Triple-negative breast cancer (TNBC), accounting for approximately 15%–20% of invasive breast cancers, is pathologically defined by the absence of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER2) expression, resulting in its aggressive behavior and poor prognosis [1,2]. The tumor microenvironment (TME), comprising tumor-infiltrating immune cells, vasculature, and extracellular matrix components, significantly influences the prognosis and treatment response of TNBC through its interaction with cancer cells [2,3]. For instance, tumor-infiltrating lymphocytes (TILs) are well-established as both prognostic and predictive biomarkers in TNBC, with their composition and spatial distribution playing critical roles [[4], [5], [6]]. Specifically, accumulating evidence highlights that tumor-infiltrating CD8+ T cells, major anti-tumor TILs, are associated with favorable prognosis and therapeutic outcomes of immunotherapy [[7], [8], [9]].

Chemokines, as chemotactic cytokines, regulate the migration and tissue infiltration of immune cells, essential for immune system function through their interaction with specific receptors [10,11]. The C-C motif chemokine ligand 25 (CCL25), also known as thymus-expressed chemokine (TECK), attracts dendritic cells, lymphocytes, thymocytes, and macrophages through its exclusive receptor, CCR9 [12,13]. The CCL25-CCR9 axis plays a pivotal role in various immune regulation and inflammatory processes, such as leukocytes development, maturation, migration, and tissue-specific homing [[14], [15], [16]]. Previously, our study demonstrated that delivery of CCL25 by a nanoparticle carrier significantly enhanced the tumor infiltration of CCR9+CD8+ T cells through chemotaxis, resulting in robust anti-tumor effects when combined with siCD47 in TNBC mouse models [17]. Thus, regulating CCL25 in the TME to influence CCR9-expressing cells may have a beneficial impact on the efficacy of tumor immunotherapy. However, direct intratumoral delivery of CCL25 alone has shown limited anti-tumor effects, primarily due to the lack of a sustained release mechanism, leading to rapid drug degradation and a short residence time. To address this issue, we engineered 4T1 tumor cells with CCL25 and observed that the CCL25-transduced cells exhibited significant tumor regression and increased tumor accumulation of CCR9+CD8+ T cells compared to untransfected cells. However, in practical applications, it is challenging to perform gene editing on tumor cells in vivo. Moreover, chemokines, as small molecular secreted proteins, are prone to degradation, and their biological activity is highly dependent on maintaining their three-dimensional structure [18]. Therefore, we need to propose a strategy that can ensure the long-term supply of chemokines, guaranteeing both sustained delivery and protection of the released factors from degradation. Furthermore, we investigated the potential of CCL25 in reshaping the immune landscape of the TME, as well as its optimal dose and timing of administration to maximize therapeutic efficacy.

In recent years, hydrogels have emerged as effective drug delivery systems for cancer treatment due to their high biocompatibility, controllability, and stability [[19], [20], [21]]. Among them, an injectable, degradable, and thermosensitive hydrogel, synthesized from methoxy-poly (ethylene glycol)-b-poly (γ-ethyl-L-glutamate) diblock copolymers (mPEG-b-PELG), has been utilized for the delivery of various biomolecules in tumor treatment [[22], [23], [24], [25]]. Our research involves the delivery of a small-molecule protein with a molecular weight of 16 KD. The preservation of the protein drug's activity during delivery is of utmost importance. Previous research has demonstrated that this hydrogel can effectively deliver proteins, such as IL-2, IL-15, and IFN-γ, which have molecular weights similar to our target protein CCL25, and ensure their full activity [26,27]. It also offers controlled drug release, which is essential for our study as it can provide a sustained and regulated supply of the protein drug. Moreover, the good biocompatibility of this hydrogel has been well-documented in the literature for its low cytotoxicity [28]. Herein, we employed this hydrogel for capsulation and sustained release of chemokine CCL25 to achieve appropriate localized delivery of chemokines for tumor immunoregulation (Scheme 1A). Our findings indicate that intratumoral injection of CCL25-loaded hydrogel (denoted as CCL25@gel) could effectively release CCL25 within tumor tissues, promoting the infiltration of CCR9+CD8+ T cells in a time-dependent and dose-dependent manner. We further discovered that optimizing delivery conditions, including dosage and timing, can facilitate CCL25 to reshape the immune landscape of the TME (Scheme 1B). In addition, intratumorally administrated CCL25@gel with low dose restrained tumor growth, reduced the number of lung metastases, and prolonged overall survival in a TNBC mouse model. Notably, CCL25@gel at an optimal dose and timing would enhance the therapeutic response to PD-1 inhibitors, credited to the activation of a T cell-dependent antitumor immunity. This work provides a promising strategy to remodel the TME for enhanced antitumor immunity utilizing a hydrogel-based chemokine delivery system capable of precise controlling of CCR9+ cells migration and highlights the great potential of this chemokine delivery system for TNBC immunotherapy and even other tumor types.