Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - SCALTIE (Single cell analysis of the lung tumour-immune ecosystem: Developing new tools for effective immunotherapy)

Teaser

Tumours evolve to evade immune surveillance through a series of immune suppression mechanisms. In line with this, cancer immunotherapies, in which the immune system is turned on against tumours, are currently revolutionizing cancer treatment. Unfortunately, still many cancer...

Summary

Tumours evolve to evade immune surveillance through a series of immune suppression mechanisms. In line with this, cancer immunotherapies, in which the immune system is turned on against tumours, are currently revolutionizing cancer treatment. Unfortunately, still many cancer patients do not respond to immunotherapies. Therefore, there is an urgent need to develop new tools for effective immunotherapy in order to increase treatment response rate. However, current methods that investigate bulk populations of cells lack the resolution to identify and analyse the diverse subpopulations of cells and the unique pathways and checkpoints they activate within the tumour ecosystem. I proposed to dissect the interface between human tumor and the immune system by combining single-cell RNA-Seq, modeling approaches, advanced functional assays and mouse models. I believe this work will lead to a better understanding of tumour immunity and the development of novel therapies tailored to individual cancer patient. Our single cell analysis of melanoma tumor immune infiltrates reveals a CD8+ T cell population that displays a continuous progression from a transitional towards a dysfunctional state and a distinct population of CD8+ cytotoxic T cells that are not connected to the dysfunctional gradient. Early dysfunctional T cells show the highest level of proliferation and dysfunctional load is associated with tumor-reactivity.

Work performed

We performed extensive transcriptional profiling of intratumoral immune infiltrates of 25 melanoma patients by massively parallel single cell RNA sequencing of 46,612 immune cells which passed quality control filtering, yielding profiles of 29,825 T and NK cells. While immune cell subtypes were largely shared across most patients, their relative abundance differed considerably between patients, even when disease stage and treatment background were matched. Notably, in spite of this variability in abundance, conserved trajectories of CD8 cells were observed, consistent with an ongoing differentiation process driven by interactions within the tumor microenvironment.
In particular, CD8 positive T cells were observed in two separate subsets, with only one of these transitioning into a dysfunctional T cell pool that is characterized by both known and novel regulatory molecules, and that includes molecules shared with CD4 Treg. Coupled single cell T cell receptor (TCR) sequencing and transcriptional profiling revealed that dysfunctional T cells display the highest level of clonal expansion. Furthermore, analysis of a cell cycle transcriptional signature, as well as flow cytometric analysis of Ki-67 expression and cell cycle progression, provided evidence for ongoing proliferation within this dysfunctional T cell compartment. This proliferative capacity was mainly associated with initial buildup of the dysfunctional program, whereas more advanced dysfunctional cells lost this proliferative signature. In contrast, a discrete pool of cytotoxic CD8 cells showed little evidence of proliferation and was unlinked to the dysfunctional T cell pool, as based on both TCR sharing data and gene module analysis. Collectively, our data suggest that the dysfunctional CD8 T cell pool should be considered a dynamically differentiating and active cell compartment that is likely to drive tumor-reactivity across patients. In-depth models of regulation of this T cell compartment should lead to the identification of novel immune modulatory pathways and optimization of T cell-based cancer therapies.
To exploit and disseminate our results, I have attended the following conferences:
• Third CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference, September 6 - 9, 2017, Germany
• SINGLE CELL GENOMICS 2017, October 16-18, 2017, Israel
• Cell-WIS Symposium: Next Gen Immunology, February 11-14, 2018, Israel
• Forth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference, 2018, USA
• SINGLE CELL GENOMICS, 2018, USA
• Cell Symposia: Single Cells: Technology to Biology, 2019, Singapore
I also published the results of the project in the Cell journal, with a statement in the Acknowledgment that “Hanjie Li is funded by the Marie Curie Individual Fellowship (EU project 746382 - SCALTIE)”. The results were reported by Weizmann Institute website and Netherland Cancer Institute website.

Final results

This project stands at the frontier of two exciting fields, single cell genomics and cancer immunotherapy. Study of the complex biological system, such as tumour-immune ecosystem, at single-cell resolution in an unbiased manner has only recently been possible. And despite its clinical benefits, many studies are still needed to increase the treatment response rate of cancer immunotherapy while minimizing its side effects. This study used single-cell genomics to dissect the interaction between the immune system and human melanoma, and identified novel biomarkers and targets for immunotherapy. This project identified subpopulations of cells specific to tumours, known and novel pathways that play crucial roles in tumour pathogenicity. A comprehensive understanding of these processes provides the basis for understanding why existing cancer (immuno)therapies are only effective in a subset of patients and will provide new potential targets for the development of precision immunotherapy, tailored to the genetic, molecular and cellular characteristics of each individual patient and tumour. This project’s technical contribution is the establishment of a single-cell genomics pipeline for dissecting complex biological systems and its application in clinical settings like cancer and other diseases. The highly innovative and up-to-date nature of the investigation allows me to stand at the forefront of biological research and global science.
I feel strongly that integration of different disciplines including single-cell genomics, bioinformatics, molecular biology, functional genomics, and mouse genetics with a close collaboration with clinics is critically important and urgent for our vision of effective immunotherapy research. Our methods and findings can provide basic insights into the cross talk of tumour and immune cells in the tumour microenvironment and provide seeds for future immunotherapy advancements towards personalized cancer treatments in Europe and world-wide. With our action as a prototype, the host lab is planning to collaborate and synergize to lead a large-scale collaborative venture for effective immunotherapy treatments around Europe.