Revolutionizing anti-tumor T-cell production with carbon monoxide

An MUSC Hollings Cancer Center research group led by Shikhar Mehrotra, Ph.D., has identified a more effective and less expensive way to prime immune cells to fight cancer. This new finding and preclinical data were published April 11 in Cancer Research and are being highlighted at the American Association for Cancer Research (AACR) annual meeting.

Adoptive cell transfer is a powerful tool against cancer. This treatment method uses donor or patients’ immune cells to fight their cancer. Adoptive cell transfer is a promising treatment option; however, this method has not worked well for long-term cancer control in patients with a high tumor burden. Scientists still are learning how to overcome the harsh conditions inside the tumor microenvironment.

“The goal is to give cancer patients immune cells that can take care of the tumor for the long term,” said Mehrotra, who is also the scientific director of the FACT-accredited Clean Cell Therapy Unit at MUSC. “Unfortunately, immune cells often get exhausted in the tumor microenvironment, and they do not perform their anti-tumor functions well enough to kill the tumor.”

Mehrotra’s research lab focuses on understanding the biological pathways that lead to a robust and long-lived anti-tumor response in T-cells. To do this, the team studies and manipulates mitochondria, which is the energy source of cells.

Before this work, strategies to boost anti-tumor T-cell function by targeting cellular communication between mitochondria and the endoplasmic reticulum (ER) had not been explored. Mitochondria are the critical cell components in terms of regulating cell metabolism and death. The endoplasmic reticulum is responsible for producing proteins but uncontrolled environmental stimuli leading to too much protein production can result in cellular stress, dysfunction and death.

“We found that if we give T-cells stress early on, then they are trained to deal with the stress inside of the tumor. As a result, the immune cells enter the tumor with the altered systems that allow them to cope with the stress.”
— Dr. Shikhar Mehrotra

“Past research has shown that persistent ER stress leads to damaged mitochondria and poor cell function. However, moderate ER stress conditions improve mitochondrial function,” said Mehrotra.

First author Paramita Chakraborty, Ph.D., said they found that the carbon monoxide treatment moderately increased the ER stress in a transient manner. This moderate ER stress in turn induced autophagy, a self-renewal mechanism that maintains healthy mitochondrial function.

Data from other labs have revealed that low-dose carbon monoxide (CO) can strengthen cells by causing mitochondrial biogenesis, which is an increase in the number of mitochondria. “We hypothesized that putting the T-cells through moderate ER stress using CO could improve mitochondrial function and therefore anti-tumor function.”

Using advanced microscopy techniques, single-cell sequencing and mouse models of melanoma with melanoma antigen-specific T-cells, Mehrotra and colleagues found that they could harness cellular stress to improve anti-tumor T-cell function.

“We found that if we give T-cells stress early on, then they are trained to deal with the stress inside of the tumor. As a result, the immune cells enter the tumor with the altered systems that allow them to cope with the stress,” said Mehrotra. Ex vivo, in-cell culture, T-cells that were briefly exposed to low doses of carbon monoxide had increased mitochondrial function. Furthermore, when those immune cells were transferred to mice with melanoma, the tumor size significantly decreased, and survival increased.

Controlling cellular stress has been an interest to Mehrotra for many years. “I was surprised that this method worked this well. Getting results in cell cultures is one thing but getting good results in vivo (taking place in a living organism) is more challenging,” he said. “We know that stress is detrimental if it continues for too long. But stress is not always bad, and once overcome, it can make things stronger. Similarly, we saw that T-cells that go through weak stress early on react positively and then do better in the long term.”

The researchers also confirmed which cellular pathways are affected by endoplasmic reticulum stress in terms of anti-tumor phenotype — the physical expression of one or more genes. Carbon monoxide exposure temporarily activated the endoplasmic reticulum stress sensor molecule called PERK. The team’s data suggested that transient stress that engages PERK could be an essential determinant in signaling between the endoplasmic reticulum and mitochondria that shapes a strong anti-tumor T-cell phenotype to improve immunotherapy.

Mehrotra said that his collaborators’ sequencing knowledge, including researcher Vamsi Gangaraju, Ph.D., from MUSC’s Department of Biochemistry and Molecular Biology, and MUSC’s core facilities helped to strengthen the in-depth technical components of this research.

“We are excited to share this work with our fellow oncologists and researchers at the AACR conference,” said Mehrotra.

Ongoing studies will test low-dose carbon monoxide exposure on human chimeric antigen receptor (CAR)-T-cells. To show proof of concept with human immune cells in the current publication, the researchers exposed immune cells taken from the tumor microenvironment and found that carbon monoxide exposure increased their mitochondrial function and anti-tumor characteristics.

Chakraborty noted that carbon monoxide, if established as an immunomodulatory agent, would be a cost effective and easily available approach for treatment.

“In the future,” Mehrotra said, “we hope this approach can be added to clinical trials to demonstrate the improved T-cell functionality and open this immunotherapy up for more patients.”

Funding: NIH R21CA198646, NIH R01CA236379, NIH R01DE030013 and NIH R41CA239952 to Mehrotra. Support from Shared Instrumentation Grant S10OD018113 and Hollings Cancer Center Shared Resources (partly supported by P30 CA138313) at MUSC is also acknowledged.

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