Tumors generate unique, temporary cell-in-cell structures to evade immunotherapy

Scientists have shown how tumor cells evade immune therapy by generating unique, temporary cell-in-cell structures, leaving the inner cells intact and able to revert to individual tumor cells.

These findings, published today in eLife, offer a new theory of how tumor cells avoid destruction by the immune system. They may also inform the development of treatments that combine immunotherapy with the timed inhibition of relevant signaling pathways in tumor cells.

“Cancer immunotherapy uses the body’s immune system to fight cancer. Despite its remarkable success, the majority of patients who receive immunotherapy will only see their tumors shrink temporarily before they recur, and these recurrent tumors are likely to be resistant to immunotherapy treatment,” said lead author Amit Gutwillig, who was a PhD student at the Carmi Lab, Tel Aviv University, Israel, at the time the study was conducted, and is now a Senior Researcher at Nucleai, Tel Aviv.

To determine how tumors relapse after immunotherapy, Carmi and team began by comparing the genetic sequences of whole genomes in primary and recurrent tumors in the same patient. Their analysis suggested that recurrent tumors do not change dramatically after immunotherapy.

Next, the team studied this process in breast cancer and melanoma, using mouse models in which immunotherapy-resistant tumors had recurred. They administered the mice cells from treated tumors and allowed these cells to reach a palpable size. The team found that the cells were equally susceptible to the same immunotherapy approach as the parent tumor, although they relapsed earlier.

To better characterize the tumor cells that survived in mice after immunotherapy, the researchers isolated and studied the living tumor cells. They found that most cells responded to the presence of T cells — a type of immune cell that targets foreign particles — by organizing into temporal formations. These consisted of clusters of different tumor cell nuclei, surrounded by a single, multilayered membrane and a network of cortical actin filaments. The innermost cell of the formation was dense and appeared to have been compacted in another cell.

To show that this result was not due to the isolation of the melanoma cells, the team also analyzed tumors with fluorescently labeled cell nuclei and membranes. They found that cell-in-cell formation was more common in immunotherapy-treated tumors, particularly in sites associated with tumor cell death. Further analysis indicated that approximately half of the tumor cells that survived immunotherapy were arranged in the cell-in-cell formation. Over time, these cells reverted to a unicellular state, with similar structural features to those of the parent cell line.

The team then tested whether this phenomenon occurs in human cancers. To do this, they incubated tumor cell lines with preactivated T cells from healthy donors. They found that the vast majority of breast, colon and melanoma tumor cells that survived T-cell killing were organized in the cell-in-cell structure. A three-day observation of T cells interacting with tumor cells showed that these structures were dynamic, with individual tumor cells constantly forming and spreading out of the structure.

Finally, they tested the clinical relevance of this discovery by analyzing cancer tissues from multiple organs from four stage 4 melanoma patients. These patients underwent surgical removal of primary and metastatic lymph nodes – that is, lymph nodes that had spread from the primary tumor. The researchers found that in all four patients, cell-in-cell formation was very abundant in the T-cell zone of the draining lymph nodes, but not in the primary tumors. In addition, in a patient with untreated recurrent melanoma, most of the cells in the primary tumor were single cells, while the recurrent tumors had an abundance of cell-in-cell formations.

This previously unknown mechanism of tumor resistance highlights a current limitation of immunotherapy. Over the past decade, many clinical trials have used immunotherapy followed by chemotherapy. But our findings suggest that timed inhibition of relevant signaling pathways should occur alongside immunotherapy to prevent the tumor from becoming resistant to subsequent treatments.”

Yaron Carmi, Southfirst author, Principal Investigator, Department of Pathology, Sackler School of Medicine, Tel Aviv University


Reference magazine:

Gutwilly, A., et al. (2022) Transient cell-in-cell formation underlies tumor relapse and resistance to immunotherapy. eLife. doi.org/10.7554/eLife.80315.

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