At Fred Hutchinson Cancer Research Center in Seattle, laboratory project manager Andrea Towlerton took in a breath and peered through the twin lenses of her microscope.
On that July morning, more than two years of careful planning and hard work were at stake. She focused on a tiny droplet of liquid gingerly pipetted onto a glass slide. “It was either going to be great, or not,” she recalled.
Towlerton knew she could be looking at a landscape of dead cells, one that would send her entire team back to the drawing board.
Quickly, she had her answer. “I have to admit I almost started crying,” she said. “I saw those big, beautiful cells were alive.”
That microscopic beauty is deceptive. These cells came from a sample of Kaposi sarcoma, or KS — a disfiguring cancer that often involves the skin, lymph nodes and other organs. They had been drawn from a tumor on a patient treated 8,800 miles away in Kampala, Uganda, where KS is a leading cause of cancer death.
Yet because Towlerton’s teammates in the U.S. and Uganda had successfully brought these cells alive to Seattle, it was now possible for Hutch scientists to analyze cancers in Africa with the same depth and detail as cancers in the United States.
Kaposi sarcoma has a fearsome reputation. On the streets of New York and San Francisco in 1981, it was the face of AIDS.
Producing large purple growths across the back, face and limbs, this cancer — almost unheard of at the time — blossomed in patients whose immune systems were destroyed by HIV, the virus that causes AIDS. Unchecked by a patient’s natural defenses, KS in that decade became a leading killer, one of the most-dreaded of AIDS-defining illnesses.
Thanks to the advent of HIV-suppressing drugs in the mid-1990s, KS is once again relatively rare in the United States. In sub-Saharan Africa, however, where nearly 26 million people are living with HIV — and four-in-10 receive no antiviral drugs — KS remains the most common AIDS-related malignancy. There, it is a widespread, cruel and lethal disease. About half of patients in that region die within a year of a KS diagnosis.
That drove researchers affiliated with Fred Hutch’s Global Oncology program to spend the last several years perfecting a way to gather tumor specimens in Kampala, where about 400 patients a year are diagnosed with advanced KS at the Uganda Cancer Institute. Their immediate goal was to bring living samples of KS tumors to the United States so they could be analyzed with state-of-the art equipment unavailable in Africa. The hope is that researchers could tease out differences that make the disease more prevalent in Uganda, and perhaps show vulnerabilities that could be targets for treatment.
At the UCI-Fred Hutch Cancer Centre in Kampala, Towlerton worked with a team of Ugandan physicians, including Dr. James Kafeero — a graduate of Global Oncology’s research fellowship program — to train medical staffers in the protocol.
It begins with a biopsy: Kafeero or a staff nurse excises a specimen about the size and shape of a pencil eraser tip from the tumor of a KS patient volunteer.
Next, the sample is chopped and bathed in enzymes so that each cell from the biopsy floats freely in a suspension. The biopsy yields a mixture of living cells — cancer cells, healthy skin cells and immune cells that have responded to the tumor — as well as viruses and bacteria. This mixture is then carefully frozen in a small vial, packaged inside a cryotube and shipped to Seattle by air freight in a liquid nitrogen container.
At the Hutch, the samples are thawed, and then the researchers there can apply to these living assortments the best analytical tools available. From mountains of data, these scientists hope to unravel the secrets of a disease that fell out of the limelight in Western countries but remains a threat both in the U.S. and abroad.
Shortly after the cells in that first sample were found viable, Towlerton and her team used a high-speed cell-sorting machine to identify each individual living cell in the specimen. The machine shunted each one into to the appropriate vial: KS cells, infection-fighting white blood cells (T cells), or antibody-producing blood cells (B cells) — all components of what biologists call the tumor microenvironment.
“These are living cells, viable and intact,” said Dr. Hootie Warren, head of the Global Oncology program. “Once you have suspensions of single cells, you can do anything you want.”
For example, the Seattle researchers subject the patient’s sorted cells to high-speed genome sequencing to record the DNA of their entire complement of genes. One goal is to find out whether all the KS cells share a common set of genetic mutations, or if they are made up of groups of malignant cells bearing slightly different sets of damaged genes.
Next, using a powerful new technology known as single-cell RNA sequencing, Warren and his collaborators will take from the samples a molecular headcount of “RNA transcripts.” These are strips of genetic code used by cells to turn gene instructions into proteins. Transcripts can be studied — like a pile of receipts — to show which genes were active or quiet at a given time. Known as transcriptional profiling, this method should help the Hutch scientists tease out important but subtle differences in the ways living cancer cells, viruses and immune cells behave inside a tumor.
“It will allow us to define KS tumor cells with unprecedented precision,” Warren said. “To my knowledge, this has never been done before.”
This is the kind of state-of-the-art tumor analysis that is becoming commonplace in advanced laboratories like the Hutch, but out of reach in most of Africa. Warren and his Global Oncology team hope their findings will lead to improved treatments for Ugandans with KS, as well as for the approximately 1,000 Americans who are diagnosed with the disease each year, primarily in areas where HIV stigma or lack of access to care delays diagnoses.
According to Dr. Tom Uldrick, deputy head of Global Oncology, the number of new KS cases in the U.S. has dropped about 80 percent since the early days of the AIDS epidemic. Yet it remains a problem for those who are HIV positive. “It is the No. 2 cancer among people with HIV (behind lymphoma) in this country,” Uldrick said.
New laboratory techniques such as single-cell RNA sequencing are needed to gain a better understanding of KS, because this cancer has proven difficult to study using the kinds of analyses that have been standard since the 1980s.
“You can’t even really study Kaposi sarcoma that well in a petri dish,” Uldrick said.
For example, KS is triggered by infection of cells lining blood and lymph vessels with human herpesvirus 8, which preys on individuals whose immune systems are weakened by HIV, age or illness. But HHV-8 will not grow in laboratory-cultured samples of KS cells.
Fred Hutch researcher Dr. Warren Phipps, medical director of the UCI-Fred Hutch collaboration and a specialist in KS, spends most of his time each year in Kampala working with Ugandan physicians and treating patients. Over his 10 years working in Uganda, Phipps has established one of the largest cohorts of KS patients in the world. He and his team perfected the art of removing KS tumor samples as they established a collection of biopsies from more than 1,000 tumors obtained from their patients in Uganda. The effort to bring living KS tumor cells from Kampala to Seattle is the latest advance building on the decade of research on KS at UCI-Fred Hutch.
Long before HIV emerged as a global pandemic and made infected people vulnerable to KS, that cancer was relatively common in Uganda, where about 70 percent of the population carries HHV-8. Today, about 10–15 percent of KS cases in Kampala are diagnosed in patients who are HIV-negative, and researchers there are seeing more HIV patients diagnosed with the cancer despite being on antiretroviral drug therapy.
“We see a lot of people presenting with advanced KS disease, and we still have relatively poor outcomes here,” Phipps said. About half the patients die within a year of diagnosis. “We need novel therapies and 21st-century technologies to understand who might respond best to which therapies,” he said.
Phipps believes the advanced research now made possible by shipping living tumor and immune cells to Seattle could be pivotal in helping researchers understand the peculiarities of KS tumors in Africa. Single-cell analysis has the potential to spotlight rare immune cells that are effective in the body’s defensive response to viruses or to damaged cells that could become malignant.
“We can see things that would otherwise be lost. We can pick out patterns that also would be lost,” Phipps said, “and what we learn from KS will have lots of application to other tumors.”
The Global Oncology team includes Towlerton and lab operations manager James Ferrenberg, based at Fred Hutch; and laboratory director Dr. Britta Flach and researchers Lazarus Okoche and Diana Basemera in Kampala. The team sees this effort to bring live tumor and immune cells to Seattle as a short-term step. Their long-term aim is to supply these highly trained Ugandan partners with their own high-speed cell-sorting and gene-sequencing equipment in Kampala, so they can pursue their own research where they feel it would bring the most benefit.
“I know our patients here are supportive,” Phipps said in a phone call from Kampala. “They are looking for better treatments. They are looking for answers.”
Sabin Russell is a staff writer at Fred Hutchinson Cancer Research Center. For two decades he covered medical science, global health and health care economics for the San Francisco Chronicle, and wrote extensively about infectious diseases, including HIV/AIDS. He was a Knight Science Journalism Fellow at MIT, and a freelance writer for the New York Times and Health Affairs. Reach him at firstname.lastname@example.org.