Reprogramming Tumor Immune Microenvironment (TIME) and

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Reprogramming Tumor Immune Microenvironment (TIME) and Metabolism via Biomimetic Targeting Codelivery of Shikonin/JQ1 Hairui Wang,†,‡ Yisi Tang,†,‡ Yuefei Fang,†,‡ Meng Zhang,‡,§ Huiyuan Wang,‡ Zhidi He,‡,§ Bing Wang,‡ Qin Xu,† and Yongzhuo Huang*,‡ †

Institute of Tropical Medical, Guangzhou University of Chinese Medicine, 12 Jichang Road, Guangzhou 510450, China State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Shanghai 201203, China § University of Chinese Academy of Sciences, Beijing 100049, China

Nano Lett. Downloaded from pubs.acs.org by UNIV AUTONOMA DE COAHUILA on 04/06/19. For personal use only.

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S Supporting Information *

ABSTRACT: Remodeling tumor immune microenvironment (TIME) is an important strategy to lift the immunosuppression and achieve immune normalization. In this work, a mannosylated lactoferrin nanoparticulate system (Man-LF NPs) is developed for dual-targeting biomimetic codelivery of shikonin and JQ1 via the mannose receptor and LRP-1 that are overexpressed in both cancer cells and tumor-associated macrophages. The Man-LF NPs can serve as multitarget therapy for inducing immune cell death in the cancer cells, repressing glucose metabolism and repolarizing tumorassociated macrophages, and consequently, lead to remodeling the TIME (e.g., promotion of dendritic cell maturation and CD8+ T cell infiltration, as well as suppression of Treg). Moreover, JQ1 is a suppressor of PD-L1, and the Man-LF NPs can also work on PD-L1 checkpoint blockage. The results reveal the synergistic combination of shikonin and JQ1 and the treatment potency of the Man-LF NPs. Importantly, it is demonstrated that the interaction between the tumor metabolism and immunity plays an essential role in immunotherapy, and the developed drug combination and nanoformulation can target the multiple components in the complicated network of TIME, providing a potential therapeutic strategy. KEYWORDS: Biomimetic delivery, glucose metabolism, immunogenic cell death (ICD), tumor-associated macrophage, shikonin, JQ1

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TIME and accounting for a portion even up to 50% in the tumor mass.5 The repolarization of TAM from the protumor M2phenotype TAM (termed TAM2) toward antitumor M1phenotype TAM (termed TAM1) can serve as a potent target for immunotherapy.6,7 Furthermore, tumor metabolism is considered as a hallmark of cancer; tumors consume the energy sources (e.g., glucose) very actively to support the rapid tumor growth, a so-called Warburg effect.8 Lactate is the major product of glucose metabolism, which is a known immunosuppressive metabolite in TIME and an important driving factor of TAM2 polarization.9,10 Suppression of glucose metabolism and lactate production can be beneficial for immunotherapy. The cancer-immunity cycle is often used to illustrate the reinstallation of tumor immunity to cancer, which can be divided into several major steps, including cancer antigen release and presentation, priming and activation, trafficking and infiltration of T cells into tumors, recognition, and killing cancer cells.11 The

mmune checkpoint blockage therapy, represented by PD-1/ PD-L1 treatment, has been used as a first line option for several types of cancer and has significantly changed the landscape in cancer therapy. The great success has directed the mainstream effort to the “immune enhancement” strategy, and in the recent decade, many therapies have focused on enhancing immune activation; however, the “immune enhancement” strategy often results in rare objective responses and frequent immune-related adverse events.1 For example, the severe side toxicities (i.e., grade >3) are common, varying from 7−16%,2 and the fatality rates reach 0.6%,3 often rendering the treatment unsustainable. Therefore, it is believed that there should be a paradigm shift from “immune enhancement” to “immune normalization”.1 The tumor immune microenvironments (TIME) represent a complicated network of various tumor composites that suppress cancer immunity and lead to tumor immune escape.4 The regulation of the immune subsets of TIME could be helpful to remodel the TIME and achieve immune normalization. For instance, tumor-associated macrophages (TAM) are abundant, representing the major population of innate immune cells at the © XXXX American Chemical Society

Received: January 3, 2019 Revised: March 26, 2019

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Figure 1. Characterization of the NPs. (A) Size distribution and TEM of the LF NPs and Man-LF NPs. (B) ζ potential measurement. (C) Stability of LF NPs and Man-LF NPs in a serum-containing PBS. (D) In vitro release of SHK and JQ1 from the LF NPs and Man-LF NPs. (E) Fluorescence images in CT26 cells after incubation with the Coumarin-6-labeled NPs (scale bar: 50 μm). (F) Quantitative analysis of cellular uptake efficiency via FACS. (G) Statistical analysis of uptake efficiency. (H) M2-subtype TAM uptake efficiency via FACS. (I) LRP-1 and MR expression.

cancer-immunity cycle thus provides a roadmap for how to modulate the TIME and normalize the immune responses. For instance, the immunogenic cell death (ICD) plays an important role in initiating antitumor immunity by release of cancer antigens, functioning as “vaccines”.12 But beyond ICD, the

activation of other steps of the cancer-immunity cycle must be followed to solidify the antitumor immunity. It is gradually realized that moving from single- to multipletarget therapy is important in the complicated and interacting cancer ecosystem.13 Combination therapy via codelivery of B

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Figure 2. Cytotoxicity in tumor cells and ICD-induced immune priming. (A) Cytotoxicity study. (B) Apoptosis assay in CT26 cells via FACS. (C) Apoptotic protein detection using Western blotting. (D) ROS levels in CT26 cells after various treatment. (E) SHK-induced CRT exposure. (F) CLSM examination (scale bar = 50 μm). (G) Quantitative assay of CRT exposure of CT26 cells. (H) Examination of HMGB1 release of CT26 cells. (I) Flow cytometric histograms of the Man-LF NPs-accelerated DC maturation. (J) Quantitative assay of matured DCs. (K) IFN-γ secretion from the CD8+ T cells primed with the ICD-induced matured DCs using the Man-LF NPs.

immunomodulators and anticancer drugs have drawn much attention.13,14 In this work, we developed a novel combination

therapy containing shikonin (SHK) and JQ1, which were encapsulated into the lactoferrin nanoparticles for biomimetic C

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Figure 3. Regulation of TAM repolarization and aerobic glycolysis. (A) CD206 expression in TAM2 after drug treatment. (B) ELISA analysis of the levels of TGF-β1 and TNF-α (C) in TAM2 after drug treatment. (D) Cytotoxicity study in CT26 cells with M1Φ or M2Φ culture medium. (E) CD206 expression in M2Φ after Lactic acid treatment. (F−H) Lactic acid inhibited DC maturation and the IFN-γ production in T lymphocytes. (I) Man-LF NPs reduced the production of lactic acid in CT26 cells. (J) PKM2 and PD-L1 expression in CT26 cells after drug treatment.

the cancer-immunity cycle via inducing ICD, as well as a regulator of TIME. JQ1 is a bromodomain and extra-terminal motif (BET) inhibitor that can reduce PD-L1 expression on tumor cells, tumor-associated dendritic cells (DC), and TAM, thus being able to coordinate CTL.19 JQ1 also possesses other positive immunoregulatory functions such as downregulation of Foxp3 and CTLA-4 in the tumor-infiltrating regulatory T cells (Tregs),20 as well as its Treg-disruptive effect along with PD-1 downregulation in potential effector T cells.21 It was expected

codelivery. SHK is a liposoluble naphthoquinone pigment, a major active compound isolated from a traditional Chinese herb Zicao (the dried root of Lithospermum erythrorhizon), and SHK possesses potential anticancer activity via multiple mechanisms such as ROS generation, induction of necroptosis, and suppression of NF-κB-regulated gene products and pyruvate kinase-M2 (PKM2).15 Attractively, SHK was able to induce ICD and elicit antitumor immunity.16,17 In addition, SHK can suppress cancer glucose metabolism and thus inhibit tumor cell proliferation.18 Therefore, SHK can serve as a trigger to initiate D

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treatment of SHK or SHK/JQ1, the CRT exposure on the CT26 tumor cells was significantly elevated, observed by flow cytometry and CLSM (Figure 2E-G), Additionally, SHK or SHK/JQ1 could also induce a significant extracellular release of HMGB1 (Figure 2H). To further evaluate the efficacy of turning the tumor cells into antigen-presenting cells via ICD, the ICD-induced DC maturation was investigated. It revealed that the tumor cells treated with the Man-LF NPs had the strongest immunogenicity and thus induced the highest percentage (83%) of matured DCs (CD80+CD86+), compared to 77% of the LF NPs and 52% of free SHK/JQ1 (Figure 2I, J). As a result, the IFN-γ secretion from the CD8+ T cells primed with the ICD-induced matured DCs using the Man-LF NPs was significantly higher than other groups (Figure 2K). Regulation of TAM Repolarization and Aerobic Glycolysis. TAM performs homeostatic functions and regulates tumor growth, which is highly plastic and can reversibly polarize between the TAM1 and TAM2 phenotypes. The preferential polarization toward TAM2 leads to the suppressive TIME and poor prognosis. Therefore, reprograming TAM is a promising therapeutic strategy to remove the immunosuppressive barricades.28 Our results revealed that the polarization toward TAM2 was reversed by the Man-LF NPs in the M2Φ/CT26 Transwell cocultures, reflecting the downregulation of the M2related markers (e.g., CD206 and legumain, Figure 3A), while the expression of STAT1 and TNF-α (TAM1 biomarkers) was increased. TGF-β secreted from TAM is a driving factor in tumor progression and exerts systemic immune suppression and inhibits host immunosurveillance.29 The NPs treatment reduced the TGF-β expression (Figure 3B) but promoted the antitumor TNF-α production (Figure 3C). Importantly, the sensitivity of the tumor cells to SHK was enhanced by exposure to the M1Φconditioned medium but reduced in the M2Φ-conditioned medium (Figure 3D). It suggested the essential regulatory function of TAM on antitumor efficacy. Lactic acid is a major factor driving the macrophages to a tumor-promoting state.30 It was found that lactic acid induced M2 polarization in the bone marrow-derived macrophages (BMDM) under normoxic condition (Figure 3E), and inhibited the IFN-γ production of T lymphocytes and DC differentiation (Figure 3F, G, H) as well. The malignant cells produce an increased amount of lactic acid, and a low pH in the tumor microenvironment inhibits the function of T lymphocytes and DC activation.31,32 Our results revealed that treatment of the Man-LF NPs reduced the production of lactic acid in the CT26 cancer cells (Figure 3I). The mechanisms of glucose metabolism regulation could involve the downregulation of pyruvate kinase M2 (PKM2), which played an important role in promoting aerobic glycolysis, and the reduction of the consequent production of lactic acid (Figure 3J). Remarkable JQ1-mediated PD-L1 downregulation was also observed in the CT26 cells treated with the Man-LF NPs (Figure 3J). In Vivo Imaging and Biodistribution. MR is an immune adhesion molecule that is typically upregulated in TAM2. Its expression is also found in the gastric cancer cells and its high level is relevant to the poor prognosis.33 LRP-1 is an important portal for lactoferrin-mediated iron transport, and highly expressed in many types of cancer cells and macrophages.22 In addition, LPR-1 also serves as an important regulator for tumor growth signals.34 Therefore, MR and LPR-1 could serve as dualtarget receptors for both the cancer cells and TAM. The tumor-

that JQ1 could solidify the subsequent response in the cancerimmunity cycle, yielding a synergistic effect along with SHK on immune normalization. We developed a lactoferrin-based nanobiomimetic system for codelivery of SHK/JQ1. Lactoferrin is a primary iron-carrying protein and specifically binds with low-density lipoprotein receptor-related protein 1 (LRP-1) that are overexpressed on the tumor vessel endothelial cells and cancer cells. 22 Interestingly, LRP-1 is also highly expressed on macrophage, which is a potential target for activating proinflammatory effect by its antagonists (e.g., lactoferrin).23 Moreover, we previously demonstrated that mannose receptors (MR) were overexpressed on both colon cancer cells and TAM2.24 Therefore, we designed a mannosylated lactoferrin nanobiomimetic system for targeting cancer cells and TAM, remodeling the TIME, and reinstalling the immunity. Preparation and Characterization of the Mannosylated Lactoferrin Nanoparticles (Man-LF NPs). The conjugation of mannosylated lactoferrin was confirmed by mass spectrum (Figure S1, Supporting Information). The ManLF NPs with coencapsulation of SHK/JQ1 were fabricated via a green method without the need of the cross-linking agents and additives, a heat-driven protein self-assembly process modified from our previous work.22 The particles size of the Man-LF NPs was slightly larger than the LF NPs without mannose modification (150 vs 110 nm) (Figure 1A); both of them yielded a ζ potential around 30 mV (Figure 1B). The NPs exhibited good stability and a sustained-release pattern (Figure 1C,D). The drug loading efficiency and drug encapsulation efficiency of the NPs were shown in Table S1. The uptake of the Man-LF NPs by the CT26 colon cancer cells was higher than that of the control LF NPs (Figure 1E−G) and the intracellular location of the Man-LF NPs was mainly at cytoplasm (Figure S2). If the cells were pretreated with free mannose, the cellular uptake of the Man-LF NPs dropped to a level similar to that of the LF NPs. The cellular uptake profile of the NPs in M2 macrophages also displayed the same pattern (Figure 1H). Both LRP-1 and MR were highly expressed in both CT26 cancer cells and M2 macrophages (Figure 1I). The result revealed the dual targeting effect of the Man-LF NPs. Effect on Cytotoxicity in Tumor Cells and ICD-Induced Immune Priming. An optimized ratio about 1:0.8 (JQ1/SHK) was selected based on the combination index (CI), (Table S2). The combination therapy of SHK/JQ1 effectively inhibited the proliferation of the CT26 cells, with the lower IC50 and higher apoptosis rate compared to the single use of SHK or JQ1 (Figure 2A). The Man-LF NPs displayed the strongest antitumor activity, with the IC50 of 0.13 μM and the apoptosis rate of 48.9% (Q2+Q3) (Figure 2B). Accordingly, after treatment of the ManLF NPs, the cleaved caspase-3 was upregulated and the antiapoptosis protein Bcl-2 reduced (Figure 2C), which were probably associated with the increased ROS production (Figure 2D). Of note, the Man-LF NP carrier displayed excellent biocompatibility to normal cells (Figure S3). ICD of tumor cells is characterized by eliciting cell surface expression of pro-apoptotic calreticulin (CRT), extracellular release of high mobility group box1 (HMGB1), and secretion of adenosine triphosphate (ATP).25 The exposure of CRT on the cancer cell surface serves as an “eat me” signal to the antigenpresentation cells (e.g., DCs and macrophages) and elicits antitumor immune responses.26 Matured DCs migrate to the ̈ T cells into effector T cells, which lymph nodes and prime naive subsequently migrate to the tumor microenvironment.27 After E

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Figure 4. In vivo imaging and biodistribution studies in the CT26-tumor models. (A) Whole body imaging. (B) In vivo radiant efficiency at tumor sites. (C) Fluorescence images of the major dissected organs, heart (H), liver (Li), spleen (S), lung (Lu), kidney (K), and tumor (T). (D) Statistical analysis of ex vivo radiant efficiency at the dissected tumors. (E) The intratumoral distribution of the Cy5-labeled NPs with staining of LRP-1 or MR (scale bar: 50 μm). (F) Quantitative assay of Cy5+ LRP-1+ cells and Cy5+ CD206+ cells.

targeting delivery was investigated by using the DiR dye-labeled NPs. Both the LF NPs and Man-LF NPs efficiently distributed to the tumor sites, and the Man-LF NPs displayed the higher

tumor accumulation (Figures 4A−D and S4). At the experimental end point, the ex vivo imaging showed that the tumor accumulation of both the NPs was also more pronounced F

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Figure 5. Antitumor treatment of the Man-LF NPs. (A) Therapeutic schedule for Man-LF NPs-mediated combination therapy. (B) Tumor growth curves in CT26-tumor-bearing mice following the indicated treatments. (C) Inhibition rate of tumor growth. (D) Survival curve. (E) CRT expression of tumor sections (scale bar: 50 μm).

(Figure 5C,D). In addition, the Man-LF NPs group displayed the longest survival time, with a median survival time of 44 days, compared to 33 days for the LF NPs group (Figure 5D). The results demonstrated the benefit of the dual targeting delivery and improvement of the anticancer efficacy. The ICD examination in the tumor sections showed the ManLF NPs dramatically promoted the CRT expression (Figure 5E). The preliminary side toxicities were evaluated. The animals did not sustain body weight loss during the treatment. There were no major pathological changes in the major organs (Figure S5). Remodeling of TIME and Glucose Metabolism. To elucidate the combination immunotherapeutic effects of the Man-LF NPs, the repolarization of TAM in the colon cancer tissues was examined. After treatment with the Man-LF NPs, the amount of TAM1 was increased while TAM2 decreased (Figure 6A,B). The Man-LF NPs treatment increased the ratio of TAM1/TAM2 by 1.5 and 1.3 folds in comparison with that of SHK and LF NPs, respectively (Figure 6C). The aerobic glycolysis is closely associated with TIME; for example, lactic acid and HIF1α promote the polarization of M2 phenotype.30 The production of lactic acid, PKM2 and HIF1α were downregulated after treatment of the Man-LF NPs (Figure 6B,D).

than in other organs, and the Man-LF NPs also had higher tumor accumulation, demonstrating the benefit of the dual targeting function. The intratumoral penetration was examined by immunofluorescent staining and flow cytometry. The confocal scanning laser microscope (CSLM) imaging showed that the intratumoral distribution of the Cy5-labeled Man-LF NPs was largely overlapped the expression of LRP-1 and MR (Figure 4E). The flow cytometric sorting showed the positive rate of the NPsCy5+/LPR-1+ cells in the tumor tissues was around 20% for both the lactoferrin NPs, but the positive rate of the NPs-Cy5+/ CD206+ cells was much higher in the group of the Man-LF NPs group than the LF NPs (Figure 4F). Our results first demonstrated the dual-targeting function of the mannosylated lactoferrin-based delivery strategy via MR and LRP-1 for dual action on both cancer cells and TAM. In Vivo Antitumor Efficacy. The antitumor efficacy of the Man-LF NPs was evaluated in an immunocompetent CT26 tumor mouse model (Figure 5A,B). The tumor-bearing mice were i.v. injected with saline, SHK (5 mg/kg), SHK + JQ1 (SHK 5 mg/kg, JQ1 15 mg/kg), and the LF NPs and Man-LF NPs (equal dose to the combo free drugs), respectively. Both the NPs efficiently inhibited the tumor growth, with inhibition rates of 68% (LF NPs) and 84% (Man-LF NPs), compared to 61% for the group of combo free drugs and 45% for the single SHK group G

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Figure 6. Remodeling of TIME and glucose metabolism. (A) MR detection using immunohistology (scale bar: 50 μm). (B) Expression of the macrophage- or glycolysis-related proteins in the tumor tissues after treatment. (C) Ratio of intratumoral TAM1 to TAM2. (D) Production of lactic acid after treatment. (E) Man-LF NPs-accelerated DC maturation in draining LNs. (F) Intratumoral infiltration of T lymphocytes. (G) Cytotoxic CD8+ T cells in draining LNs. (H) Man-LF NPs-induced Tregs detected in tumors. (I, J) Intratumoral cytokine levels of IFN-γ and TNF-α.

the CD8+ T cells in the tumor, showing 1.5- and 2.4-fold higher than the groups of the LF NPs and SHK, respectively (Figure 6F). Moreover, the CD8+ T cells in draining LNs also exhibited a similar trend after treatment (Figure 6G). The intratumoral infiltration of Tregs (CD4+Foxp3+ T cells, namely Tregs) is correlated with the immunosuppression and poor prognosis of

The ICD in the cancer cells initiated the antitumor immunity. Accordingly, the matured DCs in the draining lymph nodes (LNs) were determined by flow cytometry and the maturation rate in the Man-LF NPs group was 1.5 and 1.7 folds higher than that of the LF NPs and SHK/JQ1, respectively (Figure 6E). Meanwhile, the Man-LF NPs treatment increased the amount of H

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Nano Letters cancer patients.35 The percentage of the intratumor infiltrating Tregs after the Man-LF NPs treatment was dramatically reduced compared to other groups (Figure 6H). The immune response was also evaluated by measuring the intratumoral cytokine levels using ELISA. The Man-LF NPs treatment sustainably promoted the secretion of antitumor IFNγ and TNF-α, much higher than those in other groups (Figure 6I,J). Importantly, the intratumoral PD-L1 expression was downregulated (Figure 6B). Immunotherapy has been a mainstream cancer intervention, and numerous patients have seen pronounced treatment outcomes; however, many more patients have experienced minimal or no clinical benefit with the marketed immune drugs (e.g., PD-1/PD-L1 antibodies).4 The better understanding of TIME provides opportunities for the development of novel therapeutic targets and strategies. Cancer is viewed as the open, complex, adaptive ecosystems because they openly and freely communicate with their surroundings, and contain complex components, and each element can adaptively change over time and interact with other components.36 Therefore, the multitarget combination is the major trend. It is believed that the evolution of cancer immunotherapy relies on the development of combination therapies, and apart from T cell-based target, the innate immune cells like macrophages and NK cells should also be paid attention as potential targets for combination immunotherapies.37 We used a multiple-target immunoregulation method to address the interplay of TIME for cancer immunotherapy (Scheme 1). JQ1 has been investigated as a small-molecular candidate for PD-1/PD-L1 checkpoint blockage. Yet, its single use may not yield sufficient efficacy, and it thus is often applied in a combination with other immune drugs.20,38 We developed a novel combination of JQ1/SHK and a biomimetic codelivery strategy. Many naturally sourced compounds tend to have a broad range of effects and the relevant potential side toxicities, which used to be viewed as disadvantages against drug development. However, such “dirty” compounds are multitarget and recently have been considered to have a value of re-evaluation for polypharmacological therapy.39 SHK is a typical natural product with multiple actions. Apart from serving as a proapoptotic agent and an ICD inducer, SHK can also function as a regulator of TAM and glucose metabolism reprograming. Our previous work demonstrated that TAM played an essential role in the immunoregulation; the repolarization of TAM2 toward TAM1 can stimulate CD8+ T cells, repress Treg, and activate NK cells, as well as upregulate antitumor cytokines.40 In this work, we further revealed the interplay of TAM polarization and lactate production, and their effects on tumor immunity. Lactate is one of the most explicated TIMEsuppressive metabolites, and targeting glucose metabolism represents a promising avenue for immunotherapy.41 By dualaction on both TAM and glucose metabolism reprograming, SHK remodeled the TIME and solidified the ICD-initiated antitumor immunity. More importantly, the activated TIME further synergized the JQ1-mediated PD-1 blockage, thus augmenting the immune responses. Our results revealed that the polypharmacological activities of SHK were ideal for immunotherapy application. In particular, it demonstrated that the suppressive effect of glucose metabolism by SHK yielded a positive regulation of the cancer-immunity circle. In summary, we designed a biomimetic drug delivery nanoparticulate system containing the multitarget compound

Scheme 1. Illustration of the Man-LF NP-Based Polypharmacological Immunotherapy

SHK and a PD-L1 suppressor JQ1 that exhibited a synergistic advantage for developing novel cancer immunotherapy by simultaneously activating the ICD, repolarizing TAM2, and repressing glucometabolism. The Man-LF NPs with a combination of SHK/JQ1 showed the potent antitumor efficacy and promising potential for translation.

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* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.9b00021. Methods and Table S1 and Figures S1−S5 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(Y.H.) Telephone/Fax: +86-21-2023-1981. E-mail: yzhuang@ simm.ac.cn. ORCID

Yongzhuo Huang: 0000-0001-7067-8915 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are thankful for the support of NFSC (814022883, 81422048, 81673382, and 81521005), and we thank the I

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Strategic Priority Research Program of CAS (XDA12050307), National Special Project for Significant New Drugs Development (2018ZX09711002-010-002), the CAS Scientific Research and Equipment Development Project (YZ201437), and the Fudan-SIMM Joint Research Fund (FU-SIMM20174009) for support. We also thank the National Center for Protein Science Shanghai, CAS, for the technical support at MASS Facility.

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DOI: 10.1021/acs.nanolett.9b00021 Nano Lett. XXXX, XXX, XXX−XXX