Infoscience

Thesis

Cell lineage tracing in human tissues using somatic mitochondrial mutations

Lineage tracing is a basic concept in developmental biology, as well as diseases related to organ development. A reliable method to track cell lineages in human tissues has the potential to greatly contribute to the fields of oncogenesis, tissue renewal, ageing as well as wound healing. Inherited mitochondrial mutations have been a successful tool in the understanding of human lineage and migrations throughout history. Similarly, somatic mitochondrial mutations have been used to study the patters of cell division and renewal in some human tissues. Each cell has many copies of the mitochondrial genome, a mutation can therefore be present in any fraction of the total number of mitochondrial DNA copies. To study somatic mitochondrial mutations therefore requires mutations to detected, and the mutant fraction to be quantified. This added requirement makes the use of most sequencing techniques very difficult. The first step in the development of lineage tracing methods using somatic mtDNA mutations as a marker, as a marker, is a quantitative, fast and high throughput method of detection. A two step PCR was designed to amplify first the whole mtDNA in large fragments without amplifying nuclear DNA. Second, a set of small fragments that can be scanned for mutations using cycling temperature capillary electrophoresis. In the end, 76% of the mitochondrial genome could be scanned for mutations. Test showed that mutations could be detected and accurately quantified at fractions as low as 1%. Furthermore, the method could be applied to analyse micro-anatomical samples taken by laser capture micro dissection. Lineage tracing in human tumors was achieved by scanning the mitochondrial DNA for mutations in samples extracted from macro-anatomical samples taken across surgically removed and freshly frozen tumor pieces. Tumors carrying a mutation at a sufficient fraction were then finely sampled. The first attempt consisted in taking 286 samples across a slice of a Leydig cell tumor, in which two separate mutations were identified at a high enough fraction to justify further investigation. Both mutations strongly correlated suggesting a single lineage. Since no discernible pattern emerged, the results were validated by taking 96 samples on another slice of tissue, slightly deeper, and observing that in similar positions, similar mutant fractions could be reproduced. The natural evolution was to take multiple successive slices of tissue. On each slice 96 samples,arranged in an $8\times 12$ grid are then taken. Analysing the results from19 layers gave the three dimensional reconstruction of a tumor lineage in a tissue volume. The results show that the lineage followed is quite diffused through the volume and does not occupy its majority. A three dimensional reconstruction of a cell lineage was then established for 2 more tumors, and 3 metastasis. Under the assumption that a metastasis is seeded by a single tumor cell, the presence of the same mtDNA mutation in both primary tumor and metastasis guarantees that only cells carrying the mutation are tumor-derived. The three dimensional reconstruction of a primary tumor lineage through a metastasis is therefore the reconstruction of the patters of metastasis growth in its host tissue. Observing that most of the metastasis volume is not composed of tumor derived cells suggests that metastatic cells grow by diffusing through their host tissue and conditioning it to assume its abnormal morphology.

Fulltext

  • Thesis submitted - Forthcoming publication

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