The authors regret, 3.4. Therapeutic A-PER-p(TMZ)29 combined PER with TMZ to promote antitumor effects in U87 and GSC22-bearing mice To characterize the combined therapeutic efficacy of in the context of Akt and TMZ inhibition in vivo, we constructed an in situ glioma model and subcutaneous graft tumor model using U87 cells in male BALB/c nude mice (Fig. 4A). As shown in Fig. 4B and C, A-PER-p(TMZ)29 markedly suppressed intracranial tumor growth in BALB/c nude mice. The median survival times for U87-bearing mice were 58.5 days for A-PER-p(TMZ)29, 38.0 days for A-L-p(TMZ)29, 30.0 days for A-PER-PLGA, 30.5 days for TMZ, 26.5 days for PER and 19.5 days for PBS (Fig. 4D). Mice treated with A-PER-p(TMZ)29 exhibited significantly extended survival times compared to all other treatments. Immunohistochemical staining of p-Akt in brain tumor sections was performed to investigate PER inhibition of Akt activity. The number of p-Akt-positive cells was significantly reduced in PBS, PER, A-PER-PLGA, and A-PER-p(TMZ)29 groups, suggesting that PER indeed inhibits Akt signal activation in GBM (Fig. 4E, F). The number of Ki-67-positive cells was lowest and the number of apoptotic cells was highest in gliomas treated in situ with A-PER-p(TMZ)29 (Fig. 4G, H), further demonstrating that A-PER-p(TMZ)29 inhibits GBM growth (Fig. 4H). In a subcutaneous U87 grafted mouse model, A-PER-p(TMZ)29 yielded the strongest inhibitory effect on glioma growth (Fig. S11A, B), and had the lowest weight tumors among all groups (Fig. S11C). To explore the effect of A-PER-p(TMZ)29 on GSCs, we evaluated the tumor-suppressive effect in GSC22-derived tumor growth (Fig. 5A, B). Bioluminescence analysis revealed that tumor growth was significantly inhibited by A-PER-p(TMZ)29 compared with other treatment groups at day 24 after orthotopic seeding of GSC22 (Fig. 5C, D). Survival curves showed that A-PER-p(TMZ)29 significantly prolonged survival compared with PBS, A-PER-PLGA, and A-L-p(TMZ)29 (Fig. 5E). Thus, combined treatment with PER and poly(TMZ)29 significantly prolonged survival, demonstrating the potential therapeutic efficacy of A-PER-p(TMZ)29 in GBM. These results proved that A-PER-p(TMZ)29 combination TMZ with PER had effectively inhibited GBM and GSCs growth. The hydrophobic core of A-PER-p(TMZ)29 can be loaded with different therapeutic agents. The GBM microenvironment plays a huge role in the occurrence, development, and treatment resistance in GBM. Through the analysis of immune microenvironment, it is found that the degree of TAMs infiltration has the most significant impact on the survival period of glioma patients with chemotherapy in CGGA RNA-seq dataset (Fig. 6A, B). TAMs are the predominant immunosuppressive cells in the GBM microenvironment, and reducing their density attenuates glioma invasion and growth [10,11]. Clo selectively depletes TAMs [37–39]. Therefore, we used A-PER-p(TMZ)29 to load Clo (A-PER-p(TMZ)29/Clo) for simultaneous targeting of GBM, TAMs, and GSCs to improve therapeutic efficacy. The diameter of A-PER-p(TMZ)29/Clo was 156.98 ± 8.49 nm (Fig. S12) and TEM images of the A-PER-p(TMZ)29/Clo showed spherical particles (Fig. S13). Given our results above, we were motivated to investigate the anti-glioma effect of A-PER-p(TMZ)29/Clo on orthotopic GL261 in C57BL/6 mice (Fig. 6C). GL261 transduced with a control lentiviral firefly luciferase vector was intracranially transplanted into C57BL/6 mice. Bioluminescence imaging was used to measure in vivo growth. A-PER-p(TMZ)29/Clo group, as opposed to all other treatments, clearly retarded intracranial GBM growth, significantly reduced the luciferase signal (Fig. 6D). Brain tumor tissues were harvested on day 21, and tumor-bearing brain slices stained with hematoxylin and eosin (H&E) were imaged. Treatment with A-PER-p(TMZ)29/Clo led to maximal tumor growth inhibition compared to all other treatments (Fig. 6E, F). Moreover, A-PER-p(TMZ)29/Clo improved survival time (median 52.5 days) and decreased GL261 tumorigenicity compared to PBS (median 26 days), Clo (median 25 days), TMZ + PER (median 31 days), TMZ + PER + Clo (median 32.5 days), and A-PER-p(TMZ)29 (median 40 days) (Fig. 6G). Immunohistochemical staining of Ki67 was used to evaluate tumor cell proliferations. A-PER-p(TMZ)29/Clo yielded the fewest Ki67-positive cells compared to all other treatments (Fig. 6H). We were motivated to investigate the immune microenvironment effect of A-PER-p(TMZ)29/Clo on orthotopic GL261 in C57BL/6 mice. The effect of A-PER-p(TMZ)29/Clo on TAM depletion and regulation of the glioma immune microenvironment was assessed by flow cytometry (FACS) and immunohistochemistry of tumor transplanted with GL261 glioma cells into C57BL/6 mice. A-PER-p(TMZ)29/Clo significantly reduced the population of Cd45+ Cd11b+ macrophages in comparison to PBS, Clo, TMZ + PER, TMZ + PER + Clo, and A-PER-p(TMZ)29 (Fig. 7A, C), and increased Cd3+ Cd8+ cells (Fig. 7B, D). The ratio of Cd206-positive (M2 macrophages) cells versus Cd45+ Cd11b+ cells decreased after treatment with A-PER-p(TMZ)29/Clo compared to TMZ + PER, TMZ + PER + Clo, and A-PER-p(TMZ)29 (Fig. 7E). The Pd1-positive cell ratio in Cd45+ Cd3+ Cd8+ cells decreased (Fig. 7F), and the ratio of Ifn-γ-positive cells was increased in Cd45+ Cd3+ Cd8+ cells after treatment with A-PER-p(TMZ)29/Clo (Fig. 7G). In addition, Foxp3-positive cells (Treg cells) decreased significantly after treatment with A-PER-p(TMZ)29/Clo (Fig. 7H). The similar results were obtained on immunohistochemistry. Ionized calcium binding adaptor molecule 1 (Iba-1) is a microglia/macrophage-specific calcium-binding protein. As shown in Fig. 7I A and Fig. S14A, there was a significant decrease in Iba-1 positive cells following A-PER-p(TMZ)29/Clo treatment, suggesting that TAM number in glioma was significantly reduced. The Cd86-positive cells (M1 macrophages) increased, and the ratio of Cd163 cells (M2 macrophages) decreased significantly after treatment with A-PER-p(TMZ)29/Clo compared to PBS, Clo, TMZ + PER, TMZ + PER + Clo, and A-PER-p(TMZ)29 (Fig. 7I b, c and Fig. S14B, C). We thus conclude that TMZ and PER increase the proportion of M2 macrophages, and A-PER-p(TMZ)29/Clo significantly reduces TAM number and the proportion of M2 macrophages, achieving a synergistic effect with TMZ and PER. Cd3+ cells and Cd8+ cells positive cells were increased and Treg cells (Foxp3-positive cells) were decreased significantly after treatment with A-PER-p(TMZ)29/Clo (Fig. 7I d, e, f and Fig. S14D, E, F). Therefore, we conclude that A-PER-p(TMZ)29/Clo promotes macrophage clearance, increases T cell (Cd3+ cells) infiltration in tumor tissue, especially cytotoxic T cell (Cd8+ cells) infiltration, and decreases Treg cells (Foxp3+ cells) to modify the immune-suppressive TME of GBM to a proinflammatory/antitumorigenic immune microenvironment. These results were consistent with previous reports that indicated Clo triggers depletion of macrophages, fosters Cd8+ T cell infiltration, and suppresses tumor growth [40–42]. Compared with A-PER-p(TMZ)29, A-PER-p(TMZ)29/Clo provided the strongest tumor inhibition. Finally, the systemic biosafety of A-PER-p(TMZ)29/Clo was assessed in vivo. As shown in Fig. S15, there was no noticeable tissue damage or obvious changes in major organ morphology in animals treated with A-PER-p(TMZ)29/Clo, A-PER-p(TMZ)29, and Clo versus PBS. The liver and renal functionality indices for alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CREA) were within the normal range for all groups, suggesting that A-PER-p(TMZ)29/Clo did not cause damage to the liver or kidneys (Fig. S16). These results demonstrate that A-PER-p(TMZ)29/Clo produced negligible systemic toxicity and thus has potential value for clinical use.[Figure presented] Figure 7. In vivo immune responses after A-PER-p(TMZ)29/Clo treatment. (A) FACS analysis of Cd45+ and Cd11b+ cells in glioma tissue. (B) FACS analysis of Cd3+ and Cd8+ cells in glioma tissue. (C) Quantitative analysis of TAM cells in glioma by FACS. ***P + cells in glioma by FACS. **P + cells in glioma by FACS. ***P + cells in glioma by FACS. **P + cells in glioma by FACS. *P . The highlighted parts in blue are the sections that required amendments, and the revisions have already been made. The figures and their captions have been updated. 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