In Prof. Ido Amit's laboratory at the Weizmann Institute of Science, they identified a gene whose silencing reprograms immune system cells and can transform them from cancer supporters to cancer killers.
To thrive, cancer needs to harness the immune system to its advantage. In many cases, this is done by recruiting immune cells called macrophages and harnessing them for a range of purposes, including protecting the cancer from the rest of the immune system and helping it spread to other tissues. In a new study the published in the scientific journal Cancer Cell, researchers from Prof.'s laboratory Ido colleague Weizmann Institute of Science researchers used advanced gene editing technologies, single-cell sequencing, and artificial intelligence tools to identify the master switch that turns macrophages into cancer supporters. Based on this discovery, they developed a treatment that may offer new hope for bladder cancer patients.
"Macrophages are multifunctional cells – a kind of 'Swiss Army knife' of the immune system – that are capable of activating a variety of programs for different purposes and in different situations," explains Prof. Amit. Macrophages have the ability to be particularly effective tumor destroyers, with a variety of action programs such as creating anti-cancer inflammation or recruiting the rest of the immune system to fight the tumor. Therefore, in order to thrive, most tumors develop unique mechanisms that allow them to transfer macrophages to their side. "In this way, tumors protect themselves from the 'dangerous' side of macrophages, and also trigger the activation of programs that help them grow, such as the ability of macrophages to encourage the expansion of blood vessels that nourish the tumor with oxygen, or to suppress the activity of other cells of the immune system," he adds.
Recent studies have linked the way macrophages are activated – whether they fight or help tumors – to the survival chances of cancer patients. Given their importance, many laboratories have sought ways to reprogram macrophages to activate their anti-cancer programs. “These efforts failed because they divided them into two binary categories – tumor-supporting or tumor-suppressing,” adds Prof. Amit. “We now know that this division misses a significant part of the complexity of the picture.”
The new study, led by Dr. Paddy Siban, Dr. Truong San Pan, Dr. Kai Kun Xuy and Dr. Florian Ingelfinger, took a more complex approach. “We began the study by analyzing databases taken from cancer samples from patients and examining the various functions of macrophages. We wanted to identify which genes had the potential to influence tumor-supporting functions. We thus compiled a list of 120 genes that we suspected played a role in activating tumor-supporting programs,” says Dr. Siban.
The next step was to build a system that would allow them to examine the effect of each of the 120 suspected genes on the function of tumor-supporting macrophages to identify which of them plays a key role in the tumor's takeover of immune system cells. To this end, Prof. Amit and the research team used single-cell resolution research technologies and CRISPR-Cas9 gene editing technology. By combining the two approaches, they created a tool that allowed them to turn off a single gene from the list of suspected genes at a time and see how the change affects the properties and function of macrophages at the individual cell level.
"With this innovative platform, we were able to examine the effect of each of the 120 potential switches on the list on all genes in about 100 different macrophages," says Dr. Shiban. This created a huge database of data on how different genetic switches turn on and off a variety of macrophage action programs when they are in the cancerous environment. "It was a complete mess. With so much information, we had difficulty at first understanding which gene played a significant role and which functions it controlled," recalls Dr. Shiban.
To organize the data, Prof. Amit and the research team used an artificial intelligence tool developed by Prof. Nir Yosef, also from the Weizmann Institute's Department of Systemic Immunology. The tool is called MrVI and it allows you to simplify this type of data set and turn it into a functional map that shows how different genetic switches, each represented as a dot on the map, affect different functions of macrophages, and how similar the effect of each gene is to the effect of other genes.
"With the help of MrVI, we were able to understand which gene needed to be turned off to affect all of the macrophages' cancer-supporting functions and turn them into tumor killers. As expected, we found switches that we had studied in the past that had a major impact on macrophage activity in cancer, such as TREM2, but one gene stood out on the map due to its ability to fundamentally change macrophage activity. The gene, called Zeb2, has not been studied to date in the context of the immune system in cancer," says Dr. Shiban. "We realized that, unlike other genes on the map, a macrophage with active Zeb2 is a macrophage that activates all of its tumor-supporting programs and turns off all of the programs designed to fight cancer. Accordingly, silencing this gene can have the opposite effect." Indeed, in a series of experiments – both in cell cultures and in model animals – the researchers showed that silencing Zeb2 transforms macrophages from cancer-supporting to cancer-killing. In addition, the researchers showed through analysis of databases that patients with high levels of Zeb2 expression are at much higher risk of developing aggressive cancer.
The next step was to try to turn the discovery into a potential cancer treatment. To this end, Prof. Amit and the research team collaborated with Prof. Marcin Kortylewski from the City of Hope Medical Center in California. Prof. Kortylewski developed a unique DNA molecule designed to specifically bind to macrophages and then be ingested by them. "We used this molecule as bait and attached to it a small RNA molecule designed to specifically silence the target gene – Zeb2," says Dr. Shiban. The researchers injected the molecule into the tumor area of mice with bladder cancer and showed that the treatment reprogrammed the macrophages, made them fight the tumor and led to significant shrinkage of the tumors.
"The goal now is to develop a new cancer treatment for humans based on this approach," concludes Prof. Amit. "The study is also an example of how our advanced research technologies offer a deep, high-resolution understanding of how different players in the immune system function in different diseases, and how this understanding offers the possibility of developing new treatments for patients."
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