Sequential Method for Analysis of CTCs and Exosomes from the Same Sample of Patient Blood

Circulating tumor cells (CTCs) and exosomes, both released
from
the primary tumor into peripheral blood, are a promising source of
cancer biomarkers. They are detectable in the blood and carry a large
diversity of biological molecules, which can be used for the diagnosis
and monitoring of minimally invasive cancers. However, due to their
intrinsic differences in counts, size, and molecular contents, studies
have focused on only one type of vesicle. Herein, we have developed
an integrated system to sequentially isolate CTCs and exosomes from
a single patient blood sample for further profiling and analysis.
The CTCs are isolated using a commercial filtration method and then
the remaining blood is processed using multiple cycles of ultracentrifugation
to isolate the exosomes. The method uses two available technologies
where the eluent from CTC isolation is usually discarded and interfaces
them, so that the eluent can be interfaced to exosome isolation methods.
The CTCs are identified based on fluorescence staining of their surface
markers, while the exosomes are analyzed using transmission electron
microscopy, nanosight tracking analysis, and mass spec proteomic analysis.
This analysis showed CTCs detected by their surface markers for metastatic
hepatocellular carcinoma (HCC), while essentially none were detected
for cirrhosis. The exosome analysis resulted in the identification
of ∼500–1000 exosome proteins per sample confirmed by
detection of exosome surface markers CD9, CD63, CD81, and TSG101 in
addition to proteins related to cancer progression. Proteins enriched
in HCC exosomes were shown to be involved in the immune response,
metastasis, and proliferation.