Multiple antibodies identify Glypican-1 on serum exosomes from patients with pancreatic cancer

Abstract

Glypican-1 was found on the surface of cancer exosomes using multiple antibodies from different sources. These include anti-GPC1 antibodies from ThermoFisher (PA5-28055 and PA-5-24972)^1,2, Sigma (SAB270028), Abnova (MAB8351), EMD Millipore (MAB2600)³, SantaCruz⁴, and R&D Systems (BAF4519)². Here we report on the specific detection of Glypican-1 on the exosomes derived from the serum of pancreas cancer patients using multiple antibodies.

We previously reported on the detection of the heparin sulfate proteoglycan Glypican-1 (GPC1) on serum-derived exosomes in patients with pancreatic cancer and breast cancer¹. GPC1 expression is elevated in several cancer types and associated with poor prognosis^5,6, including in pancreas cancer cells, mouse and human pancreatic cancer^7-12. Informed by genetic studies, GPC1 is proposed to control fibroblast growth factor (FGF) signaling in mouse brain development¹³, and may be critical for pancreatic cancer cell proliferation and VEGF-A induced pancreatic tumor angiogenic response^8,9. The proteoglycan GPC1, GPI-anchored to the cell surface, acts as a co-receptor for several heparin binding growth factors, thereby implicating it in promoting tumor progression^9-12,14.

In patients with pancreatic cancer, we described the enrichment of GPC1⁺ exosomes in the circulation when compared to healthy controls¹. Exosomes are nanosized extracellular vesicles produced by all cells and abundantly found in the circulation¹⁵. The renewed interest in their biology was fueled by their potential as liquid biopsies for various pathologies, including cancer. Although there are likely distinct subpopulations of exosomes with various biological properties, exosomes are generally carriers of the nucleic acids and proteins that reflect their cell of origin¹⁵. As such, exosomes from the serum of patients emerged as an attractive approach to possibly detect cancerous lesions and monitor and predict outcome of cancer patients. Several studies have recently reported findings related to GPC1 detection on exosomes, including in breast cancer cells-derived exosomes, as well as colorectal and pancreas cancer cases^2-4,16 (Table 1). Although the methodologies differed in each study, GPC1 was indeed elevated in exosomes of patients with cancer.

Table 1

Here we report on the use of three anti-Glypican 1 antibodies for the specific detection of GPC1⁺ exosomes in the circulation of pancreas cancer patients (n=10) compared to healthy donors (n=9) and benign pancreatic diseases (BPD, n=2) using flow cytometry as a readout. The ThermoFisher antibody used in our initial report¹ (PA5-28055) is commercially available. We also utilized antibody SAB2700282 from Sigma, and MAB8351 from Abnova. Both ThermoFisher and Sigma described the immunogen as a recombinant fragment of human GPC1 corresponding to a region within amino acids 200 and 558. Abnova describes the immunogen as recombinant protein corresponding to full length human GPC1. We report on the highly reproducible and strong correlation between samples evaluated with each of these antibodies (Figure 1A-B). Notably, all 10 patients with PDAC showed elevated circulating GPC1⁺ exosomes bound-beads, in contrast with the 9 healthy donors and 2 BPD samples (Figure 1A, Table 2). We also proceeded with analyses using the ThermoFisher anti-GPC1 antibody PA5-24972, however we noted this antibody did not reproduce our initial findings (Table 3). Further, when exosomes were collected using isolation kits rather than the described ultracentrifugation procedure, the accuracy of detection was lost. Therefore, ultracentrifugation for exosomes enrichment is required to evaluate GPC1 on exosomes.

Figure 1.

A Flow cytometry analyses for the % of GPC1⁺ circulating exosomes on beads, comparing the three anti-GPC1 antibodies listed. When the Abnova antibody was used, n=6 samples were analyzed (see Table 2). B. Linear regression analysis between % of GPC1⁺ circulating exosomes on beads obtained using the ThermoFisher PA5-28055 and Sigma SAB2700282 antibodies.

Table 2

Table 3

In an effort to engage the scientific community in evaluating the utility of GPC1⁺ exosomes in early detection or monitoring of pancreas cancer patients, we provide a detailed methodology, in a step-wise manner, to accompany the findings reported therein (Table 4).

Table 4

We look forward to a productive scientific discourse on our ongoing efforts to identify GPC1 on cancer exosomes using different methods and antibodies.

Methods

We provide a detailed protocol in Table 4 that describes the procedure for exosomes purification and flow cytometry analyses of GPC1 on exosomes. The secondary antibody used was Alexa Fluor 488 goat anti-rabbit (Invitrogen A11008). The aldehyde/sulfate beads were obtained from Invitrogen (A37304).

Serum samples were obtained from patients with pancreatic cancer. Serum samples were also obtained from patients with a benign pancreas disease and from healthy donors, who had no evidence of acute or chronic or malignant disease and had no surgery within the past 12 months. The cases were obtained under an IRB-exempt protocol of the MD Anderson Cancer Center (IRB no. PA14-0154).