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While many cells in our body can accumulate oncogenic mutations, the majority of these events do not lead to the formation of tumors because these abnormal cells are eliminated by defense mechanisms. Instead, tumors arise when a mutation occurs in a particular cell type that is particularly sensitive to it. Identifying these original cancer cells is essential to properly target cancer.
For example, mutations in the retinoblastoma tumor suppressor (RB) gene, which normally blocks abnormal cell growth and division, result in retinoblastoma, a type of ocular tumor. Retinoblastoma appears in specialized retinal cells called cone cells, which collect light. Why this type of cancer always starts in conical cells is unknown. But if scientists can get a clearer and more precise view of the downstream effects of RB mutations in conical cells compared to other retinal cells, they can identify unique therapeutic targets capable of preventing or treating retinoblastoma with accuracy comparable to that of the laser.
But studying the effects of gene mutations in specific cell types is easier said than done. It is almost impossible to take pure samples composed of a single type of cell. Instead, scientists often have to use a bulk sample prepared from an entire tissue.
"This only gives a kind of average image of the expression of genes in individual cells since it groups together thousands of cells, some of which may be undesirable regardless of the purity of the cells." The results of the examination of the effects of RB mutations with the help of these types of samples do not accurately represent the incidence of an RB mutation on expression. genes in a particular cell type, "said Maxim Frolov, professor of biochemistry and molecular genetics at the University of Illinois at the Chicago College of Medicine.
A revolutionary new technology called single-cell RNA sequencing allows researchers to study gene expression in individual cells, eliminating the problem of contaminated cell samples and averaging. Frolov's lab has adapted a technology called Drop-seq, which allows researchers to isolate and genetically sequence unique cells. Drop-seq can sequence thousands of individual cells at the same time.
Frolov and his graduate student, Majd Ariss, have assembled a Drop-seq instrument to isolate eye cells from developing fruit flies, which the lab uses as a model system. They were then able to study changes in gene expression caused by mutations in the RB gene in thousands of different cells of the eye compared to gene expression in cells with normal copies of the RB gene. Their results are published in Nature Communications.
"Since it was the first time that single-cell RNA sequencing was performed in cells of the fruit fly eye, we had to create a complete map or cellular atlas, describing precisely the expression of genes in each type of normal eye cell on this atlas to determine how an RB mutation affects the gene expression of each cell type in the eye " Frolov explained.
Their analysis of ocular cells with RB mutation revealed a small but distinct cell population where the mutation altered gene expression and altered cell metabolism. Metabolic change sensitized cells to apoptosis or self-induced cell death. The propensity of cells carrying mutations of the RB gene to undergo apoptosis is a well-known phenomenon. It is finally overcome by additional mutations during the development of cancer, which is characterized by uncontrollable cell growth and division – the opposite of apoptosis.
"The metabolic changes we observed in RB mutant cells make them vulnerable in a way that could be exploited with therapeutic approaches before other mutations affect the same cell, making them resistant to cell death, "said Frolov. "Since these effects were limited to such a small group of cells, they had not been detected in the analysis of RB gene mutant ocular tissues."
The Drop-seq platform took over three months at Ariss to be built. He scrupulously followed the instructions in a 40-page manual to generate his first single-cell RNA sequencing data set.
"No one explained to us how to do single-cell sequencing because we were the first to UIC to do it," said Ariss, the newspaper's first author.
Their Drop-seq instrument is one of a kind at the UIC.
"This is a truly revolutionary technology that promises to shed new light on the origin of cancer and the reason that some cancers come from certain types of cells and not others." We can only begin to wonder why and how For a year and a half, we have performed more than one hundred experiments and generated transcriptomes of more than one hundred thousand fruit fly organ cells, mouse tumors and human cell lines. UIC researchers will use it in the future, "Frolov said.
This work was funded by National Institutes of Health grants GM93827 and GM110018.
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