Single-Cell Functional Proteomics Reveals TSC Tumor Response to CAR T Cell Treatment

Tuberous sclerosis complex (TSC) is an autosomal dominant, multisystem genetic disorder characterized by the growth of benign tumors in various parts of the body such as the skin, brain, and kidneys. This condition often causes considerable health problems, affecting over 1.5 million children and adults worldwide.1,2

TSC is caused by biallelic mutations of the TSC1 or TSC2 genes, which code for tumor suppressors.1 These mutations frequently lead to benign tumors in vital organs, which activate the mTORC1 pathway, controlling cell growth and metabolism.

Given that benign tumors can rely on ganglioside D3 (GD3), a membrane glycophospholipid that is expressed in normal brain development and malignancies, to activate the mTORC1 pathway, researchers compared GD3 expression in tissues from patients with TSC as well as controls, in a recent study published in JCI Insight. The team also performed preclinical studies with GD3-overexpressing CAR T cells due to the fact that GD3 overexpression was not accompanied by marked natural immune responses to the target molecule.

Polyfunctionality Levels Reveal Cytotoxic Subsets Responsible for Antitumor Responses

The team treated Tsc2+/- heterozygous aging mice that carried spontaneous tumors with GD3 CAR-T as well as untransduced T cells and evaluated the outcome. In the GD3 CAR-T treated mice, the majority of mice were tumor free. In the untransduced T cell treated mice, all carried tumors.

The use of Isoplexis’ single-cell functional phenotyping platform allowed for the analysis of cytokine expression profiles at the single-cell level, revealing that CD4+ GD3 CAR-T cells and CD8+ GD3 CAR-T cells had different cytokine secretion profiles, which may act synergistically to produce antitumor responses in vivo.

This study revealed that stimulated GD3 CAR-T cells had a highly polyfunctional profile, indicative of their potential to have measurable antitumor responses in vivo in TSC patients. Polyfunctionality was increased 9-fold and 10-fold among GD3 CAR-T cells in the CD4 and CD8 populations, respectively.

The results led researchers to conclude that polyfunctional CAR-T cells were cytotoxic toward GD3-overexpressing targets. The CAR-T cells substantially and durably reduced the tumor burden in Tsc2-/- tumor mice (immunodeficient) cells, as well as increased T cell infiltration. Moreover, GD3 CAR-T cells limit spontaneous tumors in aging Tsc2+/- mice.

“The high polyfunctionality profile detected in the stimulated GD3 CAR-T cells indicate the functional capacity of these cells upon antigen encounter that will likely translate into measurable in vivo antitumor responses in patients with TSC,” the authors wrote. They further added, “Polyfunctional T cells that secrete multiple cytokines sequentially or simultaneously have major implications for the efficacy of cellular therapies and are used as a criterion for the assessment of treatment responses.”

Novel CAR-T Therapy Provides New Targets for Future TSC Treatments

Researchers determined that after CAR-T cell treatment, most of the mice were tumor free, while the control mice had tumors. These results indicate that the CAR-T cell treatment had a strong treatment effect and that targeting GD3 may be successful in the treatment of TSC.1 This is encouraging news, as it could significantly improve the lives of patients who are suffering from this incurable disease.

This study is yet another example of the power of our single-cell functional phenotyping platform, which provided a method of identifying highly potent subsets of CAR-T cells within a population and the chemokines contributing to their enhanced polyfunctionality.

Contact us today to learn how we can help accelerate your therapy development with functional Proteomics.


  1. Ancy Thomas et al. “Benign tumors in TSC are amenable to treatment by GD3 CAR T cells in mice.” JCI Insight. 2021;6(22):e152014.
  2. U.S. National Library of Medicine. (2020, August 18). Tuberous Sclerosis Complex: Medlineplus genetics. MedlinePlus.
Share This Article
Subscribe to Follow the Data

Weekly email to keep you at the forefront of single-cell & multiplexed proteomics.