Transient cell-in-cell formation of tumor cells has been found to build resistance to immunotherapy

Transient cell-in-cell formation of tumor cells has been found to build resistance to immunotherapy

In a recently published study in the eLife In the journal, researchers showed that tumor cells evade immunotherapy by creating unique transient cell-in-cell structures that are resistant to chemotherapy and destruction by T cells.

Lernen: Eine vorübergehende Zell-in-Zell-Bildung liegt einem Tumorrückfall und einer Immuntherapieresistenz zugrunde. Bildnachweis: CI Photos/Shutterstock

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Despite some notable success stories, cancer immunotherapies, which use the body's own immune system to fight cancer, no longer work for many patients. It is unclear why this happens, but the way the immune system attacks cancer cells may play a role in this phenomenon.

Immunotherapies activate specialized killer T cells that trigger the immune response against tumors. These cells can identify cancer cells and inject toxic granules through their membranes to kill them. However, killer T cells are not always effective because cancer cells are naturally good at avoiding detection. During treatment, their genes tend to mutate, giving them new ways to evade the human immune system.

Interestingly, when scientists analyzed tumor cell genes, they found that many of these genes encode proteins recognized by T cells that do not change significantly, suggesting that the resistance of tumors to immunity may be physical rather than genetic.

About the study

In the present study, researchers examined tumor relapse in detail using a mouse model in which tumors recurred after complete regression. They used a combination of dendritic cell adjuvant and tumor-binding antibodies to induce highly effective T-cell immunity. A variant of this therapy is currently being tested in a multicenter phase I study. In several mouse models, treatment-induced T cell-dependent immunity completely eradicated established tumors. However, after approximately 10 days, almost 50% of the mice developed recurrent tumors that were resistant to subsequent treatments.

The researchers treated melanoma-bearing mice with splenic CD8+ T cells that express T cell receptors (TCRs) against gp100 or TRP2 melanoma antigens. Note that the antigen glycoprotein 100 (gp100) is expressed in almost 61% of melanomas, followed by tyrosinase-related protein 2 (TRP2). In addition, the team tested whether these tumor-infiltrating T cells (TIL) were active. To do this, they transferred them into naive mice and challenged them with tumor cells. In addition, they examined changes in the immunogenicity of resistant cell lines beyond gp100 and TRP2 expression.

The team established four cell lines of B16F10 that showed tumor relapse after immunotherapy to analyze their neoantigen position compared to B16F10 from untreated mice and performed whole exome analysis (WES). To assess the relevance of their findings to humans, the researchers recorded the neoantigen load detected in patients with relapsed non-small cell carcinoma and melanoma after treatment with checkpoint blockade.

The researchers also enzymatically digested treated tumors and sorted the live melanoma cells using transmission electron microscopy (TEM). To ensure that the cell structures visible in the TEM were not created by the isolation procedure, the researchers also analyzed histological sections of tumors whose nuclei and cell membranes were fluorescently labeled. Finally, the researchers compared the viability and apoptosis rates of individual tumor cells with those of cell-in-cell and which signaling cascade controlled the cell-in-cell structures.

Study results

The researchers made several important observations in the current study. First, tumor cells survived immunotherapy by organizing themselves into a transient cell-in-cell formation. Second, the parental B16F10 cell lines and their derivatives created after T cell killing were similarly susceptible to T cell killing. Taken together, these results suggest that transient in vivo mechanisms determine the resistance of recurrent tumors. Ribonucleic acid sequencing (RNAseq) analysis also showed that all established cell lines were grouped within similar major components. In contrast, the expression profile of TEM-sorted tumor cells from immunotherapy-treated mice was significantly different.

Remarkably, proteins predominantly expressed on the membrane of interferon-gamma (IFNγ)-activated T cells secreted granules that mediated cell-in-cell formation. T cell-mediated cell-in-cell formation was thus controlled by Signal Transducer and Activator of Transcription 3 (STAT3) and Early Growth Response 1 (EGR1). Confocal analyzes showed that most tumor cells are organized into constellations of many nuclei surrounded by a single plasma membrane. TEM images also showed a unique separation of the plasma membranes and cytosols of the two cells. Tumors treated with immunotherapy showed an increased prevalence of such cell structures, particularly at sites where tumor cell apoptosis occurred. Inhibiting these factors before immunotherapy could significantly improve their therapeutic effectiveness.

Conclusions

Overall, the current study revealed a significant limitation of current immunotherapy. It revealed a previously unknown resistance mechanism that allowed tumor cells to endure immune-mediated killing without compromising their immunogenicity.

The study described a novel mechanism of resistance to immune checkpoint blockade with significant implications for cancer immunotherapy that may extend to other malignancies. Gutwillig et al. Using several mouse tumor models, it was shown that tumor cells reorganize themselves when attacked by the immune system by hiding inside each other and thus getting under many layers of the cell membrane. At this point, killer T cells could identify the outer cell and inject it with toxic granules, but they cannot reach the cells inside. Gutwillig et al. identified some of the signals that killer T cells release and recognize cancer cells and showed that blocking them could make cancer cells stop hiding and make immunotherapy more effective. This novel approach could influence future research and help develop new cancer therapies or improve existing treatments.

Reference:

• Amit Gutwillig, Nadine Santana-Magal, Leen Farhat-Younis, et al. (2022). Eine vorübergehende Zell-in-Zell-Bildung liegt einem Tumorrückfall und einer Immuntherapieresistenz zugrunde. eLife. doi: https://doi.org/10.7554/eLife.80315 https://elifesciences.org/articles/80315

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