Engineered ‘Off the Shelf’ Stem Cells Target Breast Cancer that Metastasizes to the Brain

• Investigators developed a biology-driven, stem-cell therapy against key targets in a solid tumor
• Delivery occurred across the blood-brain barrier in mouse models designed to faithfully mimic metastatic cancer

Photomicrograph showing stem cells (green) tracking tumor cells (red) in a model of leptomeningeal metastasis.

Approximately 15-to-30 percent of patients with metastatic breast cancer have brain metastasis (BM), with basal-like breast cancer (BLBC) metastasizing to the brain most frequently. The prognosis for BLBC-BM patients is poor, as the blood-brain barrier (BBB) prevents most therapeutics from reaching the brain. Testing candidate therapies in clinical trials is also challenging because animal models that mimic BM are limited. In a new study, researchers from Brigham and Women’s Hospital and collaborators engineered a bimodal tumor-suppressing and killing molecule that can be delivered to the brain by stem cells. They tested the therapy in three new mouse models of BLBC-BM that imitate human cancers and found that it successfully prolonged the lifespan of the mice. Findings are published in Science Advances.

“When breast cancer patients get metastasis to the brain, there’s often not much we can do because therapeutics for brain metastatic cancer are scarce,” said corresponding author Khalid Shah, MS, PhD, of the Center for Stem Cell Therapeutics in the Brigham’s Department of Neurosurgery. “Given the urgent need and magnitude of the problem, we set out to not only work on the development of a new therapeutic approach, but also to develop it in a way that maximizes the likelihood that our findings will be clinically translatable.”

The researchers began their work by analyzing tissue from breast tumors and metastatic tumors in the brain from human patients with BM. They pinpointed key targets that appeared to drive tumor growth: an excess of epidermal growth factor receptor (EGFR) and death receptors 4 and 5 (DR4/5). With this knowledge, the investigators genetically engineered a bi-functional molecule, E_vDR_L, to target EGFR and DR4/5 simultaneously and induce cell death in the tumors.

Because breast cancer can metastasize to the brain in numerous patterns, the researchers spent three years building BM mouse models to reflect the range and complexity of human disease. One mouse mimics metastasis that takes the form of a solid tumor in the middle of the brain (macro-metastasis), the second mimics a more scattered metastasis (perivascular niche micro-metastasis), and the third mimics one that appears in the back of the brain (leptomeningeal metastases). Armed with these models, they could more accurately determine the efficacy of the therapeutic molecule, E_vDR_L.

To introduce E_vDR_L to the brains of the metastasis mouse models, the researchers developed a stem-cell-based delivery platform. Stem cells have the advantage of being able to cross the BBB and home in on tumors in the brain. Allogeneic stem cell therapies, which use donor tissue samples to create large batches of cells, can produce stem cells efficiently, making them a readily available, “off-the-shelf” technology for administering therapies. In all three mouse models, the researchers observed improved survival rates.

“Building these models was essential to testing our therapies because we wanted to mimic what happens in the patients,” Shah said. “We started our research by bringing findings in patients to mice, and now we are planning to go back to the patients.”

The U.S. Food and Drug Administration is currently reviewing a previous iteration of the researchers’ novel therapy, which uses off the shelf stem cells to zero in on one of the two proteins targeted by E_vDR_L. They hope that approval of that therapy might bolster support for clearance of the therapy developed and used in their current study.

Funding for this research was provided by NIH grants R01-CA201148 and R01-NS107857, Overseas Research Fellowships from Uehara Memorial Foundation, and Kanzawa Medical Research Foundation. Shah owns equity in and is a member of the Board of Directors of AMASA Therapeutics, a company developing stem cell–based therapies for cancer.

Paper cited: Kitamura, Y et al. “Anti-EGFR VHH-armed death receptor ligand–engineered allogeneic stem cells have therapeutic efficacy in diverse brain metastatic breast cancers” Science Advances DOI: 10.1126/sciadv.abe8671

Novel Urine Test Developed to Diagnose Human Kidney Transplant Rejection

Study demonstrates potential for a noninvasive clinical test to diagnose kidney allograft rejection and ultimately improve transplant outcomes

Patients can spend up to six years waiting for a kidney transplant. Even when they do receive a transplant, up to 20 percent of patients will experience rejection. Transplant rejection occurs when a recipient’s immune cells recognize the newly received kidney as a foreign organ and refuse to accept the donor’s antigens. Current methods for testing for kidney rejection include invasive biopsy procedures, causing patients to stay in the hospital for multiple days. A study by investigators from Brigham and Women’s Hospital and Exosome Diagnostics proposes a new, noninvasive way to test for transplant rejection using exosomes — tiny vesicles containing mRNA — from urine samples. Their findings are published in the Journal of the American Society of Nephrology. 

“Our goal is to develop better tools to monitor patients without performing unnecessary biopsies. We try to detect rejection early, so we can treat it before scarring develops,” said Jamil Azzi, MD, associate physician in the Division of Renal Transplant at the Brigham and an associate professor of Medicine at Harvard Medical School. “If rejection is not treated, it can lead to scarring and complete kidney failure. Because of these problems, recipients can face life-long challenges.”

Before this study, physicians ordered biopsies or blood tests when they suspected that a transplant recipient was rejecting the donor organ. Biopsy procedures pose risks of complications, and 70-80 percent of biopsies end up being normal. Additionally, creatinine blood tests do not always yield definitive results. Because of the limitations surrounding current tests, researchers sought alternate and easier ways to assess transplant efficacy.

In this study, researchers took urine samples from 175 patients who were already undergoing kidney biopsies advised by physicians. From these samples, investigators isolated urinary exosomes from the immune cells of the newly transplanted kidneys. From these vesicles, researchers isolated protein and mRNA and identified a rejection signature — a group of 15 genes — that could distinguish between normal kidney function and rejection. Notably, researchers also identified five genes that could differentiate between two types of rejection: cellular rejection and antibody-mediated rejection.

“These findings demonstrate that exosomes isolated from urine samples may be a viable biomarker for kidney transplant rejection,” said Azzi.

This research differs from prior attempts to characterize urinary mRNA because clinicians isolated exosomes rather than ordinary urine cells. The exosomal vesicle protects mRNA from degrading, allowing for the genes within the mRNA to be examined for the match rejection signature. In previous research, mRNA was isolated from cells that shed from the kidney into urine. However, without the extracellular vesicles to protect the mRNA, the mRNA decayed very quickly, making this test difficult to do in a clinical setting.

“Our paper shows that if you take urine from a patient at different points in time and measure mRNA from inside microvesicles, you get the same signature over time, allowing you to assess whether or not the transplant is being rejected,” said Azzi. “Without these vesicles, you lose the genetic material after a few hours.”

One limitation to this research is that these tests were done on patients undergoing a biopsy ordered by their physician, who already suspected that something was wrong. In the future, Azzi and his colleagues aim to understand whether a test such as this one can be used on kidney transplant recipients with normal kidney activity as measured in the blood to detect hidden rejection (subclinical rejection). They are currently doing a second study on patients with stable kidney function, looking to see if the same signature they identified in this current study could be used on patients without previously identified issues but still detect subclinical rejection.

“What’s most exciting about this study is being able to tell patients who participated that their effort allowed us to develop something that can help more people in the future,” said Azzi. “As a physician-scientist, seeing an idea that started as a frustration in the clinic, and being able to use the lab bench to develop this idea into a clinical trial, that is very fulfilling to me.”

Funding for this work was provided by the American Heart Association (13FTF17000018), National Institutes of Health (RO1 AI134842), Exosome Diagnostics, NIH Clinical Center Grant (F32DK11106). Azzi reports having intellectual properties and receiving royalties from Accrue Health Inc.; receiving research funding from the American Diabetes Association, American Heart Association, and Qatar Research Fund; being a scientific advisor for CareDx; and having intellectual properties in Exosome Diagnostics. Co-authors are employees of Exosome Diagnostics, a Bio-Techne brand.

Azzi, J et al. “Discovery and Validation of a Urinary Exosome mRNA Signature for the Diagnosis of Human Kidney Transplant Rejection” Journal of the American Society of Nephrology DOI:10.1681/ASN.2020060850

Identifying Patient-Specific Differences to Treat HCM with Precision Medicine

Researchers used a novel approach to study hypertrophic cardiomyopathy, looking beyond genetics for clues about the heart condition’s underlying biology

Hypertrophic cardiomyopathy (HCM) is a cardiovascular disease characterized by thickening of the left ventricle, otherwise known as the main squeezing chamber of the heart. HCM is best known for causing sudden death in athletes but can occur in persons of any age, often without symptoms. While frequently discussed in the context of genetics, most patients with HCM do not have a known genetic variant. Investigators from Brigham and Women’s Hospital uncovered a means to study the complexity of this disease beyond the identification of individual genes. This new approach offers a path toward treating HCM using individualized medicine. In a recent study, investigators analyzed the role protein-protein interactions (PPIs) play in differentiating individual cases of HCM. Their results are published in Nature Communications.

“While genes play a role in HCM, there is more information surrounding this condition that can’t be explained with genetics alone,” said corresponding author Bradley Maron, MD, a cardiologist in the Division of Cardiovascular Medicine at the Brigham. “This raises the question of whether there are other important components of the disease. With this project, we aim to provide an expanded view of the pathobiology of HCM in a way that doesn’t hinge on understanding specific gene mutations.”

The team collected tissue from 18 HCM patients recently recruited to receive myectomies, surgical procedures involving the excision of a portion of the heart muscle wall. To identify individual PPIs among the HCM cohort, Maron, co-lead author Ruisheng Wang, PhD, and colleagues analyzed tissue contents using RNA-seq, a technique that allows researchers to identify patterns of where and when genes are active. Through a series of steps, they identified patient-specific PPIs (known as reticulotypes) corresponding to the unique biological characteristics of each patient’s disease profile.

The group discovered that they could distinguish individualized protein networks in each patient in the HCM cohort.

“These findings represent a major step forward for precision medicine,” said Maron. “With the identification of patient-specific biological wiring maps, researchers may one day develop personalized treatments informed by patients’ protein networks.”

The study is unique in that researchers studied affected tissue collected directly from HCM patients, allowing for a more robust, accurate way of studying patients’ pathobiology than has been performed previously. Maron states, however, that in the future, he hopes to develop less invasive ways to perform this same test, whether it be through the collection of blood samples or through other biomarkers in the clinic. The team additionally aspires to apply this same procedure to other diseases as well, hoping to expand the number of opportunities for precision medicine.

“This study illustrates the complexity of HCM but also offers a clearer path forward for understanding the disease pathobiology with the promise of opportunity for precision medicine in this disease,” said Maron.

Funding for this work was provided by the National Institutes of Health (1R01HL155096, R56HL131787, R01HL153502, R01HL139613-01, R21HL145420, NHLBI K08128802, NHLBI R01 HL135121-01, NHLBI R01 HL132067-01A1, HG007690, HL119145, HL155107, GM107618, NHLBI: K23 HL150322-01A1), National Scleroderma Foundation, Cardiovascular Medical Research Education Foundation; McKenzie Family Charitable Trust, AHA Heart Failure Strategically Focused Research Network (16SFRN2902000), Nora Eccles Treadwell Foundation; and American Heart Association grants (D700382 and CV-19).

Maron, BA, et al. “Individualized interactomes for network-based precision medicine in hypertrophic cardiomyopathy with implications for other clinical phenotypes” Nature Communications DOI: 10.1038/s41467-021-21146-y