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Five Fred Hutchinson Cancer Research Center scientists have received the first phase of funding in a grant competition sponsored by the Immunotherapy Integrated Research Center and funded by the Bezos family Immunotherapy Initiative to conduct projects using single-cell RNA sequencing, also known as scRNA-seq. The funding supports collaborations between Fred Hutch’s Dr. Jason Bielas and the company 10x Genomics to develop a droplet-based, single-cell RNA sequencing platform. The goal of these grants is to accelerate the Hutch’s position as an early adopter of the technology.
The researchers, each of whom received $25,000 in exploratory funding, will be invited to compete for a $100,000 pilot award based on their initial findings and plans for future studies. The second phase of the competition will take place in September.
Single-cell RNA sequencing allows researchers to distinguish between different types of cells in a complex mixture and analyze data patterns among thousands of individual cells — all within a single experiment. The sequencing technology simultaneously records the tens or hundreds of thousands of gene transcripts of many thousands of cells. Much like recording the cell phone traffic of each individual in an entire city would show similarities and differences between various groups of people (teenagers, people conducting business, etc.) the platform, for example, may help scientists determine how different cell types interact in the complex environment of a tumor.
Ultimately, the goal of such sequencing in the awarded pilot project is to develop better cancer treatments while addressing fundamental questions in biology, according to Bielas, an associate member of the Public Health Sciences and Human Biology divisions at the Hutch.
The five researchers to receive funding are, in alphabetical order:
Dr. Aude Chapuis will lead a project with oncology fellow Dr. Kelly Paulson to understand the mechanisms behind the success and failure of T-cell immunotherapy in a patient with Merkel cell carcinoma, or MCC, a rare, highly aggressive skin cancer that strikes more than 2,000 in the U.S. annually. Most cases are caused by a virus, Merkel cell polyomavirus.
Chapuis, Paulson and colleagues are developing powerful new immunotherapies to enhance immune response to the virus and the cancer it causes. They will use the exploratory funding to “take a dive in unprecedented detail” into the immune responses of a single patient with advanced, metastatic MCC over the course of the patient’s treatment. The patient was treated with multiple immunotherapies, including T-cell therapy. Treatment initially was unsuccessful, but then, after adding a second immunotherapeutic agent, the patient went into a partial remission for more than a year before relapsing. Using sc-RNA-seq of the patient’s tumor samples collected over time, Chapuis’ group will simultaneously investigate the immune responses from the perspective of the patient’s tumor cells, connective-tissue cells and immune cells, and determine how these cell populations changed in response to various treatment regimens.
“It is our hope that these investigations will yield critical details as to the mechanism for immune escape that can allow us to optimize further MCC treatments,” said Chapuis, an assistant member of the Clinical Research Division. These studies will be led by Paulson, conducted in collaboration with the laboratories of the Hutch’s Bielas and Dr. Paul Nghiem at the University of Washington. If selected for the pilot phase, the researchers will expand the study to examine the mechanisms of T-cell treatment response in nine MCC patients.
Dr. Bruce Clurman, executive vice president and deputy director of Fred Hutch, studies the pathways that regulate cell growth and division. He will use scRNA-seq to investigate the progression of leukemia in mice that carry driver mutations — genetic mistakes linked to high mutation rates — that are associated with leukemia in humans.
“Through RNA-seq, we hope to gain an unprecedented molecular and phenotypic understanding of how oncogenic mutations lead to human leukemia by focusing on pre-leukemic states,” said Clurman, who holds the José Carreras/E. Donnall Thomas Endowed Chair for Cancer Research and is a member of the Clinical Research and Human Biology divisions.
Clurman will study two genes that are associated with human blood cancers and also cause leukemias in mice: c-Myc (linked with B-cell lymphoma/leukemia) and Fbw7 (associated with T-cell acutie lymphoblastic leukemia, or T-ALL). Each mutation is linked to a high risk of cancer, but with variable times to progression, which implies the role of additional genetic or epigenetic factors are at play in the progression of the disease.
“Each mouse model is driven by oncogenic transcription factor deregulation, making them ideal for studies with scRNA-seq, which will provide entirely new insights into how leukemic precursors progress to leukemia,” Clurman said.
Clurman and colleagues will conduct the research in collaboration with Bielas, who will provide analytic and experimental advice.
Dr. Hans-Peter Kiem, associate head of Transplantation Biology and a member of the Clinical Research Division at the Hutch, will lead a project that aims to improve cancer immunotherapy through the use of hematopoietic, or blood, stem cells to generate T cells, the workhorse of the immune system that can be programmed to target cancer.
Immunotherapy — particularly the use of chimeric antigen receptors (CARs) or engineered T-cell receptors (TCRs) — is revolutionizing the treatment of patients with certain blood cancers and solid tumors. One important challenge identified in these studies, however, has been the eventual “exhaustion” of engineered T cells.
Kiem’s goal is to incorporate the use of blood stem cells into immunotherapy approaches to produce a long-term, robust immune T-cell response against cancer.
“An alternative, highly appealing and exciting approach is the use of blood stem cells for the generation of engineered T cells. The use of blood stem cells would allow sustained, lifelong production of anti-cancer T cells in immunotherapy,” said Kiem, the Fred Hutch Endowed Chair for Cell and Gene Therapy and director of the Cell and Gene Therapy Program at the Hutch.
For more than three decades of research, the purification of blood stem and progenitor cells, identified as CD34+ cells, has remained the gold-standard strategy for clinical transplantation and gene therapy. However, these cells contain only a very few true blood stem cells, which limits the efficiency of current transplant approaches. The ability to identify, enrich and target “true” blood stem cells would be a major advance to guarantee lifelong production of anti-cancer T cells and, at the same time, reduce manufacturing costs, making this strategy available and affordable for more patients, Kiem said.
Kiem and colleagues recently identified a subset, or fraction, of CD34+ cells that are highly enriched with the blood stem cells required exclusively for the production of blood lineages after transplantation. The cell fraction is easy to identify and purify, making it the perfect target for blood-stem-cell-mediated immunotherapy approaches, Kiem said.
“The single-cell RNA-seq exploratory and pilot award will allow us to comprehensively analyze and study this new blood-stem-cell-enriched fraction in human stem-cell sources for gene therapy-mediated CAR-T and TCR-based approaches,” Kiem said.
Dr. Cecilia Moens, a developmental biologist and member of the Basic Sciences Division, will lead a project that aims to uncover mechanisms that drive melanoma to spread, or metastasize, throughout the body.
Melanoma can arise from what may seem like harmless moles. If it is not recognized early and the melanoma cells spread, the disease often can be fatal. “The goal of this project is to identify markers that will aid doctors in detecting harmful melanoma and also be potentially used as a target for treatment of the disease,” Moens said.
Since it is unclear how static tumor cells transition to metastatic cancer cells that travel throughout the body, Moens, and colleagues use human cells and model organisms to directly visualize tumor-cell motility, or movement.
“In our work, consistent with previous findings, we found that tumor-associated macrophages, a component of the immune system, promote tumor-cell motility. We further found that macrophages transfer cytoplasm (the fluid that fills the inside of a cell) to tumor cells and that part of this cytoplasmic transfer occurs when the macrophages and tumor cells come in contact with one another,” she said. “Remarkably, we found that tumor cells that receive cytoplasm from macrophages are more likely to metastasize than tumor cells that do not.”
Moens and colleagues propose to use the scRNA-seq grant to determine what molecules are transferred from macrophages to tumor cells and how this transfer results in increased metastasis. They will use an in-vitro system with human tumor cells and mouse macrophages so that they can detect mouse transcripts in tumor cells that have received macrophage cytoplasm as well as the resulting transcriptional response.
“We hope that this work will allow us to discover how inflammatory-response pathways regulate melanoma metastasis to better understand how immune cells and tumor cells communicate in the microenvironment,” Moens said.
Dr. Anthony Rongvaux, an assistant member of the Clinical Research Division, will also use his exploratory grant to study metastatic melanoma, a disease in which fewer than 20 percent of patients survive more than five years after diagnosis.
In considering how patients’ immune systems might help fight advanced cases of melanoma, Rongvaux and collaborator Bielas also will focus on the role of macrophages in cancer spread.
While macrophages are immune cells that play important roles in defending against infectious diseases, they also seem to support tumor growth and favor metastatic progression in melanoma, Rongvaux said.
Metastasis is a complex process that involves multiple interactions between the tumor and immune cells, and migration of cancer cells from the primary tumor to distant body sites. As an experimental model, the Rongvaux Lab uses a unique model of “humanized mice,” which are transplanted with a human immune system and a human tumor that replicates clinical observations.
“The newly developed technology of single-cell RNA sequencing will allow us to study, with previously unattainable resolution, how it is that cancer cells hijack immune macrophages and use them for their own benefit,” he said. “Our work will provide the scientific basis for future cancer therapies that target macrophage-supported tumors instead of, or in addition to, treatments that target the tumor itself.”
