Proteomic Profiling of Rhabdomyosarcoma-Derived Exosomes Yield Insights into Their Functional Role in Paracrine Signaling

Exosomes are important
intercellular communication vehicles, secreted
into body fluids by multiple cell types, including tumor cells. They
have been demonstrated to contribute to the metastatic progression
of tumor cells through paracrine signaling. Tumor exosomes contain
intact and functional proteins, mRNA and miRNA that may alter the
cellular environment to favor tumor growth. We evaluated the protein
cargo of exosomes derived from the childhood tumor rhabdomyosarcoma
(RMS) and the molecular pathways they are implicated in to decipher
their role in the progression of this aggressive disease. We conducted
a mass spectrometry analysis of exosome content isolated from five
RMS cell lines: three of embryonal RMS (ERMS) and two of alveolar
RMS (ARMS) histology and verified results by multiple reaction monitoring
and western blot analyses. Results revealed 161 common proteins in
ERMS-derived exosomes and 122 common proteins in ARMS-derived exosomes,
of which 81 proteins were common to both subtypes. Using both PANTHER
gene classification and Pathway Studio software, we assessed the perturbed
biological processes and altered pathways in which the exosomal proteins
are involved. The 81 commonly expressed proteins included those involved
in “cell-signaling,” “cell-movement,”
and “cancer.” Pathways engaging the identified proteins
revealed 37 common pathways including “integrin signaling pathway,”
“inflammation mediated by chemokine and cytokine signaling
pathway,” and “angiogenesis.” Finally, a comparison
of exosomal proteins of RMS cells with publicly available datasets
from other cancer cells revealed that 36 proteins are specific and
endogenous to the RMS-exosomes. Taken together, our results reveal
that RMS-derived exosomes carry a protein cargo that contributes to
conserved cellular signaling networks across multiple cell lines,
and we also identify RMS exosome-specific proteins that should be
further evaluated as possible novel biomarkers for this tumor.