by Duke-NUS Medical School

SPP1⁺ macrophages are increased during fibrotic disease across human tissues. (A–F) Uniform Manifold Approximation and Projection (UMAP) dimensionality reduction of all monocytes and macrophages in single-cell RNA-sequencing (scRNA-seq) of liver (A), lung (B), heart (C), skin (D), endometrium (E), and kidney (F) from fibrotic disease patients or controls. Credit: eLife (2023). DOI: 10.7554/eLife.85530

Scarring goes more than skin deep. It can occur in any organ because of injury from smoking and excessive alcohol consumption or as a byproduct of chronic conditions like endometriosis, cardiovascular disease and autoimmune disorders. While scar formation is essential in wound healing, when the scarring—or fibrosis—spins out of control, it can lead to deadly consequences, contributing to almost 2 out of 10 deaths worldwide every year.

"And we still have nothing in terms of drugs to treat fibrosis," said Jacques Behmoaras, who is the deputy director of the Center for Computational Biology and an associate professor with the Cardiovascular and Metabolic Disorders Program at Duke-NUS.

Fascinated by this carefully balanced process, Behmoaras and colleagues delved deeper, zooming in on the role of macrophages, immune cells that are known to repair injured tissues and organs, to determine if the macrophages at work in the scarring of different organs share any traits that could hold the key to a new treatment approach.

"From previous studies, including our own, we had noticed a particular molecule that kept popping up in macrophages in fibrosis, a bit of a smoking gun really," systems geneticist Enrico Petretto, who leads the Center for Computational Biology and is also an associate professor with the Cardiovascular and Metabolic Disorders Program at Duke-NUS. "But rather than focus on just this signal, we wanted to approach this question in a totally unbiased manner."

That meant characterizing all the macrophages present in healthy and fibrotic human organs—an impossible task for a single lab. So instead of reaching for a pipette, the two scientists, who share a passion for computational biology, and their teams turned to big data. Using publicly available single cell sequencing datasets, they grouped macrophages by their activation signatures in as many types of organs as they could find data for.

"We created one of the largest systematic studies, if not the largest systematic study, of macrophages in fibrosis without lifting a single pipette," said Petretto, and in the process they saved costs and eliminated the need to use animal model systems.

Among the methods the team applied were machine learning algorithms that helped them first to identify all cells catalogued in the raw data of the 15 studies that met their inclusion criteria. After filtering out the unwanted cell types, they ended up with 235,930 macrophages from healthy and fibrotic human heart, lung, liver, kidney, skin and endometrial tissue. To profile and cluster these by their different identities, without bias, they applied another set of algorithms.

"We found that the number of macrophages expressing SPP1 consistently increased in all fibrotic tissues," said Petretto.

Looking deeper still, they found that during the scarring process, the group of macrophages that had been labeled as SPP⁺ could be separated into two potentially opposing camps: one which promotes fibrosis by triggering the deposition of structural scaffolding between cells, which is essential during the early phases after an injury; and an opposing camp that will dismantle the scaffolding after the tissue has healed.

"They share the same properties but they're like yin and yang," said Behmoaras. "And it is the same in each tissue."

One organ not on the list that he would have liked to include in their paper, which was published in eLife, is muscle: "The one organ I would have liked to include is the muscle. That remains as a project to investigate in the future."

While they may have had to leave out muscle, the protocol is ready for them or anyone else to build on this analysis—a fact that the team was determined to ensure.

"To achieve this, we really had to start from scratch," recalled John Ouyang, a principal research scientist with the Cardiovascular and Metabolic Disorders at Duke-NUS, who spent some six months patiently scouring the supplementary data of the 15 papers, re-annotating them, and resolving first intra-dataset differences and then inter-dataset discrepancies.

"It took is about six months and four or five iterations before we had a protocol that it is totally reproducible. And now, seeing it working over and over—that's really exciting," he said.

Added Petretto, "We're very proud to have a protocol that anyone can run."

With this in-silico proof in hand, the team is heading back to the lab to confirm their findings, starting with the kidneys.

Petretto, who is also Associate Dean for Research Informatics at Duke-NUS, added, "We also want to investigate how to boost the 'good' population at the expense of the 'bad' guys, so that we can develop new therapies that can be effective against fibrosis in general, regardless of which organ is affected."

And it is not just the potential for new treatments of fibrotic diseases that advanced with this discovery. "If we understand scarring, we can understand healing," said Behmoaras. "If we understand healing, we can ask questions like what makes your tissue regenerate quicker?"

More information: John F Ouyang et al, Systems level identification of a matrisome-associated macrophage polarisation state in multi-organ fibrosis, eLife (2023). DOI: 10.7554/eLife.85530

Provided by Duke-NUS Medical School 

Novel therapeutic target overcomes resistance to radiation therapy

by University of Chicago Medical Center

Graphical Abstract. Credit: Journal of Clinical Investigation (2023). DOI: 10.1172/JCI172919

A new study finds that radiation therapy (RT) suppresses a key protein called bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) and activates immune suppressive cells. These effects dampen the capacity of cancer-fighting immune cells and decrease the effectiveness of radiation, inducing therapy resistance in cancer patients, according to a paper published December 15, 2023 in the Journal of Clinical Investigation.

Radiation therapy is a common cancer treatment that kills cancer cells and activates immune cells to fight cancer. Yet this process also recruits immunosuppressive cells like myeloid-derived suppressor cells (MDSCs), limiting the effectiveness of radiation therapy.

TGF-β in radiotherapy resistance

Researchers at the University of Chicago Medicine Comprehensive Cancer Center conducted a study to understand the mechanisms underlying MDSC-induced therapy resistance. MDSCs secrete a variety of immunosuppressors in response to RT. One such secreted protein, known as transforming growth factor-beta (TGF-β), plays a critical role in tumor progression. Thus, the researchers thought targeting TGF-β could be a therapeutically beneficial approach in radiation therapy-resistant patients.

"Although TGF-β is known to have an established role in immune suppression and migration of MDSCs, it is globally expressed. So, drugs that directly target TGF-β may induce unwanted side effects. Thus it is critical to understand the mechanisms that regulate the TGF-β signaling in MDSCs so we can target TGF-β indirectly and have better radiation treatment outcomes," said Ralph Weichselbaum, MD, Daniel K. Ludwig Distinguished Service Professor and Chair of Radiation and Cellular Oncology at UChicago Medicine.

Radiation reduces BAMBI levels

BAMBI is a mock receptor, or pseudoreceptor, that mimics the TGF-β receptor. It is known to suppress TGF-β signaling and is implicated in tumor suppression.

The research team, led by Liangliang Wang, Ph.D., a Research Assistant Professor in the Weichselbaum laboratory, analyzed the transcriptome data of cancer patients and found that patients with higher BAMBI expression showed prolonged overall survival in four tumor types: kidney renal clear cell carcinoma; kidney renal papillary cell carcinoma; pheochromocytoma and paraganglioma; and uterine corpus endometrial carcinoma.

Moreover, BAMBI was highly expressed in immune cells like monocytes and macrophages compared to other cell populations in melanoma and colorectal cancer patients.

BAMBI improves survival rate

"It is remarkable to see a significant reduction of BAMBI levels only in tumor-infiltrating MDSCs, not in other immune or tumor cell types following radiation treatment," said Weichselbaum. "We were interested in understanding the mechanism underlying the radiation-induced reduction of BAMBI levels in MDSCs."

Weichselbaum's team, in collaboration with Chuan He, Ph.D., the John T. Wilson Distinguished Service Professor of Chemistry at UChicago, previously published a study in Cancer Cell demonstrating increased levels of a protein known as YTH N6-methyladenosine RNA-binding protein F2 (YTHDF2) in MDSCs following radiation.

"In the current study, we were able to reproduce the similar kind of results in another cohort of patient samples. Moreover, we observed a close interaction between YTHDF2 and BAMBI in tumor-infiltrating immune cells, indicating YTHDF2 might be playing a critical role in regulating BAMBI's expression," said Weichselbaum.

The team conducted animal studies to test if overexpressing BAMBI in MDSCs could suppress the tumor infiltration of MDSCs in mice treated with radiation. As expected, viral delivery of BAMBI significantly reduced tumor growth and increased survival. Interestingly, BAMBI overexpression also further improved the outcomes of immunotherapy in the irradiated mice.

Many pharmacological interventions targeting TGF-β exist. Because many have toxic and non-specific effects, novel therapies like BAMBI—that indirectly target TGF-β and are restricted to immune suppressor cells—are especially promising, as they not only control local tumor growth but may also prevent the cancer from spreading.

The study, "Epitranscriptional regulation of TGF-β pseudoreceptor BAMBI by m6 A/YTHDF2 drives extrinsic radioresistance," was published in in Journal of Clinical Investigation on December 15, 2023.

More information: Liangliang Wang et al, Epitranscriptional regulation of TGF-β pseudoreceptor BAMBI by m6A/YTHDF2 drives extrinsic radioresistance, Journal of Clinical Investigation (2023). DOI: 10.1172/JCI172919

Journal information: Journal of Clinical Investigation  , Cancer Cell 

Provided by University of Chicago Medical Center