August 13, 2022

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Physical immune checkpoints improve the effectiveness of tumor immunotherapy?

Physical immune checkpoints improve the effectiveness of tumor immunotherapy?



 

Physical immune checkpoints improve the effectiveness of tumor immunotherapy?

 

Immunotherapy is developing rapidly, and heavy researches are emerging one after another. In the process of fighting against malignant tumors, researchers have gradually discovered that some malignant tumors can resist the effects of immunotherapeutics through a variety of ways and affect the effect of cell adoptive therapy.

 

Due to the abnormal production and cross-linking of extracellular matrix proteins, tumor tissues are usually harder than normal tissues, but single tumor cells are usually softer than non-malignant tumor cells [1,2].

Cell softness is a biomechanical property. Current studies have shown that the decrease in tumor cell stiffness is related to the transformation, malignancy and metastasis of cancer cells [3].

 

Recently, the research team led by Professor Tang Li and Professor Mahmut Selman Sakar from the Federal Institute of Technology in Lausanne, Switzerland published important research results on Nature Biomedical Engineering .

They found that tumor cell membranes are enriched in cholesterol, reduced in stiffness, and are resistant to T cell-mediated cytotoxicity; cholesterol depletion can increase tumor cell stiffness, which in turn enhances the tumor cell-mediated killing effect of T cells [4].

 

This research puts forward the concept of physical immune checkpoints for the first time and expands the scope of immune checkpoints. This new discovery can be used for the development of a new generation of targeted therapies and improve the effectiveness of tumor immunotherapy.

 

Physical immune checkpoints improve the effectiveness of tumor immunotherapy?

 

At present, the work of researchers looking for new immune checkpoints is mainly in biochemical signals, and there are few studies involving potential inhibitory pathways in biomechanical signals, such as target cell stiffness.

Studies have shown that the decrease in tumor cell stiffness is due to the softening of the cytoskeleton network and plasma membrane [5].

The physical properties of the target cell surface may affect the interaction between tumor cells and T cells [6]. However, the role of cancer cell stiffness in evading immune surveillance remains unclear .

 

T cells can exert force on the immune synapse and enhance the cytotoxicity of target cells [7].

When T cells contact the substrate surface or target cells with lower surface hardness, the cytoskeletal force and the production of effector cytokines are greatly reduced [8,9]. Inspired by the results of these studies, the researchers hypothesized that tumor cells use reduced cell stiffness as a physical immune checkpoint, by weakening the mechanical force of T cells to resist T cell-mediated cytotoxicity.

 

The researchers first proved that mouse and human tumor tissues, tumor cells isolated from these tissues, and mouse T lymphoma cell lines had significantly higher cholesterol levels through Filipin III staining and cholesterol content detection .

 

Methyl-β-cyclodextrin (MeβCD) is a widely used drug solubilizer in clinic, and it has been reported that it can remove cholesterol.

In vivo and in vitro experiments have proved that MeβCD can reduce the plasma membrane cholesterol enrichment of tumor cells, but has no direct effect on the survival ability and apoptosis of cancer cells .

 

Physical immune checkpoints improve the effectiveness of tumor immunotherapy?

▲The plasma membrane of tumor cells is rich in cholesterol

 

In order to test whether the membrane cholesterol level affects the mechanical properties of tumor cells, the researchers used a variety of methods to measure cell stiffness and found that compared with untreated cells, the surface stiffness of cholesterol-added cells was significantly reduced, the deformability was significantly increased, and cholesterol was depleted. It can significantly increase the hardness of the cell surface.

These results show that increasing cholesterol on the plasma membrane helps tumor cells soften, and depleting cholesterol through MeβCD intervention can increase the stiffness of tumor cells .

 

Physical immune checkpoints improve the effectiveness of tumor immunotherapy?

▲ Schematic diagram of the relationship between cell stiffness and membrane cholesterol levels

 

The research team next explored whether the decrease in cell stiffness would be resistant to T cell-mediated cytotoxicity.

They inoculated B16F10 cancer cells on hydrogels of different hardness and co-cultured them with activated Pmel CD8+ T cells (which can specifically recognize the gp100 antigen of melanoma B16F10 tumor cells) .

 

In vitro experiments have shown that when exogenous cholesterol is added, the survival rate of B16F10 cells is higher, indicating that the decrease in tumor cell stiffness can be used as a physical inhibitory way to affect the killing effect mediated by T cells .

In vivo experiments verified this result. It is worth noting that taking cholesterol alone has no effect on the tumor growth or survival rate of the treated mice.

 

Physical immune checkpoints improve the effectiveness of tumor immunotherapy?

▲ Reduced surface hardness of tumor cells impairs the killing effect mediated by T cells

 

In order to study whether this physical immune checkpoint can be inhibited by enhancing the stiffness of tumor cells.

The researchers prepared B16F10 cancer cells overexpressing ACAT1 (ACAT1 OE B16F10), and the membrane cholesterol level was 70% lower than that of natural B16F10 cells.

This kind of cell has higher stiffness and shows higher sensitivity to T cell-mediated cytotoxicity.

In vivo experiments show that Pmel CD8+ T cells better control the growth of ACAT1 OE B16F10 tumor cells. Mice bearing ACAT1 OE B16F10 tumors also showed prolonged survival.

 

On the basis of these findings, the researchers tried to develop an intervention that can increase the stiffness of tumor cells to enhance the effect of cancer immunotherapy.

B16F10 cells treated with MeβCD can maintain a low membrane cholesterol level within 5 hours after treatment, and the sensitivity of hardened tumor cells pretreated with MeβCD to T cell-mediated killing is significantly increased .

In animal experiments, it was found that in different tumors, after combined with MeβCD intervention, the effect of Pmel CD8+ T cell adoptive therapy alone or in combination with IL-15 super agonist (IL-15SA) was enhanced .

 

▲ MeβCd improves tumor cell stiffness and enhances the efficacy of immunotherapy

 

It is worth noting that MeβCD intervention did not cause side effects such as weight loss, splenomegaly, and increased CD8+ T cell infiltration and activation in the spleen. MeβCD alone has no therapeutic effect as a treatment method, indicating that the presence of antigen-specific cytotoxic T cells is a necessary condition for killing target cells with increased stiffness .

 

In addition, the biochemical pathways of cell-mediated cytotoxicity, such as the interaction of Fas protein and Fas ligand, the secretion of effector cytokines such as IFN-γ and TNF-α, and the particles of cytolytic proteins (such as perforin and granzyme) Exocytosis, etc., are not affected by tumor cell sclerosis.

 

According to reports, the phosphorylation of proline-rich tyrosine kinase 2 (Pyk2) is positively correlated with the cellular force exerted by primary T cells.

Researchers conducted experiments on hydrogels with different hardness and proved that Pmel CD8+ T cells produce higher mechanical stress on harder surfaces.

When co-cultured with tumor cells with enhanced cell hardness, Pmel CD8+ T cells can induce higher levels of pPyk2 .

These results indicate that T cells exert stronger cellular power on sclerotic tumor cells.

 

▲ T cell force mediates enhanced cytotoxicity to sclerosing cancer cells

 

Simply put, this study found that reduced cell stiffness can be used as a physical immune checkpoint.

By depleting tumor cell plasma membrane cholesterol to increase the hardness of cancer cells, this physical immune checkpoint can be inhibited, T cell-mediated killing effect can be enhanced, and the effect of immunotherapy can be improved.

 

▲ Mechanism of physical immunosuppression caused by reduced tumor cell stiffness

 

This discovery deepens our understanding of the multidimensional mechanisms of tumor immunosuppression, and provides research ideas for exploring new immune checkpoints and developing new tumor treatment methods.

 

 

references:

[1] Levental KR, Yu H, Kass L, et al. Matrix crosslinking forces tumor progression by enhancing integrin signaling. Cell. 2009;139(5):891-906. doi:10.1016/j.cell.2009.10.027

[2] Cross SE, Jin YS, Rao J, Gimzewski JK. Nanomechanical analysis of cells from cancer patients. Nat Nanotechnol. 2007;2(12):780-783. doi:10.1038/nnano.2007.388

[3] Swaminathan V, Mythreye K, O’Brien ET, Berchuck A, Blobe GC, Superfine R. Mechanical stiffness grades metastatic potential in patient tumor cells and in cancer cell lines. Cancer Res. 2011;71(15):5075-5080. doi:10.1158/0008-5472.CAN-11-0247

[4] Lei K, Kurum A, Kaynak M, et al. Cancer-cell stiffening via cholesterol depletion enhances adoptive T-cell immunotherapy. Nat Biomed Eng. 2021;5(12):1411-1425. doi:10.1038/s41551-021-00826-6

[5] Alibert C, Goud B, Manneville JB. Are cancer cells really softer than normal cells?. Biol Cell. 2017;109(5):167-189. doi:10.1111/boc.201600078

[6] Köster DV, Mayor S. Cortical actin and the plasma membrane: inextricably intertwined. Curr Opin Cell Biol. 2016;38:81-89. doi:10.1016/j.ceb.2016.02.021

[7] Basu R, Whitlock BM, Husson J, et al. Cytotoxic T Cells Use Mechanical Force to Potentiate Target Cell Killing. Cell. 2016;165(1):100-110. doi:10.1016/j.cell.2016.01.021

[8] Hui KL, Balagopalan L, Samelson LE, Upadhyaya A. Cytoskeletal forces during signaling activation in Jurkat T-cells. Mol Biol Cell. 2015;26(4):685-695. doi:10.1091/mbc.E14-03-0830

[9] Saitakis M, Dogniaux S, Goudot C, et al. Different TCR-induced T lymphocyte responses are potentiated by stiffness with variable sensitivity. Elife. 2017;6:e23190. Published 2017 Jun 8. doi:10.7554/eLife.23190

Physical immune checkpoints improve the effectiveness of tumor immunotherapy?

(source:internet, reference only)


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