A New Hyper-Multiplex Workflow using ZEISS Axioscan Enables High-Throughput Imaging of Fragile Tissues with 40-50 Markers over Large Regions and High Magnifications

The tumor microenvironment is comprised of cellular and non-cellular components that can engage with cancerous cells and impact tumor behavior. Professor Johanna A. Joyce leads a lab at the University of Lausanne, Switzerland, with the goal of understanding the complex cellular interactions and spatial biology of the tumor microenvironment, including in primary and metastatic brain tumors. Her team is working to find new ways to exploit the immune and stromal cell populations to develop new treatments to improve the lives of patients.

Dr. Spencer S. Watson, a Research Fellow in Professor Joyce’s lab, published an article investigating how the brain tumor microenvironment responds to radiotherapy, not just in terms of changes in different cell populations, but also regarding how the cells spatially organize themselves. In order to do this, the team worked to create a new multiplex workflow, called Hyperplexed Immunofluorescence Imaging (HIFI), to study the spatial biology of tumors before and after treatment. Their workflow, which utilizes the ZEISS Axioscan digital slide scanner, allowed them to achieve 40-50 multiplexed markers with extremely fragile tissue over large regions and high magnifications, and revealed interesting differences in spatial reorganizations post-radiotherapy treatment.

We believe that identifying consistent changes in the spatial organization of cellular superstructures after treatment can give us clues as to how tumors resist therapies. However, many cell types and phenotypes must be simultaneously assessed over many samples. Our approach for whole slide, high-dimensional imaging scales to high-throughput to derive critical, single-cell data from these images.

HIFI is based on cyclic immunofluorescence to achieve 40-50 multiplexed markers, but extensively modified and optimized to work with extremely fragile tissue types over large region sizes and high magnification. ZEISS Axioscan scales the approach to high throughput with its capability to image up to 100 slides in a single experiment. Combining the ZEISS Colibri 7 light source for low-light exposure with a gentle marker elution, the team was able to repeatedly stain and image fragile samples in a non-destructive manner. Deep-learning models automatically detect individual cells and extracellular structures in the images, and orthogonal spatial analytical approaches find consistent cellular organization features following treatment, and at the point of recurrence.