Metastasis of breast cancer to bones alters the tumor immune microenvironment

Bone metastasis occurs in 70% of patients with advanced breast cancer [1]. The skeletal-related events (SREs) associated with breast cancer bone metastasis (BCBM), such as pathological fracture, spinal cord compression, and severe pain, impair the patient’s mobility, reduce their quality of life, and increase overall morbidity [2]. Metastasis to the bone means that tumor cells leave their primary site and migrate to a new and specialized microenvironment made up of osteoblasts, osteocytes, osteoclasts, adipocytes, hematopoietic stem cells, and immune cells [3].

Current primary treatment options for BCBM include radiotherapy, osteoclasts inhibitors, endocrine therapy, chemotherapy, and supportive treatment-like analgesia. Occasionally, surgery can be applied when a single metastasis is found or when acute spinal injury happens. However, even with these options and multidisciplinary approaches, the metastases eventually develop resistance and progress. Increasing evidence suggests that immunotherapy may be a promising treatment method for primary breast cancers. Tumor-infiltrating lymphocytes (TILs) are reported to correlate with survival and therapeutic efficacy in breast cancer, especially in triple-negative and HER2 positive breast cancer subtypes [4‐7]. Moreover, another study demonstrated that TILs at metastatic sites of breast cancer also correlate with improved survival [8, 9].

The programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) pathway is an immune checkpoint pathway that suppresses immune system activation, where binding of the T cell receptor PD-1 to its ligand PD-L1 leads to downregulation of T cell proliferation, activation, and host anti-tumor function [10]. Tumor cells can exploit this checkpoint by expressing PD-L1 and, therefore, evade anti-tumor immune responses. PD-L1 expression on primary breast tumor cells, however, can be targeted with immune checkpoint inhibitors (ICIs) and subsequently correlates with immunotherapeutic benefit in the clinic and improved patient survival [11]. Moreover, ICIs have proven effective in all molecular subtypes of breast cancer [12‐14].

Despite this clinical advance in treating primary breast cancers with ICIs, limited data are available on the role of the immune microenvironment of BCBM and its impact on treatment responsiveness. A better understanding of tumor cell bone residence and interaction with the immune microenvironment may, therefore, unveil new targetable vulnerabilities and guide clinically relevant therapeutic approaches. This study aims to investigate the immune microenvironment shift between primary tumor sites and bone metastases of breast cancer by evaluating TILs, macrophages, and immune checkpoint markers.

We did a comprehensive histopathological analysis of the BCBM microenvironment. This is also the largest study to date to investigate immune microenvironment differences between primary breast cancer and its bone metastases. We observed that bone metastasis has a different distribution of stromal compartment and has a less active immune compartment compared with the primary disease site.

Stroma percentage in primary breast tumors is a proven prognostic factor; higher stroma percentage often correlates with increasing relapse rate and poorer long-term survival [19‐21]. Given this reported correlation and considering metastatic diseases are inherently advanced in nature with a likely worse prognosis, the increased percentage of stromal tissue found in the bone metastasis compared to the primary tumor site may not be surprising. However, we did not observe a significant correlation between stroma percentage of primary tumors or of the bone metastasis sites with survival. This may be due to the limited sample size of this study, or may point to other unique aspects of BCBM that influence and modulate disease progression.

Osteoclasts are the main participant in bone remodeling by secreting acid and lytic enzymes and modulating osteolytic processes [22]. Most BCBMs are osteolytic lesions. Osteoclasts display an outstanding morphological characteristic—a large multinucleate bone cell—which makes it easy to identify by microscopy [23]. In our study, we observed that all 63 BCBM lesions presented a prominent osteolytic change through the slides. Thirty-eight (60.32%) of the lesions presented osteoclasts around the cancer cells. These findings suggest that treatment targeted at osteoclasts could be effective in these patients.

Immune cells may play a crucial role in supporting bone metastasis and also have a relevant relationship with osteoclasts. Osteoclasts are derived from progenitor cells, which can also differentiate into macrophages and lymphocytes. Second, the receptor activator of nuclear factor-ƙB ligand (RANKL), which works as major regulator of osteoclasts, can be produced by immune cells [24]. In addition, bone marrow is a place, where tumor cells can direct contact with the immune system [25]. We observed that, compared to the primary site, bone metastasis sites had a less active immune environment, especially when considering TILs (Fig. 3). Studies which compared different metastatic sites also revealed that metastatic breast cancers are immunologically more inert than their corresponding primary tumors [26, 27]. Previous studies also showed that TILs correlate with survival in metastatic breast cancer sites [8, 9]. We did not observe this trend in BCBM. The luminal type breast cancer accounts for approximately 60% of the BCBMs [28, 29]. A reason for this could be that 40/63 (63.49%) of our patients had luminal type breast cancers, a molecular subtype with a prognosis that is less associated with TILs expression [4], and these luminal subtypes in our cohort showed less TILs in metastatic sites compared to the other molecular subtypes [30]. The sample size of HER2-positive and triple-negative patients were too limited to draw a conclusion that may otherwise have supported this trend. Studies on breast cancer metastasis to the lung, liver, and brain reported that high TILs correlated with better survival in triple-negative patients [27, 30]. CD4 + regulatory T cells are a known source of RANKL-induced metastases [26]. CD8 + T cells, also called cytotoxic T cells, however, are the main actor in the anti-cancer immune system that inhibits metastasis [27]. We observed a decrease in both CD4 + T cells and CD8 + T cells in bone metastasis compared with primary sites and without a significant change in the CD4 + /CD8 + ratio. This decrease aligns with a pro-metastasis tumor environment that can support BCBM, but their presence may nonetheless offer opportunities to apply certain immunotherapies and warrants further investigation.

Based on the correlation analysis we did of primary and metastasis site. We did not observer any significant correlation between primary and metastatic sites. Timepoints for the sampling collection, sampling site, and therapy regimes may possibly confound these results. We found that PD-L1 expression correlateswith TILs, CD4, CD8 especially in primary site, which is as expected. Previous clinical trials also showed that PD-L1 expression correlates with TILs and the response [31-33]. A combination of TILs and PD-L1 expression evaluation to select optimal patients for immunotherapy could be a better approach [34].

In the past few years, immunotherapy has become a promising therapy for late-stage breast cancer patients. Recent studies suggest that combination immunotherapies effectively improved the prognosis and survival of PD-L1 + patients [13, 28, 29]. In our study, 5/63 (7.94%) of patients were PD-L1 + . Three of the PD-L1 + patients had luminal type tumors who may not benefit from the PD-1 inhibitors [30]. Nevertheless, combination immunotherapy in bone metastasis patients is associated with better survival [35]. Thus, certain types of BCBM patients may benefit from the combination immunotherapy.

These results offer initial insights into primary and BCBM immune microenvironment differences and open the discussion for targeting these immune features to improve standard and alternative BCBM treatment methods. However, there are limitations to our study. First, it is a retrospective study with a limited sample size from one medical center. Staining on bone tissue also brings challenges due to technical issues which need optimization for better clarity and accuracy in quantification.

Our study shows that BCBM sites exhibit an abundance of stromal cells and an inactive immune mircroenvironment compared with paired primary breast tumor sites. Application of combination immunotherapy in select groups of BCBM patients deserves further exploration.