistant ovarian cancer cell line HeyA8-MDR, followed by porous silicon particle- or multistage vector (MSV) - enabled EphA2 siRNA therapy. Immunohistochemistry staining of tumor tissue revealed decreased expression of matrix metallopeptidase 9 (MMP-9) in mice exhibiting positive responses to MSV-EphA2 siRNA treatment. We demonstrated, via an ex vivo proteolysis assay, that C3f peptides can act as substrates of MMP-9, which cleaves C3f at L1311-L1312 into two peptides (SSATTFRL and LWENGNLLR). Importantly, we showed that these two C3f-derived fragments detected in serum were primarily generated by tumor-resident, but not blood-circulating, MMP-9. Our results suggested that the presence of the circulating fragments specially derived from the localized cleavage in tumor microenvironment can be used to evaluate therapeutic efficacy of anti-cancer treatment, assessed through a relatively noninvasive and user-friendly proteomics approach.

Figure 1. A schematic representation of peptidomics analysis for blood biomarkers of tumor activity.

(A) Proteins/peptides are cleaved by proteases within the vicinity of tumor tissues, correlating with tumor growth or response to treatment. These disease-associated proteolytic fragments are released into the bloodstream. (B) Blood/serum samples are processed through on-chip fractionation with nanopore platforms to enrich low-molecular weight peptides. (C) The target biomarkers (proteolytic products of tumor-resident proteases) are detected with mass spectrometry. The specific peptide peaks in the mass spectrum represent the expression and/or activity of their corresponding tumor-resident peptidases.

Figure 2. Administration or treatment with MSV-EphA2 siRNA decreased MMP-9 expression in tumors of mice with ovarian cancer and in cell cultures.

(A) Immunohistochemistry analysis of tumor tissue samples from mice treated with MSV-EphA2 siRNA probing for MMP-9 expression, Upper panels 10× magnification, lower panels 40× magnification. Scale bar = 20 μm. (B) Immunoblotting to examine the effect of EphA2 siRNA transfection in HeyA8-MDR cells. Equivalent volumes of conditioned media were loaded, and the blot was probed with antibody against MMP-0, EphA2 or actin. All the blots are performed under same experimental conditions, the full image of blot as shown in Figure S1. (C) Serum MMP-9 expression in sera of tumor-bearing mice showed no statistically significant difference between control and EphA2 siRNA treatments.

Figure 3. Comparison of natural C3f fragments and the cleavage site analysis after incubation with MMP-9, serum, and cell culture media indicate tumor protease specificity.

(A) Observed cleavage sites on C3f. Arrows show the observed cleavage sites. Red arrows show the cleavage sites of interest. The series of b- and y-ions in MALDI-TOF/TOF MS spectrum confirmed the amino acid sequence of these two peaks as (B) 6xHis-SSATTFRL (m/z = 1704.8) and (C) LWENGNLLR-6xHis (m/z = 1936.9). Insets show the zoomed-in view of the MS-MS spectra.

Figure 4. MMP-9 expression in cell culture media correlates with the extent of cleavage of the L₁₃₁₁ – L₁₃₁₂ peptide bond in C3f.

(A) Immunoblotting using equivalent sample volumes of conditioned media from cells transfected with control siRNA, EphA2 siRNA, or MMP-9 siRNA. To confirm MMP-9 expression levels. All gel and blots run under same condition, the full image as shown in Figure S4. (B) The relative peak intensities of two specific fragments, 6xHis-SSATTFRL (m/z = 1704.8) and (C) LWENGNLLR-6xHis (m/z = 1936.9), generated by incubating synthetic C3f with culture media under the same conditions indicated in (A). **, p < 0.01.

Figure 5. Peptide sequences and the relative expression levels of the two C3f fragments in serum (SSATTFRL and LWENGNLLR) were determined and quantified using iTRAQ.

Serum peptides from mice treated with MSV-control siRNA were labeled with 114, whereas serum peptides from mice treated with MSV-EphA2 siRNA were labeled with 115. (A) The b- and y-ion series indicate the sequence of SSATTFRL, and a zoomed-in view (B) shows its reporter ion region at m/z = 114.1 and 115.1. (C) The b- and y-ion series indicate the sequence of LWENGNLLR, and a zoomed-in view (D) shows its reporter ion region at m/z = 114 and 115.