Researchers headed by a team at the University of Texas MD Anderson Cancer Center have identified a subset of immune macrophages that obstruct treatment of glioblastoma multiforme (GBM) using PD-1 checkpoint inhibitors. An analysis of five different human tumor types discovered the macrophages, which express CD73 on their surface, uniquely in glioblastoma. The potential therapeutic utility of the findings was demonstrated through subsequent tests in which a combination of anti-PD-1 and anti-CTLA-4 immunotherapy significantly improved survival in GBM tumor-bearing mice that lacked CD73.

“By studying the immune microenvironments across tumor types, we’ve identified a rational combination therapy for glioblastoma,” said Sangeeta Goswami, MD, PhD, assistant professor of genitourinary medical oncology. Goswami is first author of the researchers’ published paper in Nature Medicine, which is titled, “Immune profiling of human tumors identifies cD73 as a combinatorial target in glioblastoma.

Immune checkpoint therapy using anti-CTLA-4 and anti-PD-1/PD-L1 antibodies has revolutionized the treatment of many solid tumors, but these treatments are only effective in subsets of patients with specific tumor types, the authors wrote. The researchers used mass cytometry (CyTOF) and single-cell RNA sequencing to profile immune cell subsets in tumor samples from a total of 94 patients with five different tumor types: non-small cell lung cancer (NSCLC), microsatellite instability-stable colorectal cancer (CRC), prostate cancer (PCa), and GBM. Their analyses compared cells in immune infiltrates that were either shared across tumor types, or were specific to individual tumors.

The most surprising finding was a metacluster of immune cells identified predominantly among the 13 glioblastoma tumors. Cells in the cluster expressed CD68, a marker for macrophages, which are immune system cells that either aid or suppress immune responses. The CD68 metacluster also expressed high levels of CD73 as well as other immune-inhibiting molecules. The scientists found the same CD73-expressing macrophages in samples from a second set of nine GBM patients. “Compared to our first GBM cohort, we found similar high frequencies of CD73hi macrophages and low T-cell numbers,” they wrote.

Single-cell RNA sequencing identified an immunosuppressive gene expression signature associated with the high-CD73-expressing macrophages. A refined gene signature for the cells was evaluated against 525 glioblastoma samples from the Cancer Genome Atlas and was found to correlate with poorer survival. “We found a significant negative correlation between overall survival and high expression of the CD73hi gene signature,” the scientists stated.

They conducted CyTOF mass cytometry cluster analysis on five glioblastoma tumors treated with the PD-1 checkpoint inhibitor pembrolizumab and seven untreated tumors. This highlighted three CD73-expressing macrophage clusters that persisted despite pembrolizumab treatment. The team suggested that this prevalence of CD73-expressing macrophages likely contributed to lack of tumor-killing T cell responses and poor clinical outcome.

To test the hypothesis that targeting CD73 might aid therapeutic strategies against GBM, the researchers investigated the effects on different treatment approaches, of knocking out CD73 in tumor-bearing mice. “We treated GBM tumor-bearing mice with anti-PD-1 antibody or with a combination of anti-PD-1 and anti-CTLA-4 antibodies.” They found that while mice with intact CD73 that were treated with both immunotherapies had increased survival over untreated mice, in animals lacking CD73, the combination of anti-PD-1 and anti-CTLA-4 immunotherapies held back tumor growth and was associated with even longer survival. “In the absence of CD73, intracranial tumor growth was impeded and mice exhibited improved survival, confirming the immunosuppressive role of CD73 in GBM,” they commented. “Importantly, following treatment with a combination of anti-PD-1 and anti-CTLA-4, CD73-/- mice had improved survival compared to WT [wild-type] GBM tumor-bearing mice.” There was no survival benefit from anti-PD-1 therapy alone.

“We’re working with pharmaceutical companies that are developing agents to target CD73 to move forward with a glioblastoma clinical trial in combination with anti-PD-1 and anti-CTLA-4 checkpoint inhibitors,” noted Padmanee Sharma, MD, PhD, professor of genitourinary medical oncology and immunology.

The approach taken by Sharma and colleagues can be thought of as reverse translation. Instead of developing hypotheses through cell line and animal model experiments that are then translated to human clinical trials, the team started by analyzing human tumors to generate hypotheses for testing in the lab. To more effectively extend immunotherapy to more cancers, researchers need to realize immune microenvironments differ from cancer to cancer, Sharma noted. “Understanding what’s different in immune niches across cancers provides clues and targets for treating tumors. That’s why we did this study.”

The team concluded, “Based on our data and earlier studies, we propose a combination therapy strategy to target CD73 plus dual blockade of PD-1 and CTLA-4,” noting that anti-CD73 antibodies have yielded promising results in early studies. “The anti-CD73 antibody has yielded promising results in preclinical as well as early clinical studies; therefore, our data have clinical applications with rapid translation of combination therapy for GBM with currently available anti-CD73 antibodies.”


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