Breast cancer (BC) is the most common cancer among females and poses a significant public health concern. Therefore, identifying circulating biomarkers is crucial for the early detection and diagnosis of BC [20].

The current study revealed that the relative mRNA expression of cathepsin-B was statistically significantly elevated in metastatic BC cases (9.2 ± 2.1-fold change) compared to localized BC cases (5.1 ± 1.8-fold change) and normal individuals (1.1 ± 0.2-fold change) (p < 0.001) (Table 2). A highly significant expression was found among patients with higher tumor stages (T3 and T4) (p = 0.02). Additionally, cathepsin-B gene expression was observed to be notably elevated among cases with lymphatic and distant metastasis compared to patients with lymph node metastasis only (p = 0.04) (Table 3).

The serum IL-6 level was statistically significantly higher among metastatic BC cases than localized BC cases and healthy individuals (p < 0.001). While CA15-3 levels were statistically significantly elevated among BC groups compared to the healthy group (p < 0.001), there was no significant difference between cases with localized or metastatic BC (p > 0.05) (Table 4).

Our results showed a strong positive association between cathepsin-B and both IL-6 and CA15-3 (r = 0.905 and 0.667, respectively), (p < 0.001) (Table 5).

When we plotted the ROC curve to investigate the ability of cathepsin-B in predicting BC and metastasis, the test showed 95.8% and 97.2% sensitivity and 94.4% and 70% specificity at a cut-off value of ≥ 1.4- and ≥ 6.1-fold change, with an area under the curve of 0.996 and 0.958, respectively (Table 6) and (Figs. 1, 2). These findings suggest that monitoring cathepsin-B expression in the blood could be a non-invasive and easy diagnostic procedure.

Although CA15-3 is widely used in clinical practice, its role in managing BC remains disputed. The most recent recommendations from both the American Society of Clinical Oncology (ASCO) and the National Comprehensive Cancer Network (NCCN) advise against the use of CA15-3 levels assessment for screening, diagnosis, or post-therapy monitoring [21].

CA15-3 levels can be elevated in healthy individuals, benign diseases, malignant illnesses, and various non-malignant conditions, including cirrhosis, hepatitis, lupus, sarcoidosis, and tuberculosis, as well as during pregnancy and breastfeeding [22].

In contrast to our results, Zhao et al. found that individuals with metastatic BC experienced more frequent CA15-3 increases than those with early BC [23].

According to Berruti et al., CA15-3 levels were found to be elevated in various metastatic sites, with patients who had visceral metastasis showing higher rates of elevated CA15-3 levels compared to those with soft tissue and bone metastases [24]. He et al. suggested that patients with higher CA15-3 levels had a higher chance of developing abdominal and bone metastases. In agreement with our results, some studies did not discover significant alterations in CA15-3 levels among various metastatic positions [25].

Cathepsin-B, a lysosomal cysteine protease, is regarded as a “multifunctional enzyme in cancer” and primarily contributes to the proteolytic cascades involved in cancer progression, invasion, and metastasis. Cathepsins are typically found in lysosomes, but when cancer occurs, they often move to the cell surface or are even secreted [5]. In BC, cathepsin-B overexpression can break down the extracellular matrix, allowing cancer cells to migrate and invade surrounding tissues. Additionally, it can activate pro-angiogenic factors, promoting the formation of new blood vessels that feed the tumor [16, 17].

Lah et al. conducted one of the first studies on cathepsin-B in a large group of BC patients. They demonstrated that cathepsin-B levels were significantly higher in breast carcinomas compared to healthy breast tissues, which is consistent with our findings [26].

Decock et al. discovered that early-stage BC patients with poorly differentiated tumors had lower blood cathepsin-B levels than patients with well or moderately differentiated carcinomas [27].

Maguire et al. observed that cathepsin-B levels were significantly higher in both primary carcinomas and metastatic (nodal metastasis) tissues compared to benign tumors (fibroadenomas), aligning with our study results. However, they found no significant difference between cathepsin-B levels in primary cancers and metastatic specimens, and these levels did not correlate with tumor stage or nodal status unlike our findings [28].

Rudzinska-Radecka et al. reported significantly higher cathepsin-B expression in laryngeal cancer compared to nearby normal tissue, with no notable differences between patients with or without lymph node involvement or cancer stage [7].

Cathepsin-B serum concentrations were elevated in hepatoma and cirrhotic liver cases compared to normal individuals, but no distinction was observed between hepatoma and cirrhotic liver cases [29].

IL-6 is a key cytokine present in the cancer microenvironment produced by adipocytes and monocytes that are linked to BC tissues. The effect of IL-6 on BC cell growth varies based on the activation level in the Jak/Stat3 signaling pathway and hormone receptor status. In some instances, IL-6 has been shown to stimulate BC cell growth, while in others, it can inhibit growth. Studies have indicated that IL-6 can play various tumor-promoting roles, including enhancing proliferation and suppressing apoptosis in BC cells [30]. IL-6 can stimulate the production of vascular endothelial growth factor (VEGF), which induces angiogenesis and tumor vascularization [31]. Furthermore, IL-6 can induce the expression of genes involved in epithelial-to-mesenchymal transition (EMT), which enables cancer cells to acquire a more aggressive, invasive phenotype [32].

In accordance with our findings, Kozlowski et al. reported that patients with BC exhibited significantly elevated levels of interleukin-6 (IL-6) in their bloodstream compared to healthy women, which was consistent with the patient's clinical information [33].

Furthermore, Mohamed et al. demonstrated that IL-6 can increase the expression of cathepsin-B in response to soluble factors released by BC cells, and showed, through western blotting and enzymatic activity analyses, that counteracting antibodies against IL-6 could reduce cathepsin-B secretion and activity stimulated by 231-conditioned medium [16].

Ibrahim et al. investigated how the cathepsin-B protein expression in BC cells was influenced by IL-6 at different concentrations. They revealed that IL-6 is higher in carcinoma tissues of human hormonal receptor-positive (HRP) BC and that it correlates with the expression of cathepsin-B. Moreover, they found that IL-6, either alone or together with cathepsin-B, are significant therapeutic targets for patients with HRP-BC and positive lymph node patients [11]. Additionally, Knüpfer and Preiss revealed that cathepsin-B and IL-6 were markers associated with poor prognosis in BC patients [34].

Our results, in line with the outcomes of previous studies, suggest that elevated levels of both cathepsin-B and IL-6 are associated with more aggressive BC, poorer prognosis, and resistance to treatment. Consequently, targeting either cathepsin-B or IL-6 has been explored as a promising therapeutic approach in BC. It is influential to note that the association between cathepsin-B and IL-6 in BC is complex and context-dependent, with other factors, such as other proteases, signaling pathways, and interactions with the immune system, also influencing their interplay [16]. Further research is needed to fully elucidate the intricacies of their relationship and its therapeutic implications. Moreover, the current study has key limitations that should be acknowledged, including a small sample size and the fact that the study comprised only Egyptian participants. We suggest that subsequent genetic assessment of the cathepsin-B/IL-6 axis with other proteases, such as matrix metalloproteinases (MMPs), and signaling pathways, such as NF-κB and STAT3, in diverse molecular subtypes of BC with larger sample sizes is necessary to gain a better understanding of their role in BC development and to evaluate the significance between them.