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Exosomal Proteomic Studies Suggest That Integrins Determine Organotropic Metastasis

NEW YORK (GenomeWeb) – A team led by scientists at Weill Cornell Medical College has found that a family of proteins called integrins that determine where exosomes are sent to prepare a new site for metastasis.

Through exosome proteomics, they found specific exosomal integrin expression patterns, involving specific alpha (α) and beta (β) pair combinations, that directed exosomes from human cancer cells to specific sites in mice to prepare an area for metastasis. The researchers also found that integrins upregulated S100 proteins, which promoted pro-migratory and pro-inflammatory activity.

The researchers described their work in a paper published this week in Nature. David Lyden, one of the lead authors on the paper and a professor of pediatrics and cell and developmental biology at Weill Cornell Medical College, told GenomeWeb that this study was able to uncover a great deal of information about exosome and integrin function in tumor cells that hadn't been known before.

Understanding metastatic organotropism has remained one of cancer's greatest mysteries, since Stephen Paget's original 1889 hypothesis, called the 'seed and soil' idea, that essentially states that when cancer cells spread to distant sites around the body they don't just stick to the first tissue they come across, but instead have preferred sites. These sites also appeared to have been made somehow more receptive to tumor growth. 

When Lyden and his colleagues first began their research over ten years ago, they found the first piece of the puzzle to answer Paget's question by discovering that primary tumor cells secrete exosomes that act as little messengers and essentially tag sites where the cancer can metastasize in the future. After that, scientists hypothesized that the action of exosomes in metastasis is gene-driven. However, the study published this week clearly finds that integrins direct exosomes to these sites and dictate the number of S100 proteins contributing to inflammation. 

Cell lines and cell cultures used in the study — which included cancer cells from breast, lung, liver, brain, and pancreatic cancer patients and/or mice — were provided by multiple international institutions. The researchers purified exosomes from the cells by sequential centrifugation, using electron microscopy for verification. They then analyzed exosome size and particle number using Malvern Instruments' NanoSight LM10 or DS500 nanoparticle characterization system. 

The researchers then used nano-liquid chromatography mass spectrometry (LC-MS/MS) at the Rockefeller University Proteomics Resource Center to analyze the exosomes, followed by proteolytic digestion. They performed LC-MS/MS on the Thermo Scientific Ultimate 3000 coupled to QExactive. They performed semi-quantitative analysis by searching MS/MS spectra against Uniprot complete human or mouse proteome databases, and label-free quantitative LC-MS/MS analysis on MaxQuant (version and Perseus (version software.

They then assessed the distribution of exosomes in lung, liver, bone, and brain tissue by injecting the exosomes into mice. Purified exosomes were fluorescently labeled to measure exosome uptake by specific cell types. The labeled exosomes were injected 24 hours before tissue was collected and analyzed for exosome-positive cells using the Li-Cor Biosciences Odyssey imaging system.

Using the data from their research, Lyden and his colleagues were able to identify the different specific integrins that determined where new cancer tumors would form and how they directed the exosomes to these sites to prepare them for metastases. They found that exosomes that expressed integrin αvβ5 bind to Kupffer cells that mediate liver tropism, while integrins α6β4 and α6β1 bind to lung-resident fibroblasts and epithelial cells that govern lung tropism.

Interestingly, bone-tropic exosomes expressed a limited integrin repertoire, suggesting that integrin-independent mechanisms may mediate vascular leakiness and exosome involvement in bone metastasis.

The researchers then conducted gene expression analysis on mouse cells to determine that integrin uptake activated Src phosphorylation and pro-inflammatory S100 expression. To do this they used pre-designed Applied Biosystems TaqMan assays from Thermo Fisher Scientific performed on an Applied Biosystems 7500 Fast Real-Time PCR System.

Upon analysis, the researchers found that S100A4, S100A6, S100A10, S100A11, S100-A13, and S100A16 were upregulated in lung fibroblasts; while S100P and S100A8 were upregulated in Kupffer cells.

The investigators also noted that they were able to implement integrin-blocking decoy peptides, specifically RGD and HYD-1, which successfully destroyed the possibility of tumor exosome adhesion. Lyden said that this research proves that it would be possible to develop peptide-derived drugs that can prevent exosomes from creating viable sites for tumors. 

"You could actually develop a kit to measure exosomal integrins [in the clinic]," Lyden said, adding that knowing how many exosomal integrins from tumor secretion are circulating could help physicians better predict the types of treatment patients may need and determine how closely a patient may need to be monitored for disease progression. "At the same time it may tailor [patient] therapy," he said. "So that would mean that if you didn't find the integrin, [patients] could have surgery alone, without going to chemo radiation therapy. You could really improve treatment."

Lyden and his colleagues said in the paper that they hope future studies will focus on identifying exosomal integrins and proteins that could dictate metastasis to other organs, as well as further exploring the potential of exosomal integrin α2β1 as marker and driver of all cancer metastasis.