Neighborhood Analysis Highlights Role Of Macrophages In ER+ Breast Cancers

By Deborah Borfitz 

March 22, 2023 | When it comes to breast cancer, the most commonly occurring estrogen receptor-positive (ER+) variety is a poor candidate for treatment with conventional immunotherapies designed to help the patient’s own immune system fight back. Unlike triple-negative breast cancer, ER+ tumors don’t have many infiltrating T cells so potentiating their function alone doesn’t make much of a difference, according to Sayali Onkar, who performed this research as a Ph.D. trainee at University of Pittsburgh School of Medicine. 

But after spending the last five years identifying and investigating macrophages, another major player in the immune arsenal, she has some leads regarding the kind of immunotherapies that could improve patient outcomes. The strategies might need to differ depending on whether the diagnosis is invasive (infiltrating) lobular carcinoma (ILC) or invasive (infiltrating) ductal carcinoma (IDC), which until recently were lumped into the same category and treated as the same disease, she says. 

Macrophages are in fact the dominant immune cell infiltrating both ILC and IDC tumors, reports a study newly published in Nature Cancer (DOI: 10.1038/s43018-023-00527-w) where Onkar was the lead author. The IDC samples also tended to have a relatively higher proportion of T cells in the tumor microenvironment (TME), while almost 80% of all immune cells in the stromal compartment of the ILC samples were macrophages. 

A detailed spatial analysis of the TME found distinct immune cell “neighborhoods” associated with the two ER+ breast cancer subtypes as well as disease recurrence, says Onkar. With IDC, the bioinformatics approach known as neighborhood analysis further revealed an interplay between macrophages and T cells was linked to longer disease-free survival. 

The study took a comprehensive approach looking at not just the relative quantity of immune cells within the TME but where they were located “because immune cells are capable of communicating with each other and coordinating with each other to mount a response together,” Onkar says. The types of macrophages inhabiting the TME also matter, since their function and phenotype are highly context-dependent, she adds, noting that fewer of the pro-inflammatory, tumor-fighting M1-like macrophages and more of the tumor-promoting M2-like macrophages—which play more of an immune surveillance role—were in the ER+ ILC than IDC patient samples.  

The datasets provide a rich resource for further exploration of immune cell dynamics in ER+ breast cancer and macrophages as a potential treatment target, says Steffi Oesterreich, Ph.D., co-leader of the cancer biology program at the University of Pittsburgh Medical Center (UPMC) Hillman Cancer Center and co-senior author of the study. This might logically include efforts to treat ILC by removing harmful M2-like macrophages or reprogramming them to become M1-like macrophages, or to come up with new therapies combining macrophages and T cells. 

Repolarization of macrophages using epigenetic enzymes is but one of the promising paths forward, says Onkar, noting that work in this area has been ongoing for close to a decade now and “not just for breast cancer.” Preliminary observations of the research team also point to the type of checkpoint blockades that might potentiate the function of T cells and be most beneficial in the differential treatment of ILC and IDC patients.   

But the phenotypic, transcriptional, and functional analyses were done on limited sample sizes (94 and 87 treatment-naïve IDC and ILC tumors, respectively), requiring that these results be interpreted with caution until they are validated in larger studies, she adds. If the findings are confirmed, the next step would be to test different checkpoint blockades, in combination with other therapies, in the two ER+ patient subtypes “rather than having a one-size-fits-all approach.” 

Cross-Disciplinary Work 

The study, conducted at UPMC, serves to strengthen the evidence base highlighting the importance of macrophages in the development and progression of breast cancer, says Onkar. It also calls attention to the plight of patients with ER+ IDC and ILC tumors who don’t respond well to existing immunotherapies focused on T cells. 

T cells are central effectors of the immune response that ultimately act to kill tumor cells or shrink tumors. But before T cells “get to the scene,” explains Onkar, many other cells are involved in the immune cascade, starting with its activation. 

For ER+ breast cancers, scientists in the field have come to realize that targeting these other populations of immune cells is critical, she continues. Macrophages haven’t been the first pick because they are a “plastic population that exist on a spectrum of function from tissue surveillance, wound healing, and tumor-fighting to tumor- and metastasis-promoting cells” and thus “tricky to work with.”  

The research focus has traditionally been on characterizing the immune infiltration in triple-negative breast cancer, although ER+ disease accounts for roughly two-thirds of all breast cancer cases and most disease-related deaths, notes Oesterreich. “However, it is pretty clear from earlier studies on ER+ tumors that the fraction of cold tumors [having less T cell infiltration] ... is much higher.” 

Use of neighborhood analysis—which Oesterreich likens to “houses next to a grocery store”—is also a relatively novel approach to surveying the immune landscape to learn which clusters of cell types are in residence and their association with patient outcomes, she says. The methodology revealed a higher prevalence of macrophages and CD8+ T cells in IDC than ILC that were also associated with improved disease-free survival. This suggests that the two cell types, when appearing together near the tumor, are somehow keeping it in check. 

Oesterreich has been studying breast cancer for the past 25 years and holds the Shear Family Endowed Chair in Breast Cancer Research and co-directs the Women’s Cancer Research Center (a partnership between UPMC Hillman and Magee-Womens Research Institute). Immunology was added to the mix in more recent times thanks to her collaboration with Dario Vignali, Ph.D., distinguished professor and interim chair of the Pitt School of Medicine’s department of immunology and associate director for scientific strategy and co-leader of the cancer immunology and immunotherapy program at UPMC Hillman. Cross-disciplinary studies like this one are the only way to make significant progress in precision oncology, she says.  

“We started out being unbiased, looking at the global infiltrate of these two diseases [ILC and IDC] but then zoomed in a little more and tried to gauge their function,” says Onkar. It took two years just to learn that macrophages were a major player in ER+ tumors, and another three to complete the study.  

“To derive clinical strategies based on these findings, we would need more analysis in studies similar to ours and more myeloid-targeting therapies in the clinic, which are largely lacking at present,” says Vignali. “In the future, we plan to use systems immunology approaches to further interrogate clinical samples from treatment-naïve patients and those treated with immunotherapies to obtain greater granularity of the complex interactions that occur within the breast cancer-immune microenvironment.”  

Role for AI 

Unquestionably, “spatial organization matters” when it comes to immune cells in the TME, says Oesterreich. Neighborhood analysis provides information that would otherwise be missed by the pathologist’s eye alone when peering at a tumor biopsy sample. 

This has been demonstrated on a small scale, she emphasizes, pointing to a growing role for artificial intelligence to decipher interactions between hundreds if not thousands of different immune cells to better understand these neighborhoods. The Nature Cancer paper generated an enormous amount of data from single-cell sequencing, which Oesterreich hopes will now be tapped by others to make additional discoveries.  

“If the macrophage makeup is different between the two different histologies, the ductal and the lobular cancer ... how can we actually target them?” she rhetorically asks. “Other scientists may have a drug for this.”