Take Immune Cells Back on Track: Glycopolymer-Engineered Tumor

Letter Cite This: ACS Macro Lett. 2019, 8, 337−344

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Take Immune Cells Back on Track: Glycopolymer-Engineered Tumor Cells for Triggering Immune Response Qi Liu,†,‡ Shuaibing Jiang,† Bing Liu,† You Yu,§ Zhen-Ao Zhao,§ Chao Wang,∥ Zhuang Liu,∥ Gaojian Chen,*,†,‡ and Hong Chen*,†

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State and Local Joint Engineering Laboratory for Novel Functional Polymeric Materials, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, 199 Ren’ai Road, Suzhou 215123, People’s Republic of China ‡ Center for Soft Condensed Matter Physics and Interdisciplinary Research, Soochow University, Suzhou 215006, People’s Republic of China § Institute for Cardiovascular Science and Department of Cardiovascular Surgery of the First Affiliated Hospital, Soochow University, Suzhou 215000, People’s Republic of China ∥ Institute of Functional Nano and Soft Materials (FUNSOM), Jiangsu Key Laboratory for Carbon-Based Functional Materials and Devices, Soochow University, Suzhou, Jiangsu 215123, People’s Republic of China S Supporting Information *

ABSTRACT: The “self-homing” of cancer cells to primary or metastatic tumor sites indicates that they could serve as vehicles for self-targeted cancer therapy; this suggests a promising method for treating end-stage cancer. Inspired by this, we propose that engineering cancer cells to carry efficient “coup” molecules for in situ activation of immune cells in or near tumor sites to attack tumors is a promising strategy for cancer therapy. Therefore, herein we explored the potential of engineered tumor cells to enhance their anticancer activity by stimulating immune cells. We armed tumor cell surfaces with specific glycopolymer−ligands that bind to lectins on macrophages or dendritic cells by combining HaloTag protein (HTP) fusion technique with reversible addition−fragmentation chain transfer (RAFT) polymerization. We demonstrated that two synthetic well-defined glycopolymers containing, respectively, N-acetylglucosamine and N-acetylmannosamine units, were introduced and stably presented on the cell surfaces via the stable covalent binding of chloroalkane-terminated polymers with membrane-bound HTP. Furthermore, it was shown that the glycopolymer-engineered HeLa cells with HTP anchors increased expression of the typical marker for M1-type macrophages (CD86) and upregulated secretion of pro-inflammatory cytokines (IL-12p70, TNF-α, and iNOS), thereby accelerating HeLa cell lysis. The maturation of dendritic cells was also promoted. This study demonstrates the strong potential of glycopolymer-engineered tumor cells in cancer immunotherapy.

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homing properties to enhance immune responses in tumor sites is a valid approach for cancer therapy. To the best of our knowledge, the concept of engineering tumor cells to enhance their anticancer activity by stimulating immune cells has not been explored. Among the immune cells recruited to tumor sites in the tumor microenvironment, macrophages are major species that infiltrate both primary and secondary tumors and are present at all stages of tumor progression. These cells are termed tumorassociated macrophages (TAMs).12,13 Macrophages are remarkably plastic cells and can acquire two classical phenotypes: (1) M2-type which enhance tumor cell invasion, motility, and intravasation; (2) M1-type which are efficient

major obstacle in the treatment of end-stage cancer is local and distant tumor cell metastasis, with recurrence of the cancer.1,2 Recent studies suggest that the self-homing property of circulating cancer cells enhances tumor progression and metastasis, a process that involves cell dissemination into the vascular system from the primary or metastatic lesions and subsequent retargeting of the cells to their tumor of origin.3,4 Based on this mechanistic insight, cancer cells have been engineered ex vivo to serve as vehicles for tumor self-targeted therapy, in which the therapeutic cancer cells that produce tumor necrosis factor alpha (TNF-α) or secrete death receptor-targeting ligands home to tumors, contributing to direct damage to tumor cells in both primary tumors and metastatic colonies.5,6 In the past decade, immunotherapy by which long-term anticancer immune responses are promoted, has taken center stage as a cancer treatment strategy.7−11 Therefore, we propose that engineering tumor cells with self© XXXX American Chemical Society

Received: January 17, 2019 Accepted: March 8, 2019

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Scheme 1. (a) Strategy for Displaying Synthetic Well-Defined Glycopolymers on HeLa Cell Membranes Using HTP Anchors; (b) HeLa Cells Stably Remodeled with Well-Defined Glycopolymers on the Cell Surface for Enhancing the Recognition of Macrophages (CBDs: carbohydrate-binding domains); (c) Molecular Structures of Chloroalkane-Conjugated Chain Transfer Agent (Left) and Well-Defined Glycopolymers with Chloroalkane End Groups (Right) Used in This Work

Scheme 2. Synthesis Procedure for Well-Defined Glycopolymers with Chloroalkane End Groups

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were stained with FITC-avidin for 1 h at 4 °C for imaging. As shown in Figure 1a, strong green fluorescence was observed on

immune effector cells that are able to secrete directly products harmful to cancer cells, such as oxygen radicals and tumor necrosis factor, and produce high levels of T-cell stimulatory cytokines, or present antigens to help kill tumor cells in an indirect manner.14−17 Therefore, redirecting M2-type macrophages to the M1 type can slow or even stop tumor growth.18 Accordingly we reasoned that directing TAMs to the M1 phenotype and enhancing macrophage-related immune responses using engineered cancer cells hold promise as an approach to cancer therapy. To “engineer” cancer cells with increased immunological effects, selected glycopolymer ligands were introduced onto the cancer cell surfaces for binding to macrophage surface lectins (Scheme 1a,b). The best-characterized lectins on macrophage surfaces, that is, the mannose receptor (MR) and complement receptor three (CR3), are both able to induce intracellular killing mechanisms when bound to their specific glycan ligands mannose and N-acetyl-glucosamine-containing oligosaccharide, respectively.19 Based on this knowledge, herein, we engineered tumor cell membranes with two synthetic welldefined glycopolymers containing, respectively, N-acetylglucosamine and N-acetylmannosamine derivatives, namely, poly(Nmethacryloylglucosamine) (pMAG) and poly(N-methacryloylmannosamine) (pMAM; Scheme 1c, right). To implement this concept, we designed a “chem-bio” strategy to bond well-defined glycopolymers to cell membranes by combining protein fusion technique (PFT) with reversible addition−fragmentation chain transfer (RAFT) polymerization. The membrane-bound HaloTag proteins (HTP) were chosen as anchors for the long-lived cell surface engineering20 considering the required long period of circulation time for the engineered cancer cells to present their antitumor efficacy in vivo.5 It should be noted that although the lipid-anchored glycopolymers has been generated for modifying cell surface, their plasma membrane residence half-lives are limited to a few hours.21,22 Therefore, to achieve more stable attachment of glycans and to facilitate the study of events occurring on longer time scales, such as the long-time circulation for the engineered cancer cells in vivo, a strategy based on membrane-bound HTP as anchors has been developed. First we synthesized the chloroalkane-conjugated chain transfer agent, 5-((2-(2-((6chlorohexyl)oxy)ethoxy)ethyl)amino)-2-cyano-5-oxopentan-2yl benzodithioate (Scheme 1c, left) via the reaction between N-hydroxysuccinimide groups and a primary amine (Scheme S1). The RAFT polymerization of the sugar monomers, MAG and MAM (synthesized as reported previously23−25), was then carried out to generate well-defined glycopolymers containing a chloroalkane linker for conjugation to HTP (Scheme 2b,c). The 1H NMR spectra shown in Figures S6 and S7 and size exclusion chromatography (SEC) traces (Figure S9) demonstrate the successful preparation of well-defined glycopolymers pMAG (5740 Da, PDI = 1.17) and pMAM (5220 Da, PDI = 1.21). Also a biotin-labeled poly(MAG) with chloroalkane end groups (pMB, Scheme 2a, see also the Supporting Information, Figure S8) was also prepared to test the binding of glycopolymers to cells. The conversions and molecular weights of the three obtained glycopolymers were shown in Table S1. Then HTP fused to the platelet-derived growth factor receptor (PDGFR) transmembrane domain was stably expressed in human cervical cancer cells (HeLa) membranes. Cells were incubated in a solution of pMB (6010 Da, PDI = 1.25) for 1 h at 37 °C, then washed with phosphate-buffered saline (PBS) to remove excess unbound glycopolymers. After that, the cells

Figure 1. Confocal fluorescence images of HeLa cells modified with glycopolymers. (a) After incubating with synthetic glycopolymer solution (pMB) for 1 h at 37 °C, HeLa cells stably expressing HTP were stained with FITC-avidin (green) for 1 h at 4 °C. (b) After washing twice with DPBS, HeLa cells expressing HTP stably functionalized with synthetic glycopolymers were incubated in complete medium for specified times (1, 3, and 7 days). The cells were stained with FITC-avidin for 1 h for detection (blue: DAPI).

the cell membranes after pMB incubation, indicating the successful incorporation of glycopolymers onto the surfaces of HeLa cells expressing HTP (HTP-HeLa). The fluorescence images of multiple cells are shown in Figure S12. In contrast, no fluorescence was observed in HeLa cells lacking HTP expression or in cells stably expressing HTP without pMB incubation (Figure S13). Furthermore, green fluorescence persisting for 7 days on HeLa cell membranes was also observed (Figure 1b). These results demonstrate the successful specific conjugation of chloroalkane-glycopolymer to HTP and the long-term, that is, stable, attachment of exogenous glycopolymers on HeLa cell membranes. We next investigated whether the glycopolymer-engineered cells could promote M1 polarization of macrophages and further enhance their anticancer activity. HTP-HeLa cells were incubated with pMAG or pMAM at 37 °C for one hour to prepare pMAG-HeLa or pMAM-HeLa cells, respectively. Human monocyte-like U937 cells (TAM model cells26−29) expressing CR3 on the cell membranes19,30 were chosen to investigate polarization provoked by the modified HeLa cells. Coculture of U937 with pMAG/pMAM-HeLa cells was carried out. First, the cell−cell interactions between U937 and modified HeLa cells were visualized through live content imaging. As shown in Figure 2a, glycopolymer-engineered HeLa cells were damaged over 14 h and even polarized 339

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Figure 2. Glycopolymer-engineered HeLa cells enhanced the stimulation of U937. (a) Images of cell−cell contacts between U937 and modified HeLa cells through live content imaging. Yellow arrows point toward U937 cells, while the adhered cells are HeLa cells. (b) After coculture with HTP-HeLa, pMAG-HeLa, and pMAM-HeLa cells for 4 days, macrophages were harvested and CD86 expression was measured by flow cytometry. Flow cytometry data giving percentage of CD86-expressing cells. (c) Flow cytometric analysis of expression of CD86 and CD206. HTP-HeLatreated cells were taken as control. The MFIs in all groups were normalized relative to that of CD86 in HTP-HeLa-treated cells. Data are expressed as the mean ± SD of three independent experiments (**p < 0.01 by t test).

cells can promote M1 polarization with much higher efficiency than HTP-HeLa cells without glycopolymers-modification by up-regulation expression of CD86, whereas low expression of M2-specific marker CD206. Moreover, the promotion of M1 polarization and the immunological effect in the glycopolymer-engineered HeLastimulated groups were further investigated by cytokine secretion using enzyme-linked immunosorbent assays (ELISA). The data show that macrophages in all three groups secreted extremely low quantities of interleukin 10 (IL-10, a typical M2-type cytokine14; Figure 3a). Indeed, the concentrations of IL-10 in the pMAG/MAM-HeLa-treated groups were close to zero when the initial seeding density ratio of the two cell types was 1:1. In contrast, as shown in Figure 3b, the levels of interleukin 12 (IL-12p70, a typical M1-type cytokine14) were significantly greater after pMAG/pMAMHeLa stimulation. The concentrations of IL-12p70 in the two groups were ∼3.4- and ∼3.2-fold higher than in the HTPHeLa-treated group at initial seeding ratio 1:1, and ∼2.8 and ∼2.6 times higher at initial seeding ratio 2:1 (U937:HeLa). These data (IL-12p70 high, IL-10 low) demonstrate that glycopolymer-engineered HeLa stimulation directed macrophages to the M1 phenotype and enhanced the secretion of the proinflammatory cytokine IL-12p70. Another characteristic proinflammatory cytokine with anticancer activity, tumor necrosis factor α (TNF-α), can be

macrophages with pseudopodia shapes can be observed in the pMAM-HeLa-stimulated group. Also, the percentage of cell lysis determined by the lactate dehydrogenase (LDH) release assay showed that ∼1.5- and ∼2.0-fold increase in cell lysis were observed for pMAG-HeLa and pMAM-HeLa-treated groups compared with HTP-HeLa-treated group (Figure S14). The typical marker for M1-type macrophages, CD86, and CD206 for M2, expressions were determined.31−33 To quantify the CD86 and CD206-positive cells in the different groups, the cocultured cells were harvested, labeled with PE antihuman CD86 and CD206 antibody, and analyzed by flow cytometry. With the untreated U937 cells taken as negative control, the CD86+ cells in the pMAG-HeLa and pMAM-HeLa-treated groups were approximately 1.4-fold as abundant as in the HTP-HeLa-treated group (Figure 2b). It should be noted that CD86 positive cells were much more abundant in the HTPHeLa-treated group than in the negative control group. This is believed to be due mainly to the various antigens on HTPHeLa cells which also cause U937 polarization. The mean fluorescence intensity (MFI) of cells in different groups was further quantitated by FlowJo. As shown in Figure 2c, the quantitative CD86 expression in the glycopolymer-engineered HeLa-stimulated groups were ∼36% and ∼34% more abundant, respectively, than in the HTP-HeLa-treated group, while expression of CD206 in all three groups exhibited low levels. It can be concluded that glycopolymer-engineered HeLa 340

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Figure 3. Glycopolymer-engineered HeLa cells altered the cytokine secretion of macrophages. After coculture of U937 with HTP-HeLa, pMAGHeLa and pMAM-HeLa cells, respectively, for 6 days, the levels of interleukins secreted into the supernatants were determined: IL-10 (a), IL-12p70 (b), TNF-α (c) and iNOS (d). Data are expressed as the mean ± SD of three independent experiments (*p < 0.05, **p < 0.01, and ***p < 0.001 by t test).

Figure 4. Glycopolymer-engineered HeLa cells promote DC maturation. (a) After coculture of DC2.4 cells with HTP-HeLa, pMAG-HeLa, and pMAM-HeLa cells, respectively, for 2 days, DCs were harvested and CD80 and CD86 expressions were measured by flow cytometry. Flow cytometry data giving percentage of CD86 and CD80-expressing cells. (b) The coculture supernatants were collected and the secretion of IL-12p70 was determined by ELISA. Data are expressed as the mean ± SD of three independent experiments (**p < 0.01 and ***p < 0.001 by t test).

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glycopolymer-engineered HeLa cells upregulate expression of M1-specific cell surface antigen CD86 and secretion of the proinflammatory cytokines IL-12, TNF-α, and iNOS, promoting M1 polarization of macrophages and enhance DC maturation. A versatile method to prepare well-defined glycopolymerengineered cancer cells was developed and the potential of these cells to enhance the immune response and damage tumor cells was demonstrated. Further studies are contemplated to demonstrate the efficacy of glycopolymer-engineered cancer cells with self-homing properties for enhancing immune responses and antitumor activity in vivo in different murine models and in models of tumors at different stages. Furthermore, to increase the safety of the system in vivo, the glycopolymer-engineered tumor cells should be further engineered to express a suicide gene, thus, avoiding secondary tumor formation while conserving antitumor efficacy.

produced by activated M1 macrophages and plays a critical role in damaging the tumor vascular endothelia.34−37 With this in mind the secretion of TNF-α in the coculture supernatants was measured using ELISA. It was shown that the secretion of TNF-α also increased significantly after stimulation with pMAG/pMAM-HeLa cells (Figure 3c). For the pMAG-HeLa and pMAM-HeLa groups, the levels were, respectively, ∼16.3and ∼9.4-fold greater than in the HTP-HeLa-treated group at a 1:1 cell seeding ratio (U937:HeLa). At a 2:1 ratio, the TNFα concentrations in the pMAG- and pMAM-HeLa-stimulated groups were, respectively, about 1.4- and 1.5-fold higher than at the 1:1 ratio, suggesting that TNF-α secretion was positively correlated to the U937 cell concentration. It was also observed that the secretion of TNF-α in the pMAG-HeLa-treated group at the two seeding ratios was, respectively, ∼1.7- and ∼1.6-fold greater than in the pMAM-HeLa-treated group. This may be because the pMAG glycopolymers on HeLa cell surfaces behave more like the natural ligands of the CR3 receptor (e.g., β-D-glucans).38 M1 polarization of macrophages also leads to secretion of the reactive oxygen molecule, nitric oxide (NO), which promotes the destruction of tumor cells.39,40 Owing to the instability of NO, it is better to detect the inducible nitric oxide synthase (iNOS) production responsible for generating NO.41 Accordingly the iNOS activity in the supernatants collected from cocultures was determined. As shown in Figure 3d, similar to TNF-α secretion, the iNOS activity in the pMAG/ pMAM-HeLa and U937 cells coculture supernatants was greater than in the HTP-HeLa-treated group. The data in Figure 3 taken together show that the glycopolymerengineered HeLa cells increased the secretion of proinflammatory cytokines, indicating that the synthetic glycopolymers anchored on the HeLa cell membrane can enhance the recognition between HeLa cells and U937 cells and promote the activation of U937 cells to M1 macrophages. Given the similarities of DCs and macrophages with respect to certain functions and the abundantly expressed mannose receptors on DC cell surfaces,42,43 it was of interest to investigate whether the glycopolymer-engineered HeLa cells could promote DC maturation. To this end, the coculture of pMAG/pMAM-HeLa and DC2.4 cells was carried out over 2 days. DC maturation will lead to the coexpression of CD86 and CD80;44,45 therefore, the CD86 and CD80 expressions on DCs were detected to quantify the percentage of matured DCs. As shown in Figure 4a, the efficiency for promoting DC maturation in the glycopolymer-engineered HeLa-stimulated groups were ∼34.8% and ∼44.4% higher, respectively, than in the HTP-HeLa-treated group. In addition, since mature DCs can also enhance the production of IL-12 cytokines, IL-12p70 secretion in the coculture supernatants was determined. Similarly to the macrophage experiments, significantly increased IL-12p70 concentrations in the pMAG/pMAMHeLa and DC2.4 coculture supernatants compared to that in the HTP-HeLa-treated group were observed and were DC density-dependent (Figure 4b). Also, the IL-12p70 level in the pMAM-HeLa-treated group was somewhat greater than that in the pMAG-HeLa-treated group. These data indicate that the glycopolymer-engineered HeLa cells can promote DC maturation with much higher efficiency than HTP-HeLa cells. The present study shows that synthetic glycopolymers with chloroalkane end groups can be incorporated into HeLa cell surfaces by conjugation with membrane-bound HTP, and that they persist in the surface for at least 7 days. The

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.9b00046. Additional text with details on the experimental procedures and general methods, synthesis and characterization of the chloroalkane-conjugated chain transfer agent and glycopolymers, construction of HeLa cells stably expressing HTP, confocal fluorescence images of glycopolymer-engineered HeLa with multiple cells, and percentage of cell lysis (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Zhuang Liu: 0000-0002-1629-1039 Gaojian Chen: 0000-0002-5877-3159 Hong Chen: 0000-0001-7799-4961 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the National Natural Science Foundation of China (Nos. 21674074, 21774084, and 21704072) and the Natural Science Foundation of Jiangsu Province (No. BK20161208) for financial support. We thank Prof. John L. Brash for the valuable advice and for proofreading the manuscript.

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REFERENCES

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DOI: 10.1021/acsmacrolett.9b00046 ACS Macro Lett. 2019, 8, 337−344

Letter

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DOI: 10.1021/acsmacrolett.9b00046 ACS Macro Lett. 2019, 8, 337−344