M1 Macrophage-Derived Nanovesicles Potentiate the Anticancer

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M1 Macrophage-Derived Nanovesicles Potentiate the Anticancer Efficacy of Immune Checkpoint Inhibitors Yeon Woong Choo, Mikyung Kang, Han Young Kim, Jin Han, Seokyung Kang, Ju-Ro Lee, Gun-Jae Jeong, Sung Pil Kwon, Seuk Young Song, Seokhyeong Go, Mungyo Jung, Jihye Hong, and Byung-Soo Kim ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b02446 • Publication Date (Web): 22 Aug 2018 Downloaded from http://pubs.acs.org on August 23, 2018

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M1 Macrophage-Derived Nanovesicles Potentiate the Anticancer Efficacy of Immune Checkpoint Inhibitors Yeon Woong Choo,1,† Mikyung Kang,2,† Han Young Kim,1 Jin Han,1 Seokyung Kang,1 Ju-Ro Lee,1 Gun-Jae Jeong,1 Sung Pil Kwon,1 Seuk Young Song,1 Seokhyeong Go,2 Mungyo Jung,1 Jihye Hong,2 Byung-Soo Kim1,2,3,* 1

School of Chemical and Biological Engineering, Seoul National University, Seoul 08826,

Republic of Korea 2

Interdisciplinary Program for Bioengineering, Seoul National University, Seoul 08826,

Republic of Korea 3

†

Institute of Chemical Processes, Seoul National University, Seoul 08826, Republic of Korea

These authors contributed equally to this work.

*Byung-Soo Kim, Ph.D., E-mail: [email protected]

Key Words: cancer immunotherapy • checkpoint inhibitor • macrophage polarization • nanovesicle • tumor-associated macrophage

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ABSTRACT Cancer immunotherapy modulates immune cells to induce antitumor immune responses. Tumors employ immune checkpoints to evade immune cell attacks. Immune checkpoint inhibitors such as anti–PD–L1 antibody (aPD–L1), which are being used clinically for cancer treatments, can block immune checkpoints so that the immune system can attack tumors. However, the immune checkpoint inhibitor therapy may be hampered by polarization of macrophages within the tumor microenvironment (TME) into M2 tumor-associated macrophages (TAMs), which suppress antitumor immune responses and promote tumor growth by releasing anti-inflammatory cytokines and angiogenic factors. In this study, we used exosome-mimetic nanovesicles derived from M1 macrophages (M1NVs) to repolarize M2 TAMs to M1 macrophages that release pro-inflammatory cytokines and induce antitumor immune responses, and investigated whether the macrophage repolarization can potentiate the anticancer efficacy of aPD–L1. M1NV treatment induced successful polarization of M2 macrophages to M1 macrophages in vitro and in vivo. Intravenous injection of M1NVs into tumor-bearing mice suppressed tumor growth. Importantly, injection of a combination of M1NVs and aPD–L1 further reduced the tumor size, compared to the injection of either M1NVs or aPD–L1 alone. Thus, our study indicates that M1NV injection can repolarize M2 TAMs to M1 macrophages and potentiate antitumor efficacy of the checkpoint inhibitor therapy.

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Cancer immunotherapy is a treatment used to activate immune cells, such as T cells, B cells, and macrophages, to induce immune responses against cancer.1 Cancer immunotherapy has focused on T cell activation that results in tumor cell necrosis.2 However, tumors employ immune checkpoints, such as programmed cell death protein–1 (PD–1), programmed death– ligand 1 (PD–L1), and cytotoxic T-lymphocyte-associated protein 4 (CTLA–4), to protect themselves from T cell attacks.3 For example, signal transduction induced by the interaction between PD–L1 on cancer cells and PD–1 on T cells inhibits the T cell proliferation and induces apoptosis of the T cells.3,4 Recently, the inhibitors of immune checkpoints has been identified as a promising cancer immunotherapeutic approach.1,3,5 Antibodies targeted against the immune checkpoints block the inhibitory signaling of the adaptive T cell immune response, which allows the anticancer activity of the cytotoxic T cells.6,7 For example, antibodies against PD–L1 (aPD–L1) can interact with PD–L1 on cancer cells to prevent the PD–L1 from interacting with PD–1 on T cells, thereby allowing T cell activation and facilitation of antitumor immune response.8 Although the antibodies against PD–1, PD–L1, and CTLA–4 have been approved by Food and Drug Administration (FDA) and are being used clinically for cancer treatment,9 the minority population of patients respond to such therapy.10 A potential limitation of current immune checkpoint inhibitors is the immunosuppressive tumor microenvironment (TME) that attenuate the activation of effector T cells.11 Recently, researchers have targeted macrophages for cancer immunotherapy,12-15 since macrophages constitute the major immune regulators that interact with cytotoxic T cells in the TME.16 TME is composed of multiple components including immune cells, fibroblasts, cytokines, extracellular matrix, and cancer cells.17 Cancer cells release signaling cytokines 3


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and extracellular vesicles in TME to suppress immune cell activation and promote their own growth.18-20 Macrophages in TME, which are tumor-associated macrophages (TAMs), are exposed to IL–4, IL–10, TGF–β1, and lactic acid, and are polarized to alternatively activated M2 type.21,22 M2 TAMs promote tumor growth by secreting molecules that suppress antitumor immune responses and promote angiogenesis.16,21,23-25 Importantly, antitumor activation of T cells by aPD–L1 may be downregulated by M2 TAMs, since M2 TAMs recruit regulatory T cells and release anti-inflammatory cytokines, suppressing the immune response to tumor and destabilizing the anticancer function of T cells.23,26 In contrast, classically activated M1 macrophages suppress tumor growth by releasing pro-inflammatory factors, inducing stromal destruction, and normalizing the tumor vasculature.16,27,28 There have been efforts to induce repolarization of M2 TAMs to M1 TAMs for tumor suppression. The repolarization methods include anti–CD40 antibody,29 anti–MARCO antibody,13 BTK inhibitor,30 iron oxide nanoparticles,12 and expression of immunomodulatory histidine-rich glycoprotein.15 Herein, we propose that the repolarization of M2 TAMs to M1 macrophages may enhance the antitumor efficacy of aPD–L1. We postulate that exosomes derived from M1 macrophages may repolarize M2 macrophages to M1 macrophages and be subsequently used to potentiate the anticancer efficiency of immune checkpoint inhibitors such as aPD–L1. Exosomes are subpopulations of extracellular vesicles that are naturally secreted from cells, have sizes in the range of 40–150 nm, and serve as biomolecular carriers for intracellular communication in vivo.31,32 Exosomes contain RNAs and proteins that originate from their parent cells and transfer the biomolecules to recipient cells, thereby inducing phenotypic changes in the recipient cells.33 It has been reported that exosomes isolated from differentiated cells can be used to induce differentiation 4


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of stem cells into specific lineage.34,35 Furthermore, exosomes can be exploited as drug delivery vehicles in several investigation.36-39 However, the quantity of exosomes released from cells is low.32 Given the current limitation of low-yield exosome production, several studies have suggested nanovesicles (NVs) as a substitute for exosomes.32,40 NVs, which are prepared by the serial extrusion of cells, have cell membranes and sizes similar to those of exosomes, have higher production-yield, and are more enriched in proteins and RNAs than exosomes.24,32 Thus, it is expected that NVs may be more effective in the intercellular transfer of biomolecules and in the induction of subsequent phenotypic changes in the recipient cells than exosomes. Herein, we investigated whether NVs derived from M1 macrophages (M1NVs) can be used as an immune regulator to repolarize M2 TAMs to M1 TAMs for cancer immunotherapy. Also, we investigated whether repolarizing M2 TAMs to M1 TAMs with M1NVs can potentiate the antitumor efficacy of aPD–L1 (Figure 1). The previous study demonstrated that exosomes derived from M1 macrophages enhance the antitumor effects of lipid calcium phosphate-based peptide vaccine and investigated interactions between dendritic cells and T cells in the lymph nodes were examined.41 Here, we will discuss that M1NVs potentiate antitumoral efficacy of anti-PD-L1 antibody through the interaction between activated T cells and M1 macrophages in the TME.

RESULTS AND DISCUSSION Characterization of M1NVs. M1NVs were obtained from LPS-treated RAW264.7 cells 5


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(M1 macrophages) by serial extrusion through polycarbonate membranes with pore sizes of 1 µm, 400 nm, and 200 nm (Figure 1). Nanoparticle tracking analyses of M1NVs showed a size distribution with a mean diameter of 189.7 ± 2.5 nm (Figure 2A). Electrophoretic light scattering (ELS) of M1NVs revealed a zeta potential of -17.6 ± 0.4 mV (Figure 2A). M1NVs and M1 macrophage-derived exosomes had similar size distribution and zeta potentials (Figure 2A). Analysis with energy-filtering transmission electron microscopy showed the exosome-mimetic morphology of M1NVs (Figure 2B). We examined the stability of the M1NVs in PBS and 10% (v/v) serum as measured by DLS (Figure 2C). The data showed no significant difference in hydrodynamic diameter over 72 h. The number and protein amount of M1NVs produced from macrophages was approximately four and three times higher compared to those of exosomes produced from the same number of macrophages, respectively (Figure 2D). Since M1NVs were prepared by serially extruding M1 macrophages, we evaluated mRNA quantity of pro-inflammatory factors and M1 macrophage markers, such as CD86, IL–6, TNF–α, and iNOS in the parent cells and the parent cellderived NVs via quantitative real-time polymerase chain reaction (qRT–PCR) (Figure 2E). We compared the values with those of the nonpolarized M0 macrophages and M0 macrophage-derived NVs (M0NVs). The analysis revealed that M1 macrophages and M1NVs contained higher amounts of mRNA of pro-inflammation factors and M1 markers compared to M0 macrophages and M0NV, respectively. These data indicate that RNAs in macrophages were included in NVs derived from macrophages.42 Since a previous study revealed that microRNAs (miRNAs) in dendritic cell exosomes are functional in recipient dendritic cells following cellular uptake and regluate the phenotype of the recipient dendritic cells,43,44 we evaluated the difference in the miRNA amount between M1NVs and M0NVs via microarray assay (Supporting Information Figure 1). The 6


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M1 and M2 macrophage-related miRNA expressions of M1NVs are shown as compared to those of M0NVs in Figure 2E. Substantially, M1 macrophage-related miRNAs are upregulated and M2 macrophage-related miRNAs are downregulated in M1 macrophages.4548

Importanly, upregulated expressions of miR-155, miR-125, and miR-21 are known to

polarize macrophage to M1 type, and downregulated expressions of miR-34a, let-7c, and let7f are known to polarize macrophage to M2 type.44,47,49 Thus the miRNA profiles of M1NVs (Figure 2E) confirm that M1NVs have potential to polarize M2 TAM to M1 macrophage. In Vitro Cellular Uptake and Cytotoxicity of M1NV. Cancer cells and M2 TAMs are abundant in TME. In vitro uptake of M1NVs by M2 macrophages and cancer cells was compared to determine whether the uptake of M1NVs is better for M2 macrophages compared to cancer cells. To obtain M2 macrophages, bone marrow-derived macrophages (BMDM) were treated with 20 ng/mL IL–4 for 24 h. Subsequently, M2 macrophages and CT26 colon carcinoma cells were treated with DiI–conjugated M1NV for 4 h. Fluorescence imaging and FACS analyses revealed that 80.4 % and 12.0 % of M1NVs were taken up by M2 macrophages and cancer cells, respectively (Figure 3A and B). The exact mechanism for the higher cellular uptake of M1NV by M2 macrophages rather than cancer cells is not clear, but the phagocytic activity of macrophages may be responsible for the higher cellular uptake.38 To determine whether M1NVs affect cell viability after the cellular uptake, a cell viability assay was performed using the cell counting kit-8 (CCK-8) on CT26 cells and M2 macrophages cultured for 24 h in the presence of 0, 10, 50, and 100 µg/mL M1NVs. Figure 3C shows that M1NVs do not induce cellular toxicity in either M2 macrophages or CT26 cells. In addition, M1NVs have no effects on tumorigenesis-related gene expressions of CT26 cells (Figure 3D). mRNA expression of PD–L1 in CT26 cells after M0NVs or M1NVs 7


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treatment was also evaluated and the data showed no significant difference among groups (Supporting Information Figure 2). These data revealed that the M1NV treatment itself does not induce cell death in cancer cells and macrophages. Comparison of the Therapeutic Effects between M1NVs and M1 Macrophages in vitro. To determine whether M2 macrophages that have been repolarized to M1 type affect cancer cell growth, a CCK8 assay was performed on CT26 cells co-cultured with M2 macrophages. M2 macrophages treated with M1NVs (group 4) significantly suppressed the cancer cell growth compared to no treatment (group 1, negative control), untreated M2 macrophages (group 2) and M2 macrophages co-cultured with M1 macrophages (group 3), but showed relatively less inhibitory effect than M1 macrophage (group 5, positive control, Figure 4A). We postulated that M2 macrophages were repolarized to M1 macrophage by either co-culturing with M1 macrophages or M1NV treatment. The data suggest that repolarization of anti-inflammatory M2 macrophages to pro-inflammatory M1 macrophages hindered the cancer cell proliferation by M1 macrophage-secreted cytokines.12,13 To examine how M1 macrophages are affected by M2 macrophages and cancer cells, both of which are the most abundant cell types in tumors, M1 macrophages were co-cultured with M2 macrophages or CT26 cells for 24 h. Subsequently, the mRNA expressions of the M1 (CD86) and M2 markers (CD206 and Fizz–1) in the M1 macrophages were evaluated by qRT–PCR (Figure 4B). It is shown that mRNA expression of the M1 marker was decreased and that of the M2 markers were increased in M1 macrophages co-cultured with M2 macrophages or CT26 cells. The data indicate that M1 macrophages can be polarized to M2 macrophages through interactions with M2 macrophages and cancer cells, both of which are abundant in tumor. Thus, implantation of M1 macrophages to the tumor site in order to supply M1 macrophages in the TME for cancer therapy may result in polarization of the 8


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implanted M1 macrophages to M2 TAMs and ultimate stimulation of tumor growth by the M2 TAMs.50 Additionally, the mRNA expressions of the M1 (IL–6) and M2 markers (Fizz–1) in the M2 macrophages co-cultured with M1 macrophages or treated with M1NVs were evaluated by qRT–PCR (Figure 4C). The mRNA expression of the M2 marker was shown to be decreased in both groups, and that of the M1 marker was significantly increased only in the M1NV treatment group. Importantly, the data revealed that M1NV treatment is more effective in the polarization of M2 macrophages, which are abundant in TME, to M1 macrophages than direct treatment with M1 macrophages. Also, we compared distribution of M1 macrophages and M1NVs in tumor-bearing mice with Vivotrack 680 (Supporting Information Figure 3). The graph shows relative intensity of each organ over the sum of intensities of the major organs in each group. Differences exist in the sum of fluorescence intensities of the major organs between M1NV- and M1 cell-treated mice, which might be due to the difference in circulation time caused by the different size between M1NVs and M1 macrophage cells.51 The fluorescent intensity comparison in tumor tissue of both groups showed no significant difference. In terms of tumor tissue targeting, neither of them have superiority. M1NV Polarizes M2 to M1 Macrophages. To determine whether M1NVs can polarize M2 macrophages to M1 macrophages, we evaluated mRNA expression changes in the M1 (IL–6, iNOS, and TNF–α) and M2 (Fizz–1, IL–4 and IL–10) markers in M2 macrophages after 24 h of treatment with M1NVs (Figure 5A). No-treatment and M0NVtreatment groups were used as controls. The data show that the mRNA levels of the M1 markers were significantly higher in the M1NV treatment group, whereas those of M2 markers were lower compared to those of the no-treatment and the M0NV-treatment groups. The mRNA expressions of the angiogenesis factor VEGF and metastatic factor CCL–18 were 9


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also evaluated in M2 macrophages after 24 h treatment with M1NVs. No significant difference was observed in mRNA expression of VEGF among the three groups and significant decrease was observed in mRNA expression of CCL–18 of M1NV-treatment group compared to other groups (Figure 5B). Previous studies demonstrated that PD-L1 expression on TAMs contributes to immunosuppressive activity by mediating checkpoint regulation, in which TAMs impair the antitumor activity of T cells via signals produced by the interaction between PD-1 on T cells and PD-L1 on TAM.52-54 We therefore evaluated PDL1 mRNA expression of M2 macrophages after treatment with M1NVs by qRT-PCR. The M1NVs treatment to M2 macrophages did not make significant changes in the PD-L1 mRNA expression (Supporting Information Figure 4). The detailed mechanism of NV internalization is still not well elucidated. A number of previous studies revealed that cellular uptake of exosomes or NVs occurs via multiple routes, including clathrin- or caveolin-mediated endocytosis, phagocytosis, and cell surface membrane fusion.43,55-62 Recent studies have suggested that some cellular entry pathways of larger particles (~ 200nm), including NV, bypass lysosomal degradation63 and can deliver cargos into the cytosol of the target cells.55,56,59 Thus, we concluded that M1NV treatment can repolarize M2 macrophages to M1 macrophages and reduce expression of CCL–18, a metastatic factor, in M2 macrophages. The polarization of M2 macrophages to M1 macrophages enhanced the pro-inflammatory cytokine (IL–6) secretion and reduced the secretion of anti-inflammatory cytokine (IL–4) (Figure 5C). In addition, immunofluorescence analysis of M2 macrophages after 24 h treatment with M1NVs showed a higher expression of CD86 (M1 marker) and lower expression of CD206 (M2 marker) compared to the no-treatment group and the M0NVtreatment group (Figure 5D). This suggests that M1NVs may effectively polarize M2 TAMs 10


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to M1 macrophages. The NV treatment itself did not considerably affect polarization since the mRNA expression changes elicited by the M0NV treatment were not more prominent than those of the M1NV treatment. These data also suggest that the content including mRNAs, miRNAs in NV is responsible for polarization of M2 macrophages to M1 macrophages as we compared contents of M1NVs and M0NVs. In addition, the data of Figure 4A suggest that polarization of M2 macrophages to M1 macrophages may suppresses cancer cell proliferation, since M1 macrophages are known to suppress tumor growth by secreting chemokines.27,50,64 In Vivo Tumor Targeting of M1NV. Leukocytes are known to elicit homing affinities to inflammatory and tumor sites.65 The interaction between ICAM–1 on endothelial cells and leukocyte-derived adhesion molecules such as lymphocyte function-associated antigen 1 (LFA–1) on leukocytes enables leukocytes to adhere to the inflamed endothelium.66,67 ICAM–1 is known to be overexpressed in the inflamed endothelium of TME.68,69 Previous studies utilized macrophage membranes containing leukocyte-derived adhesion molecules and developed nanoparticles that selectively targets inflamed endothelium.40,70 We investigated whether the expression of LFA–1 was maintained on M1NVs. As a control group, trypsin-treated M1NVs (T-M1NVs) were used, since trypsin can remove the externally exposed proteins (e.g., LFA–1) on the cellular membrane. To analyze the presence of LFA–1, M1NVs and T-M1NVs were adsorbed to latex beads and stained with DiI fluoresceinconjugated antibodies against CD11a/CD18 (LFA–1). FACS analyses showed that LFA–1 was highly expressed on M1NVs compared to T-M1NVs (Figure 6A), thereby indicating that trypsin treatment removed the leukocyte-derived adhesion molecules on T-M1NVs. Subsequently, we assessed the tumor-targeting efficiency of M1NVs and T-M1NVs in 11


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vivo. To evaluate distribution of M1NVs in tumor-bearing mice, M1NVs and T-M1NVs labeled with Vivotrack 680, and phosphate buffered saline (PBS) were intravenously injected into CT26 colon carcinoma-bearing BALB/c mice. Major organs (heart, lung, spleen, kidney, and liver) and tumor were then excised. Fluorescence intensities of the organs and tumors were calculated by subtracting the fluorescence intensity of the corresponding tissues in the PBS-treatment group (Figure 6B). Tumors in the M1NV injected mice elicited higher fluorescence signals than those from the T-M1NV injected mice, confirming the accumulation of M1NVs at the tumor site by the externally exposed proteins on the cellular membrane that have the homing affinity to tumor sites. Next, we examined M1NV toxicity on the major organs (Figure 6C and D). Fifty µg of M1NVs derived from RAW264.7 cells of BALB/C mice were intravenously injected in to the same stain mice. The histological analyses of the major organs (heart, liver, lung, kidney and spleen) 1 day after the last injection showed no in vivo toxicity of M1NV (Figure 6C). Furthermore, the levels of hepatic enzyme (aspartate aminotransferase (AST) and alanine aminotransferase (ALT)) in the serum of the mice were determined 1 day before and 1, 6, 13 and 20 days after the intravenous injection of PBS or MINVs (Figure 6D). The ALT and AST levels in the serum of the M1NV-treated mice were not significantly different from those of the PBS-treated mice. Previous studies demonstrated that cell-derived NVs are biocompatible and not significantly toxic or immunogenic since NVs can evade immune phagocytosis and complement system, are originated from cells and retain the membrane topology of the cells and maintain stability in serum.32,62,71,72 Also, a previous study reported that NVs derived from RAW264.7 cells showed no cytotoxicity.73 The amount of IgG monoclonal antibodies bound to their immunogenic substance and form aggregates, which is of great concern for drug immunogenicity.74 For in vivo assessment of immunogenicity, we 12


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also determined the levels of anti-M1NV IgG in serum of the mice treated with either PBS or M1NVs (Supporting Information Figure 5). The results showed that no significant difference in the IgG titer observed between PBS- and M1NV-treated mice. M1NV Enhances Efficacy of Anti PD–L1 Immune Checkpoint Inhibitor Therapy. Encouraged by the results of the in vitro experiments, we then investigated the antitumor effects of M1NVs in CT26-bearing BALB/c mice. When the tumor size reached 70 mm3 approximately, 50 µg total protein of M1NV was injected intravenously every three days for three times in total, and the tumor volume was monitored over 15 days (Figure 7A). We found that the M1NV injection yielded a significant inhibition of tumor growth compared to PBS injection (Figure 7B). We then examined whether the M1NV injection could enhance the antitumor efficacy of immune checkpoint inhibitors. CT26-bearing mice were injected with either M1NVs, aPD– L1, or a combination of M1NVs and aPD–L1. The tumor volumes of the group treated with the combination of M1NVs and aPD–L1 were significantly smaller than those from the M1NVs or aPD–L1groups (Figure 7C), thereby indicating that M1NVs potentiate the antitumor efficacy of the immune checkpoint inhibitor therapy using aPD–L1. Since M1 macrophages have PD-L1 on the cell membrane,75 aPD-L1 could be captured by M1NVs and then uptaken by TAM. However, Figure 7C shows that M1NVs and aPD-L1 combination treatment resulted in a significant decrease in tumor volume compared to either of M1NV or aPD-L1 treatment, suggesting negligible PD-L1 antibody capture by M1NVs. The antitumor effects were analyzed by histological examinations of the tumor tissues collected at 24 h after the last treatment. TdT-mediated dUTP nick-end labeling (TUNEL) staining indicated that mice injected with the combination of M1NVs and aPD–L1 led to the largest number of apoptotic cells in the tumor, compared to the significantly fewer apoptotic cells found in the 13


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other groups (Figure 7D). Hematoxylin and eosin (H&E) staining of the tumor tissues showed decreased nucleus-to-cytoplasm ratios, which were related to the decreased number of cancer cells, in mice injected with the combination of M1NVs and aPD–L1 (Figure 7E). To evaluate the polarization effects of M1NVs, we compared the mRNA expressions of the immunosuppressive M2 markers (Arg–1, Fizz–1, and IL–10) and pro-inflammatory M1 markers (CD86, iNOS, and TNF–α) in the tumor tissues in all the studied groups. Correspondingly, qRT–PCR analysis showed that the expressions of pro-inflammatory genes were significantly upregulated in the combination of M1NVs and aPD–L1 group, while those of the anti-inflammatory genes were downregulated (Figure 8A). Interestingly, the mRNA expression of CD86, a well-known M1 macrophage marker,76 was increased by more than 200 times compared to the aPD–L1 group. Additionally, the mRNA expressions of angiogenesis-related factors (VEGF and MMP9) were significantly decreased in the combination of M1NVs and aPD–L1 group (Figure 8B). M1 and M2 macrophages in the tumor tissues were evaluated by immunofluorescence assays. The polarization of M2 TAMs to M1 macrophages was indicated by the increased M1 marker (CD86, iNOS and CD80) protein expression (Figure 8C and Supporting Information Figure 6) and the decreased M2 marker (CD206, CD163 and Arg-1) protein expression (Figure 8D and Supporting Information Figure 7). M1NV injection significantly inhibited the tumor growth compared to PBS injection (Figure 7C), but there was no significant difference in the apoptotic cell number and M1 macrophage marker mRNA expression in the tumor between the M1NVinjected mice and the PBS-injected mice (Figure 7D and 8A). Thus, the tumor growth inhibition by M1NV is likely due to inhibition of TAM polarization into M2 macrophages (Figure

8A,

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tumor

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immunosuppression.78 14


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angiogenesis77

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We determined total number of macrophages in the tumor of mice by a pan-macrophage marker (CD68) expression (Supporting Information Figure 8).72 The data show that the combination of M1NVs and aPD-L1-treated mice have a higher number of macrophages than the other groups. It is may be due to the higher inflammatory cytokine (TNF-α) expression in the combination of M1NVs and aPD-L1-treated mice (Figure 8A), as TNF-α is known to stimulate monocyte recruitment and infiltration.79 The increased number of M1 marker (iNOS)-positive macrophages in tumor after the combination treatment (Figure 8C) may be partially caused by the monocyte recruitment. Meanwhile, our in vitro data (Figure 5) indicate that M1NV treatment directly polarize M2 macrophages to M1 macrophages. The injection of the combination of M1NVs and aPD–L1 exhibited successful synergistic effects for the polarization of M2 TAMs to M1 macrophages. The triggering of the synergistic mechanism associated with the combined presence of aPD–L1 and M1NVs may be attributed to the fact that T cells, which are activated by aPD–L1, and M1 TAMs, which are polarized by M1NVs, interact with each other and elicit type 1 T helper (Th1) cell immune responses favoring antitumor effects.24,27,80 PD-L1 expression on cancer cells contributes to immunosuppressive activity by mediating checkpoint regulation. Cancer cells impair the antitumor activity of T cells via signals produced by the interaction between PD-1 on T cells and PD-L1 on cancer cells.81 Blockade of the PD-1/PD-L1 interaction by anti-PD-L1 antibody treatment restores CD8+ T cells function82,83 and enhances secretion of IFN-γ that induces M1 polarization of macrophages.8487

M1 macrophages stimulate the infiltration of type 1 T helper (Th1) cells and promote

antitumor

T

cell

responses.27,85,88

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addition,

M1

macrophages

hinder

the

immunosuppressive function of regulatory T cells (Tregs), such as inhibition of antitumor T 15


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cell function and induction of immunosuppressive M2 TAMs.89,90 Previous studies demonstrated that M1 macrophages produce iNOS that converts oxygen to nitric oxide, which preferentially induces type 1 T helper (Th1) cell differentiation.91,92 We therefore analyzed the expression of iNOS in the tumor tissue of the CT26 tumor-bearing mice after various treatments. Immunohistochemistry showed that the combination of M1NV and aPDL1 treatment group exhibited higher protein expression of iNOS than the other groups (Figure 8C). To analyze the antitumoral activity of T cells, we measured the populations of tumor-infiltrating CD3+ CD4+ T cells, CD3+ CD8+ T cells, and CD3+ CD4+ Foxp3+ T cells (Tregs) in the tumor tissues. We found increased ratios of CD4+ T cells to Tregs in the tumor tissues of the combination of M1NV and aPD-L1 treatment group than other groups (Figure 8E). The combination group showed higher ratio of and CD8+ T cells to Tregs than the aPDL1 group (Figure 8E). Relative IFN-γ mRNA expression in the tumor tissues was also evaluated. The combination of M1NV and aPD-L1 treatment group showed significantly increased IFN-γ mRNA expression compared to the other groups (Supporting Information Figure 9). The low therapeutic efficacy of M1NVs than that of the combination of M1NVs and aPD-L1 is likely due to lower antitumor activity of T cells that interact with PD-L1 of cancer cells in the M1NV group and higher T cell activity in the combination therapy. We also determined the possibility of using primary cells (e.g., BMDM) as an alternative to RAW264.7 cell line. In a clinical study, ex vivo education of macrophages collected from blood of patients to a suitable phenotype and subsequent injection of the educated macrophages to the patients have long been used as a macrophage-based therapy.93 Thus, we evaluated the feasibility of using the same method in the present study by assessing repolarization of M1 BMDM-derived NVs (M1 BMNVs, Supporting Information Figure 10). The data showed that M1 BMNVs retain mRNA of M1 BMDM, and that M1 BMNV 16


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treatment significantly increased M1 marker mRNA expression in M2 macrophages. Thus, our approach can be applied to autologous or primary cells. Taken together, the best antitumor effect elicited by the injection of the combination of M1NVs and aPD–L1 may be attributed to the synergistic effects between the aPD–L1mediated inhibition of T cell deactivation in TME and the M1NV-mediated repolarization of M2 TAMs to M1 macrophages. CONCLUSION M2 TAMs and cancer cells deactivate T cells and polarize macrophages toward the M2 phenotype.16,23 Our data demonstrate that M1NV treatment polarized protumor M2 type macrophages to antitumor M1 type macrophages, which potentiated the antitumor efficacy of aPD–L1. Leukocyte-derived adhesion molecules such as LFA–1 on M1NVs enabled intravenously injected M1NVs to target tumors. Injection of a combination of M1NV and aPD–L1 into tumor-bearing mice induced the repolarization of M2 TAMs to M1 macrophages and led to a significant suppression of tumor growth, as compared to the injection of either M1NVs or aPD–L1. This study suggests that M1NVs can be used as an immune regulator in the TME to potentiate immune checkpoint inhibitor therapies for cancer treatment.

METHODS Cell Culture: The macrophage cell line RAW264.7 and the CT26 colon carcinoma cancer cell line were purchased from the Korean cell line bank (Seoul, Korea). They were cultured in Dulbecco’s modified eagle’s medium (DMEM, Gibco, NY, USA) supplemented 17


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with 10 % (v/v) fetal bovine serum (FBS, Gibco, NY) and 1 % (v/v) penicillin/streptomycin (Gibco). The cells were incubated at 37 °C with 5 % CO2. To obtain mouse bone marrowderived macrophages (BMDMs), bone marrow was collected from the femurs of six-weekold female BALB/c mice (Orient bio, Seoul, Korea) as described previously.94 In brief, hindlimbs were removed from mice and muscle tissue was discarded. Tibia and femur bones were then flushed with PBS using 5 mL syringes and 25 gauge needles to obtain bone marrow cells. The cells differentiated into macrophages within an incubation period of 7 days in 10 mL of macrophage differentiation medium for 100mm2 petri dish, which was highglucose DMEM supplemented with 10 % (v/v) FBS, 1 % (v/v) penicillin/streptomycin, and 10 % (v/v) L929 cell-conditioned medium. L929 cell-conditioned medium was prepared by growing L929 cells in high-glucose DMEM containing 10 % (v/v) FBS, and 1 % (v/v) penicillin/streptomycin. At day 3, 5 mL of additional macrophage differentiation medium was added to the BMDM culture. BMDMs were collected at day 7 for in vitro experiments. M1 macrophages were induced by the addition of 100 ng/mL lipopolysaccharide (LPS). M2 macrophages were induced by the addition of 20 ng/mL IL–4 (Invitrogen, CA, USA). Preparation of M1NVs: NVs were prepared from M1 macrophages as described previously.32 In brief, LPS-treated RAW264.7 cells (M1 macrophages) were suspended at a concentration of 5×106 cells/mL in PBS. They were sequentially extruded 11 times through polycarbonate membrane filters (Whatman) with pore sizes of 1 µm, 400 nm, and 200 nm using a mini-extruder (Avanti Polar Lipids) to obtain nano-sized extracellular vesicles. The extracellular vesicles were then ultracentrifuged in a density gradient formed by 10 and 50% OptiPrep layers at 100,000g for 2 h at 4°C. To obtain M1NVs, extracellular vesicles obtained from the interface of the layers were further ultracentrifuged at 100,000g for 2 h at 4°C. The protein concentration of the isolated M1NVs was quantified using Bradford reagent (Sigma– 18


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Aldrich, USA) according to the manufacturer’s protocol. Preparation of Exosomes: Exosomes were prepared from M1 macrophages as described previously with modification.95 Exosome-depleted FBS was prepared by ultracentrifugation at 100,000g for 16 h at 4°C. LPS-treated RAW264.7 cells (M1 macrophages) were incubated in DMEM medium containing 10% (v/v) of exosome-depleted FBS and 1% (v/v) penicillin/streptomycin for 24 h. The spent medium was collected, and cells and debris were eliminated by serial centrifugation at 500g for 10 min, and 3000g for 15 min, at 4 °C. Exosomes were collected by ultracentrifugation at 100,000g for 2 h at 4°C. The protein concentration of the isolated exosomes was quantified using the Bradford reagent (Sigma– Aldrich) according to the manufacturer’s protocol. Physicochemical Characterization of M1NVs and Exosomes: The size distributions of M1NVs and macrophage-derived exosomes were assessed by nanoparticle tracking analyses using the Nanosight LM10 system (Malvern, Worcestershire, UK). M1NVs and exosomes were dispersed in PBS at 500 ng total proteins/mL, and the NV particle sizes and numbers were determined with a CDD camera at level 14, a slide shutter of 1500, and a slider gain of 420. The chamber temperature was maintained at 23 °C. The measurements were obtained in triplicate. The duration of each individual measurement lasted 1 min. The data were analyzed using a nanoparticle tracking analysis software version with a detection threshold of 5 (multiple), and autoset to blur and max jump distance. ELS Z-1000 (Otsuka Electronics, Osaka, Japan) was used to determine the zeta potential of M1NVs and exosomes. The morphology of M1NVs and exosomes was examined using a transmission electron microscope (LIBRA 120, Carl Zeiss, Germany). A drop of M1NVs and exosomes at a concentration of 4 µg⁄mL was deposited onto a glow-discharged carbon-coated grid. Three minutes later, a drop of 1% uranyl acetate stain was added to the grid. The grid was rinsed 19


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with five drops of distilled water and dried. M1NVs were then visualized using a LIBRA-120 120 kV energy-filtering transmission electron microscope. mRNA Quantification of NVs and Cells: Total RNA was extracted from M1 macrophages, M1NVs, and M0NVs using 1 mL of Trizol (Qiagen Valencia, CA, USA). The total RNA concentration was determined using a NanoDrop spectrometer (ND-2000, NanoDrop Technologies, USA). Six hundred nanograms of total RNA from each sample were reverse-transcribed into cDNAs, and SYBR green-based qRT–PCR was performed using a Step One Plus real-time PCR system (Applied Biosystems, USA) with TOPreal qPCR 2X PreMIX (Enzynomics, Finland). Cycling conditions were the following: initial denaturation at 95°C for 15 min, followed by 45 cycles at 95°C for 10 s, 60°C for 15 s, and 72°C for 30 s. All of the data were analyzed using the comparative Ct method.96 Three samples were analyzed per group. MicroRNA Profiling Assay: M1NVs and M0NVs are prepared as mentioned early. Total RNA was prepared from M1NVs and M0NVs using Trizol reagent and quantified by NanoDrop spectrometer. miRNA expression profiling with Affymetrix GeneChip® miRNA 4.0 assay are conducted by a commercial service (BioCore, Seoul, Korea). All the experimental results were saved as Microsoft Excel files. In Vitro Cellular Uptake of M1NVs: M2 macrophages were prepared by the IL–4 treatments of BMDMs. M2 macrophages and CT26 cells were allowed to attach to culture plates containing 10% (v/v) FBS-containing medium for 24 h. Subsequently, DiI-labeled M1NVs were added into the culture at a concentration of 50 µg/mL and incubated for 4 h. Cellular uptake of M1NVs was evaluated using fluorescence microscopy. Additionally, 1 h after the treatment with DiI–labeled M1NVs, M2 macrophages and CT26 cells were washed with PBS and analyzed with FACS. qRT–PCR was performed to determine the expression 20


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of PD-L1 in CT26 cells after 50 µg/mL M1NVs or M1NVs treatment. Three samples were analyzed per group. Viability of Cells after Treatment with M1NVs: Various concentrations (10, 50, and 100 µg/mL) of M1NVs were added to cultures of M2 macrophages and CT26 cells, and the cells were incubated for 24 h. The number of live cells was determined with the use of a cell counting kit-8 (CCK-8) assay (DoGenBio Co., LTD) after 24 h, according to the manufacturer’s protocol (n=3 per group). After replenishing with fresh medium, CCK-8 solution was added into each well of 24 well plate, and the cells were incubated for 2 h. Absorbance (= relative viable cell number) was measured at 450 nm using Powerwave X340 (Bio-Tek Instruments, Winooski, Vermont, U.S). The relative viable cell number was expressed relative to the relative viable cell number of the no treatment group. qRT–PCR was performed to determine the mRNA expression of angiogenic factors (FGF–2, VEGF, and PDGFβ). Three samples were analyzed per group. Transwell Assay: M2 macrophages were co-cultured with M1 macrophages using transwell, or treated with 50 ng/mL M1NVs for 24 h. CT26 cells were cultured alone, cocultured with either untreated M2 macrophages, M2 macrophages co-cultured with M1 macrophages, M2 macrophages treated with M1NVs or M1 macrophages for 24 h. The CCK8 assay was performed on cancer cells to evaluate cancer cell growth, as mentioned earlier. M1 macrophages were co-cultured with either M2 macrophages or CT26 cells for 24 h. M2 macrophages were co-cultured with M1 macrophages or treated with M1NVs. Moreover, qRT–PCR was performed to determine the expression levels of the M1 (CD86 and IL–6) and M2 marker genes (CD206 and Fizz–1). Three samples were analyzed per group. In Vitro Analyses of Macrophage Polarization: M2 macrophages were induced as mentioned earlier. Concentrations of 50 ng/mL of either M0NVs or M1NVs were used to 21


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treat M2 macrophages for 24 h for repolarization. Moreover, qRT–PCR was performed to determine the expressions of M1 (CD86, IL–6, iNOS, and TNF–α), M2-associated genes (CD206, Fizz–1, IL–4, and IL–10) and PD-L1, as mentioned earlier. Three samples were analyzed per group. The protein expressions of the M1 macrophage marker (CD86) and M2 macrophage marker (CD206) were evaluated by an immunocytochemistry assay. Cells were fixed with 4% paraformaldehyde for 10 min at RT and washed in PBS. Primary antibodies against CD86 (Santa Cruz Biotechnology, CA, USA) and CD206 (Abcam, Cambridge, UK) were used for staining. The samples were then incubated in PBS containing Rhodamineconjugated secondary antibodies (Jackson-Immunoresearch) for 1 h at RT. All samples were mounted with mounting solution containing 4',6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA, USA) to stain the nuclei, and were imaged using a fluorescent microscope (Olympus, Tokyo, Japan). To further confirm the production of the M1 marker IL–6 and suppression of the M2 marker IL–4 in M2 macrophages treated with 50 µg/mL M1NVs for 1 day, cytokine secretion was analyzed using mouse IL–6 and IL–4 ELISA kits (R&D Systems, MN, USA) following the manufacturer’s instructions. In Vitro Macrophage Polarization by M1 BMNV treatment: M1 BMDM were induced by 100 ng/mL LPS treatment to unpolarized BMDM (M0 BMDM). qRT–PCR was performed to determine M1 marker (CD86 and IL-12) expression in M0 BMDM and M1 BMDM. BMNVs were produced from M0 BMDM and M1 BMDM by the same method for M1NV production. qRT-PCR was performed to determine the M1 marker (IL-6, iNOS, and TNF-α) mRNA expression in M2 macrophages after treatment with 50 µg of M0 BMNVs and M1 BMNVs for 24 h. Three samples were analyzed per group. LFA–1 Protein Analysis: For the analysis of LFA–1 protein on the M1NV surface, M1NVs were passively adsorbed onto 4 µM aldehyde/sulfate latex beads (Invitrogen) for 22


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visualization by FACS, as previously described.37 For the comparison group, we prepared trypsin-treated M1NVs (T-M1NVs) in which the externally exposed proteins on the M1NV membrane were removed by trypsin treatment. Briefly, 30 µg of M1NVs or T-M1NVs were incubated in a bead volume of 100 µl at room temperature (RT) for 15 min. Subsequently, 900 µl of PBS were added, and the mixture was incubated at RT for 2 h to ensure that the beads were coated with M1NVs and T-M1NVs. The bead-M1NV reaction was terminated by the addition of 100 mM glycine. The M1NV- and T-M1NV-coated beads were collected by centrifugation at 3,000g for 10 min, and were then stained with DiI fluorescein-conjugated CD11a/CD18 (LFA–1) antibodies (Biolegend, San Diego, California, U.S.) at the antibody concentration recommended by the manufacturer. The stained M1NV- and T-M1NV-coated beads were incubated at 4°C for 1 h, washed three times with PBS, and analyzed with FACS. In Vivo Imaging of M1NVs: The biodistributions of plain M1NVs, M1 macrophages and T-M1NVs were investigated using an IVIS Spectrum computed tomography system (PerkinElmer, Waltham, MA, U.S.) after intravenous injections of M1NVs, M1 macrophages and T-M1NVs into CT26-bearing mice. Briefly, 100 µg M1NVs, T-M1NVs, or 2×106 of M1 macrophages were labeled with VivoTrack 680 NIR fluorescent imaging agent (PerkinElmer, Waltham, MA, U.S.) according to the manufacturer's instructions, suspended in 100 µL of PBS, and intravenously injected via the tail vein (n=3 mice per group). At 24 h after injection, the mice were sacrificed, and VivoTrack 680 fluorescence images of the major organs (heart, lung, liver, kidney, and spleen) and tumor were acquired at a 670 nm excitation wavelength and a 720 nm emission wavelength. Then the relative fluorescence intensities of the organs and tumor were quantified using the software Living Image 3.1. Organ Toxicity and In Vivo Immunogenicity Analysis: For organ toxicity analysis, whole blood was collected from five CT26 tumor-bearing mice in each group 1 day before 23


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and 1, 6, 13 and 20 days after the intravenous administration of 100 µL PBS or 50 µg M1NV suspended in 100 µL of PBS. The supernatant (serum) from the whole blood was collected after centrifugation. The levels of AST and ALT in each collected serum were determined using AST and ALT activity kit (Sigma-Aldrich) according to the manufacturer’s instructions. For histological analysis, major organs (liver, lung, spleen, heart and kidney) were retrieved at 20 days after the first injection and fixed with 4% paraformaldehyde in PBS. The samples were embedded in OCT compound, sectioned at a thickness of 10 µm, stained using hematoxylin and eosin and imaged using an optical microscope. To investigate the immunogenicity of M1NVs, serum samples from the mice were used to determine the circulating IgG antibody levels by enzyme linked immunosorbent assay (ELISA) as previously described.97 Briefly, flat-bottom 96-well plates were first coated with 1 mg of M1NV overnight, blocked with 1% BSA. The level of IgG specific to M1NVs was determined using goat anti-mouse IgG antibody (BioLegend). In Vivo Tumor Growth: Animal experiments were performed using six-week-old female BALB/c mice (Orient Bio, Korea). The animal study was approved by the Institutional Animal Care and Use Committee of Seoul National University (SNU–170922–1). Mice were anesthetized with injection of rompun (10 mg/kg) and ketamine (100 mg/kg), and CT26 cells (5×106 cells in 100 µL PBS per mouse) were subcutaneously injected into a flank of the mice. The tumor volume (V) was estimated according to an ellipsoidal calculation, whereby V = a×b2×0.5, where a is the largest and b is the smallest diameters of the tumor ellipsoid. When the tumor size reached 70 mm3, either 50 µg M1NVs, 100 µg anti-PD–L1 mAb (aPD–L1, BioXcell), or a combination of 50 µg M1NVs and 100 µg aPD–L1 were injected intravenously every three days for a total of three times. The tumor volume was determined every three days. 24


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Tumor Tissue Assessments: To compare mRNA expressions of M1 (CD86, iNOS, and TNF–α), M2 macrophage-associated genes (Arg–1, Fizz–1, and IL–10), VEGF, MMP9 and IFN-γ in the tumor tissues, tumor tissues were removed from euthanized mice 1 day after the last injection and were homogenized after grinding with a scalpel. In addition, qRT–PCR was performed as mentioned earlier. For immunohistochemistry and H&E staining, tumor tissues were fixed with formaldehyde, embedded in paraffin, and sliced at a thickness of 4 µm. The sections of the tumor tissues were stained with H&E and were examined using an optical microscope (Olympus, Tokyo, Japan). Apoptotic cells in the tissue sections were determined using an DeadEnd™ Fluorometric TUNEL System (Promega) according to the manufacturer's instructions. Immunohistochemistry staining for M1 and M2 markers was performed. Sections were treated with sodium citrate buffer (10 mM Sodium citrate and 0.05% Tween 20, pH 6.0) for 10 min at 85oC for antigen retrieval after hydration. Prior to staining, the tissue sections on slides were blocked with 5% (v/v) normal goat serum (Gibco) and incubated with primary antibodies against CD86 (Santa Cruz Biotechnology), iNOS (Abcam), CD80 (BioLegend), arginase 1 (Abcam), CD163 (Abcam), CD206 (Abcam) and CD68 (BioLegend) for 18 h at 4°C. The sections were washed three times with PBS and were incubated for 1 h with rhodamine-conjugated secondary antibodies. After washing with PBS, the slides were mounted with a mounting medium (VectaMount mounting medium, Vector Labs Inc., Burlingame, CA, USA). Flow Cytometry Analyses of Intratumoral T Cells: Tumor tissues were made into single-cell suspensions and tumor-infiltrating lymphocytes were quantitatively analyzed by flow cytometry after immunofluorescence staining. In brief, tumor tissues were harvested from euthanized mice 1 day after the last injection and chopped into small pieces using a 25


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razor blade, and digested enzymatically with collagenase Ⅳ (1 mg/mL) and DNase (0.1 mg/mL) at 37°C for 60–80 min. Cells were collected, stained by the addition of a cocktail of fluorescence-conjugated antibodies and analyzed by FACS AriaII (BD Biosciences) installed at the National Center for Inter-university Research Facilities(NCIRF) at Seoul National University. The CD3 positive T cells were initially identified and then further gated by the expression of CD4 and CD8. The following monoclonal anti-mouse antibodies were used: FITC anti-mouse CD3, APC anti-mouse CD4, PE anti-mouse CD8a and PE anti-mouse FOXP3 Antibody (Biolegned). Statistical Analyses: Data were presented as mean ± standard deviation (SD). Statistical comparisons were performed using unpaired Student’s t-test for two group comparisons, oneway analysis of variance (ANOVA) for comparisons of more than three groups with the Turkey’s significant difference post hoc test or two-way analysis of variance (ANOVA) for comparisons of two independent variables with the Bonferroni post-tests using Prism 6 (GraphPad software). Differences were considered statistically significant when p < 0.05.

ASSOCIATED CONTENT The Supporting Information is available free of charge via the Internet at http://pubs.acs.org. The authors declare no competing financial interest.

AUTHOR INFORMATION Corresponding Author Byung-Soo Kim, Ph.D., School of Chemical and Biological Engineering, Seoul National University, Seoul 08826, Republic of Korea. 26


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Tel: +82-2-880-1509; Fax: 82-2-888-1604; E-mail: [email protected] ORCID Mikyung Kang: 0000-0002-0592-1648 Byung-Soo Kim: 0000-0001-5210-7430 Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

†

Y. W. Choo and M. Kang contributed

equally to this work.

ACKNOWLEDGMENTS This

study

was

supported

by

the

National

Research

Foundation

of

Korea

(2017R1A2B3005842).

REFERENCE (1)

Emens, L. A.; Middleton, G. The Interplay of Immunotherapy and Chemothera

py: Harnessing Potential Synergies. Cancer Immunol. Res. 2015, 3, 436-443. (2)

Wang, M.; Yin, B.; Wang, H. Y.; Wang, R. F. Current Advances in T-cell-ba

sed Cancer Immunotherapy. Immunotherapy 2014, 6, 1265-1278. (3)

Brusa, D.; Serra, S.; Coscia, M.; Rossi, D.; D'Arena, G.; Laurenti, L.; Jaksic,

O.; Fedele, G.; Inghirami, G.; Gaidano, G.; Malavasi, F.; Deaglio, S. The PD-1/PD-L 1 Axis Contributes to T-cell Dysfunction in Chronic Lymphocytic Leukemia. Haemato logica 2013, 98, 953-963. (4)

Gordon, S. R.; Maute, R. L.; Dulken, B. W.; Hutter, G.; George, B. M.; Mc 27


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Cracken, M. N.; Gupta, R.; Tsai, J. M.; Sinha, R.; Corey, D.; Ring, A. M.; Connolly , A. J.; Weissman, I. L. PD-1 Expression by Tumour-Associated Macrophages Inhibits Phagocytosis and Tumour Immunity. Nature 2017, 545, 495-499. (5)

McClanahan, F.; Hanna, B.; Miller, S.; Clear, A. J.; Lichter, P.; Gribben, J. G

.; Seiffert, M. PD-L1 Checkpoint Blockade Prevents Immune Dysfunction and Leukem ia Development in A Mouse Model of Chronic Lymphocytic Leukemia. Blood 2015, 126, 203-211. (6)

Singh, P. P.; Sharma, P. K.; Krishnan, G.; Lockhart, A. C. Immune Checkpoi

nts and Immunotherapy for Colorectal Cancer. Gastroenterol. Rep. (Oxf) 2015, 3, 289297. (7)

Sagiv-Barfi, I.; Kohrt, H. E.; Czerwinski, D. K.; Ng, P. P.; Chang, B. Y.; Le

vy, R. Therapeutic Antitumor Immunity by Checkpoint Blockade is Enhanced by Ibrut inib, An Inhibitor of Both BTK and ITK. Proc. Natl. Acad. Sci. U.S.A. 2015, 112, E 966-972. (8)

Pardoll, D. M. The Blockade of Immune Checkpoints in Cancer Immunotherap

y. Nat. Rev. Cancer 2012, 12, 252-264. (9)

Alsaab, H. O.; Sau, S.; Alzhrani, R.; Tatiparti, K.; Bhise, K.; Kashaw, S. K.;

Iyer, A. K. PD-1 and PD-L1 Checkpoint Signaling Inhibition for Cancer Immunothera py: Mechanism, Combinations, and Clinical Outcome. Front. Pharmacol. 2017, 8, 561. (10)

Postow, M. A.; Chesney, J.; Pavlick, A. C.; Robert, C.; Grossmann, K.; McD

ermott, D.; Linette, G. P.; Meyer, N.; Giguere, J. K.; Agarwala, S. S.; Shaheen, M.; Ernstoff, M. S.; Minor, D.; Salama, A. K.; Taylor, M.; Ott, P. A.; Rollin, L. M.; Ho rak, C.; Gagnier, P.; Wolchok, J. D.; Hodi, F. S. Nivolumab and Ipilimumab versus I pilimumab in Untreated Melanoma. N. Engl. J. Med. 2015, 372, 2006-2017. 28


Page 28 of 57

Page 29 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

(11)

Kavanagh, B.; O'Brien, S.; Lee, D.; Hou, Y.; Weinberg, V.; Rini, B.; Allison,

J. P.; Small, E. J.; Fong, L. CTLA4 Blockade Expands FoxP3+ Regulatory and Acti vated Effector CD4+ T Cells in a Dose-dependent Fashion. Blood 2008, 112, 1175-11 83. (12)

Zanganeh, S.; Hutter, G.; Spitler, R.; Lenkov, O.; Mahmoudi, M.; Shaw, A.;

Pajarinen, J. S.; Nejadnik, H.; Goodman, S.; Moseley, M.; Coussens, L. M.; DaldrupLink, H. E. Iron Oxide Nanoparticles Inhibit Tumour Growth by Inducing Pro-inflam matory Macrophage Polarization in Tumour Tissues. Nat. Nanotechnol. 2016, 11, 986994. (13)

Georgoudaki, A. M.; Prokopec, K. E.; Boura, V. F.; Hellqvist, E.; Sohn, S.;

Ostling, J.; Dahan, R.; Harris, R. A.; Rantalainen, M.; Klevebring, D.; Sund, M.; Bra ge, S. E.; Fuxe, J.; Rolny, C.; Li, F.; Ravetch, J. V.; Karlsson, M. C. Reprogrammin g Tumor-Associated Macrophages by Antibody Targeting Inhibits Cancer Progression a nd Metastasis. Cell Rep. 2016, 15, 2000-2011. (14)

Pyonteck, S. M.; Akkari, L.; Schuhmacher, A. J.; Bowman, R. L.; Sevenich,

L.; Quail, D. F.; Olson, O. C.; Quick, M. L.; Huse, J. T.; Teijeiro, V.; Setty, M.; Le slie, C. S.; Oei, Y.; Pedraza, A.; Zhang, J.; Brennan, C. W.; Sutton, J. C.; Holland, E. C.; Daniel, D.; Joyce, J. A. CSF-1R Inhibition Alters Macrophage Polarization and Blocks Glioma Progression. Nat. Med. (N. Y., NY, U. S.) 2013, 19, 1264-1272. (15)

Rolny, C.; Mazzone, M.; Tugues, S.; Laoui, D.; Johansson, I.; Coulon, C.; Sq

uadrito, M. L.; Segura, I.; Li, X.; Knevels, E.; Costa, S.; Vinckier, S.; Dresselaer, T.; Akerud, P.; De Mol, M.; Salomaki, H.; Phillipson, M.; Wyns, S.; Larsson, E.; Buyss chaert, I.; et al. HRG Inhibits Tumor Growth and Metastasis by Inducing Macrophage Polarization and Vessel Normalization through Downregulation of PlGF. Cancer Cell 29


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

2011, 19, 31-44. (16)

Chanmee, T.; Ontong, P.; Konno, K.; Itano, N. Tumor-Associated Macrophage

s as Major Players in The Tumor Microenvironment. Cancers (Basel) 2014, 6, 1670-1 690. (17)

Wang, M.; Zhao, J.; Zhang, L.; Wei, F.; Lian, Y.; Wu, Y.; Gong, Z.; Zhang,

S.; Zhou, J.; Cao, K.; Li, X.; Xiong, W.; Li, G.; Zeng, Z.; Guo, C. Role of Tumor Microenvironment in Tumorigenesis. J. Cancer 2017, 8, 761-773. (18)

Schreiber, R. D.; Old, L. J.; Smyth, M. J. Cancer Immunoediting: Integrating

Immunity's Roles in Cancer Suppression and Promotion. Science 2011, 331, 1565-1570 . (19)

Zou, W. Immunosuppressive Networks in The Tumour Environment and Their

Therapeutic Relevance. Nat. Rev. Cancer 2005, 5, 263-274. (20)

Azmi, A. S.; Bao, B.; Sarkar, F. H. Exosomes in Cancer Development, Metas

tasis and Drug Resistance: A Comprehensive Review. Cancer Metastasis Rev. 2013, 3 2, 623-642. (21)

Burkholder, B.; Huang, R. Y.; Burgess, R.; Luo, S.; Jones, V. S.; Zhang, W.;

Lv, Z. Q.; Gao, C. Y.; Wang, B. L.; Zhang, Y. M.; Huang, R. P. Tumor-induced P erturbations of Cytokines and Immune Cell Networks. Biochim. Biophys. Acta 2014, 1 845, 182-201. (22)

Colegio, O. R.; Chu, N. Q.; Szabo, A. L.; Chu, T.; Rhebergen, A. M.; Jairam

, V.; Cyrus, N.; Brokowski, C. E.; Eisenbarth, S. C.; Phillips, G. M.; Cline, G. W.; Phillips, A. J.; Medzhitov, R. Functional Polarization of Tumour-Associated Macropha ges by Tumour-derived Lactic Acid. Nature 2014, 513, 559-563. (23)

Dannenmann, S. R.; Thielicke, J.; Stockli, M.; Matter, C.; von Boehmer, L.; 30


Page 30 of 57

Page 31 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Cecconi, V.; Hermanns, T.; Hefermehl, L.; Schraml, P.; Moch, H.; Knuth, A.; van de n Broek, M. Tumor-Associated Macrophages Subvert T-cell Function and Correlate wi th Reduced Survival in Clear Cell Renal Cell Carcinoma. Oncoimmunology 2013, 2, e 23562. (24)

Mosser, D. M.; Edwards, J. P. Exploring The Full Spectrum of Macrophage

Activation. Nat. Rev. Immunol. 2008, 8, 958-969. (25)

Riabov, V.; Gudima, A.; Wang, N.; Mickley, A.; Orekhov, A.; Kzhyshkowska,

J. Role of Tumor Associated Macrophages in Tumor Angiogenesis and Lymphangiog enesis. Front Physiol. 2014, 5, 75. (26)

Liu, J.; Zhang, N.; Li, Q.; Zhang, W.; Ke, F.; Leng, Q.; Wang, H.; Chen, J.;

Wang, H. Tumor-Associated Macrophages Recruit CCR6+ Regulatory T Cells and Pr omote The Development of Colorectal Cancer via Enhancing CCL20 Production in Mi ce. PLoS ONE 2011, 6, e19495. (27)

Mills, C. D.; Lenz, L. L.; Harris, R. A. A Breakthrough: Macrophage-Directed

Cancer Immunotherapy. Cancer Res. 2016, 76, 513-516. (28)

Hanahan, D.; Coussens, L. M. Accessories to The Crime: Functions of Cells

Recruited to The Tumor Microenvironment. Cancer Cell 2012, 21, 309-322. (29)

Beatty, G. L.; Chiorean, E. G.; Fishman, M. P.; Saboury, B.; Teitelbaum, U.

R.; Sun, W.; Huhn, R. D.; Song, W.; Li, D.; Sharp, L. L.; Torigian, D. A.; O'Dwyer , P. J.; Vonderheide, R. H. CD40 Agonists Alter Tumor Stroma and Show Efficacy Against Pancreatic Carcinoma in Mice and Humans. Science 2011, 331, 1612-1616. (30)

Gunderson, A. J.; Kaneda, M. M.; Tsujikawa, T.; Nguyen, A. V.; Affara, N. I

.; Ruffell, B.; Gorjestani, S.; Liudahl, S. M.; Truitt, M.; Olson, P.; Kim, G.; Hanahan , D.; Tempero, M. A.; Sheppard, B.; Irving, B.; Chang, B. Y.; Varner, J. A.; Cousse 31


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ns, L. M. Bruton Tyrosine Kinase-Dependent Immune Cell Cross-talk Drives Pancreas Cancer. Cancer Discov. 2016, 6, 270-285. (31)

Vlassov, A. V.; Magdaleno, S.; Setterquist, R.; Conrad, R. Exosomes: Current

Knowledge of Their Composition, Biological Functions, and Diagnostic and Therapeu tic Potentials. Biochim. Biophys. Acta 2012, 1820, 940-948. (32)

Jang, S.; Kim, O.; Yoon, C.; Choi, D.; Roh, T.; Park, J.; Nilsson, J.; Lötvall,

J.; Kim, Y.; Gho, Y. Bioinspired Exosome-Mimetic Nanovesicles for Targeted Delive ry of Chemotherapeutics to Malignant Tumors. ACS Nano 2013, 7, 7698-7710. (33)

Oh, K.; Kim, S.; Kim, D.; Seo, M.; Lee, C.; Lee, H.; Oh, J.; Choi, E.; Lee,

D.; Gho, Y.; Park, K. In Vivo Differentiation of Therapeutic Insulin-Producing Cells f rom Bone Marrow Cells via Extracellular Vesicle-Mimetic Nanovesicles. ACS Nano 2 015, 9, 11718–11727. (34)

Narayanan, R.; Huang, C. C.; Ravindran, S. Hijacking the Cellular Mail: Exos

ome Mediated Differentiation of Mesenchymal Stem Cells. Stem Cells In.t 2016, 2016, 3808674. (35)

Takeda, Y. S.; Xu, Q. Neuronal Differentiation of Human Mesenchymal Stem

Cells Using Exosomes Derived from Differentiating Neuronal Cells. PLoS ONE 2015, 10, e0135111. (36)

Kim, M. S.; Haney, M. J.; Zhao, Y.; Mahajan, V.; Deygen, I.; Klyachko, N.

L.; Inskoe, E.; Piroyan, A.; Sokolsky, M.; Okolie, O.; Hingtgen, S. D.; Kabanov, A. V.; Batrakova, E. V. Development of Exosome-encapsulated Paclitaxel to Overcome MDR in Cancer Cells. Nanomedicine 2016, 12, 655-664. (37)

El-Andaloussi, S.; Lee, Y.; Lakhal-Littleton, S.; Li, J.; Seow, Y.; Gardiner, C.;

Alvarez-Erviti, L.; Sargent, I. L.; Wood, M. J. A. Exosome-Mediated Delivery of si 32


Page 32 of 57

Page 33 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

RNA in vitro and in vivo. Nat. Protoc. 2012, 7, 2112. (38)

Tan, S.; Wu, T.; Zhang, D.; Zhang, Z. Cell or Cell Membrane-based Drug De

livery Systems. Theranostics 2015, 5, 863-881. (39)

Soltani, F.; Parhiz, H.; Mokhtarzadeh, A.; Ramezani, M. Synthetic and Biologi

cal Vesicular Nano-Carriers Designed for Gene Delivery. Curr. Pharm. Des. 2015, 21, 6214-6235. (40)

Molinaro, R.; Corbo, C.; Martinez, J. O.; Taraballi, F.; Evangelopoulos, M.;

Minardi, S.; Yazdi, I. K.; Zhao, P.; De Rosa, E.; Sherman, M. B.; De Vita, A.; Tole dano Furman, N. E.; Wang, X.; Parodi, A.; Tasciotti, E. Biomimetic Proteolipid Vesic les for Targeting Inflamed Tissues. Nat. Mater. 2016, 15, 1037-1046. (41)

Cheng, L.; Wang, Y.; Huang, L. Exosomes from M1-Polarized Macrophages P

otentiate the Cancer Vaccine by Creating a Pro-inflammatory Microenvironment in Th e Lymph Node. Mol. Ther. 2017, 25, 1665-1675. (42)

Jo, W.; Kim, J.; Yoon, J.; Jeong, D.; Cho, S.; Jeong, H.; Yoon, Y. J.; Kim,

S. C.; Gho, Y. S.; Park, J. Large-scale Generation of Cell-derived Nanovesicles. Nano scale 2014, 6, 12056-12064. (43)

Montecalvo, A.; Larregina, A. T.; Shufesky, W. J.; Stolz, D. B.; Sullivan, M.

L.; Karlsson, J. M.; Baty, C. J.; Gibson, G. A.; Erdos, G.; Wang, Z.; Milosevic, J.; Tkacheva, O. A.; Divito, S. J.; Jordan, R.; Lyons-Weiler, J.; Watkins, S. C.; Morelli, A. E. Mechanism of Transfer of Functional microRNAs between Mouse Dendritic Ce lls via Exosomes. Blood 2012, 119, 756-766. (44)

Squadrito, M. L.; Etzrodt, M.; De Palma, M.; Pittet, M. J. MicroRNA-mediate

d Control of Macrophages and Its Implications for Cancer. Trends Immunol. 2013, 34, 350-359. 33


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(45)

Yamasaki, T.; Seki, N.; Yoshino, H.; Itesako, T.; Yamada, Y.; Tatarano, S.; H

idaka, H.; Yonezawa, T.; Nakagawa, M.; Enokida, H. Tumor-suppressive microRNA-1 291 Directly Regulates Glucose Transporter 1 in Renal Cell Carcinoma. Cancer Sci. 2 013, 104, 1411-1419. (46)

Zhang, Y.; Zhang, M.; Zhong, M.; Suo, Q.; Lv, K. Expression Profiles of mi

RNAs in Polarized Macrophages. Int. J. Mol. Med. 2013, 31, 797-802. (47)

Essandoh, K.; Li, Y.; Huo, J.; Fan, G. C. MiRNA-Mediated Macrophage Polar

ization and Its Potential Role in The Regulation of Inflammatory Response. Shock 20 16, 46, 122-131. (48)

Self-Fordham, J. B.; Naqvi, A. R.; Uttamani, J. R.; Kulkarni, V.; Nares, S. M

icroRNA: Dynamic Regulators of Macrophage Polarization and Plasticity. Front Immun ol. 2017, 8, 1062. (49)

Wu, X. Q.; Dai, Y.; Yang, Y.; Huang, C.; Meng, X. M.; Wu, B. M.; Li, J.

Emerging Role of microRNAs in Regulating Macrophage Activation and Polarization i n Immune Response and Inflammation. Immunology 2016, 148, 237-248. (50)

Sica, A.; Larghi, P.; Mancino, A.; Rubino, L.; Porta, C.; Totaro, M. G.; Rimo

ldi, M.; Biswas, S. K.; Allavena, P.; Mantovani, A. Macrophage Polarization in Tumo ur Progression. Semin. Cancer Biol. 2008, 18, 349-355. (51)

Yoo, J.-W.; Chambers, E.; Mitragotri, S. Factors that Control The Circulation

Time of Nanoparticles in Blood: Challenges, Solutions and Future Prospects. Curr. Ph arm. Des. 2010, 16, 2298-2307. (52)

Williams, C. B.; Yeh, E. S.; Soloff, A. C. Tumor-Associated Macrophages: U

nwitting Accomplices in Breast Cancer Malignancy. NPJ Breast Cancer 2016, 2, 1502 5. 34


Page 34 of 57

Page 35 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

(53)

Sato-Kaneko, F.; Yao, S.; Ahmadi, A.; Zhang, S. S.; Hosoya, T.; Kaneda, M.

M.; Varner, J. A.; Pu, M.; Messer, K. S.; Guiducci, C.; Coffman, R. L.; Kitaura, K. ; Matsutani, T.; Suzuki, R.; Carson, D. A.; Hayashi, T.; Cohen, E. E. W. Combinatio n Immunotherapy with TLR Agonists and Checkpoint Inhibitors Suppresses Head and Neck Cancer. JCI Insight 2017, 2, e93397. (54)

Kuang, D. M.; Zhao, Q.; Peng, C.; Xu, J.; Zhang, J. P.; Wu, C.; Zheng, L.

Activated Monocytes in Peritumoral Stroma of Hepatocellular Carcinoma Foster Immu ne Privilege and Disease Progression through PD-L1. J. Exp. Med. 2009, 206, 1327-1 337. (55)

Mulcahy, L. A.; Pink, R. C.; Carter, D. R. Routes and Mechanisms of Extrac

ellular Vesicle Uptake. J. Extracell. Vesicles 2014, 3, 24641. (56)

Prada, I.; Meldolesi, J. Binding and Fusion of Extracellular Vesicles to The Pl

asma Membrane of Their Cell Targets. Int. J. Mol. Sci. 2016, 17, 1296. (57)

Tian, T.; Yan- Liang, Z.; Fei- Hu, H.; Yuan- Yuan, W.; Ning- Ping, H.; Zhon

g- Dang, X. Dynamics of Exosome Internalization and Trafficking. J. Cell. Physiol. 2 013, 228, 1487-1495. (58)

Feng, D.; Zhao, W.-L.; Ye, Y.-Y.; Bai, X.-C.; Liu, R.-Q.; Chang, L.-F.; Zhou,

Q.; Sui, S.-F. Cellular Internalization of Exosomes Occurs through Phagocytosis. Traf fic 2010, 11, 675-687. (59)

Parolini, I.; Federici, C.; Raggi, C.; Lugini, L.; Palleschi, S.; De Milito, A.; C

oscia, C.; Iessi, E.; Logozzi, M.; Molinari, A.; Colone, M.; Tatti, M.; Sargiacomo, M. ; Fais, S. Microenvironmental pH Is A Key Factor for Exosome Traffic in Tumor Ce lls. J. Biol. Chem. 2009, 284, 34211-34222. (60)

Escrevente, C.; Keller, S.; Altevogt, P.; Costa, J. Interaction and Uptake of Ex 35


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

osomes by Ovarian Cancer Cells. BMC Cancer 2011, 11, 108-117. (61)

Nanbo, A.; Kawanishi, E.; Yoshida, R.; Yoshiyama, H. Exosomes Derived fro

m Epstein-Barr Virus-Infected Cells Are Internalized via Caveola-Dependent Endocytos is and Promote Phenotypic Modulation in Target Cells. J. Virol. 2013, 87, 10334-103 47. (62)

Goh, W. J.; Zou, S.; Ong, W. Y.; Torta, F.; Alexandra, A. F.; Schiffelers, R.

M.; Storm, G.; Wang, J.-W.; Czarny, B.; Pastorin, G. Bioinspired Cell-Derived Nano vesicles versus Exosomes as Drug Delivery Systems: a Cost-Effective Alternative. Sci. Rep. 2017, 7, 14322. (63)

Kou, L.; Sun, J.; Zhai, Y.; He, Z. The Endocytosis and Intracellular Fate of

Nanomedicines: Implication for Rational Design. AJPS 2013, 8, 1-10. (64)

Mantovani, A.; Sica, A.; Sozzani, S.; Allavena, P.; Vecchi, A.; Locati, M. Th

e Chemokine System in Diverse Forms of Macrophage Activation and Polarization. Tr ends Immunol. 2004, 25, 677-686. (65)

Lee, H. W.; Choi, H. J.; Ha, S. J.; Lee, K. T.; Kwon, Y. G. Recruitment of

Monocytes/Macrophages in Different Tumor Microenvironments. Biochim. Biophys. Act a 2013, 1835, 170-179. (66)

Astrof, N. S.; Salas, A.; Shimaoka, M.; Chen, J.; Springer, T. A. Importance

of Force Linkage in Mechanochemistry of Adhesion Receptors. Biochem. 2006, 45, 15 020-15028. (67)

Chesnutt, B. C.; Smith, D. F.; Raffler, N. A.; Smith, M. L.; White, E. J.; Le

y, K. Induction of LFA-1-dependent Neutrophil Rolling on ICAM-1 by Engagement o f E-selectin. Microcirculation 2006, 13, 99-109. (68)

Melis, M.; Spatafora, M.; Melodia, A.; Pace, E.; Gjomarkaj, M.; Merendino, 36


Page 36 of 57

Page 37 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

A. M.; Bonsignore, G. ICAM-1 Expression by Lung Cancer Cell Lines: Effects of Up regulation by Cytokines on The Interaction with LAK Cells. Eur. Respir. J. 1996, 9, 1831-1838. (69)

Zhang, P.; Goodrich, C.; Fu, C.; Dong, C. Melanoma Upregulates ICAM-1 Ex

pression on Endothelial Cells through Engagement of Tumor CD44 with Endothelial E -selectin and Activation of a PKCalpha-p38-SP-1 Pathway. FASEB J. 2014, 28, 45914609. (70)

Parodi, A.; Quattrocchi, N.; van de Ven, A. L.; Chiappini, C.; Evangelopoulos

, M.; Martinez, J. O.; Brown, B. S.; Khaled, S. Z.; Yazdi, I. K.; Enzo, M. V.; Isenh art, L.; Ferrari, M.; Tasciotti, E. Synthetic Nanoparticles Functionalized with Biomimet ic Leukocyte Membranes Possess Cell-like Functions. Nat. Nanotechnol. 2013, 8, 61-6 8. (71)

Kooijmans, S. A.; Vader, P.; van Dommelen, S. M.; van Solinge, W. W.; Sch

iffelers, R. M. Exosome Mimetics: A Novel Class of Drug Delivery Systems. Int. J. Nanomedicine 2012, 7, 1525-1541. (72)

Kim, O. Y.; Lee, J.; Gho, Y. S. Extracellular Vesicle Mimetics: Novel Altern

atives to Extracellular Vesicle-based Theranostics, Drug Delivery, and Vaccines. Semin. Cell Dev. Biol. 2017, 67, 74-82. (73)

Luan, X.; Sansanaphongpricha, K.; Myers, I.; Chen, H.; Yuan, H.; Sun, D. En

gineering Exosomes as Refined Biological Nanoplatforms for Drug Delivery. Acta Pha rmacol. Sin. 2017, 38, 754. (74)

Filipe, V.; Jiskoot, W.; Basmeleh, A. H.; Halim, A.; Schellekens, H.; Brinks,

V. Immunogenicity of Different Stressed IgG Monoclonal Antibody Formulations in I mmune Tolerant Transgenic Mice. mAbs 2012, 4, 740-752. 37


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(75)

Haribhai, D.; Ziegelbauer, J.; Jia, S.; Upchurch, K.; Yan, K.; Schmitt, E. G.;

Salzman, N. H.; Simpson, P.; Hessner, M. J.; Chatila, T. A.; Williams, C. B. Alternat ively Activated Macrophages Boost Induced Regulatory T and Th17 Cell Responses d uring Immunotherapy for Colitis. J. Immunol. 2016, 196, 3305-3317. (76)

Jablonski, K. A.; Amici, S. A.; Webb, L. M.; Ruiz-Rosado, J. d. D.; Popovic

h, P. G.; Partida-Sanchez, S.; Guerau-de-Arellano, M. Novel Markers to Delineate Mu rine M1 and M2 Macrophages. PLoS ONE 2015, 10, e0145342. (77)

Corliss, B. A.; Azimi, M. S.; Munson, J. M.; Peirce, S. M.; Murfee, W. L.

Macrophages: An Inflammatory Link Between Angiogenesis and Lymphangiogenesis. Microcirculation 2016, 23, 95-121. (78)

Hao, N. B.; Lu, M. H.; Fan, Y. H.; Cao, Y. L.; Zhang, Z. R.; Yang, S. M.

Macrophages in Tumor Microenvironments and The Progression of Tumors. Clin. Dev. Immunol. 2012, 2012, 948098. (79)

Arango Duque, G.; Descoteaux, A. Macrophage Cytokines: Involvement in Im

munity and Infectious Diseases. Front Immunol. 2014, 5, 491. (80)

Teixeira, L. K.; Fonseca, B. P. F.; Vieira-de-Abreu, A.; Barboza, B. A.; Robb

s, B. K.; Bozza, P. T.; Viola, J. P. B. IFN-gamma Production by CD8+ T Cells De pends on NFAT1 Transcription Factor and Regulates Th Differentiation. J. Immunol. 2 005, 175, 5931-5939. (81)

Wang, D.; DuBois, R. N. Immunosuppression Associated with Chronic Inflam

mation in The Tumor Microenvironment. Carcinogenesis 2015, 36, 1085-1093. (82)

Yu, P.; Steel, J. C.; Zhang, M.; Morris, J. C.; Waldmann, T. A. Simultaneous

Blockade of Multiple Immune System Inhibitory Checkpoints Enhances Antitumor Ac tivity Mediated by Interleukin-15 in A Murine Metastatic Colon Carcinoma Model. Cl 38


Page 38 of 57

Page 39 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

in. Cancer Res. 2010, 16, 6019-6028. (83)

Herbst, R. S.; Soria, J.-C.; Kowanetz, M.; Fine, G. D.; Hamid, O.; Gordon,

M. S.; Sosman, J. A.; McDermott, D. F.; Powderly, J. D.; Gettinger, S. N.; Kohrt, H . E. K.; Horn, L.; Lawrence, D. P.; Rost, S.; Leabman, M.; Xiao, Y.; Mokatrin, A.; Koeppen, H.; Hegde, P. S.; Mellman, I.; et al. Predictive Correlates of Response to T he Anti-PD-L1 Antibody MPDL3280A in Cancer Patients. Nature 2014, 515, 563. (84)

Mediavilla-Varela, M.; Page, M. M.; Kreahling, J.; Antonia, S. J.; Altiok, S.

Anti-PD1 Treatment to Induce M1 Polarization of Tumor Infiltrating Macrophages in A 3D ex vivo System of Lung Cancer Patients. J. Clin. Oncol. 2017, 35, e23090-e23 090. (85)

Biswas, S. K.; Mantovani, A. Macrophage Plasticity and Interaction with Lym

phocyte Subsets: Cancer as A Paradigm. Nat. Immunol. 2010, 11, 889. (86)

Duluc, D.; Corvaisier, M.; Blanchard, S.; Catala, L.; Descamps, P.; Gamelin,

E.; Ponsoda, S.; Delneste, Y.; Hebbar, M.; Jeannin, P. Interferon-gamma Reverses The Immunosuppressive and Protumoral Properties and Prevents the Generation of Human Tumor-Associated Macrophages. Int. J. Cancer 2009, 125, 367-373. (87)

Shankaran, V.; Ikeda, H.; Bruce, A. T.; White, J. M.; Swanson, P. E.; Old, L

. J.; Schreiber, R. D. IFNgamma and Lymphocytes Prevent Primary Tumour Develop ment and Shape Tumour Immunogenicity. Nature 2001, 410, 1107-1111. (88)

Lanitis, E.; Dangaj, D.; Irving, M.; Coukos, G. Mechanisms Regulating T-cell

Infiltration and Activity in Solid Tumors. Ann. Oncol. 2017, 28, xii18-xii32. (89)

Liu, C.; Workman, C. J.; Vignali, D. A. Targeting Regulatory T Cells in Tum

ors. FEBS J. 2016, 283, 2731-2748. (90)

Li, X.; Kostareli, E.; Suffner, J.; Garbi, N.; Hammerling, G. J. Efficient Treg 39


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Depletion Induces T-cell Infiltration and Rejection of Large Tumors. Eur. J. Immunol. 2010, 40, 3325-3335. (91)

Niedbala, W.; Wei, X. Q.; Piedrafita, D.; Xu, D.; Liew, F. Y. Effects of Nitri

c Oxide on The Induction and Differentiation of Th1 cells. Eur. J. Immunol. 1999, 2 9, 2498-2505. (92)

Sinha, P.; Clements, V. K.; Ostrand-Rosenberg, S. Reduction of Myeloid-Deriv

ed Suppressor Cells and Induction of M1 Macrophages Facilitate the Rejection of Est ablished Metastatic Disease. Eur. J. Immunol. 2005, 174, 636-645. (93)

Lee, S.; Kivimae, S.; Dolor, A.; Szoka, F. C. Macrophage-based Cell Therapie

s: The Long and Winding Road. J. Controlled Release 2016, 240, 527-540. (94)

Weischenfeldt, J.; Porse, B. Bone Marrow-Derived Macrophages (BMM): Isolat

ion and Applications. Cold Spring Harbor Protoc. 2008, 2008, pdb-prot5080. (95)

Shin, H.; Han, C.; Labuz, J. M.; Kim, J.; Kim, J.; Cho, S.; Gho, Y. S.; Taka

yama, S.; Park, J. High-yield Isolation of Extracellular Vesicles Using Aqueous Two-p hase System. Sci. Rep. 2015, 5, 13103. (96)

Schmittgen, T. D.; Livak, K. J. Analyzing Real-time PCR Data by The Comp

arative CT method. Nat. Protoc. 2008, 3, 1101. (97)

Gao, J.; Kou, G.; Wang, H.; Chen, H.; Li, B.; Lu, Y.; Zhang, D.; Wang, S.;

Hou, S.; Qian, W.; Dai, J.; Zhao, J.; Zhong, Y.; Guo, Y. PE38KDEL-loaded Anti-HE R2 Nanoparticles Inhibit Breast Tumor Progression with Reduced Toxicity and Immun ogenicity. Breast Cancer Res. Treat. 2009, 115, 29-41.

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Figure 1. Schematic of the preparation of M1 macrophage-derived nanovesicles (M1NVs) and the therapeutic effects of M1NVs in terms of their ability to potentiate the anticancer efficacy of checkpoint inhibitors (anti PD–L1 antibody) in cancer therapy. M2 TAMs secrete anti-inflammatory cytokines that suppress T cells activation by aPD–L1 and promote tumor growth (Left). When M1NVs polarize M2 TAMs to M1 macrophage type, M1 macrophage secrets pro-inflammatory cytokines that activate T cells and attacks cancer cells. Also, activated T cell secret cytokines that induce M1 activation (Right).

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Figure 2. Characterization of M1NVs. (A) Size distribution and zeta potential of M1NVs and

M1 macrophage-derived exosomes, as analyzed by nanoparticle tracking (n=3) and electrophoretic light scattering (n=3), respectively. (B) Transmission electron microscopic images of M1NVs and M1 macrophage-derived exosome. (C) The size profiles of M1NVs in PBS and 10% serum (v/v) over 72 h, as evaluated by DLS measurements (n=3-6). The data represent mean ± SD. (D) Comparison of the number and protein amount of M1NVs and exosomes, both of which were produced from the same number of M1 macrophages (*p < 0.05 vs. exosome). (E) Relative mRNA amounts of pro-inflammatory M1 macrophage markers (CD86, IL–6, iNOS, and TNF–α) in M0 phenotype macrophages (M0 cell), M1 phenotype macrophages (M1 cell), M0NV (nanovesicles derived from M0 cells) and M1NV. The amounts of mRNA from genes of interest were normalized to the GAPDH amount. (F) Microarray analysis of M1, M2 macrophage-related miRNA expressions in M1NVs compared to M0NVs. Red and green indicate upregulation and downregulation, respectively, in the miRNA expressions of M1NVs as compared to those of M0NVs. The number indicates log2 ratio of M1NV miRNA to M0NV miRNA. Only miRNAs whose difference in the amount between M1NVs and M0NVs is over 1.5 fold are represented. (D)–(E) The data represent mean ± SD. (Student’s t-test).

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Figure 3. Cellular uptake and cytotoxicity of M1NVs in vitro. (A) Fluorescent image analysis

and (B) FACS analysis for M1NV uptake by M2 macrophages and cancer cells. M1NVs were labeled prior to the M1NV (50 µg/mL) treatments with a red fluorescent dye (DiI) for fluorescent imaging (4 h) and FACS analyses (1 h) (n=3, *p < 0.05 vs. cancer cell). NS: no staining. (C) Relative viable cell number of cancer cells and M2 macrophages treated with various doses of M1NV for 1 d, as evaluated by the CCK8 assay (n=3, *p < 0.05 vs. 0 µg/mL). (D) Effects of M0NV or M1NV treatments for 24 h on the angiogenic gene expressions in cancer cells. Amounts of mRNA from genes of interest were normalized to the GAPDH amount (n=3, ns: not significant). (B)–(D) One-way ANOVA, Tukey’s significant difference post hoc test. The data represent mean ± SD.

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Figure 4. Comparison of the therapeutic effects and macrophage polarization induction

effects between M1 macrophages and M1NVs. (A) Relative viable cancer cell number cocultured with untreated M2 macrophages (group 2), M1 macrophage-cocultured-M2 macrophages (group 3), M1NV treated-M2 macrophages (group 4) or with M1 macrophages (group 5, positive control) for 1 day was examined by CCK-8 assay and compared to that of cancer cell only group (group 1, negative control). n=8-10, ns: not significant. The data represent mean ± SD.*p < 0.05 vs. group 1, †p < 0.05 vs. group 2, ‡p < 0.05 vs. group 3. Twoway ANOVA and Bonferroni post-tests were used for comparison. (B) Reduced M1 marker gene expression and enhanced M2 marker gene expression in M1 macrophages co-cultured with either M2 macrophages (Group 2) or cancer cells (Group 3), as compared to the control group (M1 macrophages only, Group 1). (C) Relative mRNA expressions of M1 and M2 markers in M2 macrophages after 24 h of co-culture with M1 macrophages (Group 2) or after 24 h of M1NV treatment (Group 3), as compared to the control group (M2 macrophages only, Group 1). (B)–(C) n=3, *p < 0.05 vs. Group 1, and †p < 0.05 vs. Group 2 One-way ANOVA, Tukey’s significant difference post hoc test. The data represent mean ± SD. The mRNA expression from genes of interest was normalized to GAPDH and expressed as a relative change.

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Figure 5. Polarization of M2 macrophages to M1 macrophages in vitro induced by the

M1NV treatment. M2 macrophages were treated with either M0NVs or M1NVs for 24 h. (A) Relative expressions mRNA in M1 (iNOS, TNF–α, and IL–6) and M2 macrophages (IL–4, IL–10, and Fizz–1), as evaluated by qRT–PCR. NT denotes no treatment. (B) Angiogenic gene expressions in M2 macrophages after M0NV or M1NV treatments for 24 h. The mRNA expressions from genes of interest were normalized to GAPDH. (C) ELISA assay assessment for secretion of pro-inflammatory cytokine (IL–6) and anti-inflammatory cytokine (IL–4) from M2 macrophages treated with either M0NVs or M1NVs for 24 h. ND denotes no detection. (D) Immunofluorescence staining for CD86 (red, M1 marker) and CD206 (red, M2 marker) of M2 macrophages treated with either M0NVs or M1NVs for 24 h. Nuclei were stained with DAPI (blue). (A)–(D) n=3 per group, *p < 0.05 vs. NT, †p < 0.05 vs. M0NV (One-way ANOVA, Tukey’s significant difference post hoc test). The data represent mean ± SD.

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Figure 6. In vivo tumor targeting of M1NVs. (A) Membrane protein characterization of T-

M1NV and M1NV. The APC anti-mouse CD11a/CD18 (LFA-1) expression on T-M1NV and M1NV were shown by flow cytometry using latex beads. NS: no staining. (B) Representative fluorescence image showing biodistributions of M1NVs and T-M1NVs in the tumor tissues and major organs (heart, lung, spleen, kidney, and liver) 1 day after intravenous injections of 50 µg M1NV and 50 µg T-M1NV labeled with the Vivotrack 680 NIR fluorescent agent into CT26 tumor-bearing-mice (n=3 per group), and quantification of accumulated fluorescence signals in organs and tumor tissues (*p < 0.05 vs. T-M1NV, Student’s t-test). The graph shows relative intensity of each organ over the sum of intensities of the major organs in each group. Differences exist in the sum of fluorescence intensities of the major organs between M1NVand T-M1NV-treated mice, which might be due to the difference in cell adhesion protein (e.g., LFA-1) expression between T-M1NVs and M1NVs (Figure 6A), allowing higher adhesion of M1NVs on the endothelium of organs. (C) Representative H&E staining images of the major organs (heart, liver, lung, kidney and spleen) of the PBS- and M1NV-injected CT26 tumor bearing mice 1 day after the last treatment. Scale bars = 50 µm. (n=5 mice per group). (D) Hepatic enzyme analysis of the serum of CT26 tumor-bearing mice after intravenous injection of PBS or M1NVs. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels of the mice were monitored and the levels showed no significant difference between the PBS and M1NV groups (n=5 per group, ns: not significant). Two-way ANOVA and Bonferroni post-tests were used for statistical comparison. The data represent mean ± SD.

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Figure 7. M1NVs potentiate antitumor effects of PD–L1 inhibitors (aPD–L1) in a tumor

mouse model. (A) Schematic diagram of CT26 colon carcinoma tumor model fabrication and treatments with M1NVs and anti-PD–L1 antibody (aPD–L1). Tumor-bearing mice were treated with intravenous injection of either 50 µg M1NV, 100 µg aPD–L1, or a combination of 50 µg M1NV and 100 µg aPD–L1 every three days for a total of three times. (B) Tumor growth profiles in tumor-bearing mice treated with injections of either PBS or M1NV (n=3–5 mice per group). *p

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