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Drop-seq: Key to Why Cancers Develop in Certain Cells

Retinoblastoma.

The body undergoes numerous cell divisions every minute. With each cell division, the possibility of a mutation occurring is perpetual. Usually, these mutations are destroyed by various defense mechanisms, but sometimes, the mutations develop into cancer. To accurately target cancer, understanding why mutations transform into cancer in some cells but not others is critical.

Retinoblastoma (RB), a type of eye cancer, forms when mutations in the RB tumor suppressor gene stop correctly performing their job of blocking abnormal cell growth and division. Retinoblastoma only occurs in cone cells in the retina, and scientists are unsure why these cells specifically develop cancer while other retinal cells do not. A clearer view of why cancer forms in cone cells could lead to a more targeted therapy to combat RB.

Unfortunately, it is almost impossible to collect samples with only one specific cell type. Instead, when samples are collected, they are comprised of several different cell types from an entire tissue.

"This only gives a sort of average picture of gene expression in individual cells since it pools thousands of cells, some of which may be unwanted no matter how much the sample is purified," explained Maxim Frolov, PhD, Professor of Biochemistry and Molecular Genetics at the University of Illinois at Chicago (UIC) College of Medicine. "The results of examining the effects of RB mutations using these kinds of samples don't accurately represent how an RB mutation affects gene expression in a particular cell type."

Dr. Frolov and his research time found a solution to this problem by developing Drop-seq, a technology that allows for single cell analysis through isolation and genetic sequencing that has the capability to sequence thousands of individual cells simultaneously.

Dr. Frolov and graduate student Majd Ariss, lead author of the study, which was published in Nature Communications, used Drop-seq to analyze isolated eye cells in developing fruit flies. Gene expression of mutations of the RB gene were compared to gene expression of normal copies of the RB gene.

"Since this is the first time single-cell RNA sequencing has been performed in cells of the fruit fly eye, we had to create a comprehensive map or cell atlas, accurately describing gene expression in each cell type in the normal eye," explained Dr. Frolov. "We then relied on this atlas to determine how an RB mutation affects gene expression of each cell type in the eye."

The researchers discovered an area of cells where mutation distorts gene expression and cell metabolism. After the alteration, cells become more sensitive to apoptosis (programmed cell death). However, that sensitivity is short-lived. Once more mutations occur, the mutated cells develop a resistance to apoptosis, allowing them to develop into cancer.

"The metabolic changes we observed in RB mutant cells make them vulnerable in ways that might be exploited with therapeutic approaches before additional mutations hit the same cell, making them resistant to cell death," Dr. Frolov expounded. "Since these effects were limited to such a small group of cells, they were previously missed when whole RB mutant eye tissue was analyzed."

"It is a truly revolutionary technology that promises to shed new light on the origin of cancer and why certain cancers originate in certain cell types and not others. Only now can we begin to investigate why and how," added Dr. Frolov. "For the past year and a half, we performed over a hundred experiments and generated transcriptomes of more than a hundred thousand cells from fruit fly organs, mouse tumors, and human cell lines. We hope that more UIC researchers will use it going forward."

For More Information

Ariss MM, Islam ABMMK, Critcher M, et al (2018). Single cell RNA-sequencing identifies a metabolic aspect of apoptosis in Rbf mutant. Nat Com. [Epub ahead of print] DOI:10.1038/s41467-018-07540-z

Image Courtesy of Orphanet Journal of Rare Diseases

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