Discovery points to new drug targets that could prevent cancer spread

Any cancer cell migrating from a tumor to set up shop elsewhere in the body will face a brutal attack from an immune system programmed to seek and destroy abnormal cells. But two recent studies from Stanford Medicine show that the hearty few that manage to infiltrate nearby lymph nodes carry out a stunning biological coup — convincing the body's defense system to accept them as part of its own tissues. This savvy rebranding gives tumor cells a free pass to easily metastasize to any site in the body and significantly worsen cancer prognoses.

The studies, conducted in laboratory mice, human cells and human tissue samples from cancer patients, upend the idea that lymph nodes — often the first site of metastasis — are simply passive downstream harbors for circulating cancer cells that have broken loose from nearby tumors.

"It's not just plumbing," said Edgar Engleman, MD, a professor of pathology and of medicine at the Stanford School of Medicine, clarifying the nodes' dubious distinction as the first agents in  spreading cancer. "It's something more nefarious. Once in the nodes, the cancer cells reprogram the immune system to not just stop the attack, but to directly aid metastasis. This concept is game-changing in our understanding of how tumors spread and how we might intervene in that process."

Engleman is the senior author of one of the papers, published online May 26 in Cell. Nathan Reticker-Flynn, PhD, instructor of medicine, is the first author of the Cell paper. The second paper was published June 2 in Nature Methods. Weiruo Zhang, PhD, research engineer, is the first author of the Nature Methods study, and Sylvia Plevritis, PhD, professor and chair of biomedical data science, is the senior author.

A closer look at lymph nodes

The research combines work from Engleman's team that shows how cancer cells gain the ability to metastasize with a new imaging platform developed in the Plevritis lab. The analytical platform can precisely identify individual cell types, including healthy versus cancerous, in cross sections of human tissue removed during cancer surgeries or biopsies.

When we began these studies, we really wanted to know if something happens in the nodes to facilitate metastasis. Are there early changes that suppress the immune system's response body-wide? And are these changes dependent on the spatial organization of individual cell types in the nodes?"

Sylvia Plevritis, PhD, professor and chair of biomedical data science

Lymph nodes are small chambers, or glands, in the armpits, neck, abdomen and groin that are stuffed with immune cells. They are part of the lymphatic system, which collects excess fluid that naturally accumulates in tissues as blood circulates. Lymphatic vessels deliver the fluid, called lymph, to the nodes, which trap any bacteria, abnormal cells and other detritus. The filtered lymph is then returned to the circulatory system via a vein near the heart. The nodes serve as surveillance headquarters, training grounds and mobilization centers for immune cells, which monitor the lymph contents and spring into action at the first sign of danger to attack cancers or infected cells.

Oddly, although the nodes are frequently one of the first sites of metastasis for nearby tumors, it has been unclear exactly why. It was once believed that they might serve as natural downstream harbors for circulating cancer cells, which then gradually accumulate additional mutations that further increase their metastatic ability. But the latest studies suggest that once metastasis is established in the nodes, the nodes play a far more active role in enabling the cancer cells to metastasize to other organs.

Engleman and Reticker-Flynn wondered what changes a cancer cell must undergo to split from the original tumor and successfully colonize a lymph node. To investigate, they started with a skin cancer called melanoma that, in mice, does not often metastasize. Reticker-Flynn implanted the cells into laboratory mice and watched to see which, if any, cancer cells managed to reach the animals' lymph nodes. These cancer cells were removed from the nodes and transplanted into another animal, and the process was repeated. Over the course of nine iterations, the cells became more and more skilled at metastasizing — evolving into a supercharged version of their previous selves.

The researchers then compared the original cells with their footloose descendants to learn how the cells escaped patrolling immune cells called natural killer, or NK cells.

'It's devious'

"It turns out it's not an easy ride for cancer cells to make it to a node," Engleman said. "There is an intense attack by the immune system. To avoid this, the tumor cells increase the production of molecules on their surfaces that say to the NK cells 'Do not kill me.' It's devious."

Once in the node, the cancer cells pivot from avoiding to actively engaging the immune system, the researchers found. In particular, the cancer cells' arrival boosts the number of another type of immune cell called regulatory T cell, or Tregs (pronounced tee-regs), in the node. Much like a school monitor roaming the halls to break up fights, Tregs tamp down inappropriate or overactive immune responses in the node that would result in an attack on the body's own cells and tissues.

While researchers in Engleman's lab continued their investigation into the types of proteins and cells in the nodes, Plevritis, Zhang and their colleagues mapped cellular neighborhoods in nodes in tissue samples from patients with head and neck cancers. Building on an existing technology that provides a picture of single cells in tissue samples, they developed a machine-learning tool called CELESTA that labels each cell with its cell type. Imagine zooming in on a Google Earth map that initially shows only town names to generate an enhanced version that stakes out the location of individual cars, houses, pools and barbecue grills. The results of the computerized tool are far more informative, and generated more quickly, than was previously thought possible.

"Automatically labeling items in images makes it easier for computers to further process an image to answer more specific questions like whether barbecue grills are more common in homes with pools," Plevritis said. "Even for an experienced pathologist, this kind of analysis would take weeks or months. But CELESTA can deliver an answer within minutes."

Because, like human neighbors, adjacent cells are likely to talk frequently or share resources, the cells' relative locations hint at their relationships. "The ability to use imaging and computational methods to infer crosstalk between cell types in health and disease allows us to better understand basic biological properties of human tissues," Plevritis said.

Disinformation campaign

CELESTA's analysis of the human nodes confirmed what Engleman and Reticker-Flynn were finding in their mouse studies. The cancer cells sidle up to Tregs in the nodes and begin a campaign of disinformation, asserting their harmlessness though a series of biological eye winks, whispered messages and elbow nudges. The bamboozled Tregs grant an all-access pass to the cancer cells, moving them in one fell swoop off the immunological Most Wanted list to body-wide VIP status.

"Immune cells all over the body communicate with each other, and cancer cells take advantage of this conversation, which begins in the lymph nodes," Engleman said. "Essentially, the conditioned Tregs circulate to distant tissues and prepare them for the arrival of their new 'friends,' the cancer cells. It's the induction of what we call metastatic tolerance, and it mirrors naturally occurring immune tolerance."

Although piggybacking on a critical tolerance pathway is a clever way to dodge some potential anti-cancer therapies, the researchers' discovery points to some new drug targets that could prevent cancer spread.

"Our studies have pinpointed hundreds of genes that the cancers use to escape the immune system," Engleman said. "These all represent potential targets, and I expect many laboratories will now be looking into new ways to intervene in the metastatic process. Like viruses, cancers have learned to take advantage of what nature has offered them, and the fact that tumors universally seem to use this mechanism makes this process an appealing target for cancer researchers."

Researchers at the UC San Francisco and Tel-Aviv University also contributed to the study.