Surfaceome profiling enables isolation of cancer-specific exosomal cargo in liquid biopsies from pancreatic cancer patients

Abstract

Background: Detection of circulating tumor DNA can be limited due to their relative scarcity in circulation, particularly while patients are actively undergoing therapy. Exosomes provide a vehicle through which cancer-specific material can be enriched from the compendium of circulating non-neoplastic tissue-derived nucleic acids. We carried out a comprehensive profiling of the pancreatic ductal adenocarcinoma (PDAC) exosomal 'surfaceome' in order to identify surface proteins that will render liquid biopsies amenable to cancer-derived exosome enrichment for downstream molecular profiling.

Patients and methods: Surface exosomal proteins were profiled in 13 human PDAC and 2 non-neoplastic cell lines by liquid chromatography-mass spectrometry. A total of 173 prospectively collected blood samples from 103 PDAC patients underwent exosome isolation. Droplet digital PCR was used on 74 patients (136 total exosome samples) to determine baseline KRAS mutation call rates while patients were on therapy. PDAC-specific exosome capture was then carried out on additional 29 patients (37 samples) using an antibody cocktail directed against selected proteins, followed by droplet digital PCR analysis. Exosomal DNA in a PDAC patient resistant to therapy were profiled using a molecular barcoded, targeted sequencing panel to determine the utility of enriched nucleic acid material for comprehensive molecular analysis.

Results: Proteomic analysis of the exosome 'surfaceome' revealed multiple PDAC-specific biomarker candidates: CLDN4, EPCAM, CD151, LGALS3BP, HIST2H2BE, and HIST2H2BF. KRAS mutations in total exosomes were detected in 44.1% of patients undergoing active therapy compared with 73.0% following exosome capture using the selected biomarkers. Enrichment of exosomal cargo was amenable to molecular profiling, elucidating a putative mechanism of resistance to PARP inhibitor therapy in a patient harboring a BRCA2 mutation.

Conclusion: Exosomes provide unique opportunities in the context of liquid biopsies for enrichment of tumor-specific material in circulation. We present a comprehensive surfaceome characterization of PDAC exosomes which allows for capture and molecular profiling of tumor-derived DNA.

Keywords: exosomes; liquid biopsies; next-generation sequencing; pancreatic cancer; proteomics.

Figure 1.

Cancer-specific exosomal biomarker selection and validation. (A) Heat map representation of proteins that are expressed on the surface of pancreatic ductal adenocarcinoma exosomes that are not expressed (or expressed at very low levels) on the surface of HPNE and CAF19 exosomes. (B) Western blot validation of identified candidate biomarkers: protein expression analysis of cell lysates (left) compared with exosomes (right) of neoplastic (Pa01C, Pa03C, and Pa04C) and non-neoplastic (CAF19 and SC2) cell lines. CD63 and TSG101 are used as antibody controls for identification of exosome populations. Most selected biomarkers show enriched specificity towards being present in cancer exosomes versus normal exosomes.

Figure 2.

exoDNA KRAS mutant detection in circulation. (A) Percent of patients with detectable mutant KRAS in exoDNA among those patient samples that did and did not undergo capture enrichment. When comparing the percentages of patients with detectable KRAS in the pulldown-cohort versus the total exosome cohort, the pulldown-cohort consistently detects KRAS in a higher proportion of patients across stages. This increase in call-rate was statistically significant in resectable patients (P = 0.024) where pulldown samples were 4.11 (95% CI: 1.14–17.19) more likely to have KRAS detected. (B) KRAS mutant allele frequency (MAF) comparisons of captured exosomes versus total exosomes, there was a statistically significant difference showing increased KRAS MAFs from the captured exosomes for resectable and metastatic patients (P = 0.003 and 0.015, respectively, using one-sided Wilcoxon Rank Sum tests).

Figure 3.

(A) Clinical course of a pancreatic ductal adenocarcinoma patient who underwent prior pancreatic tumor resection, and subsequently progressed while on Parp-1 inhibitor therapy. ExoDNA enrichment led to capture of tumor derived material which was not previously detectable. (B) Relevant mutations found in the metastatic tissue and liquid biopsies 6 months (lbx_02) and 9 months (lbx_03) after tissue biopsy. Of note is the presence of a stopgain BRCA mutation (L583*) that was correlated to her prolonged response to PARP1 inhibitor therapy. (C) A subsequent mechanism of resistance was detected in the liquid biopsies and confirmed in the tissue in the form of a BRCA2 splice site variant which splices out the aberrant stopgain mutation. SLD sum of the largest dimension; exoDNA MAF represent the KRAS mutant allele fraction. ABR, abraxane (nab-paclitaxel); CIS, cisplatin; GEM, gemcitabine.