Isolation of Colorectal Cancer Stem Cell Exosomes in Order to Use in Tumor Cell Invasion Studies

Background and Aim: Colorectal cancer (CRC) is the second mostcommon cancer in the world. Cancer stem cells (CSCs) have beenattributed to mediate chemo-resistance, recurrence, invasion, andmetastasis in cancer. Tumor-derived exosomes are nanometer-sizedvesicles (30-130 nm) which proposed to have a role in invasion anddrug resistance by transferring proteins and RNA cargo (mRNAs andmicroRNAs). Therefore, a better understanding of exosomes functionwill propose new opportunities in diagnostic or therapeutic strategiesagainst cancer. This study designed to evaluate colorectal CSCs derivedexosomes role on cancer invasion and drug resistance.Methods: In this study, HT-29 cell line colonspheres were cultured byhanging drop method in the serum-free media under non-adherentconditions (poly-HEMA coated plates). Putative colorectal CSC markersincluding CD166, CD133, CD44, and DCKL-1 were evaluated using flowcytometry analysis. In addition, the expression of well-known stemnessgenes (OCT-4, SOX2, KLF-4) were assessed by real-time PCR. ExosomePurification Kit was used for isolation and purification of intact exosomesfrom CSCs culture medium. CSCs derived exosomes were characterizedand confirmed by scanning electron microscopy (SEM) analysis anddynamic light scattering (DLS) measurement.Results: HT29 derived spheroids made by hanging drop method after10 days culture. Flow cytometry analysis showed higher expressionof CD166, CD133, CD44 and DCKL-1 markers in spheroid cells thanparental cells. Moreover, relative to control parental cells, the expressionof stemness genes including OCT4, SOX2 and KLF-4 were increased inthe spheroid cells. Scanning electron microscopic examination of thepurified exosomes indicated that they have a spherical shape with adiameter of ~30– 150 nm. Further exosome size evaluation by dynamiclight scattering showed a single bell-shaped size distribution with a peakat ~135 nm.Conclusion: This purified and confirmed exosomes could be facilitatedsubsequent studies in cancer and CSCs fields such as drug resistance,invasion, and metastasis. Moreover, exosomal contents assessment maybe useful for detection of promising CRC biomarkers involved in invasionand drug resistance.

Keywords:

Colorectal cancer , Cancer stem cells , Exosome