Liver cancer cells that rub shoulders with a class of immune cells called macrophages promote treatment-resistant recurrence of the cancer, according to a study led by Stanford Medicine researchers. Two drugs that restrain that cozy relationship eliminated the liver cancer cells in two mouse models of recurrent liver cancer and slowed or prevented tumor recurrence in the animals.
The study used techniques developed at Stanford Medicine to precisely map the location of more than 1 million single cells in more than 100 human liver tumor samples after initial chemotherapy treatment. Although this form of treatment, called chemoembolization, is often successful - leading to no evidence of cancer - the tumors frequently return within a few months.
"We wanted to know where these residual tumor cells are hiding," said Renumathy Dhanasekaran, MD, assistant professor of gastroenterology and hepatology. "You can't see them on a scan, and there are no biomarkers in the blood we can track. Our study shows that the location of the cells in the tumor - their biological ZIP code - matters. We found that tumor cells enlist nearby macrophages to shield them from attack by other immune cells."
Sheltering in place, the tumor cells function as cancer stem cells, lying dormant and undetectable for weeks or months before waking to promote the growth of a new tumor.
Dhanasekaran, who is a member of the Stanford Cancer Institute, and professor of medicine and of pathology Dean Felsher, MD, PhD, are the senior authors of the study, which was published Sept. 20 in Nature Cancer. Postdoctoral scholars Lea Lemaitre, PhD; former medical student Nia Adeniji, MD; and former undergraduate student Akanksha Suresh are the lead authors of the paper.
Sifting through a million cells
The researchers had to overcome several obstacles to conduct the study. Studying residual disease in a solid tumor such as liver cancer is difficult because the tissue remaining after treatment is mostly made up of dead cells, so many samples are needed. And identifying not just the precise location of every cell relative to one another - a cellular milieu known as the tumor microenvironment - but also the genes and proteins each cell is producing has been made possible only by recent computational and machine-learning advances.
The researchers drew on biopsies of 108 previously treated human liver tumors conducted at Stanford Medicine, comprising more than 1 million cancer cells, and used a spatial mapping technique developed at Stanford Medicine called co-detection by indexing (CODEX) to identify 11 major cell types in the tumors. They picked three cell types - tumor cells, macrophages and the immune cells known as T cells - for further study.
Renumathy Dhanasekaran
The researchers then subdivided these cell types based on proteins on their surfaces and compared their prevalence and locations in liver tumors that hadn't been treated with tumors that had been treated with chemotherapy. They found that the two groups were quite different.
The cells remaining in treated tumors included more cancer cells associated with tamping down the immune system than were found in untreated liver tumors. A type of macrophage called M2-like was also more prevalent in the residual tumors. Additionally, the cancer cells remaining in the residual tumors had markers on their surfaces that suggested a more stem-like phenotype - including a resistance to chemotherapy - than untreated cancer cells.
"We found that, although these stem-cell-like cancer cells were surrounded by macrophages, these macrophages were not attacking the tumor," Dhanasekaran said. "Instead, the macrophages had flipped their personalities to become pro-tumor - protecting the cancer cells from attacks by T cells."
A closer look at the communication pathways between the cancer stem cells and the M2-like macrophages revealed a molecular conversation mediated by two known cancer-associated molecules: TGF beta and PDL1.
PDL1, a molecule found on the surface of some cells, inhibits T cells. PDL1's job is an important one, as it prevents autoimmune diseases sparked by an overly enthusiastic immune response. But it can also blunt the immune response to cancers. TGF beta is a member of a molecular signaling pathway that can be activated by PDL1 to promote cancer growth - also by dampening the immune response.
"We hypothesized that the macrophages expressing PDL1 promote tumor growth by activating TGF beta in the cancer cells, and causing T cell exhaustion," Dhanasekaran said.
Indeed, studies in mouse models of recurrent liver cancer showed that blocking the activity of both PDL1 and TGF beta increased the animals' immune response to the cancer, eliminated the cancer cells in residual disease and prevented recurrence of the cancer.
'Very promising'
"This approach appears very promising," Dhanasekaran said, noting that the drugs to block PDL1 and TGF beta have already been tested in humans for other diseases. "If we can eliminate the small fraction of residual liver cancer cells that remain after treatment, we could significantly reduce the risk of recurrence."
The relationship between PDL1 and TGF beta would not have been discovered without identifying the proximity of residual tumor cells to M2-like macrophages in the tumor microenvironment.
Dhanasekaran said that researchers knew PDL1 expression played a role in cancer, but she noted, "It's not sufficient to just see it in circulation, or in a tumor generally. This study highlights how PDL1-expressing macrophages interact physically with cancer cells to promote immune escape and cancer recurrence. It's more than just the individual players; it's how they interact spatially. This is a beautiful example of how preserving and studying the spatial orientation of cells within a tumor can identify previously unknown molecular mechanisms that promote tumor growth and suggest new therapeutic approaches."
Researchers from Enable Medicine and Aarhus University contributed to the study.
The research was supported by the National Institutes of Health (grants CA222676, CA208735 and CA253180), the Cancer League and the American College of Gastroenterology.
