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Transcript Sequencing in Mouse Model Offers Insights Into Cancer Editing By Immune System

NEW YORK (GenomeWeb News) – In a twist on the tumor-sequencing studies done in the past, an international team led by investigators at Washington University used targeted sequencing of expressed tumor transcripts to explore ways in which the host immune system "immunoedits" cancer cells.

As they reported online today in Nature, the researchers used massively parallel sequencing to look for complementary DNA mutations in cell lines developed from two sarcomas that had been induced with a carcinogenic chemical called methylcholanthrene in immune deficient mice.

After transplanting one of the sarcoma cell lines into normal mice, they did similar cDNA sequencing experiments to look at genetic patterns in cell lines that were or were not destroyed by the mouse immune system. By analyzing mutations in these lines using an algorithm that predicts interactions between antigen epitopes and class I major histocompatibility complex proteins, they tracked down a mutation in the antigen spectrin-beta2 that seem to make tumor cells more prone to destruction by the immune system.

Together, results from the study indicated that T cells in the immune system efficiently destroy tumors with easily recognizable "rejection antigens" — in the case of the mouse sarcomas, a mutated form of spectrin-beta2.

"Many of the cancer genome projects now under way are looking for the 'driver' mutations, or the mutations that cause the cancers," senior author Robert Schreiber, a pathology and immunology researcher at Washington University and co-leader of the Alvin J. Siteman Cancer Center's tumor immunology program, said in a statement. "Our results suggest there may be additional information in the sequencing data that can help us make the immune system attack cancers."

"The idea would be to make a vaccine that helps the immune system recognize and attack six or seven of these mutated proteins in a cancer," he added.

In studies published over the past decade or so, researchers have proposed an immunoediting function for the immune system, which prunes some tumor cells and influences the immune profiles of the tumor cells that remain.

As it removes some cancer cells, for example, the immune system may leave behind cancerous cells with less recognizable antigens that can linger in the body or continue to grow into so-called "escape tumors."

It's not altogether clear how this happens, Schreiber and his co-authors explained, even though some immune system players in the immunoediting process have been described. That's because researchers typically only see the end products of this process, rather than tumor cells before or during the immunoediting process.

"[O]ur current understanding of tumor antigens comes largely from analyses of cancers that develop in immunocompetent hosts and thus may have already been edited," they explained. "Little is known about the antigens expressed in nascent tumor cells, whether they are sufficient to induce protective anti-tumor immune response or whether their expression is modulated by the immune system."

The team got around this problem by doing studies of immune-deficient mice. After inducing sarcomas in mice with methylcholanthrene, they produced cell lines from the two of the tumors and used a modified form of exome sequencing with the Illumina GAIIx to look at transcripts expressed by these cells.

Using this data, researchers found more than 3,700 somatic, non-synonymous mutations in one of the cell lines, dubbed d42m1, and 2,677 in the other cell line. Almost all of these were specific to one line or the other, with just 119 missense mutations shared between the two.

To see how intact mouse immune systems would react to the sarcoma cell lines, the team then injected d42m1 sarcoma cells into wild type mice. From wild type mice that did develop escape tumors, the researchers generated additional cell lines.

These cell lines produced tumors in other wild type mice, they reported. But the immune systems of wild type mice could easily recognize and remove d42m1 cells that had been grown in immune-deficient mice prior to transplantation.

Capture sequencing experiments on cDNA from cell lines, clones, and escape tumors stemming from the d42m1 sarcoma line revealed a comparable number of mutations in each, researchers reported, though there was variability in the specific mutations present.
Using an Immune Epitope Database and Analysis Resource algorithm that predicts the consequences of missense mutations on epitope-MHC class I molecule interactions, the team found evidence that the cell lines that are suppressed by the immune system in wild type mice contain mutated versions of spectrin-beta2. This rejection antigen appears to be efficiently recognized and destroyed by cytotoxic T cells — a notion that the team verified through follow-up experiments.

On the other hand, the spectrin-beta2 mutation was not found in escape tumors, apparently because cells containing the antigen had already been weeded out by the immune system, the study authors explained.

They argue that the ability to track down antigens that prompt this sort of pronounced immune response may eventually lead to treatments that help marshal the immune system against a given tumor.

"Although more work is needed to determine whether and how frequently this [immunoediting] process occurs during development of spontaneous and carcinogen-induced human cancers," they concluded, "it is tempting to speculate that a genomics approach to tumor antigen identification could, in the future, facilitate the development of individualized cancer immunotherapies directed at tumor-specific — rather than cancer-associated — antigens."

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