Using super-resolved in situ sequencing to reveal the cellular encoding of immune-tumour contact events

Project Information

TouchCancer

Grant agreement ID: 101117324

DOI

10.3030/101117324

EC signature date 22 August 2023

Start date 1 September 2023

End date 31 August 2028

Funded under

• European Research Council (ERC)

Total cost

€ 2 124 896,00

EU contribution

€ 2 124 896,00

Coordinated by

BAR ILAN UNIVERSITY
Israel

Periodic Reporting for period 1 - TouchCancer (Using super-resolved in situ sequencing to reveal the cellular encoding of immune-tumour contact events)

Reporting period: 2023-09-01 to 2026-02-28

Summary of the context and overall objectives of the project

The human immune system has the remarkable ability to recognise and destroy cancer cells. This principle lies at the heart of modern immunotherapy, which has transformed cancer treatment for many patients. Yet, most individuals still do not respond to immunotherapy, and the reasons remain unclear. One major gap in knowledge is how immune cells and tumour cells interact directly within human tissues — at the level of individual cells making physical contact. Understanding these microscopic interactions is essential for uncovering how cancers evade immune attack and for developing more effective, personalised treatments.

The TouchCancer project addresses this challenge by combining cutting-edge imaging and molecular biology to study cancer at unprecedented spatial resolution. The project builds on a technology called Expansion Sequencing (ExSeq), which enables sequencing of RNA molecules directly inside tissues while preserving their exact 3D locations. Using this approach, the project identifies how immune and tumour cells influence each other when they are in close proximity — effectively reading out the molecular “conversation” between them.

The research proceeds in two major directions. First, it maps the genes and pathways activated when immune and tumour cells touch, revealing the molecular programs that drive either immune surveillance or tumour evasion. Second, it develops a new method to detect tumour-specific immune receptors directly in patient biopsies, paving the way toward identifying the exact T and B cells that recognise a tumour.

By uncovering how cancer cells and immune cells interact at single-cell and molecular scales, TouchCancer provides fundamental insights into the mechanisms of immune control and escape in human tumours. The expected impact of this work is twofold: advancing the scientific understanding of cancer-immune communication, and laying the technological foundations for next-generation diagnostic and therapeutic strategies in cancer immunology.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The TouchCancer project investigates how immune and tumour cells interact directly inside human tissues and how these local encounters influence cancer progression and immune response. The project combines advanced imaging and sequencing technologies with new computational tools to study these interactions at single-cell resolution.

During the reporting period, the project achieved its first major milestone—completion of Aim 1. Using Expansion Sequencing (ExSeq), the team successfully mapped gene expression across intact human biopsies with nanoscale precision. A dedicated image-analysis tool, InSituSeg, was developed to automatically detect individual cells and their physical contacts within three-dimensional tissue environments. These combined methods revealed that gene expression in immune, stromal, and tumour cells changes gradually with physical distance and local cell density, uncovering complex multicellular influences on immune activity within tumours.

The findings indicate that cellular behaviour in cancer is influenced not only by cell type but also by the immediate spatial context of neighbouring cells. This approach provides a detailed and quantitative perspective on how proximity between immune and tumour cells shapes local gene expression patterns. The results of this work led to two manuscripts—one already published and another in final preparation.

Building on this foundation, the project has initiated Aim 2, which focuses on developing a new spatial sequencing method to identify tumour-specific T- and B-cell receptors directly in human biopsies. Core experimental steps—including RNA anchoring, amplification, and combined in situ/ex situ sequencing—have been successfully validated, confirming the feasibility of receptor mapping at single-cell resolution.

Together, these achievements establish a robust technological and analytical platform for linking immune-cell behaviour to molecular signatures in human cancers, paving the way for improved understanding of immune surveillance and for future precision immunotherapy strategies.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The TouchCancer project has generated new technologies and biological insights that go beyond the current state of the art at the intersection between spatial genomics and cancer immunology. Traditionally, studies of the tumour microenvironment relied on dissociated cells or low-resolution imaging, which destroyed the spatial information necessary to understand how immune and tumour cells communicate. TouchCancer overcomes this limitation by combining super-resolution imaging with in situ RNA sequencing, enabling measurement of gene expression directly within intact human biopsies at nanoscale precision.

A key technological advance is the integration of Expansion Sequencing (ExSeq) with an image analysis pipeline termed InSituSeg. This combination allows precise identification of single cells and the detection of immune–tumour contact events in three dimensions. Using this approach, the project revealed that gene expression in immune, stromal, and tumour cells changes gradually with distance and local cell density, reflecting complex multicellular regulation within the tumour environment. These results extend previous spatial transcriptomic methods by capturing the continuous nature of molecular gradients and interactions between physically neighbouring cells.

In parallel, the project is developing a new spatial sequencing framework to identify tumour-specific T- and B-cell receptors directly inside human tissues. This approach aims to determine the spatial position of immune receptors that recognise tumour antigens, thereby linking immune specificity with tissue context. Once established, this technology could serve as a foundation for new diagnostic tools that characterise patient-specific immune responses in situ.

Together, these advances bridge molecular imaging and immunogenomics, providing a unified view of how immune surveillance and tumour evasion occur at the cellular level. The expected long-term impact includes improved understanding of immune-tumour interactions, better biomarkers for predicting therapy response, and new avenues for personalised immunotherapy design based on spatially resolved immune profiling.