The impact of crown-like structures and metabolic intervention on immune responses in patients with primary breast cancer

Abstract

Obesity can initiate, promote, and maintain systemic low-grade inflammation partly via metabolic reprogramming of macrophages that encircle adipocytes termed crown-like structures (CLS). The tumour microenvironment can also exert metabolic stress on both innate and adaptive immunity, leading to local immunosuppression. Metformin has demonstrated antitumorigenic properties both in pre-clinical and clinical studies. Nevertheless, its immunomodulating properties in patients with breast cancer are largely unknown. Tumour-associated macrophages (TAM) and CLS are a diverse and heterogenous population of cells. The prognostic significance of CLS and their association with the spatial distribution of TAM and T-cells in breast cancer are still not clear.

First, the effect of metformin on tumour infiltrating immune cells was investigated in two independent cohorts of 31 and 47 patients each using immunohistochemistry. Metformin administration was associated with higher densities of tumour-infiltrating CD68+ macrophages, tumour-infiltrating CD8+ T-cells and lower density of regulatory T-cells suggesting that metformin may enhance antitumour immune responses. Then, to assess the effect of systemic inflammation on metformin-related immune responses, patients were stratified by circulatory levels of serum leptin. Elevated levels of leptin were associated with inactivated T-cell populations which were restored with the administration of metformin. These changes were confined to the tumour islands suggesting that there is an interplay between tumour and immune cells which may be partly mediated via metabolic competition. Then, the prognostic significance of CLS in a cohort of 69 trastuzumab-naïve and 117 adjuvant trastuzumab-treated patients with primary HER2+ breast cancer was evaluated. This showed that CD32B+ CLS at the adipose-tumour border were strongly correlated to BMI≥25 kg/m2 and were an independent prognostic factor for reduced time to metastatic disease in trastuzumab treated patients suggesting that adiposity potentially plays an important role in therapeutic responses.

To elucidate the immune mechanisms involved, multiplexed immunohistochemistry for various immune markers on CLS, TAM and T-cells was performed using the BEGIN cohort (Investigating outcomes from breast cancer: Correlating genetic, immunological, and nutritional predictors) (n=134). Analysis of the BEGIN cohort showed that CLS were associated with a differential effect on the densities and spatial distribution stromal immune cells and fibroblasts that is tumour subtype specific. These results imply that the interplay between CLS and tumour biology may be associated with an adaptation in the tumour microenvironment that promotes polarization of macrophages towards immunosuppressive phenotypes and T-cell inhibition in high histological grade breast tumours. Whilst CLS were associated with activated macrophage and T-cell phenotypes in low histological grade tumours. To further understand the effect of CLS on the tumour-immune microenvironment, targeted RNA sequencing of 36 samples with matched multiplexed immunohistochemistry data using the Precision Immuno-Oncology Panel in HTG EdgeSeq platform was done. Targeted RNA sequencing showed that gene sets that are associated with cell cycle progression were upregulated, whereas gene sets involved in the regulation of anticancer immune responses were downregulated in patients with high CLS that is consistent with immunohistochemistry findings.

In summary, the presence of CLS may be associated with inflammatory signatures at the tumour border and an aggressive molecular phenotype in the core that may promote tumour growth and immune suppression. Metabolic interventions such as metformin may play a role in the metabolic reprogramming of T-cell and macrophages populations that are dysregulated by systemic inflammation.

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Identifiers

ORCID for Constantinos Savva: orcid.org/0000-0003-0805-4719

ORCID for Stephen Beers: orcid.org/0000-0002-3765-3342

ORCID for Peter Johnson: orcid.org/0000-0003-2306-4974

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