Investigating What Keeps Metastatic Breast Cancer in Check

New evidence suggests that inflammation may help prevent growth of tumor cells that have spread, with important implications for clinical trials

At the time of initial diagnosis, most patients with breast cancer show no signs that their cancer has spread elsewhere in the body. Yet, up to 30 percent of patients will ultimately experience metastasis, with breast cancer taking root and growing at other sites in the body, sometimes months, years or even decades later. Sandra McAllister, PhD, of the Division of Hematology at Brigham and Women’s Hospital, and her colleagues are working to understand what happens when cancer cells escape from the primary tumor, and what, if anything, can be done to intervene before cancer recurs. In a paper published online in Nature Cell Biology on Aug. 27, McAllister and colleagues reveal an unlikely protector against metastatic growth: inflammation.

“Our findings flip the current thinking on its head,” said McAllister, co-senior author of the study. “Many people study primary tumors and the assumption has been that metastases grow the same way. But our work suggests that, while inflammation can help tumor cells escape and land elsewhere in the body, if inflammation is there when they land, it keeps the cells in check. When inflammation is suppressed, the cells grow out.”

McAllister and lead author Zafira Castano, PhD, an instructor in the McAllister lab, and colleagues carried out their study in mouse models of metastatic breast cancer and confirmed findings using samples from patients. In the mouse model, the researchers observed that primary tumors triggered an inflammatory reaction that kept tumor cells in the lung from growing. The team reports that levels of interleukin-1β (IL-1β) were especially high in the lungs of mice. When IL-1β was suppressed, the metastases in the lungs grew. When examining samples from patients with breast cancer, the team found that patients who had high levels of IL-1β had lower risk of metastatic tumors.

Inhibitors of IL-1β, such as the drug canakinumab, are currently being tested in clinical trials for the treatment of numerous diseases, including forms of cancer.

“An important implication of our study is that therapies that may prevent the growth of primary tumors may not confer the same beneficial effects for preventing the growth of metastases and further research is required,” said McAllister.

While inflammation appeared to prevent the growth of metastatic cancer, it did not appear to kill the cancer cells that had spread.

“This new research has yielded that rare thing: A clue from the cancer itself about how we can fight its spread,” said Christine Chaffer of the Garvan Institute for Medical Research, who co-led the study with McAllister. In a separate paper, published recently in Cancer Research, McAllister and colleagues, with lead author Jessalyn Ubellacker, PhD, a former graduate student in the McAllister lab, uncovered a connection between metastasis and a common drug used to treat osteoporosis. In that study, the team found that bisphosphonates could affect bone marrow cells in a way that stopped the growth of breast cancer cells that had spread to the bones. However, they also found evidence that a specific protein – granulocyte-colony stimulating factor (G-CSF) – can help cancer cells overcome this effect. Women with high levels of G-CSF did worse on bisphosphonates than women with low levels of G-CSF.

Both studies may help inform clinical trial design and the selection of patients most likely to benefit from drugs such as IL-1β inhibitors and bisphosphonates.

Funding for this work was provided by the International Mentoring Foundation for the Advancement of Higher Education; Center for Stem Cell Bioinformatics at the Harvard Stem Cell Institute; Samuel Waxman Cancer Research Foundation, Breast Cancer Research Foundation, and Ludwig Center for Molecular Oncology; Advanced Medical Research Foundation and Nelune Foundation; National Institutes of Health (NCI) RO1 CA166284, Presidential Early Career Award for Scientists and Engineers, American Cancer Society Research Scholar award, and Department of Defense BCMRP Era of Hope Scholar Award W81XWH-14-1-0191.

Paper cited: Castaño, Z et al. “IL-1β inflammatory response driven by primary breast cancer prevents metastasis-initiating cell colonization” Nature Cell Biology DOI: 10.1038/s41556-018-0173-5

One Step Closer to Bioengineered Replacements for Vessels and Ducts

Researchers bioprint complex tubular tissues to replace dysfunctional vessels and ducts in the body

A team of Brigham and Women’s Hospital researchers have developed a way to bioprint tubular structures that better mimic native vessels and ducts in the body. The 3-D bioprinting technique allows fine-tuning of the printed tissues’ properties, such as number of layers and ability to transport nutrients. These more complex tissues offer potentially viable replacements for damaged tissue. The team describes its new approach and results in a paper published on Aug. 23 in Advanced Materials.

“The vessels in the body are not uniform,” said Yu Shrike Zhang, PhD, senior author on the study and an associate bioengineer in BWH’s Department of Medicine. “This bioprinting method generates complex tubular structures that mimic those in the human system with higher fidelity than previous techniques.”

Many disorders damage tubular tissues: arteritis, atherosclerosis and thrombosis damage blood vessels, while urothelial tissue can suffer inflammatory lesions and deleterious congenital anomalies.

To make the 3D bioprinter’s “ink,” the researchers mixed the human cells with a hydrogel, a flexible structure composed of hydrophilic polymers. They optimized the chemistry of the hydrogel to allow the human cells to proliferate, or “seed,” throughout the mixture.

Next, they filled the cartridge of a 3D bioprinter with this bio-ink. They fitted the bioprinter with a custom nozzle that would allow them to continuously print tubular structures with up to three layers. Once the tubes were printed, the researchers demonstrated their ability to transport nutrients by perfusing fluids.

The researchers found that they could print tissues mimicking both vascular tissue and urothelial tissue. They mixed human urothelial and bladder smooth muscle cells with the hydrogel to form the urothelial tissue. To print the vascular tissue, they used a mixture of human endothelial cells, smooth muscle cells and the hydrogel.

The printed tubes had varying sizes, thicknesses and properties. According to Zhang, structural complexity of bioprinted tissue is critical to its viability as a replacement for native tissue. That’s because natural tissues are complex. For instance, blood vessels are comprised of multiple layers, which in turn are made up of various cell types.

The team plans to continue preclinical studies to optimize the bio-ink composition and 3D-printing parameters before testing for safety and effectiveness.

“We’re currently optimizing the parameters and biomaterial even further,” said Zhang. “Our goal is to create tubular structures with enough mechanical stability to sustain themselves in the body.”

Co-senior author of the paper is Ali Khademhosseini, PhD, professor at University of California, Los Angeles, who collaborated on the project.

This study was funded by the National Cancer Institute of the National Institutes of Health (AR057837, DE021468, AR068258, AR066193, EB022403, EB021148, HL137193, EB021857, EB024403 and CA201603), the Presidential Early Career Award for Scientists and Engineers (PECASE), China Scholarship Council (201406235036), the National Natural Science Foundation of China (21673197 and 31570947), Young Overseas High-level Talents Introduction Plan, the 111 Project (B16029), the Fundamental Research Funds for the Central Universities of China (20720170050), the CONACYT postdoctoral fellowship (291018-173853), Swiss National Science Foundation, American Fund for Alternatives to Animal Research (AFAAR) and the New England Anti-Vivisection Society (NEAVS).

Paper cited: Q. Pi et al. (2018). Digitally Tunable Microfluidic Bioprinting of Multilayered Cannular Tissues. Advanced Materials. DOI: 10.1002/adma.201706913.

New Insights into What Drives Organ Transplant Rejection

Subset of cells appear to trigger rejection of skin grafts; pre-treating organs could have positive implications for face transplants

When it comes to transplant rejection, some organs are far trickier than others. Some transplantable organs, such as the liver, are readily accepted by the recipient’s immune system, rarely triggering an immune response and rejection. But the skin is a very different matter: Skin grafts have a high rate of rejection for unknown reasons.

Investigators from Brigham and Women’s Hospital set out to understand why, hoping to capitalize on new biological insights to not only explain why skin transplants provoke the immune system but also what treatments can be given to an organ prior to transplantation to decrease the likelihood of rejection. Their findings, with implications for the future of face transplantation, are published in Nature Communications.

“Our work in the lab is driven by clinical observation,” said co-senior author Leonardo V. Riella, MD, PhD, medical director of the Vascularized Composite Tissue Transplant Program at BWH. “Skin grafts have such a high rate of rejection compared to other organs. We wanted to find out why.”

Using a mouse model of skin transplantation, the team identified a novel, specific subset of cells in donor transplanted organs that triggered rejection. Known as CD103+ dendritic cells, this cell type appeared to play a major role in rejection. In its absence, researchers observed fewer signs that the recipient’s immune system had been triggered and found that the transplanted skin survived longer.

In addition, the team was able to mitigate rejection by treating organs prior to transplantation. When skin grafts were incubated with an anti-inflammatory mycobacterial protein, the grafts survived longer after transplantation. This effect was mediated by March-1, an enzyme that can inhibit the maturation of CD103+ dendritic cells in donor grafts. In addition to these observations in mouse models, the team also tested the effect of adding the mycobacterial protein to human skin grafts, finding signs that this treatment may help prevent an immune reaction.

Pre-treating skin grafts or other organs prior to transplantation represents a new, complementary strategy for preventing organ rejection.

“Today, most treatment focuses on the recipient side, using immunosuppressive drugs to try to prevent rejection,” said Riella. “What we propose is a complementary strategy to tame the rejection process through direct treatment of the donor organ prior to transplantation. This could offer many benefits since we’re focusing on an arm of the immune system – innate immunity – that immune suppressors cannot regulate.”

The researchers plan to continue testing safety and effectiveness of this strategy in preclinical models and, if successful, will conduct phase 1 clinical trial in humans in the years ahead.

Funding for this work was provided by Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS) Grant 11/0903-1 and Financiadora de Estudos e Projetos (FINEP) Grant 01.08.0600-00, Research Grant 12FTF120070328 from the American Heart Association, RT150078 and RT150081 from the US Department of Defense and NIH grant number RO1CA119045.

Paper cited: Borges, TJ et al. “March1-dependent modulation of donor MHC II on CD103+ dendritic cells mitigates alloimmunity” Nature Communications DOI: 10.1038/s41467-018-05572-z

Glycans at the “I” of the Storm in Humoral Immunity and Melanoma Progression

Charles J. Dimitroff, PhD, left, helps to train the next generation of immune/cancer research scientists. His laboratory is focused on the glyco-pathological basis of immunity, inflammation and cancer.

Back-to-back papers detail novel findings on cell surface carbohydrates that regulate human B cell function and human melanoma progression

Two new studies have unveiled how a peculiar molecule impacts how antibody-producing cells develop and function as well as how normal melanocytes progress to melanoma malignancy.

“These findings on fundamental immunology and melanoma development originate from totally different areas of research, though have intersected at the bench,” said Charles Dimitroff, PhD, of the Department of Dermatology at Brigham and Women’s Hospital.

The Dimitroff lab, along with collaborators at Imperial College London, recently published two back-to-back-articles in the journal, Nature Communications, detailing novel findings on cell surface carbohydrates (‘glycans’) regulating human B cell function and human melanoma progression – two scientific areas seemingly at the opposite ends of the research spectrum.  Over a five-year period in the Dimitroff laboratory, Nicholas Giovannone, PhD, and Jenna Geddes Sweeney, PhD, who were investigating the global glycan features of human B cells at various stages of differentiation and of normal and malignant melanocytes, independently discovered that a distinct glycan feature known as blood group I-antigen or “I-branches” was remarkably central to glycan-mediated processes regulating both human B cell signaling/activation and melanoma aggressiveness.  Precisely how these I-branch features control B cell differentiation and humoral immunity or drive melanoma progression are still under intense investigation and likely to reveal new targets for immunomodulation or anti-cancer treatments.

Papers cited:  Gionvannone, N et al. “Galectin-9 suppresses B cell receptor signaling andis regulated by I-branching of N-glycans.” Nature Communications DOI: 10.1038/s41467-018-05770-9

Sweeney GS et al. “Loss of GCNT2/I-branched glycans enhancesmelanoma growth and survival” Nature Communications DOI: 10.1038/s41467-018-05795-0

Investigators Validate New Potential Targets for Immunotherapy

Study suggests that cancer and other disorders can trigger the overexpression of T cells’ own co-inhibitory (“checkpoint”) receptors that deactivate immune defenses, revealing potential targets for future immunotherapies

The immune system constantly regulates itself, avoiding the extremes of underreaction on one side and autoimmunity on the other. Two molecule types that immune cells use to achieve this balance are cytokines and co-inhibitory, or checkpoint, receptors. Cytokines are signaling proteins that can activate immune cells, while checkpoint receptors are molecules that appear on cell membranes to shut T cells off once they’ve fought off a threat.

Some tumors, however, can gain the ability to turn off T cells. According to a study recently published in Nature, tumors achieve this in part by secreting a cytokine normally dispatched by immune cells to prevent autoimmunity. Too much of this cytokine, called IL-27, triggers overexpression of checkpoint receptors on T cells, disabling them and making them dysfunctional.

“The tumors are the smartest immunologists,” said Vijay Kuchroo, PhD, an immunologist at Brigham and Women’s Hospital who led the study. “They know exactly how to trick the immune system.”

Kuchroo, along with his BWH colleagues, Norio Chihara, MD, PhD, Asaf Madi, PhD, and Ana Anderson, PhD, and investigators at the Broad Institute, Aviv Regev, PhD and Orit-Rozenblatt-Rosen, PhD, observed that melanoma tumors in mice increased cytokine IL-27 levels, which triggered overexpression of checkpoint receptors on CD8+ and CD4+ T cells. This same process also occurred in many other contexts where T cells malfunction, such as other cancers and during chronic viral infection.

Using proteomics and single-cell RNA sequencing, the team identified 57 genes induced by the cytokine IL-27 implicated in T cell dysfunction. By looking at the molecules arising from the IL-27-induced genes, the researchers identified new potential targets for immunotherapies, or treatments that enlist the body’s immune system to fight invaders like cancer. Out of many novel checkpoint receptors they identified, they experimentally validated two, called protein C receptor (Procr) and podoplanin (Pdpn). They observed significantly delayed growth of melanomas in Procr and Pdpn receptor knockout mice.

They also pinpointed two transcription factors, called Prdm1 and c-Maf, that together seem to drive checkpoint receptor expression. When they studied double Prdm1 and c-Maf knockout mice, they saw significant delays in tumor growth.

“There is a huge need for additional immunotherapy targets,” said Madi. “That’s why examining these novel molecules is so important.”

Unlike radiation and chemotherapy, tumors treated with immunotherapy show durable responses. Currently, however, immunotherapy succeeds only for certain patients and cancers – Kuchroo’s team seeks to uncover why that is. Potential treatments arising from their findings include administration of antibodies that bind to checkpoint receptors and block or otherwise modify their function.

“The molecules we’ve identified are not unique to cancer,” said Anderson. “Our findings provide a set of genes that can be modulated therapeutically in different ways for a range of diseases. In cancer, you want to block co-inhibitory receptors – in autoimmune disorders, you want to increase their function.”

Kuchroo and his team will continue validating other checkpoints receptors encoded by the identified genes, their interactions in the T cell membrane, and the pathways that induce their expression. They also plan to examine the roles of checkpoint receptors and cytokines in other immune disorders and cancers, like colorectal carcinoma.

“Different cancers use different co-inhibitory receptors from the ones we identified,” said Kuchroo. “So knowing all the players in the game is equally important. These will be the new targets for immunotherapy.”

This work was supported by grants from the National Institutes of Health, the American Cancer Society, the Melanoma Research Alliance, the Klarman Cell Observatory at the Broad Institute, and the Howard Hughes Medical Institute.

Study Cited: Norio Chihara, Asaf Madi, Takaaki Kondo, Huiyuan Zhang, Nandini Acharya, Meromit Singer, Jackson Nyman, Nemanja D. Marjanovic, Monika S. Kowalczyk, Chao Wang, Sema Kurtulus, Travis Law, Yasaman Etminan, James Nevin, Christopher D. Buckley, Patrick R. Burkett, Jason D. Buenrostro, Orit Rozenblatt-Rosen, Ana C. Anderson, Aviv Regev & Vijay K. Kuchroo. (June 13 2018). Induction and transcriptional regulation of the co-inhibitory gene module in T cells. Nature 558, 454-459.