Breast cancer has been the world's mostly commonly-diagnosed cancer since 2020 [1]. In China alone, it accounts for approximately 20% of all cancer incidences [2]. Metabolic reprogramming is a hallmark of breast cancer [3]. As a case in point, glycolysis is the primary pattern of energy generation in breast cancer cells, by which glucose is mainly converted into lactate even in an oxygenated condition [4]. Abundant glycolytic activity is associated with poor human triple-negative breast cancer (TNBC) outcomes [5], and glycolysis restriction can prolong experimental animal survival by reducing myeloid-derived suppressor cells (MDSCs) and enhancing T cell immunity [5]. Therefore, targeting glycolysis has shown promise in TNBC treatment. Currently, glycolysis regulation strategies for tumor cells are mainly divided into two categories for drug intervention: transporters (e.g., glucose transporters and monocarboxylate transporters) and metabolic enzymes (e.g., hexokinase II, phosphor-fructokinase, lactate dehydrogenase, and pyruvate kinase) [6]. Monocarboxylate transporters (MCT) play an important role in promoting tumor growth by transporting L-lactic acid and pyruvate [7,8]. Inhibition of MCT-1 can arrest the growth of breast cancer cells [9]. Some MCT inhibitors are in clinical trials, such as AZD3965 (phase I) for diffuse large B-cell lymphoma and Burkitt lymphoma therapy [10]. In addition, lactate, the end product of glycolysis, serves as an essential regulatory metabolite in the tumor immune microenvironment (TIME) for reprogramming energy production and promoting angiogenesis and immune evasion [11]. For example, regulatory T (Treg) cells actively absorbed lactate through MCT-1 to enhance the expression of PD-1, thus limiting T cell activation and further forming the immune escape [12]. Moreover, lactate can decrease the chemotherapy efficacy and inhibiting lactate production can reverse chemoresistance and rescue the DNA mismatch repair (MMR) system [13]. Lactate from the cancer cells can also stabilize HIF-1α form degradation, and induce VEGF expression by TAM and subsequent neovascularization [14]. Therefore, targeting lactate metabolism is helpful in anticancer therapy [15].

Quercetin (QU), a plant-derived flavonoid, has been reported to have anticancer activity through several mechanisms [16]. The combination therapy of QU and chemotherapeutics has been demonstrated with a synergistic anti-tumor effect [17,18]. Notably, QU was revealed to be able to inhibit lactate transport in cancer cells [19], and QU-mediated lactate reduction can relieve the immunosuppressive status in TIME [20]. Thus, we proposed an immune-metabolic regulation using QU in combination therapy with doxorubicin (DOX). DOX is a potent immunogenic cell death (ICD) inducer to release damage-associated molecular patterns (DAMPs, e.g., calreticulin and high mobility group box 1, HMGB1) [21]. DAMPs can effectively stimulate the maturation of DC cells and subsequently activate strong anti-tumor T cell immunity [21,22]. It was expected that QU could suppress lactate production by targeting both glycolysis and MCT-1 and thus relieve the tumor immunosuppression. Meanwhile, DOX can induce ICD to stimulate anticancer immunity. Therefore, the combination of QU and DOX may provide multifaceted effects on TNBC therapy.

To benefit the combination therapy, we aimed to design a codelivery system of QU and DOX, because codelivery can facilitate the simultaneous action of the combined drugs in a spatial-temporal synchronization manner [22]. Legumain is an asparagine endopeptidase that promotes breast cancer progression [23]. Legumain overexpression is related to poor overall survival in TNBC patients [24]. Legumain can specifically recognize the peptide substrates containing asparagine (Asn, N) residues to cleave the carboxyl end of Asn and the most reported substrate sequences were proline-threonine-asparagine (PTN) and alanine-alanine-asparagine (AAN) [25,26]. KC26 peptide (sequence: ke5Ne4GPTN2R9C, k: d-lysine; e: D-glutamate) is a legumain responsive hairpin-structured cell-penetrating peptide (polyarginine) derivative developed in our lab based on the molecular-dynamics-simulation-driven design [27], which can be applied for tumor-specific targeted delivery by responding to the overexpressed legumain in TIME.

Herein, we reported a legumain-responsive liposome (termed KC26-Lipo) with codelivery of QU&DOX for modulation of breast tumor metabolism and TIME. The KC26-Lipo will be specifically activated in the legumain-overexpressed tumor microenvironment, and the exposed cell-penetrating polyarginine would then mediate intratumoral and intracellular penetration, thus achieving enhanced treatment outcomes.