Shende, S. | Search bioRxiv

bioRxiv2023

Horizontally transferred cell-free chromatin particles function as miniature predatory genomes and vehicles for transposable elements within host cells

BackgroundHorizontal gene transfer (HGT) plays an important evolutionary role in prokaryotes, but it is thought to be less frequent in mammals. We previously reported that cell-free chromatin particles (cfChPs) - chromosomal fragments released from the billions of dying cells that circulate in human blood - are horizontally transferred to healthy cells with biological effects. However, the underlying mechanism and function of these effects remained unclear. MethodsWe treated NIH3T3 mouse fibroblasts cells with cfChPs isolated from human serum and serially passaged the cells. The intracellular activities of cfChPs were analysed using chromatin fibre fluorography, cytogenetic analysis, immuno-fluorescence and fluorescent in situ hybridisation. ResultsWe discovered that the internalised cfChPs were almost exclusively comprised of non-coding DNA, and the disparate DNA sequences contained within them had randomly combined to form complex concatemers some of which were ostensibly multi-mega base pairs in size. The concatemers exhibited variable and bizarre spatial relationships with the host cell interphase DNA with many remaining in the cytoplasm and others aligning themselves with the mouse chromosomal DNA. The concatemers autonomously performed many functions attributable to the nuclear genome. Being associated with DNA polymerase, the concatemers could synthesize DNA and autonomously replicate themselves without heed to the mitotic cycle of the host cell. They could synthesise RNA, RNA polymerase, ribosomal RNA, ribosomal proteins, and generate numerous human proteins, including oncogenes, within the mouse cells which manifested as complex and highly amplified multi-peptide fusion proteins. The concatemers harboured human LINE-1 and Alu elements, which being associated with DNA polymerase, reverse transcriptase and transposes could markedly amplify themselves and increase their copy number with time in culture with the potential to rearrange themselves within the mouse genome. The above findings were reproducible in four other cell lines derived from different species suggesting that horizontal transfer of cfChPs may be a universal phenomenon. ConclusionsOur results lead us to propose that: 1) a cell simultaneously harbours two autonomous genome forms: one that is inherited (hereditary genome) and numerous others that are acquired (satellite genomes); 2) satellite genomes may potentially have evolutionary functions given their ability to serve as vehicles for transposable elements and to generate a plethora of novel proteins; 3) transposable elements are "foreign" genetic elements that are acquired from dying cells via HGT; 4) non-coding DNA has many hidden biological functions that remain dormant but are activated following cellular apoptosis to become detectable in association with the cfChP concatemers; 5) "within-self" HGT occurs in mammals on a massive scale via the medium of cfChP concatemers that have undergone extensive and complex modifications resulting in their behaviour as "foreign" genetic elements. Video Abstracthttps://drive.google.com/file/d/1I6NbrYmT9BRM63ywYj47AU0O9WJ_NrcR/view?usp=drive_link

evolutionary biology↗

bioRxiv2023

Metastases arise as new cancers from cells of target organs transformed by cell-free chromatin particles released from dying cancer cells

IntroductionBased on our earlier finding that cell-free chromatin particles (cfChPs) released from dying cancer cells are potentially oncogenic, we hypothesised that metastases arise as new cancers from the cells of target organs transformed by cfChPs released from dying cancer cells. MethodsWe fluorescently dually-labelled MDA-MB-231 human breast cancer cells and A-375 human melanoma cells in their DNA with BrdU and in their histones with CellLight(R) Histone 2BGFP. One hundred thousand dually-labelled cells were intravenously injected into SCID mice. In other experiments unlabelled cells were injected for detection of lung metastasis. Also intravenously injected were 700 ng of purified cfChPs isolated from radiation-killed MDA-MB-231 cells. ResultsWe observed that the fluorescently dually-labelled MDA-MB-231 and A-375 cells died upon reaching the lungs and released dually-labelled fluorescent chromatin particles that accumulated in the nuclei of lung cells at 48 h. Injection of unlabelled cfChPs led to the activation of 10 hallmarks of cancer and immune checkpoints in lung cells at 72 h, suggesting that the lung cells had rapidly transformed into incipient cancer cells. Fluorescent in situ hybridisation analysis of lung metastases that subsequently developed using mouse and human specific DNA probes revealed that the tumour cells contained both mouse and human DNA in almost equal proportions. Similarly, immune-fluorescence analysis using species specific mAbs revealed that the tumour cells co-expressed mouse and human specific proteins. Metaphase preparations and single-cell clones developed from cell cultures of lung metastases were found to contain chimeric chromosomes containing both mouse and human DNA, and the cells to co-express both human- and mouse-specific proteins. The Intravenously injected purified cfChPs isolated from radiation-killed MDA-MB-231 cells also induced lung metastasis which predominantly contained mouse DNA strongly suggesting that the metastatic tumours had arisen from the mouse lung cells. ConclusionThese results provide strong evidence that cfChPs released from dying cancer cells integrate into genomes of target lung cells to transform them into new cancers that masquerade as metastasis. They support our hypothesis that metastases arise from the cells of target organs and not from those of the primary tumour. These findings have implications for the principles of cancer therapy.

cancer biology↗