Department of Experimental Tumor Biology, Institute of Carcinogenesis, N.N. Blokhin National Medical Research Center of Oncology of the Ministry of Health of Russia, 115522 Moscow, Russia.
Laboratory of Medical Genetics, Institute of Molecular Medicine, I.M. Sechenov First Moscow State Medical University (Sechenov University), 119991 Moscow, Russia.
Molecules. 2021 Apr 25;26(9):2499. doi: 10.3390/molecules26092499.
Exosomes are the small vesicles that are secreted by different types of normal and tumour cells and can incorporate and transfer their cargo to the recipient cells. The main goal of the present work was to study the tumour exosomes' ability to accumulate the parent mutant DNA or RNA transcripts with their following transfer to the surrounding cells. The experiments were performed on the MCF7 breast cancer cells that are characterized by the unique coding mutation in the gene. Using two independent methods, Sanger sequencing and allele-specific real-time PCR, we revealed the presence of the fragments of the mutant DNA and RNA transcripts in the exosomes secreted by the MCF7 cells. Furthermore, we demonstrated the MCF7 exosomes' ability to incorporate into the heterologous MDA-MB-231 breast cancer cells supporting the possible transferring of the exosomal cargo into the recipient cells. Sanger sequencing of the DNA from MDA-MB-231 cells (originally bearing a wild type of ) treated with MCF7 exosomes showed no detectable amount of mutant DNA or RNA; however, using allele-specific real-time PCR, we revealed a minor signal from amplification of a mutant allele, showing a slight increase of mutant DNA in the exosome-treated MDA-MB-231 cells. The results demonstrate the exosome-mediated secretion of the fragments of mutant DNA and mRNA by the cancer cells and the exosomes' ability to transfer their cargo into the heterologous cells.
外泌体是由不同类型的正常和肿瘤细胞分泌的小囊泡，可以整合并将其货物转移到受体细胞。本工作的主要目的是研究肿瘤外泌体积累亲本突变 DNA 或 RNA 转录本的能力及其随后向周围细胞的转移。该实验是在 MCF7 乳腺癌细胞上进行的，这些细胞的特点是在 基因中存在独特的编码突变。使用两种独立的方法，Sanger 测序和等位基因特异性实时 PCR，我们在 MCF7 细胞分泌的外泌体中发现了突变 DNA 和 RNA 转录本的片段。此外，我们还证明了 MCF7 外泌体能够将自身包裹的内容物转入异源 MDA-MB-231 乳腺癌细胞，支持外泌体货物向受体细胞转移的可能性。用 MCF7 外泌体处理的 MDA-MB-231 细胞（原本携带野生型 ）的 DNA 的 Sanger 测序显示没有可检测到的突变 DNA 或 RNA 量；然而，使用等位基因特异性实时 PCR，我们从突变等位基因的扩增中检测到了一个微弱的信号，表明外泌体处理的 MDA-MB-231 细胞中突变 DNA 略有增加。这些结果表明肿瘤细胞通过外泌体介导的方式分泌突变 DNA 和 mRNA 的片段，以及外泌体将其货物转移到异源细胞的能力。
