Boosting anti-PD-1 therapy with metformin-loaded macrophage-derived microparticles

The main challenges for programmed cell death 1(PD-1)/PD-1 ligand (PD-L1) checkpoint blockade lie in a lack of sufficient T cell infiltration, tumor immunosuppressive microenvironment, and the inadequate tumor accumulation and penetration of anti-PD-1/PD-L1 antibody. Resetting tumor-associated macrophages (TAMs) is a promising strategy to enhance T-cell antitumor immunity and ameliorate tumor immunosuppression. Here, mannose-modified macrophage-derived microparticles (Man-MPs) loading metformin (Met@Man-MPs) are developed to efficiently target to M2-like TAMs to repolarize into M1-like phenotype. Met@Man-MPs-reset TAMs remodel the tumor immune microenvironment by increasing the recruitment of CD8+ T cells into tumor tissues and decreasing immunosuppressive infiltration of myeloid-derived suppressor cells and regulatory T cells. More importantly, the collagen-degrading capacity of Man-MPs contributes to the infiltration of CD8+ T cells into tumor interiors and enhances tumor accumulation and penetration of anti-PD-1 antibody. These unique features of Met@Man-MPs contribute to boost anti-PD-1 antibody therapy, improving anticancer efficacy and long-term memory immunity after combination treatment. Our results support Met@Man-MPs as a potential drug to improve tumor resistance to anti-PD-1 therapy.

Programmed cell death 1(PD-1)/PD-1 ligand (PD-L1) checkpoint blockade is a promising clinical anticancer treatment modality by blocking the binding of PD-L1 on tumor cells to PD-1 on activated T cells to reactivate T-cell-mediated antitumor immunity1,2,3. However, the durable response rate to anti-PD-1/PD-L1 therapy remains relatively low in most cases4,5. The major factors attributed to the resistance to anti-PD-1/PD-L1 therapy include the lack of infiltrating T lymphocytes in tumor tissues characterizing the so-called “cold tumor”6, and the presence of tumor immunosuppressive microenvironment, such as regulatory T (Treg) cells and myeloid-derived suppressor cells (MDSCs) to inhibit the anti-PD-1/PD-L1 antibody-regenerated anti-tumor cytotoxic T lymphocytes and Th1 cell response7,8. In addition, the aberrant vascular architecture, elevated interstitial fluid pressure, and compact extracellular matrix (ECM) in tumor tissues hamper the tumor accumulation of anti-PD-1/PD-L1 antibody and subsequent deep penetration into tumor parenchyma9,10, which limits the therapeutic effects of anti-PD-1/PD-L1 antibody. Therefore, efficient T lymphocyte tumor infiltration, improved tumor immunosuppressive microenvironment, and enhanced tumor accumulation and penetration of anti-PD-1/PD-L1 antibody are required to increase the potential responses to anti-PD-1/PD-L1 antibody.

Tumor-associated macrophages (TAMs), one of the most abundant tumor-infiltrating leukocytes in various tumors, are commonly educated by tumor microenvironment to different phenotypes11,12. M1-like TAMs, recognized as classically activated macrophages, exhibit anticancer activity by releasing nitrogen oxide (NO) and stimulating naïve T cells to make a Th1/cytotoxic response13. In contrast, M2-like TAMs, alternatively activated macrophages predominantly present in tumors, promote tumor growth, angiogenesis, metastasis and tumor immune escape14. M2-like TAMs can directly or indirectly inhibit T cell functions by expressing T cell immune checkpoint ligands (such as PD-L1 and PD-L2)15,16, secreting inhibitory cytokines (such as IL-10 and transforming growth factor-β)17, and recruiting immunosuppressive cells (such as Treg cells)18. Therefore, repolarization of M2-like TAMs to M1 phenotype is a promising strategy to improve the T cell-mediated antitumor immunity and ameliorate the immunosuppressive tumor microenvironment, which might be beneficial for the enhanced anticancer activity of anti-PD-1/PD-L1 antibody.

Metformin (Met), a popular drug used to treat diabetes due to its good hypoglycemic effect, few side effects, and low cost19, exhibits anticancer activity20. A large number of studies have shown that Met can regulate tumor metabolism21, arrest tumor cell cycle22, inhibit angiogenesis23, and kill cancer stem cells by activating adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK)24. Recently, Met has been found to efficiently repolarize M2-like TAMs to M1 phenotype to inhibit tumor growth and metastasis through the AMPK-NF-κB signaling pathway25,26. However, how to achieve the targeted delivery of Met to M2-like TAMs to further improve the therapeutic effects remains a big challenge.

Cellular microparticles (MPs) are extracellular vesicles with a diameter of 100–1000 nm that are shed by cells in response to various endogenous or exogenous stimuli27,28,29. MPs hold great potential as drug-delivery systems due to the unique property to deliver messenger molecules, enzymes, and genetic material (DNAs, RNAs) between cells30,31,32, and the superior circulation stability, high biocompatibility, low immunogenicity and toxicity33,34,35. Considering that TAMs are derived from monocytes or tissue-resident macrophages recruited by various signals in the tumor microenvironment (such as cytokines, chemokines, ECM components, and hypoxia, etc.)14,36, macrophage-derived MPs have natural tumor-targeting capacity37. M2-like TAMs highly express mannose receptor CD206/MRC138,39. In this study, mannose (Man)-modified macrophage-derived MPs are used as carriers for targeted delivery of Met (denoted as Met@Man-MPs) to M2-like TAMs. Met@Man-MPs efficiently reset TAMs toward M1 phenotype to inhibit tumor growth. Importantly, Met@Man-MPs significantly improve tumor immunosuppressive microenvironment, and enhance CD8+ T cell infiltration into tumor interiors by the reset macrophages-induced recruitment of CD8+ T cells and Man-MPs-induced tumor ECM degradation since macrophages express matrix metalloproteinases (MMPs)40,41. Meanwhile, Met@Man-MPs efficiently enhance tumor accumulation and penetration of anti-PD-1 antibody. These unique features of Met@Man-MPs contribute to the anticancer treatment of anti-PD-1 antibody, resulting in strong anticancer efficacy and the generation of long-term memory immunity after co-administration (Fig. 1a).

Preparation and characterization of Met@Man-MPs

M2-like macrophages highly expressed Mrc1 gene (Supplementary Fig. 1), which encoded mannose receptor CD206/MRC1. To acquire Man-modified MPs to achieve M2-like macrophage targeting, murine RAW264.7 macrophages were first incubated with DSPE-PEG-Man to obtain Man-engineered cells by virtue of the natural membrane phospholipid exchange of cells42. RAW264.7 cells and Man-engineered RAW264.7 cells were then treated with Met (2 mg mL−1), followed by ultraviolet irradiation for Met-packaging MPs and Man-MPs (denoted as Met@MPs and Met@Man-MPs, respectively) induction34,35. Lectin recognition assay confirmed the successful modification of Man in Man-MPs and Met@Man-MPs (Supplementary Fig. 2). Dynamic light scattering (DLS) analysis showed that Met@MPs and Met@Man-MPs have a similar size of 440 nm (Fig. 1b) and zeta potential of −11.6 mV (Fig. 1c), respectively. Transmission electron microscopy (TEM) revealed that both Met@MPs and Met@Man-MPs were monodisperse and irregularly spherical (Fig. 1d). High-performance liquid chromatography (HPLC) analysis showed that the drug loading capacity for both MPs and Man-MPs was about 0.69 μg of Met per μg protein (MPs and Man-MPs were quantified according to the protein content). Furthermore, Met@MPs and Met@Man-MPs showed a pH-responsive sustained drug release (Fig. 1e). Met@MPs and Met@Man-MPs did not exhibit significant changes of the size or zeta potentials in phosphate-buffered saline (PBS) with or without 10% fetal bovine serum (FBS) after 7 days (Supplementary Fig. 3), suggesting that Met@MPs and Met@Man-MPs are relatively stable.

Man-MPs efficiently target to M2-like TAMs

To investigate the M2-like TAM-targeting capacity of Man-MPs, we first evaluated the uptake efficiency of PKH67-labeled MPs and Man-MPs by RAW264.7 cells (M0 macrophages), LPS- and IFN-γ-conditioned RAW264.7 cells (M1-like macrophages), IL-4-conditioned RAW264.7 cells (M2-like macrophages), murine dendritic DC2.4 cells which are another main phagocytes expressing mannose receptor CD20643, and murine H22 hepatocarcinoma cells by flow cytometry (Fig. 2a). The strongest intracellular PKH67 fluorescence was detected in M2-like macrophages treated with Man-MPs among all the groups, suggesting a selective M2-like macrophage targeting of Man-MPs. Consistently, more Met@Man-MPs were internalized into M2-like macrophages compared with Met@MPs and free Met (Supplementary Fig. 4). The M2-like macrophage targeting capability of Man-MPs was confirmed in IL-4-conditioned murine bone marrow-derived macrophages (M2-like BMDMs, Fig. 2b). Pretreatment with free Man significantly decreased the intracellular accumulation of Man-MPs in M2-like macrophages (Fig. 2c), revealing that the M2-like macrophage targeting ability of Man-MPs was mediated by mannose receptor CD206 expressed in M2-like macrophages.

The M2-like TAM-targeting capacity of Man-MPs was further confirmed in vivo. H22 tumor-bearing mice were intravenously injected with Cy5-labeled MPs or Man-MPs. Biodistribution analysis showed that at 24 h after administration, MPs and Man-MPs were mainly accumulated in tumors, revealing the tumor-targeting capacity of macrophages-derived MPs (Fig. 2d, e). However, more Man-MPs were accumulated in tumor tissues than MPs (Fig. 2d, e). Meanwhile, the highest Met accumulation in tumor tissues was also detected in Met@Man-MPs-treated group (Supplementary Fig. 5). Furthermore, green fluorescent protein (GFP)-expressing H22 tumor-bearing mice were intravenously injected with PKH26-labeled Man-MPs or MPs. At 24 h after administration, the tumor tissues were digested into single-cell suspensions and the intracellular PKH26 fluorescence in different types of cells was evaluated by flow cytometry (Fig. 2fand Supplementary Fig. 6). M2-like TAMs captured a much greater amount of Man-MPs than MPs. In addition, significantly more Man-MPs were enriched in M2-like TAMs compared with those in other cells, including GFP-positive tumor cells, M1-like TAMs, dendritic cells (DCs), CD4+ T cells, CD8+ T cells, MDSCs, and Tregs, suggesting the superior M2-like TAM-targeting capability of Man-MPs. Confocal microscopic images also confirmed that more Man-MPs were colocalized with M2-like TAMs compared with other immune cells in tumor tissues (Supplementary Fig. 7). Due to the tumor-targeting capacity of macrophages-derived MPs and high CD206 expression in TAMs (Supplementary Fig. 8), PKH26 fluorescence in TAMs was significantly higher than that in Kupffer cells, splenic macrophages, splenic DCs, and pulmonary macrophages at 24 h after intravenous injection of PKH26-labeled Man-MPs (Fig. 2g), excluding the targeting accumulation of Man-MPs in other tissue-resident macrophages and phagocytes. Meanwhile, Man-MPs were found to be mainly colocalized with CD206+ cells in tumor tissues (Supplementary Fig. 9) although CD209, a C-type lectin, can also bind mannose residues44.

Met@Man-MPs efficiently repolarize M2-like macrophages to M1 phenotype

The repolarization of M2-like macrophages to M1 phenotype by Met@Man-MPs derived from RAW264.7 cells was evaluated in IL-4-conditioned RAW264.7 cells (Fig. 3a–e and Supplementary Fig. 10), which expressed typical M2-like macrophage markers (Supplementary Fig. 11a). Compared with PBS, MPs, Man-MPs, and free Met, treatment with Met@MPs significantly enhanced the mRNA expression of TNFα and iNOS, and protein expression of CD80 and CD86 (M1-related markers), while decreased the mRNA expression of Arg1, Mrc1 and Mgl1, and protein expression of CD206 (M2-related markers). However, Met@Man-MPs exhibited the strongest M2 macrophage repolarization activity, even better than the high dosage of free Met (Fig. 3a–e and Supplementary Fig. 10), which was confirmed in IL-4-conditioned BMDMs (Supplementary Fig. 12). In addition, Met@Man-MPs derived from BMDMs also showed the excellent M2 macrophage repolarization activity in IL-4-conditioned BMDMs (Supplementary Fig. 13). A similar phenomenon was further confirmed using Met@Man-MPs derived from human monocytic THP-1-originated macrophages and human peripheral blood monocyte-derived macrophages (MDMs) in IL-4-conditioned THP-1-originated macrophages (Supplementary Fig. 14) and MDMs (Supplementary Fig. 15), respectively. These results suggest that the M2-like macrophage repolarization activity of Met@Man-MPs is universal. Compound C (CC), an AMPK inhibitor significantly abrogated Met@Man-MPs-induced increase in M1-related markers (TNF-α and iNOS) and decrease in M2-related markers (Mrc1 and Arg1) (Supplementary Fig. 16), suggesting that similar to Met, Met@Man-MPs repolarized M2-like macrophages into M1 phenotype through an AMPK signaling pathway. Caveolin-, clathrin-dependent endocytosis and phagocytosis were involved in the cellular uptake of Man-MPs in M2-like macrophages (Supplementary Fig. 17). Intracellular tracing analysis of Rhodamine B (a model drug)-loaded Man-MPs (RhB@Man-MPs) showed that drug and Man-MPs entered M2-like macrophages together and then colocalized with lysosomes, followed by drug release over time (Supplementary Fig. 18).

The classic pro-inflammatory M1-like macrophages possess antitumor activity by secreting proinflammatory cytokines and reactive nitrogen and oxygen species13. To further confirm the M2-like macrophage repolarization activity of Met@Man-MPs, we determined the anticancer effects of the supernatants of IL-4-conditioned RAW264.7 cells pretreated with MPs, Man-MPs, free Met, Met@MPs or Met@Man-MPs, or high dosage of free Met. As expected, the supernatants of M2-like macrophages significantly increased the cell viability of murine hepatocellular carcinoma H22 cells, human hepatocellular carcinoma HepG2 cells, and murine 4T1 breast cancer cells (Supplementary Fig. 11b). However, the supernatants of Met@Man-MPs-educated M2-like macrophages exhibited strong inhibition in the cell viability of these cancer cells compared with those of free Met- or Met@MPs-treated group, even more effective than high dosage of free Met group (Fig. 3f-h). The strong cytotoxicity of the supernatants of Met@Man-MPs-educated M2-like macrophages might be due to the secreted TNF-α, as evidenced by the fact that TNF-α content in the supernatants of Met@Man-MPs-treated M2-like macrophages was significantly higher than other groups (Supplementary Fig. 19a), and treatment with etanercept (Etan), an inhibitor of TNF-α significantly decreased the cytotoxicity of the supernatants of Met@Man-MPs-treated M2 macrophages against H22 (Supplementary Fig. 19b) and 4T1 cells (Supplementary Fig. 19c). Meanwhile, Met@Man-MPs-treated M2-like macrophages exhibited stronger phagocytosis of H22 (Supplementary Fig. 20a) and 4T1 cells (Supplementary Fig. 20b). Furthermore, we subcutaneously co-implanted these educated M2-like macrophages and H22 cells into the flanks of BALB/c mice and then assessed the potential of tumor formation. Consistently, the lowest tumor formation ratio (Fig. 3i), the slowest tumor growth (Fig. 3j) and the smallest tumor weight (Fig. 3k) were observed in the group of co-transplantation of Met@Man-MPs-treated M2-like macrophages and H22 cells. Collectively, these data indicate that Met@Man-MPs efficiently repolarize M2-like macrophages to M1 phenotype.

Met@Man-MPs significantly inhibit tumor growth and ameliorate tumor immune microenvironment

To determine the biological function of Met@Man-MPs, the in vivo antitumor activity of Met@Man-MPs was determined in BALB/c mice bearing subcutaneous H22 tumors. Free Met, Met@MPs or Met@Man-MPs at Met dosage of 10 mg kg−1, or high dosage of Met at 100 mg kg−1 were intravenously injected into H22 tumor-bearing mice once every other day for six times. Compared with free Met (10 mg kg−1), Met@MPs significantly reduced the tumor growth, with 41.6 and 39.9% reduction in tumor volume and weight (Fig. 4a, b). However, Met@Man-MPs exhibited the strongest anticancer activity, achieving 70.2 and 63.2% reduction in tumor volume and weight, even better than a high dosage of Met (Fig. 4a, b). Kaplan–Meier survival analysis showed that 86% of mice were still alive in the Met@Man-MPs-treated group after 63 d, much higher than Met@MPs- and high dosage of Met-treated groups (Fig. 4c). Hematoxylin-eosin (H&E) staining (Supplementary Fig. 21a) and TUNEL analysis (Supplementary Fig. 21b, c) of tumor tissues revealed the minimum number of tumor cells and the maximum number of apoptotic cells in the Met@Man-MPs-treated group, respectively, further confirming the good anticancer activity of Met@Man-MPs. Similar anticancer activity of Met@Man-MPs was found in mice bearing orthotopic 4T1 breast tumors. Met@Man-MPs exhibited the strongest capability to reduce tumor growth (Supplementary Fig. 22a) and prolong the survival time (Supplementary Fig. 22b) compared with free Met, Met@MPs, and high dosage of Met. Moreover, significantly fewer lung metastatic nodules were detected in the Met@Man-MPs-treated group by counting nodule numbers (Supplementary Fig. 22c) and H&E staining analysis of lungs (Supplementary Fig. 22d). In addition, body weight change (Supplementary Fig. 23), H&E staining of major organs (Supplementary Fig. 24), and serological analysis (Supplementary Fig. 25) showed that Met@Man-MPs did not cause obvious toxicity, although high dosage of Met induced nephrotoxicity (Supplementary Figs. 24 and 25).

The tumor immune microenvironment was then investigated in H22 tumor-bearing mice after intravenous injection of free Met, Met@MPs or Met@Man-MPs at Met dosage of 10 mg kg−1, or high dosage of Met at 100 mg kg−1 once every other day for six times. Consistently, Met@Man-MPs exhibited the strongest ability to increase the number of M1-like TAMs (Fig. 4d) and decrease the number of M2-like TAMs (Fig. 4e) in tumor tissues. Moreover, Met@Man-MPs significantly enhanced the numbers of CD8+ T cells (Fig. 4f), activated CD8+CD69+ T cells (Fig. 4g), CD4+ T cells (Fig. 4h), and activated CD4+CD69+ T cells (Fig. 4i), while markedly decreased the numbers of MDSCs (Fig. 4j) and Tregs (Fig. 4k) in tumor tissues compared with free Met, Met@MPs and even high dosage of Met. Meanwhile, Met@Man-MPs treatment markedly increased the levels of pro-inflammation cytokines IFN-γ (Fig. 4l), TNF-α (Fig. 4m), and IL12p70 (Fig. 4n), while decreased the levels of anti-inflammation cytokine TGF-β (Fig. 4o) in tumor tissues. The improved tumor immune microenvironment induced by Met@Man-MPs was also confirmed in 4T1 tumor-bearing mice (Supplementary Fig. 26a–h), suggesting that Met@Man-MPs efficiently enhance the antitumor immunity and ameliorate the tumor immunosuppression.

To determine whether macrophages were involved in the Met@Man-MPs-mediated antitumor effect, we used clodronate liposomes to deplete macrophages in H22 tumor-bearing mice. As expected, macrophages were effectively depleted by clodronate liposomes (Supplementary Fig. 27a), whereas other immune cells, such as CD8+ T cells (Supplementary Fig. 27b), CD8+CD69+ T cells (Supplementary Fig. 27c), CD4+ T cells (Supplementary Fig. 27d), CD4+CD69+ T cells (Supplementary Fig. 27e), MDSCs (Supplementary Fig. 27f) and Tregs (Supplementary Fig. 27g) were not affected. The macrophage depletion disrupted the antitumor effect of Met@Man-MPs (Supplementary Fig. 27h, i). Correspondingly, the improved tumor immune microenvironment induced by Met@Man-MPs was abrogated after macrophage depletion (Supplementary Fig. 27b–g), suggesting that the antitumor effect and improved tumor immune microenvironment of Met@Man-MPs is macrophage-dependent. It is well known that M1-like macrophages have the ability to recruit T cells and promote Th1 responses45. The improved efficacy following macrophage depletion is often dependent upon enhanced recruitment or function of cytotoxic CD8+ T cells46,47. Depletion of CD8+ T cells using anti-CD8 antibody markedly impaired the anticancer ability of Met@Man-MPs (Supplementary Fig. 28a, b). Furthermore, Met@Man-MPs did not exhibit significant tumor inhibition in H22 tumor-bearing nude mice (Supplementary Fig. 28c, d), suggesting that CD8+ T cells are involved in the anticancer activity of Met@Man-MPs.

Man-MPs efficiently degrade tumor collagen

Tumor ECM, consisting of a collagen network and proteoglycans, provides a natural barrier against monocyte infiltration and therapeutic drug accumulation and penetration, resulting in the immune evasion and resistance of antitumor therapy48. Similar to RAW264.7 macrophages, MPs and Man-MPs derived from RAW264.7 macrophages overexpressed MMP9 and MMP14 proteins (Supplementary Fig. 29a). Meanwhile, MPs and Man-MPs exhibited high MMP activity as detected by the MMP activity assay kit, and loading Met did not affect their MMP activity (Supplementary Fig. 29b). In addition, pretreatment with batimastat (Bati, a broad-spectrum MMP inhibitor) significantly decreased the MMP activity of Met@Man-MPs (Supplementary Fig. 29b), further confirming the MMP activity of Man-MPs. The high MMP activity was also detected in Man-MPs derived from THP-1-originated macrophages (Supplementary Fig. 29c). To evaluate whether MPs and Man-MPs can degrade tumor ECM by MMP activity, PKH26-labeled MPs or Man-MPs pretreated with or without Bati or 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF, an inhibitor of serine proteases, such as plasminogen activation system involved in the activation of a number of MMPs49) were incubated with collagen I film. Collagen I was efficiently degraded by MPs or Man-MPs as evidenced by no colocalization between collagen I and MPs or Man-MPs (Supplementary Fig. 30). Pretreatment with Bati or AEBSF significantly decreased MPs- and Man-MP-induced collagen I degradation (Supplementary Fig. 30), suggesting that MMP was involved in the MPs- and Man-MPs-induced collagen degradation. The collagen content determination also supported that MPs and Man-MPs efficiently degraded collagen in a concentration- (Supplementary Fig. 31) and MMP-dependent manner (Fig. 5a). Furthermore, H22 tumor-bearing mice were intravenously injected with PBS, MPs, Man-MPs, free Met, Met@MPs or Met@Man-MPs at the Met dosage of 10 mg kg−1, or high dosage of Met at 100 mg kg−1 every other day for different times. Second-harmonic generation (SHG) microscopy was used to analyze the fibrillar collagen structure in tumors (Fig. 5b, c). A gradual increase in the SHG signal of tumors was observed in the PBS- and free Met-treated groups. However, MPs and Man-MPs significantly decreased the deposition of collagen fiber network, revealing the collagen-degrading capability of MPs and Man-MPs. Consistently, loading Met into MPs or Man-MPs did not affect their collagen-degrading activity. Immunofluorescence analysis of collagen I in H22 tumor tissues further confirmed that MPs, Man-MPs, Met@MPs, and Met@Man-MPs significantly decreased the collagen area in tumor tissues (Fig. 5d, e).

Met@Man-MPs induce CD8+ T cell infiltration into tumors by recruiting CD8+ T cells and degrading ECM

CD8+ T cells are recruited by M1-like macrophages by secreting some chemokines and cytokines, such as CXCL-9, CXCL-10, CXCL-11, and TNF-α50,51,52. Transwell analysis showed that Met@Man-MPs-treated M2-like macrophages can efficiently recruit CD8+ T cells (Supplementary Fig. 32). However, TNF-α inhibitor Etan significantly decreased the recruitment of CD8+ T cells by Met@Man-MPs-treated M2-like macrophages (Supplementary Fig. 32), suggesting that TNF-α, upregulated expression and secretion by Met@Man-MPs-treated M2-like macrophages, is responsible for the recruitment of CD8+ T cells. Besides the recruitment of CD8+ T cells into tumor tissues, degrading tumor ECM contributes to the infiltration of CD8+ T cells into tumor parenchyma53. Immunofluorescence analysis showed that Met@Man-MPs significantly enhanced the CD8+T cell number in the interior of tumor tissues (Fig. 6b, c). To determine whether Met@Man-MPs-induced CD8+ T cell infiltration was due to the recruitment by the repolarization of M2-like TAM to M1 phenotype and the degradation of tumor ECM, H22 tumor-bearing mice were intravenously injected with Met@Man-MPs pretreated with or without Bati or/and intraperitoneal injection of Etan (Fig. 6a). Pretreatment with Bati significantly decreased the Met@Man-MPs-induced collagen degradation in tumor tissues by SHG microscopy (Supplementary Fig. 33a, b), and Etan treatment significantly decreased TNF-α content in tumor tissues (Supplementary Fig. 33c). Etan or pretreatment with Bati significantly abrogated the Met@Man-MPs-induced CD8+ T cell increase in the interiors of tumor tissues (Fig. 6b, c). In addition, Met@Man-MPs markedly increased the numbers of CD8+ T (Fig. 6d), CD8+CD69+ T (Fig. 6e), and CD8+IFN-γ+ T cells (Fig. 6f) in tumor tissues by flow cytometry. Etan or pretreatment with Bati markedly decreased the Met@Man-MPs-induced increase in the numbers of these immune cells (Fig. 6d–f). The lowest numbers of these immune cells were detected in Bati-preincubated Met@Man-MPs- and Etan-treated group (Fig. 6d–f). Correspondingly, the anticancer activity of Met@Man-MPs was significantly decreased after pretreatment with Bati/and Etan (Fig. 6g, h). These results suggest that Met@Man-MPs-induced recruitment of CD8+ T cells by the repolarization of M2-like TAMs to M1 phenotype and degradation of tumor ECM are responsible for the increased CD8+ T cell infiltration in the interior of tumor tissues, resulting in the improved anticancer activity of Met@Man-MPs.

Met@Man-MPs efficiently enhance tumor accumulation and penetration of anti-PD-1 antibody

The condensed ECM in tumor tissues critically affects the tumor accumulation and penetration of anti-PD-1 antibody54. On the basis of the collagen-degrading capability of Man-MPs, the effects of Met@Man-MPs on the tumor accumulation of anti-PD-1 antibody was assessed in H22 tumor-bearing mice. The mice were intravenously injected with PBS or Met@Man-MPs pretreated with or without Bati every other day for two times, followed by intraperitoneal injection of Cy5-conjugated anti-PD-1 antibody, and the Cy5 fluorescence in different tissues was determined at 24 h after injection (Fig. 7a, b). Met@Man-MPs significantly increased the tumor accumulation of anti-PD-1 antibody. However, pretreatment with Bati abrogated the Met@Man-MPs-induced tumor accumulation of anti-PD-1 antibody, suggesting that the collagen degradation capability of Met@Man-MPs is responsible for the enhanced tumor accumulation of anti-PD-1 antibody.

Next we evaluated the effects of Met@Man-MPs on the tumor penetration of anti-PD-1 antibody. H22 tumor-bearing mice were intravenously injected with PBS or Met@Man-MPs pretreated with or without Bati every other day for two times, followed by intraperitoneal injection of Cy5-conjugated anti-PD-1 antibody. At 24 h after injection, the tumor tissues were stained with fluorescein isothiocyanate (FITC)-conjugated CD31 antibody and then analyzed by confocal microscopy (Fig. 7a, c, d). Anti-PD-1 antibody was strongly colocalized with CD31-labeled tumor vessels due to the dense tumor ECM, although the tumor vessels were permeable as evidenced by the fact that endogenous IgG was distributed throughout the entire tumor tissues (Supplementary Fig. 34). Met@Man-MPs treatment resulted in the wide distribution of anti-PD-1 antibody in tumor tissues, suggesting that anti-PD-1 antibody was extravasated from tumor vessels and then penetrated into tumor tissues. However, the improved tumor penetration of anti-PD-1 antibody was compromised in Bati-pretreated Met@Man-MPs group, revealing that the tumor penetration of anti-PD-1 antibody was achieved by the Met@Man-MPs-induced collagen degradation in tumor tissues. Furthermore, the dorsal skin window chamber tumor model was constructed to directly observe the extravasation and tumor penetration of anti-PD-1 antibody (Fig. 7e, f). When the tumors were formed, the mice were intravenously injected with PBS or Met@Man-MPs pretreated with or without Bati every other day for two times. The tumor vessels were labeled by intravenous injection of fluorescein-conjugated tomato lectin, followed by intravenous injection of Cy5-conjugated anti-PD-1 antibody. The real-time observation showed that although anti-PD-1 antibody was mainly retained within or around blood vessels, treatment with Met@Man-MPs resulted in the quick extravasation of anti-PD-1 antibody from the blood vessels and subsequent distribution in the extravascular region over time. However, pretreatment with Bati significantly decreased the Met@Man-MPs-induced extravasation and tumor penetration of anti-PD-1 antibody, confirming that Met@Man-MPs efficiently enhance the tumor penetration of anti-PD-1 antibody by collagen degradation in tumor tissues.

Met@Man-MPs efficiently improve the anticancer activity of anti-PD-1 antibody

In view of the reshaped tumor immune microenvironment, such as the enrichment of CD8+T cells, the downregulation of the immunosuppressive cells, the upregulated PD-L1 expression (Supplementary Fig. 35), and the enhanced tumor accumulation and penetration of anti-PD-1 antibody by Met@Man-MPs, the effects of Met@Man-MPs on the anticancer activity of anti-PD-1 antibody were first evaluated in H22 tumor-bearing mice (Fig. 8a–i). As expected, treatment with Met@Man-MPs or anti-PD-1 antibody alone exhibited significant anticancer activity. Man-MPs treatment significantly increased the anticancer activity of anti-PD-1 antibody, which might be due to the enhanced tumor accumulation and penetration of anti-PD-1 antibody induced by the collagen degradation of Man-MPs. A combination of Met@Man-MPs and anti-PD-1 antibodies generated the strongest synergistic anticancer effects with no significant toxicity (Supplementary Fig. 36), resulting in 60% of mice being tumor-free. Kaplan–Meier survival analysis showed that 100% of mice were still alive in the Met@Man-MPs and anti-PD-1 antibody-treated group after 70 d, better than any of the other groups. Importantly, compared with other groups, the CD8+effector memory T cells in spleens were significantly increased in Met@Man-MPs and anti-PD-1 antibody-treated group (Fig. 8j). To further investigate the effects of co-administration of Met@Man-MPs and anti-PD-1 antibodies on the memory T-cell response in vivo, we used a tumor rechallenge model. When the mice with complete response (CR) in the Met@Man-MPs and anti-PD-1 antibody-treated group were again inoculated with H22 cells at 70 d after initial treatment, 100% of the mice rejected the tumor rechallenge up to 25 d, while continuous tumor growth was observed in naïve mice inoculated in parallel with the same number of cells (Fig. 8k). Meanwhile, 100% of tumor formation was observed in these mice with CR when inoculated with 4T1 cells (Fig. 8l). These results suggest that the combined treatment with Met@Man-MPs and anti-PD-1 antibody induces long-term antitumor effects by generating immunological memory. The excellent synergistic antitumor activity of the combination of Met@Man-MPs and anti-PD-1 antibody was further confirmed in 4T1 orthotopic tumor model with high malignancy. Anti-PD-1 antibody did not generate significant inhibition in tumor growth and lung metastasis of 4T1 tumor-bearing mice (Supplementary Fig. 37a–e). However, a combination of Man-MPs and anti-PD-1 antibodies significantly halted the development of the tumor mass (Supplementary Fig. 37b) and inhibited the lung metastasis (Supplementary Fig. 37c–e). Combination of Met@Man-MPs and anti-PD-1 antibody generated the strongest synergistic anticancer effects, achieving 75.0% tumor shrinkage (Supplementary Fig. 37b), 81.2% metastasis inhibition (Supplementary Fig. 37c, d) and longer survival time (Supplementary Fig. 37f).

The improved anticancer activity and tumor immune microenvironment induced by the combination of Met@Man-MPs and anti-PD-1 antibody were further confirmed in azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced colitis-associated cancer (CAC) mouse model (Fig. 9a). Consistently, Met@Man-MPs significantly decreased the number of large tumors (Fig. 9b and Supplementary Fig. 38). The anticancer activity of anti-PD-1 antibody was significantly improved by Man-MPs (Fig. 9b and Supplementary Fig. 38). The combination of Met@Man-MPs and anti-PD-1 antibody generated the strongest synergistic inhibition in large tumors (Fig. 9b and Supplementary Fig. 38). H&E staining also confirmed the strongest anticancer effects of combination of Met@Man-MPs and anti-PD-1 antibody (Supplementary Fig. 39). Combination of Met@Man-MPs and anti-PD-1 antibody-treated mice lost less body weight (Supplementary Fig. 40). Meanwhile, tumor immune microenvironment analysis showed that Met@Man-MPs significantly increased the percentages of M1-like TAMs (Fig. 9c), CD8+ T cells (Fig. 9e), activated CD8+ T cells (Fig. 9f), and activated CD4+ T cells (Fig. 9h), while decreased the percentages of M2-like TAMs (Fig. 9d), MDSCs (Fig. 9i) and Tregs (Fig. 9j). However, the combination of Met@Man-MPs and anti-PD-1 antibodies exhibited the strongest capacity to reshape tumor immune microenvironment (Fig. 9c–j).

To determine whether the combination of Met@Man-MPs and anti-PD-1 antibody could promote the antitumor immunity in situ (Supplementary Fig. 41), we employed an organotypic ex vivo slice culture model of fresh human tumors. The organotypic slices from one liver cancer patient-derived tumor were incubated with peripheral blood mononuclear cells (PBMCs) from the same patient in the presence of Met@Man-MPs derived from THP-1-originated macrophages, anti-PD-1 antibody or both Met@Man-MPs and anti-PD-1 antibody. Consistently, Met@Man-MPs significantly induced apoptosis in tumor cells (Fig. 10a). Meanwhile, Met@Man-MPs markedly reshaped the tumor immune microenvironment, as evidenced by the increased M1-like TAM percentages (Fig. 10b), the decreased M2-like TAM percentages (Fig. 10c), and the enhanced infiltration and activation of CD8+ T cells (Fig. 10d–g) in tumor tissues. However, the strongest apoptosis induction in tumor cells (Fig. 10a) and the strongest CD8+ T cell activation (Fig. 10d–f) were observed in the Met@Man-MPs and anti-PD-1-treated group, which was further confirmed in organotypic slices from another liver cancer patient-derived tumor (Supplementary Fig. 42). These results strongly support the notion that Met@Man-MPs efficiently reshape the tumor microenvironment and improve the anticancer activity of anti-PD-1 antibody.

In this study, we developed Man-modified macrophage-derived MPs loading Met (Met@Man-MPs) to boost the anticancer activity of the anti-PD-1 antibody. Met@Man-MPs were highly accumulated in M2-like TAMs compared with other immune cells and tumor cells in tumor tissues and other tissue-resident macrophages, resulting in the repolarization of M2-like TAMs into M1 phenotype. The Met@Man-MPs-reset macrophages not only directly killed and phagocytosed tumor cells to inhibit tumor growth, but also probably generated tumor antigen to increase the tumor recognition capability of cytotoxic T cells, potentiating anti-PD-1 treatment efficacy. Meanwhile, Met@Man-MPs-reset macrophages efficiently reconstructed antitumor immune microenvironment to improve anti-PD-1 therapy by decreasing the numbers of Tregs and MDSCs and increasing the recruitment of CD8+ T cells in tumor tissues, as evidenced by the fact that macrophage depletion using clodronates liposomes significantly abrogated the improved tumor immune microenvironment induced by Met@Man-MPs. The recruitment of CD8+ T cells into tumor tissues might be due to the secreted chemokines and cytokines by Met@Man-MPs-reset macrophages, such as TNF-α, since TNF-α expression and secretion was significantly upregulated in Met@Man-MPs-treated M2-like macrophages, and Etan, an inhibitor of TNF-α, significantly decreased the recruitment of CD8+ T cells by Met@Man-MPs-treated M2-like macrophages by transwell migration assay. More importantly, intraperitoneal injection of Etan markedly abrogated the enhanced number of CD8+ T cells in tumor tissues induced by Met@Man-MPs in H22 tumor-bearing mice. However, whether other chemokines and cytokines were involved in the Met@Man-MPs-induced recruitment of CD8+ T cells remains to be elucidated.

Besides enhanced tumor immunogenicity, reshaped tumor immunosuppressive microenvironment, and increased CD8+ T cell recruitment, the infiltration of CD8+ T cells into tumor interiors and the enhanced tumor accumulation and penetration of PD-1 antibody are also important for the improved anti-PD-1 therapy6,9,10. Man-MPs overexpressed MMPs, such as MMP9 and MMP14 proteins, leading to the efficient degradation of tumor collagen. The Man-MPs-triggered degradation of tumor collagen favored the infiltration of CD8+ T cells into tumor interiors and tumor accumulation and penetration of anti-PD-1 antibody since pretreatment with Bati, an MMP inhibitor, markedly abrogated these effects induced by Met@Man-MPs. Importantly, H&E staining analysis showed that the Man-MPs-triggered degradation of tumor collagen did not induce tumor metastasis in H22 tumor-bearing mice, showing good biosafety. However, which MMP plays an important role in Man-MPs-triggered degradation of tumor collagen needs further clarification. Meanwhile, Bati, like its derivative marimastat having a collagen-mimicking hydroxamate structure, can bind to off-target metalloproteinases that are not MMPs55. Besides MMPs, whether other metalloproteinases are involved in the biological function of Met@Man-MPs remains to be elucidated.

In view of the above facts, Met@Man-MP efficiently integrated all features to improve the therapeutic efficacy of anti-PD1 antibody, generating remarkable anticancer activity after coadministration in different tumor models, including H22 tumor-bearing mice, 4T-1 tumor-bearing mice, AOM/DSS-induced CAC mice, and even an organotypic ex vivo slice culture model of fresh human tumors. Here, Met@Man-MPs derived from RAW264.7 cells, BMDMs, THP-1-originated macrophages, and human MDMs showed similar repolarization activity of M2-like macrophages to M1 phenotype, and expressed MMP, showing that the features of MPs derived from different macrophages are universal. To maximize the treatment efficacy of Met@Man-MPs, several areas should be further explored. Particle size critically affects the pharmacokinetics and tissue biodistribution. Developing Man-MPs with a narrower size distribution would be ideal for the intravenous delivery of Met; Recently, M1-like macrophage-derived exosomes were reported to repolarize M2-like TAMs to M1 phenotype56, and be uptaken by DCs and macrophages to induce the release of a pool of Th1 cytokines57. Thus, the development of Man-modified MPs derived from M1-like macrophages to load Met might exert better anticancer activity and improved tumor microenvironment to potentiate the therapeutic effects of anti-PD-1 antibody; Optimization of Met loading content and the dosing schedule is also required.

Despite the encouraging efficacy and satisfactory safety of a combination of Met@Man-MPs and anti-PD-1 antibody in immunocompetent mouse models, great care has been taken to translate this treatment platform from bench to human clinical trials. First, the macrophage resource for Man-MP fabrication would be a critical issue. For clinical application, it would be better to use human MDMs rather than immortalized cells as the donor cells to prepare Met@Man-MPs because cell lines would not have the necessary “self” characteristics to avoid recognition by the immune system58. However, the availability of human MDMs and the development of a robust, scalable process would be a challenge. MPs derived from immortalized tumor cell lines have been applied to cancer patients in clinical settings59. Addressing the potential risks of the immune response induced by allogeneic cell-derived MPs needs to be further elucidated. Second, quality control standards for MP constitution and content, and drug encapsulation efficiency and loading content in the GMP production department are essential.

In summary, the data in this study show that Met@Man-MPs efficiently target to M2-like TAMs to repolarize into M1-like phenotype, resulting in the recruitment of CD8+ T cells into tumor tissues and the ameliorated tumor immunosuppressive microenvironment. In addition, the collagen-degrading capacity of Man-MPs contributes to the infiltration of CD8+ T cells into tumor interiors and the enhanced tumor accumulation and penetration of anti-PD-1 antibody. With these unique features, Met@Man-MP efficiently improve the anticancer activity of anti-PD1 antibody therapy, generating long-term memory immunity. These findings reveal that Met@Man-MPs may add to the therapeutic tools for tumor resistance to current anti-PD-1 therapy.