GBM is the most common and aggressive brain tumor, affecting three to five individuals in every 100 000 people. The average age at diagnosis is 60 years and available treatments, involving surgery followed by radio- and chemotherapy, are associated with a median survival of only 15 months. In addition, GBM generally develops resistance to therapy, and only a few patients experience prolonged survival.
The goal of this project is to understand how the tumor microenvironment (TME), particularly immune system components, differ between tumors sampled at diagnosis and at recurrence, and how this correlates with patient response to standard and experimental treatments. In addition, patients with extraordinarily long survival will be analyzed and compared with patients experiencing a shorter course of disease.
Work on this project is supported by Wellcome Leap as part of the Delta Tissue program and is led by the Wyss Center, in collaboration with the University of Geneva, Geneva University Hospital and the Paris Brain Institute.
The problem
Brain tumors are not usually analyzed as a whole. As a result, the complexity of the tumor and its microenvironment is not captured in its entirety
Investigations are typically performed on very thin GBM slices, only about 10 microns thick, and fail to capture the 3D complexity of the TME. In addition, standard analyses rarely analyze how the TME changes over time upon treatment. GBM comprise many different types of cells, each with their own genetic makeup. This tumor cell diversity enables cells to adapt to their environment.
A contributing factor to GBM resistance to current treatments is likely the immunosuppressive TME. The TME is composed of immune cells, blood vessels, molecules released by tumor cells as well as hypoxic areas. Altogether, this milieu is immunosuppressive, allowing the tumor to escape the immune response and progress.
“To address this challenge, we are bringing together a dynamic group of scientists. Our colleagues at the Paris Brain Institute are experts in tissue preparation and computational analysis and, at the Wyss Center, we have deep expertise with fast and high-resolution lightsheet microscopy. Along with our collaborators from the University of Geneva and Geneva University Hospital, our aim is to understand the role of specific genes and proteins in the development of the tumor.”
A new approach
Revealing the 3D structure of large GBM samples to find correlates of prolonged survival
The team is using high-resolution 3D microscopy to map the spatial distribution of immune cells, tumor cells, genetic markers, blood vessels and hypoxic zones in entire human GBM.
By accurately mapping the TME in tumors from the same patient obtained before and after treatment, the goal is to build a model to help predict response to therapies. In addition, by comparing patients with short versus long survival, it will help identify parameters of the TME associated with longer survival, opening avenues for the development of novel therapies.
The experimental pipeline: A series of technologies, not yet applied to human brain tumors, will be used to map the spatial distribution of molecular markers, cells and blood vessels
Tissue preparation and labelling
Tissue Clearing
To enable 3D microscopy of large tumor samples that are millimeters or even centimeters thick, the tissue must be made completely transparent using a process known as clearing.
Clearing removes opaque lipids, including fats, and replaces or modifies them to make the sample transparent.
ELAST
The team is exploring a new method known as entangled link-augmented stretchable tissue-hydrogel (ELAST). This involves embedding the sample in an elastic polymer gel that allows it to deform and reshape. Stretching the sample over hundreds of cycles while it bathes in a solution of antibodies makes labeling of thick samples more efficient.
Labeling
To differentiate between different cell types, blood vessels and other elements of the TME under the microscope, the former are tagged with different fluorescent molecules.
The team uses immunolabeling - a technique in which modified antibodies, that glow under certain wavelengths of light, target proteins of interest. For example, targeting proteins that occur in immune cells allows visualization of these cells throughout the entire sample.
Labeling specific proteins enables the researchers to visualize and understand the interplay between the different components of the tumor and TME.
Spatial transcriptomics
Spatial transcriptomics determines the location of expressed genes within an intact piece of tissue.
The team is using an approach which binds and identifies RNAs from specific genes thought to be involved in tumor development or progression.
This detailed genetic information could be used to target personalized therapies based on the genetics of individual tumors.
Imaging of immune cells (labeled with CD3 in purple - an antibody coupled to fluorescent particles and specific for T lymphocytes) and cellular nuclei (labeled with DAPI in Cyan) in a human glioblastoma sample.
RNA probes detect known genes thought to be involved in tumor development.
High resolution 3D imaging
The team is using microscopes optimized for high spatial resolution (COLM), high speed (mesoSPIM) and large fields of view (ClearScope) to capture the molecular and cellular content of clarified human GBM in 3D without slicing or damaging the samples.
COLM - Clarity Optimized Lightsheet microscope (high spatial resolution)
MesoSPIM – (high speed)
ClearScope (Large fields of view, optimized for large samples)
Data analysis and modelling
Data on the spatial organization of molecular markers, cell populations and vasculature in a tumor will be integrated with clinical data. Together, the information will be used to train models and predict tissue states over time.
Ultimately, the goal is to be able to understand what parameters are modified over time upon treatment and are possibly associated with better outcomes. This could help optimize treatment for patients or help to develop novel treatment modalities.
Star-shaped astrocytes in a human GBM. Astrocytes play many roles in brain homeostasis – in which the brain self regulates in a changing environment – but are transformed upon tumorigenesis to give rise to GBM. Image acquired with a confocal microscope on a 100 µm thick human GBM slice.
Experimental therapies
The local immunosuppressive effect of the GBM TME is thought to play a role in tumor progression. A new generation of immunotherapies attempts to reverse this immunosuppression by boosting the immune response. However, these strategies do not yet benefit the majority of patients.
A phase I/II clinical trial testing a GBM-derived peptide vaccine combined with an immune checkpoint inhibitor is enrolling patients with recurrent GBM at the University of Geneva and Geneva University Hospital. This immunotherapy trial aims at stimulating the patient’s immune system, and in particular T cells, towards tumor cells.
The team will map tumors from two patient cohorts in the trial, one being treated with the peptide vaccine alone and one with the vaccine combined with the immune checkpoint inhibitor pembrolizumab, an antibody used to potentiate vaccine-elicited T cell responses. This will help understand the effects of the peptide vaccine on the GBM TME and potentially identify parameters associated with patient outcome.
"We hope that the project will lead to a better understanding of the role of the tumor microenvironment in patients’ response to standard or experimental intervention and help to identify those patients likely to respond."
The insights provided by this project will provide a better understanding of the role of the TME in the response of patients to standard or experimental treatments, help to identify those patients with prolonged survival and open new avenues for developing novel treatment options.
Detection of GFAP RNA molecules (grey dots) showing their location within a human glioblastoma sample. GFAP is a protein found in glial cells (astrocytes), one of the constituent cell types of tumors. Here, probes specifically bind to RNA molecules which code for GFAP, they are copied multiple times – known as amplification – and labelled with fluorescent ‘tags’. Cell nuclei: blue dots (labeled with DAPI), dark space indicates an area with no GFAP expression. Scale bar: 100µm
