Exosomes shed by melanoma cells play a key role in promoting metastasis by mobilizing and instructing bone marrow progenitors to develop pro-vasculogenic and pro-metastatic phenotypes, researchers claim. Studies by a Weill Cornell Medical College-led team showed that this cross-talk between exosomes and bone marrow-derived cells (BMDCs) is effected through transfer of the receptor tyrosine kinase MET and appears to be distinct from tumor-promoting cues signaled by tumor-derived growth factors, extracellular proteins, and chemokines.
Moreover, suggests David Lyden, M.D. and colleagues, levels of specific proteins within melanoma exosomes may represent quantitative biomarkers for predicting disease stage, likelihood of metastasis, and patient survival. The researchers report their findings in Nature Medicine in a paper titled “Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET.”
BMDCs are critical to generating the required microenvironment both for primary tumor formation and the development of metastasis. Secreted factors are already known to contribute to the recruitment of BDMCs to primary tumor and premetastatic niches, but the role of tumor-derived exosomes hasn’t yet been evaluated.
The Weill-Cornell researchers first identified a link between exosome protein content and disease stage. When they compared circulating exosomes taken from patients with different stages of melanoma, they found that those from stage 4 disease displayed much higher protein content than those from earlier stage melanomas. Even among Individuals with stage 4 disease, those with the least protein-rich exosomes tended to have longer survival than stage 4 patients with exosomes demonstrating higher protein content.
Mass spectrometry analyses indicated that levels of specific exosome proteins could also represent predictors of disease stage, prognosis, and survival. For example, exosomes from stage 4 disease contained elevated levels of TYRP2 (a melanoma-specific protein), VLA-4, and HSP70. Stage 3 disease exosomes, on the other hand, only expressed raised levels of TYRP2. And even among stage 3 patients, the amount of exosome TYRP2 was increased in individuals who went on to develop metastatic disease. Interestingly, the exosomes of 70% of patients with stage 4 disease contained an HSP90 isoform that has previously been linked with cellular transformation.
Exosomes from highly malignant melanoma cell lines including B16-F10, SK-Mel-28 and SK-Mel-202, also expressed two-to ten-fold higher protein levels than exosomes from either the poorly metastatic cell line B16-F1 or less metastatic lung, breast, and colon cancer cells. To evaluate the relevance of this in vivo, they injected mice with exosomes from either B16-F10 cells or from nonmetastatic cells. The results were striking, in that animals receiving the B16-F10 exosomes started to exhibit increased lung endothelial permeability within 24 hours, whereas this didn’t occur in animals injected with exosomes from less aggressive cell lines. Lung tissue in the B16-F10 exosome animals also differentially expressed some 130 genes, many of which are involved in extracellular matrix remodeling, inflammation, and premetastatic niche formation.
To evaluate the influence of exosomes on tumor growth, mice were given repeated injections of either B16-F10 exosomes or B16-F1 exosomes, and were then implanted with luciferase-tagged B16-F10 tumor cells. While there was no difference between the cohorts in terms of primary tumor growth, animals receiving either the B16-F10 exosomes or exhibited a comparatively 240-fold higher lung metastatic tumor burden and developed metastases in a wider range of tissues including bone. “These data suggest that qualitative differences in exosome content can mediate metastatic potential and organotropism,” the authors state.
They moved on to see whether tumor-derived exosomes could educate BMDCs and influence metastatic progression. Lethally irradiated mice were given transplants of bone marrow from animals that had previously been treated with either B16-F10 exosomes or synthetic exosomes. After bone marrow reconstitution the transplant recipients were then injected with mCherry-expressing B16-F10 cells. In comparison with mice receiving the synthetic exosome-treated control bone marrow, those transplanted with exosome-educated bone marrow exhibited increased primary tumor growth, increased BMDC recruitment and tumor vascular density in the primary tumor, and a much greater metastatic tumor burden at both typical and atypical sites.
Further analyses indicated that in comparison with the effects of B16-F1 exosomes on bone marrow progenitors, B16-F10 exosome education led to an increase in the frequency of proangiogenic cells but not other bone-marrow derived cell populations that are mobilized by growth factors and chemokines.
Interestingly, when the team looked for proteins that were produced at much higher levels in B16-F10 exosomes compared with B16-F1 exosomes, the Met oncoprotein stood out. “We hypothesized that exosomes could horizontally transfer MET from melanoma to bone marrow progenitor cells and that this transfer could be a previously unknown mechanism for promoting metastatic progression,” they note.
This notion was supported by the observation that Met expression was increased in the bone marrow progenitor cells of mice that had received the B16-F10 exosomes. But not in the bone marrow progenitor cells of mice given injections of B16-F1 exosomes. Moreover, only bone marrow cells pretreated with B16-F10 exosomes demonstrated activation of the MET pathway, as evaluated by measuring phosphorylation of downstream mediators of hepatocyte growth factor (HGF)-MET signaling.
The functional consequences of HGF-MET pathway signaling were assessed by implanting B16-F10 tumor cells in mice that had been pretreated with either unmodified B16-F10 exosomes or shRNA-knockdown B16-F10 exosomes that exhibited markedly reduced MET expression. While both cohorts of animals demonstrated similar primary tumor growth, those injected with the B16-F10 exosomes expressing reduced MET developed fewer lung and bone metastases and exhibited lower levels of vasculogenic and hematopoietic precursors in their bone marrow and peripheral blood.
To try and give these collective findings clinical relevance, the researchers looked at the amounts of MET and pMET in circulating exosomes taken from melanoma patients with different stages of disease. Mirroring data from the initial studies that looked at total exosome protein levels, the MET analyses showed that circulating exosomes from patients with stage 3 or 4 disease contained much higher levels of MET and pMET than normal controls. MET protein expression was in addition much higher in both bone marrow progenitor cells and provasculogenic cells in the blood of subjects with stage 4 disease than those with earlier stage disease.
“Our study is the first, to our knowledge, to show that transfer of the MET oncoprotein from tumor-derived exosomes to bone marrow progenitor cells promotes the metastatic process in vivo,” Drs. Lyden et al. conclude. “In addition, we identified an exosome-specific melanoma signature with prognostic and therapeutic potential comprised of TYRP2, VLA-4, HSP70, an HSP90 isoform and the MET oncoprotein.”