Construction of enzyme-displaying protein nanoparticles through LOX or LOX and CAT ligation on a protein nanoparticle
To develop multiple enzyme-displaying protein nanoparticles, we utilized our previously established AaLS-ST comprising 60 identical subunits as a template [32, 33]. AaLS subunits self-assemble to form an icosahedral structure having outer and inner diameters of 16 and 9 nm, respectively [36]. They also effectively displayed various functional proteins through a ST/SC ligation system [32, 33]. SC was genetically fused to the N-termini of both LOX and CAT to form SC-LOX and SC-CAT, respectively. As a result, the fusion proteins were successfully overexpressed and purified (Additional file 1: Figs. S1 and S2a). The subsequent comparison of size exclusion chromatography (SEC) elution profiles with molecular weight standards revealed that both SC-LOX and SC-CAT maintained their tetrameric configuration (Additional file 1: Fig. S2b, c). In addition, SC fusion proteins were eluted slightly earlier than wild-type proteins, indicating a size increase due to the SC fusion (Additional file 1: Fig. S2b). Therefore, we conducted band intensity analyses of sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) of individual proteins and ligated forms to determine and control the relative amount of displayed proteins on a AaLS, using the NIH Image J, which is one commonly used analytical technique to determine the relative band intensities of multiple components in SDS-PAGE [37, 38]. Results from ST/SC ligations between AaLS-ST and either SC-LOX or SC-CAT showed that while a successful covalent connection with both proteins exhibited new bands around 100 kDa in SDS-PAGE (Additional file 1: Fig. S2d, e), spontaneous protein aggregation was induced by an ST/SC ligation between AaLS-ST and SC-CAT (AaLS/CAT). Although approximately 40% of ligated proteins were precipitated upon ligation between AaLS-ST and SC-CAT and removed by centrifugation, no apparent precipitation was observed upon ST/SC ligation between AaLS-ST and SC-LOX (AaLS/LOX) (Additional file 1: Fig. S2d, e). Structural analyses based on the crystal structures of LOX and homologous CAT suggested that the LOX tetramer had a C4 symmetry, causing all N-termini of LOX subunits to face the same direction (Additional file 1: Fig. S3a, red-colored spheres) [39] and the CAT tetramer was tetrahedrally arranged with two oppositely facing N-termini of four CAT subunits (Additional file 1: Fig. S3b, blue-colored spheres) [40]. Thus, multiple SC-LOXs could interact with only one AaLS-ST nanoparticle, decorating the surface without forming further clusters with nascent nanoparticles (Fig. 1a), whereas one SC-CAT was able to interact with two or more AaLS-ST particles to form a large interparticle network, leading to protein aggregation and subsequent precipitation. Therefore, SC-CAT tetramers were first partially pre-blocked with the ST-fused small collagen-binding domain (ST-CBD) afterward to prevent interparticle aggregation, and subsequently ligated to AaLS-ST (Additional file 1: Fig. S2f) and SDS-PAGE analyses of prior reactions between ST-CBD and SC-CAT, and the subsequent reaction with AaLS-ST, revealed that the partial passivation of SC-CAT with ST-CBD rescued protein aggregation and precipitation (Additional file 1: Fig. S2g, h).
Subsequently, LOX-displaying protein nanoparticles were generated through the ligation of SC-LOX with AaLS-ST to form AaLS/LOX. However, LOX-and-CAT-dual enzyme-displaying protein nanoparticles were formed through the ligation of CBD-passivated SC-CAT (SC-CAT from here for simplicity) to AaLS/LOX to form AaLS/LOX/CAT (Fig. 2a). Equivalent amounts of CBD-passivated SC-CAT and SC-LOX were ligated to AaLS-ST and approximately 20% of AaLS-ST subunits remained unmodified (Fig. 2b, black arrow). Nevertheless, the ligation reactions between AaLS-ST and SC-LOX or AaLS-ST and SC-CAT, revealed new bands of approximately 85 or 100 kDa in the SDS-PAGE which were well matched with a molecular weight of the ligated AaLS-ST and SC-LOX subunits (84.6 kDa, Fig. 2b, orange arrow) and those of the ligated SC-CAT and AaLS-ST subunits (95.8 kDa) or SC-CAT and ST-CBD subunits (98.8 kDa) (Fig. 2b, blue arrow). Nonetheless, the molecular weights of ST-CBD (26.2 kDa) and AaLS-ST subunits (29.2 kDa) were similar to each other (Fig. 2b, black arrow), and the ligated products with SC-CAT subunits exhibited similar molecular weights (Fig. 2b, blue arrow). SC-LOX ligation and subsequent SC-CAT ligation onto the AaLS-ST resulted in increased hydrodynamic diameters (22.9, 31.5, 41.9 nm, respectively) in dynamic light scattering (DLS) analyses and an earlier elution in SEC, suggesting that the particle size increased upon enzyme immobilization (Figs. 2c, d), as illustrated in Fig. 2a. Additionally, transmission electron microscopic (TEM) images showed stable spherical nanoparticle architectures in each protein and both AaLS/LOX and AaLS/LOX/CAT exhibited extra-electron densities around the nanoparticles (Fig. 2e), as illustrated in Fig. 2a. Notably, however, the TEM images of both LOX and CAT showed smaller particles of approximately 5 nm (Additional file 1: Fig. S3c), similar to the extra densities observed in AaLS/LOX and AaLS/LOX/CAT (Fig. 2e and insets). The zeta potential values of AaLS-ST, AaLS/LOX, and AaLS/LOX/CAT were − 13.6 mV, − 13.2 mV, and − 14.9 mV, respectively, (Additional file 1: Fig. S2j) and there was no significant difference among them observed. Therefore, these results collectively indicate that both LOX and CAT simultaneously and successfully ligated and displayed onto the surface of the AaLS-ST to form dual-enzyme-displaying nanoparticles.
AaLS/LOX and AaLS/LOX/CAT efficiently consumed lactate in the lactate-containing buffer
We also evaluated the enzymatic activities of LOX and CAT variants. First, we prepared and measured each enzyme’s typical Michaelis–Menten kinetic parameters. Then, the enzymatic activities of LOX variants (SC-LOX and AaLS/LOX) were evaluated by monitoring the amounts of produced H2O2 that underwent subsequent reactions with Amplex Red, using horseradish peroxidase (HRP) to develop colorimetric signals [41]. On the other hand, the enzymatic activities of CAT variants (SC-CAT and AaLS/CAT) were determined by measuring ammonium molybdate’s consumption amounts, reacting with residual H2O2, generating colorimetric signals at 405 nm [42]. Although SC-LOX and AaLS-LOX or SC-CAT and AaLS-CAT exhibited similar enzyme kinetic parameters, no significant enzymatic activity alterations were observed even after the ligation to AaLS-ST (Fig. 3a, b). Furthermore, compared with the reported LOX and CAT kinetic parameters [41, 43], while CAT showed similar enzyme kinetic behavior [43], LOX showed a slightly reduced enzymatic activity, probably due to the N-terminal fusion of SC [41].
Next, we tested the lactate consumption capability of each sample in the buffer. First, 10 mM lactate was added to phosphate-buffered saline (PBS) at pH 6.7 to mimic the TME, after which lactate consumption was monitored under normoxic and hypoxic conditions. Under normoxic condition, all LOX-containing samples (SC-LOX, SC-LOX & SC-CAT, AaLS/LOX, and AaLS/LOX/CAT) rapidly consumed lactate and completed reactions within 12 h, in an almost identical pattern. In contrast, no noticeable lactate consumption was observed in the samples treated with AaLS-ST or SC-CAT (Fig. 3c). Under hypoxic condition, however, LOX-containing samples showed delayed lactate consumption profiles, probably due to the limited oxygen supply (Fig. 3d). We also observed that the samples treated with a combination of LOX and CAT (SC-LOX & SC-CAT and AaLS/LOX/CAT; blue triangles and black squares, respectively) exhibited increased lactate consumption levels compared with those of the LOX-only variants (SC-LOX and AaLS/LOX). These results suggest that co-existing CAT supplies additional oxygen by converting produced H2O2 to water and oxygen, allowing LOX to more efficiently convert lactate, even during a limited oxygen supply.
AaLS/LOX induced drastic necrotic cell death but AaLS/LOX/CAT did not alter cell viability with complete lactate consumption
LOX not only consumes lactate but also generates H2O2, damaging and eventually killing cells. However, CAT converts highly toxic H2O2 to oxygen and water, which are beneficial to cells. Therefore, we evaluated the lactate consumption capability and cytotoxic effect of AaLS/LOX and AaLS/LOX/CAT in a cell culture system. First, we prepared a CT26 mouse colorectal carcinoma cell-line and monitored lactate concentrations in the cell culture medium under normoxic conditions at various incubation times (Additional file 1: Fig. S4a). Lactate concentrations gradually increased from 4 mM at 24 h to 12 mM at 48 h, reaching approximately 18 mM after a 72-h culture (Additional file 1: Fig. S4a). Next, CT26 cells were cultured for 48 h to maintain the lactate rich environment followed by treating with samples (AaLS-ST, AaLS/LOX, or AaLS/LOX/CAT) in a concentration-dependent manner. After an additional culturing period of 24 h (72 h total), lactate concentrations and cell viabilities were evaluated (Fig. 4a–c). Although the lactate concentrations of CT26 cells treated with AaLS-ST remained unchanged, even at a high concentration (1 μM) (Fig. 4a, blue squares), almost all lactate produced by CT26 cells were consumed upon treatment with 4 nM or higher concentrations of either AaLS/LOX (Fig. 4b, blue squares) or AaLS/LOX/CAT (Fig. 4c, blue squares). Furthermore, as expected, CT26 cells treated with 4–100 nM of AaLS/LOX were almost completely killed (Fig. 4b, red squares) and their cell morphology was significantly altered (Additional file 1: Fig. S4b). However, 250 nM and higher concentrations of AaLS/LOX allowed the survival of CT26 cells (Fig. 4b, red squares). We hypothesized that the recovered cell viability at 250 nM and higher concentrations of AaLS/LOX-treated cells was due to the explosive conversion of lactate to pyruvate and H2O2, which subsequently reacted each other to produce acetate, CO2, and water, resulting in a rapid reduction of toxic H2O2 in the culture media [44]. External addition of pyruvate to the culture media indeed completely rescued AaLS/LOX-treated CT26 cells from necrotic cell death (Additional file 1: Fig. S4c), supporting the hypothesis that pyruvate effectively scavenges H2O2 to protect cells from excessive oxidative stress. We also used a thiol-containing antioxidant, N-Acetyl-L-cysteine (NAC), that can indirectly scavenge H2O2 by maintaining reduced glutathione levels [45, 46] and observed that NAC partially rescued AaLS/LOX-induced cell death (Additional file 1: Fig. S4c). However, other reactive oxygen species (ROS) scavengers, superoxide dismutase (SOD) mimetics Mn(III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) and Cu(II) diisopropylsalicylate (CuDIPS) did not rescue CT26 cells from AaLS/LOX-induced cell death (Additional file 1: Fig. S4c). Although AaLS/LOX produced significant amounts of H2O2 leading to necrotic CT26 cell death, AaLS/LOX/CAT did not induce noticeable cell death, even with effective lactate consumption (Fig. 4c). Furthermore, morphological changes were not detected in CT26 cells treated with AaLS/LOX/CAT (Additional file 1: Fig. S4b). Therefore, we concluded that the maintenance of the cell viability of CT26 cells treated with AaLS/LOX/CAT was due to the additional CAT activity, converting toxic H2O2 to nontoxic oxygen and water, even with the complete consumption of lactate. Fluorescence microscopy using the cell-permeable ROS indicator, 2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA), also showed that while the AaLS/LOX treatment dramatically increased ROS levels, the AaLS/LOX/CAT treatment did not produce ROS in CT26 cells, indicating the effective H2O2-removing capability of AaLS/LOX/CAT (Fig. 4d).
Subsequently, we investigated the cell death mode induced by AaLS/LOX, using CT26 cells stained with annexin V and 7-amino-actinomycin D (7-AAD) after treatment with AaLS-ST, AaLS/LOX, or AaLS/LOX/CAT. The population of AaLS/LOX-treated CT26 cells began to shift to the 7-AAD positive population after six hours post-sample treatment. Furthermore, they finally shifted to both 7-AAD and annexin V-positive populations at 12 h post-sample treatment, indicating the induction of necrotic cell death (Fig. 4e). However, the population of AaLS/LOX/CAT-treated CT26 cells did not shift to either annexin V or the 7-AAD positive population, similar to that of AaLS-ST-treated CT26 cells (Fig. 4e). Additionally, live cell imaging capture results showed that while AaLS-ST- or AaLS/LOX/CAT-treated CT26 cells maintained their cell morphology and membrane integrity (Figs. 4f, h), AaLS/LOX-treated CT26 cells were extensively swelled, and their membranes were rapidly ruptured (Fig. 4g). These results suggest that although AaLS/LOX treatment produces a large amount of H2O2 by rapidly consuming the surrounding lactate, inducing drastic necrotic cell death, AaLS/LOX/CAT converts toxic H2O2 to nontoxic oxygen and water, rescuing cells from ROS-induced cell death even with complete lactate consumption.
AaLS/LOX and AaLS/LOX/CAT consumed lactate similarly under hypoxic conditions but exhibited different cell cytotoxicity
Since the TME was lactate-rich but oxygen-deficient, the lactate consumption and H2O2 conversion patterns by LOX and CAT would be different from those under normoxic conditions. Therefore, to mimic the hypoxic TME condition, we cultured CT26 cells under 1% O2 condition at different time intervals and monitored their lactate production capabilities (Fig. 5a, green bars). CT26 cells produced more lactate when initially cultured under the normoxic condition and transferred to hypoxic condition (Fig. 5a; purple and yellow bars). Subsequently, we hypothesized that initial tumor formation occurs in vivo under normoxic condition and then the environment could transform to a hypoxic condition as the tumor grows to form a unique TME. Therefore, we initially cultured CT26 cells under a normoxic condition for 24 h and then transferred them to a hypoxic chamber and subjected them to incubation for a further 24 h. After, we treated these cells concentration-dependently with each sample and incubated them for an additional 24 h under the same hypoxic condition (Fig. 5b). Microscopic images of either AaLS/LOX- or AaLS/LOX/CAT-treated CT26 cells showed different cell morphologies from those of AaLS-ST-treated CT26 cells at different concentrations (Fig. 5c). Although AaLS/LOX/CAT and AaLS/LOX consumed lactate from CT26 cell cultures under hypoxic condition (Fig. 5e, f, blue squares), larger LOX amounts were required to consume lactate completely than those under normoxic condition (Fig. 4b, c), suggesting that the limited oxygen supply under hypoxic condition might slow down the LOX activity and more LOX was required to effectively consume lactate. Interestingly, in contrast to normoxic condition, AaLS/LOX/CAT-treated CT26 cells under hypoxic condition showed reduced cell viability throughout whole concentration ranges and the lowest cell viability was observed around 100 nM of AaLS/LOX/CAT (Fig. 5f, red squares). Moreover, fluorescence microscopy using CM-H2DCFDA of AaLS-ST, AaLS/LOX, and AaLS/LOX/CAT-treated CT26 cells under the hypoxic condition revealed that while AaLS/LOX/CAT induced weak ROS generation after 12 h treatment, AaLS/LOX markedly increased ROS levels after 4 h treatment, subsequently killing most of the cells (Additional file 1: Fig. S5). The reduced cell viability of AaLS/LOX/CAT treated-CT26 cells at medium concentration (10–100 nM) under hypoxic condition may result from the different enzyme kinetic behavior of LOX and CAT. Both Vmax and Km values of CAT were higher than those of the LOX (Fig. 3a, b), suggesting that H2O2 produced by LOX was incompletely removed by CAT, and this residual H2O2 sufficiently accumulated enough to damage cells under hypoxic condition, leading to cell death. Collectively, these results imply that AaLS/LOX/CAT can effectively consume lactate to produce small amounts of cytotoxic H2O2, inducing cell death under the hypoxic TME while reducing its side effects, such as burst production of toxic H2O2 production, possibly driven by AaLS/LOX to normal cells under normoxic condition.
Local administration of AaLS/LOX/CAT suppressed tumor growth and altered the TME
Finally, we explored the translational implications of our findings by examining the efficacy of AaLS/LOX and AaLS/LOX/CAT in CT26 tumor-bearing allografted mice. Since the significant H2O2 amount generated by AaLS/LOX caused severe necrotic cell death in vitro, we first tested whether AaLS/LOX and AaLS/LOX/CAT can be used in vivo without severe side effects. Various AaLS/LOX or AaLS/LOX/CAT concentrations were locally injected to the tumor site every two or three days for six injections to the CT26 tumor-bearing mice (Fig. 6a). As controls, PBS and AaLS were also administrated based on the same schedule. After the CT26 tumor-bearing mice were treated with either 100 μL of 5 μM or 10 μM AaLS/LOX (molar concentration of LOX subunits) samples, mice begun to die a day after administration (Additional file 1: Fig. S6a; purple and green lines). These results suggest that the significant H2O2 amount generated by AaLS/LOX caused severe necrotic cell death around the tumor site to kill mice. In contrast, none of the mice died or lost body weight after administration of the same amount of AaLS/LOX/CAT (Additional file 1: Fig. S6a, b; blue and red lines), showing moderate tumor suppressive efficacy (Additional file 1: Fig. S6c). Therefore, we decided not to use AaLS/LOX for in vivo efficacy evaluation any further and increase the AaLS/LOX/CAT doses to enhance tumor suppression efficacy.
We further tested 10 and 20 μM AaLS/LOX/CAT and obtained effective tumor suppression efficacy (Additional file 1: Fig. S6d, e). These AaLS/LOX/CAT concentrations maintained the capability of tumor suppression but did not show serious adverse effects (Additional file 1: Fig. S6d, e). Therefore, we decided to use 10 μM AaLS/LOX/CAT for in vivo efficacy evaluation. CT26 allografted mice were locally injected to the tumor site every two or three days with 100 μL of 10 μM AaLS/LOX/CAT for six injections (Fig. 6a). While AaLS did not affect tumor growth, notably, AaLS/LOX/CAT treatments significantly suppressed tumor growth (Fig. 6b). Additionally, no weight loss and mouse death were observed (Fig. 6c), and there was no noticeable tissue damage in major organs (Fig. 6d). These results suggest that AaLS/LOX/CAT successfully inhibits tumor growth by depleting lactate in the TME without severe side effects.
Numerous publications over the past decades have shown that lactate in the TME modulates tumor cells as well as the immune microenvironment to promote tumor growth [19, 47,48,49,50,51,52]. Indeed, tumor-derived lactate in the TME can impair the functions of anti-tumor immune cells, including CD8+ T and NK cells, supporting the generation of immunosuppressive tumor-associated macrophages (TAMs) [19, 48, 49]. To investigate whether lactate consumption in the TME could impact the alteration of immune cell populations, we collected tumor masses and profiled immune cells from CT26 tumor-bearing mice injected with PBS, AaLS, or AaLS/LOX/CAT. Freshly resected tumors were collected and analyzed by a flow cytometry to define total macrophages, TAMs (M2-like), neutrophils, T cells, and NK cells (Fig. 6e and Additional file 1: Fig. S7a). We observed that while AaLS alone increased CD64+ (total) macrophages, AaLS/LOX/CAT further increased infiltrating CD64+ macrophages (Fig. 6e, top row). These results suggest that even though protein nanoparticles alone did not affect tumor growth inhibition, they affected the recruitment of macrophage. Additionally, lactate removal further increased macrophage intratumoral intrusion. Of note, we observed that the depletion of lactate by AaLS/LOX/CAT significantly reduced CD206+, F4/80+ tumor-supportive M2-like TAMs (Fig. 6e, second row), indicating that lactate-induced TAM production was strongly suppressed [51]. In contrast, AaLS moderately increased TAMs associated with poor tumor suppressive efficacy and both AaLS and AaLS/LOX/CAT did not affect M1 activation. Consistently, the depletion of lactate by AaLS/LOX/CAT in mouse bone marrow-derived macrophages significantly reduced the expression of Arg1 and Vegf, which are well-known markers of TAMs (Additional file 1: Fig. S8). Therefore, we proposed that a decrease in TAMs and increased total macrophages, including M0, contributed to the tumor suppressive efficacy. Next, we investigated the proportion of neutrophils (Ly6G+) with potent cytotoxic and anti-tumor activities [47]. Interestingly, only AaLS/LOX/CAT highly increased neutrophils (Fig. 6e, bottom row) and these findings indicate that AaLS/LOX/CAT specifically affected the neutrophils’ activity. Furthermore, the proportion of T cells was changed. Treatments with AaLS or AaLS/LOX/CAT increased CD4+ T cells and decreased CD8+ T cells (Additional file 1: Fig. S7b). Specifically, AaLS/LOX/CAT showed the highest increase in the CD4+/CD8+ ratio associated with increased immune function. However, the proportion of NK cells was insignificantly affected (Additional file 1: Fig. S7b). Overall, our findings suggest that the specific clearance of lactate by AaLS/LOX/CAT altered the immune environment in favor of anticancer functions, such as neutrophil increase and TAMs decrease, showing clear translational implications for tumor therapy.