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Nano-engineered lymphocytes for alleviating suppressive tumor immune microenvironment
Applied Materials Today 16 (2019) 273–279
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Short communication
Nano-engineered lymphocytes for alleviating suppressive tumor immune microenvironment Lisha Liu 1 , Qinjun Chen 1 , Chunhui Ruan, Xinli Chen, Xi He, Yu Zhang, Yujie Zhang, Yifei Lu, Qin Guo, Wenxi Zhou, Chao Li, Tao Sun, Chen Jiang ∗ Key Laboratory of Smart Drug Delivery, Ministry of Education, State Key Laboratory of Medical Neurobiology, Department of Pharmaceutics, School of Pharmacy, Fudan University, Shanghai 201203, China
a r t i c l e
i n f o
Article history: Received 30 April 2019 Received in revised form 20 June 2019 Accepted 21 June 2019 Keywords: Tumor-infiltrating lymphocytes Doxorubicin Albumin conjugate Tumor immune microenvironment Drug delivery
a b s t r a c t The intrinsic trafficking ability of host cells to infiltrate various disease sites and act as living chaperones for chemotherapeutics alone or chemotherapeutics-loaded nanoparticles to actively transmigrate the endothelial barrier within solid tumor and achieve efficient tumor accumulation is being explored in a quantity of contexts. Here, we leveraged tumor-infiltrating T lymphocytes as delivery vehicle to increase the tumor apoptosis and immunogenic cell death (ICD) of doxorubicin to efficiently boost immune response and eliminate the tumor growth. First we decorated the redox responsive doxorubicin-albumin conjugates (DOX-A) onto the surface of T lymphocytes (DOX-A@T cell) via a pH sensitive bond, which allows for stable retention of DOX-A on the surface of T lymphocytes in blood circulation but the responsive release of DOX-A within the weak acid tumor microenvironment. The released DOX-A exerted deep tumor penetration, efficient tumor cell uptake and rapid intracellular drug release via redox responsive cleavage. We showed that DOX-A can be stably conjugated to the surface of T lymphocytes for at least 6 h without any detectable toxicity or interference with intrinsic cell functions. DOX-A@T cell had the strong tumor infiltration and penetration both in vitro and in vivo. More significantly, this DOX-A@T cell actively efficiently augmented the DOX-induced ICD efficacy through activation of dendritic cells (DCs) and consequent activation of specific T cell response, thus potentiating immune response in tumor microenvironment. © 2019 Published by Elsevier Ltd.
Although immunotherapy and chemotherapy fight against cancer via targeting different module of tumor-cell survival and their combined effects, the fact is that potent chemotherapy is well known to blunt immune systems long perplexes the successful application of this combination [1]. However, over the past few years accumulating evidence indicates the positive immunologic effects of chemotherapeutic agents [2]. Among, immunogenic cell death (ICD) is a unique modality of cell death in response to some antineoplastic agents such as anthracyclines and oxaliplatin. Unlike normal apoptosis mechanism, ICD involves changes in the composition of the cell surface as well as releases a large number of immune-stimulatory signals, including calreticulin (CRT) exposure, high mobility group box 1 (HMGB1) translocation and heat shock protein (HSP) expression, into tumor immune microenvironment (TIME), then induce an effective antitumor immune
∗ Corresponding author. E-mail address: [email protected] (C. Jiang). 1 These authors contributed equally to this paper. https://doi.org/10.1016/j.apmt.2019.06.009 2352-9407/© 2019 Published by Elsevier Ltd.
response through activation of dendritic cells (DCs) and consequent activation of specific T cell response [3,4]. Recently the increasing preclinical evidences have demonstrated that ICD induced by chemotherapeutics efficiently broke down the immune tolerance and arrested tumor growth in animals [5,6]. However, effective dose of ICD drug, which is required to break immune tolerance to the tumor meanwhile avoid undesired host autoimmune outcomes, is usually a tremendous challenge to most of active targeting nanoparticles, in view of insufficient tumor accumulation and inferior penetration within the tumor microenvironment [7]. Therefore, how to design a smart drug delivery system, which can actively transmigrate the endothelial barrier of many solid tumors with higher densities of blood vessels, efficiently accumulate in tumor tissues and improve the deep penetration of nanoparticle into deep tumor region, still encounter a major challenge. Cell engineering strategies showed their promising application for targeted drug delivery, cancer immunotherapy and blood glucose regulation [8,9]. Among T lymphocytes play a central role in immune protection against cancer. Their infiltration in tumor site is usually associated with a favorable prognosis and may predict
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outcome of therapies with drugs that block immune inhibitory receptors. A recent meta-analysis reported that tumor-infiltrating lymphocytes (TIL) can be identified in 48% of all breast cancers. Interestingly, triple-negative breast cancers show the highest incidence of lymphocyte predominance [10]. The intrinsic trafficking ability of T lymphocytes to infiltrate tumor sites and act as living chaperones for therapeutics has been explored in a number of contexts [11–16]. Those outcomes demonstrated that leveraging T lymphocytes as drug carriers greatly enhanced the amount of drug that can be delivered to tumor, achieving the quantity in the tumor that are orders of magnitude greater than that which can be delivered by nanoparticles alone or even most of active targeted nanoparticles [11,14]. In those previous works, there were two methods for T lymphocytes to load therapeutic cargoes, such as indirect way and direct way. The indirect way conjugated the surface of drug-loaded nanoparticles with an antibody against antigen of T lymphocytes, which showed specific T lymphocytes binding and then effective receptor-mediated internalization by T lymphocytes [15]. The direct way used complementary chemistry-based approaches to conjugate drug-loaded nanoparticles directly to the plasma membrane of T lymphocytes, where nanoparticles were not internalized and slowly released their cargo compounds. This retention of nanoparticles on the surfaces of chaperon cells was shown to avoid the premature degradation of nanoparticles carrier or cargo owing to internalization into degradative intracellular compartments [12–14,16]. However, both internalized drug cargos (indirect T lymphocyte drug loading) and local permeation of drug cargos released from nanoparticles conjugated on the surface of T lymphocyte through the plasma membrane to the cytosol (direct T lymphocyte drug loading) might lead to some adverse effects and dampen the intrinsic functions of T lymphocytes. In addition, the lack of control on drug release from nanoparticles might lead to some premature prior to target sites or insufficient release of drug cargos within target cells, which affect the drug accumulation in the target sites or drug concentration-dependent antitumor efficacy. To avoid the internalization of drug-loaded nanoparticles and control the drug release from nanoparticles, here a pH sensitive covalent bond was used to decorate the surface of T lymphocytes by novel ICD drug-albumin conjugates containing a redox cleavable bond. We expected that drug-albumin conjugates would stably reside on the surface of T lymphocytes and then autologous T lymphocytes would act as “Trojan horses” to efficiently deliver doxorubicin-albumin conjugates to tumor tissue, which efficiently accumulated in peripheral cells of the tumor mass but not tumor core region. Under the more acidic (pH 6.5) tumor microenvironment, the doxorubicin-albumin conjugates would be automatically dislodged from T lymphocytes by the pH sensitive covalent bond. In comparison to the T lymphocytes drug carriers, the ultra-smaller size enables doxorubicin-albumin conjugates to reach the tumor core region and achieve the deep tumor penetration. We also expect that our doxorubicin-albumin conjugates exhibit avid uptake by tumor cells owing to the natural fast-growing demand. Once being internalized into the cytoplasm, the redox cleavable bond between the doxorubicin-albumin conjugate trigger the efficient doxorubicin release under the action of abundant intracellular redox species. This high concentration of doxorubicin within tumor cells exerts potent therapeutic benefits with absence of systemic toxicity via both concentration-dependent chemotherapy and immunogenic cell death. In order to fulfill our hypothesis, we first devised a hyperbranched self-immolative polymers with dendrimer like structure (termed as Dendron), which has multiple chemotherapeutics coupling points on the branch end and a disulfide bond on the capping end. Next, we used a bi-functional polyethylene glycol space to couple four doxorubicin (DOX) molecules onto one
Dendron molecule and then hooked the Dendron-DOX conjugate to the amine group of albumin to form the DOX-albumin conjugate (DOX-A). We then synthesized another PEG derivative consisting of a pH sensitive bond and a maleimide end group as the linkage to hook DOX-A to the free thiol groups T lymphocytes plasma membrane, which is termed as DOX-A@T cell. Herein, we investigated DOX-A@T cell as an innovative drug delivery platform substantially implementation in chemotherapy-mediated tumor immune microenvironment (TIME) modulation and antitumor efficacy. In summary, we developed here a novel approach for active targeting of therapeutics (drug-albumin conjugates) to tumor sites, using autologous T lymphocytes as “Trojan horses” (Scheme 1). As designed, DOX-A@T cells consist of two sections, DOX-A conjugate synthesis and engineered DOX-A@ T cells construction (Fig. 1a). As for the conjugate part, we noted that dendrimerbased nanomaterials possessed higher drug loading efficiency and smaller particle size, which endowed with remarkable enrichment in tumor tissues, in the light of our previous work [17,18]. Unfortunately, there’s still unmet criteria primarily in controllable drug release pattern. Considering the unique biological condition in tumor microenvironment, stimuli-responsive trigger could be applied into the design of nanocarriers. A smart self-immolative polymers with branched structure (termed as Dendron) were initially synthesized step by step (Scheme S1–S2, Figure S1–S12). Then DOX could be conjugated with hydroxyl group of Dendron (four branches) while the disulfide linker was introduced into the head group of Dendron. Hence, drug disassembled from Dendron could follow like domino fashion, in other words, like a self-immolative reaction resulting in controllable release [19]. Dendron-DOX derivative was water insoluble due to its structure without any hydrophilic block adding. To increase the hydrophilicity of Dendron-DOX derivative and construct the bridge linking to the T lymphocytes, albumin was chosen to act as the bridge protein because of the following merits [20]: albumin is the most naturally abundant plasma protein in blood and has been success® fully approved as drug delivery vehicle like Abraxane ; albumin conjugation could increase the in vivo stability and facilitate the tumor transportation of small chemotherapeutics. Albumin could be simply bridged with Dendron-DOX via a bi-functional PEG linker, termed as DOX-albumin conjugate (termed as DOX-A). The morphology of DOX-A conjugate showed near spheroid particle under normal condition while collapsed immediately in the media supplied with 10 mM DTT solution, where the disulfide bond was cleaved by reductive reagent, such as DTT or GSH, thus leading to a complete break up in Dendron molecule. Dynamic light scattering (DLS) analysis further validated the structure transformation under the redox condition (Fig. 1b). Consistent with those expectations, DTT as reducing reagent apparently accelerated the release of DOX from DOX-A in a manner dependent on the cleavable disulfide bond, while DOX-A remains intact in pH 7.4 PBS buffer (Fig. 1c). We postulated that this redox-responsive release could occur in tumor cells. To further construct the engineered DOX-A@ T cells, T lymphocytes were freshly isolated from mouse spleen and stimulated via anti-CD3/anti-CD28 activation kit [21]. Activated T lymphocyte could be sorted out through detecting the CD3 expression for the subsequent nanoparticle labeling (Figure S13). Driven by the phenomenon that activated T lymphocyte have elevated levels of free thiol groups on cell membrane [12], we attempted to link DOX-A to carrier cell through insertion of maleimide-activated linkers into the DOX-A structure, for coupling the thiol groups on cell surface. More importantly, to achieve the deep penetration of therapeutics into hypoperfusion area in tumors, we synthesized a novel bi-functional PEG linker containing pH sensitive bond which could be cleaved synthesized regarding the weakly acidic tumor microenvironment (Scheme S3). Thus, DOX-A was backpacked to
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Scheme 1. Construction of DOX-A@T cells for bolstering up ICD within tumors.
Fig. 1. Design of DOX-A@T cell. Construction of engineered DOX-A@T cells include DOX-A conjugate synthesis and T cells labeling strategy (a); TEM images and size distribution of HDD in the absence and presence of 10 mM DTT, scale bar = 200 nm (b); in vitro DOX release from DOX-A triggered by 10 mM DTT or not in PBS 7.4. Data were presented as mean ± SD (n = 3) (c); representative dot plots of CD3+ T lymphocyte after activation (upper panel) and IFN-␥ production from CD8+ T lymphocyte (lower panel), in comparison between endogenous T lymphocyte and engineered DOX-A@T cell (d); colocalization of DOX-A and T cells after conjugate labeling, membrane stained with WGA (green) and Bodipy labeled DOX-A (red) were imaged by confocal microscopy after 1 h and 6 h, respectively. Scale bar = 5 m (e). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
the plasma membrane of T lymphocyte through this PEG linker, constructing DOX-A@T cell. Next we scrutinized the total CD3 expression level of T cells, occupation of CD8+ effector T cells and secreted level of IFN-␥ from endogenous T cells and engineered
DOX-A@T cells, respectively (Figure S15). There were no obvious difference in endogenous T lymphocyte and DOX-A@T cell, suggesting that this chemical conjugation exhibited no toxicity or interference with intrinsic cell functions (Fig. 1d). DOX-A should
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Fig. 2. In vitro study of DOX-A@T cell. In vitro penetration of Bodipy labeled DOX-A@T cell on 4T1 tumor spheroids with 2 h incubation under pH 6.5 acidic environment. Scale bar 100 m, z-axis depth 20 m (a); in vitro killing of 4T1 cells by free DOX, DOX-A conjugate and DOX-A@T cells, unconjugated T cells as control. Data were presented as means ± SD (n = 4) (c and d); (c). Cell apoptosis of 4T1 cells induced by free DOX, DOX-A, DOX-A@T cells and unconjugated T cells were determined by FACS analysis after 12 h treatment (d); observation of immunogenic cell death induced by free DOX, DOX-A, DOX-A@T cell and unconjugated T cells after 12 h treatment, cell membrane surface CRT exposure were detected by immunofluorescence with antibodies against CRT, followed by FITC-conjugated secondary antibodies (green) and DAPI (blue) staining nucleus under two-photon laser microscopy. Scale bar: 10 m (e). Release of Hsp70 in the whole cell lysate was measured using western blot, and -actin was selected as the control protein. Data are presented as means ± SD (n = 3) (f). Significance is defined as ns, no significance, *p < 0.05, **p < 0.01, ***p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
localize on the membrane of T cells instead of internalization. Thus, the presence of Bodipy-labeled DOX-A at the surface of T lymphocyte was visualized by confocal microscopy. The images captured in Fig. 1e showed that the red signal of Bodipy co-localized with the green signal T cell membrane indicating that the DOX-A were predominantly retained on the T cell membrane through thiol ether bond without endocytosis into T cells for at least 6 h (Fig. 1e), which was in consistent with L. Tang’s work [13]. In addition, covalent coupling the hyper-branched Dendron-DOX to the albumin particle also maximized the loading efficiency per cell, we also confirmed the drug loading efficiency of this complex through cell lysis approach, which approximately calculated as 6.5 g DOX/106 cells. Given that the in vivo microenvironment of many solid tumors is slightly acidic (pH 6.5) [22,23], we anticipated that incubating DOX-A@T cell with tumor spheroid under culture medium with different pH values could mimic the tumor extracellular environment in vitro. At the meantime, a pH insensitive PEG linker was synthesized as control. Compared with DOX-A@T cells without pH sensitive linker, DOX-A@T cells we fabricated could be able to cleave under weakly acidic (pH 6.8) media with more Bodipy signal distributed the inner core of tumor spheroid, enabling the deep penetration behavior of DOX-A (Fig. 2a). Besides, the microsize of DOX-A@T cell impeded penetration under physiological condition, suggesting the stability of DOX-A conjugate conserved with the “Trojan horse” role of T cells (Figure S15). Next we adopted several assays to investigate the killing efficacy of DOX-A@T cells complex in vitro. To begin with, the inhibitory role of DOX-A@T cells on the viability of tumor cells was detected using MTT assay. As shown in Fig. 2b, the inhibition of tumor cell viability was concentration-dependent. There were no obvious difference among the three DOX formulations, respectively (Table S2). Previous results showed that endogenous T cells origin from mouse
spleen with very subtle CD8+ T cells population. We next conversed the drug concentration of DOX-A@T cells into different T cells/4T1 cell ratio comparing with the same ratio under endogenous T cells incubation. The killing efficacy of plain T cells was quite marginal, compared with DOX-A@T cells, suggesting that endogenous T cells possess the inferior CD8 and less interferon gamma secretion primarily attributed to the devoid stimulation of peculiar tumor antigen. Low dose of doxorubicin, similar to platinum-based drug, reckons on their capacity as ICD inducer to promote apoptosis of tumor cells [4]. Key events in this pathway include early translocation to the tumor cell surface of calreticulin protein (CRT), which could be recognized as an “eat-me” signal for DCs phagocytosis and tumor antigen presentation [24]. Meanwhile, molecular chaperones, such as Hsp70 appear on the tumor cell surface, could stimulate DC maturation [25]. Additionally, the post-apoptotic release signal like the nuclear chromatin binding protein HMGB1 released into the extracellular matrix, might contribute to the antigen presentation by DCs as well as activation of CD8+ T cells [4]. To investigate the ICD inducing apoptosis in vitro, cell apoptosis assay via Annexin V/PI staining is the most intuitive way. In agreement with previous antitumor efficacy in vitro, endogenous T lymphocytes merely generate anti-tumor efficacy probably due to the undifferentiated state isolated from mouse spleen. DOX-A and DOX-A@T cell induced significantly apoptosis than their free compound counterparts in virtue of the high concentration of intracellular reducing agents, such as glutathione (GSH) accelerating the drug release from DOX-A or DOX-A@T cell (Fig. 2d). Of note, the detachment of DOX-A@T cell should be conducted artificially under weakly acidic condition to ensure the subsequent uptake from tumor cells and the similar responsiveness with DOX-A conjugate.
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Fig. 3. In vivo study of DOX-A@T cell. In vivo fluorescence imaging of mice post injection of DOX-A and DOX-A@T cell (a); ex vivo fluorescence images of normal organs and tumors collected at 48 h after i.v. administration (b); colocalization between SPARC protein (green) and Bodipy labeled DOX-A or DOX-A@T cell after 36 h injection. SPARC protein were detected by immunofluorescence method with antibodies against SPARC, followed by FITC conjugated secondary antibodies (green) and DAPI (blue) staining nucleus. Scale bar: 100 m (c); antitumor efficacy of saline, free DOX, DOX-A and DOX-A@T cell evaluated on body weight and tumor volume. Data are presented as means ± SD (n = 6) (d and e); representative histological images of 4T1 tumor xenografts of different DOX-based treated groups using the TUNEL assay. Green: apoptosis cells. Green, apoptosis cells; original magnification = 100×. Scale bar: 100 m (d). Significance is defined as *p < 0.05, **p < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
As described previously [26], the exposure of some ICD signals is essential for ICD mediated anti-tumor efficacy and rescue effect on suppressive TIME (Fig. 2e and f). To analyze the membrane translocation of CRT protein, post-treatment cells were labeled with anti-CRT antibodies and fluorescent secondary antibodies for observation by confocal microscopy. As expected, the non-ICD inducer, unconjugated T lymphocyte had extremely weak effects on triggering CRT exposure. Treatment with DOX, DOX-A and DOXA@T cell could increase CRT exposure on the cell membrane, in which exhibiting obvious aggregation and shrinkage of nucleus. Furthermore, the expression of Hsp70 was enhanced after free DOX, DOX-A and DOX-A@T cell treatment due to the ICD effect, suggesting the possibility of DC maturation and effector T cell infiltration in tumor immune microenvironment. Inspired by above findings in vitro, we next investigated the effect of DOX-A@T cells on cancer treatment in vivo, via establishing xenograft mouse model of breast cancer. We anticipated that the homing feature of T cells could enhance the tumor targeting of DOX-A@T cells. Bodipy as fluorescent probe was labeled into DOX-A and DOX-A@T cells, respectively. The biodistribution in vivo was monitored at different time after treatment with either of two vehicles. As illustrated in Fig. 3a, the mice injected with DOX-A@T cells exhibited stronger near-infrared (NIR) signal at tumor sites (indicated with the red circle) than the one injected with DOXA conjugate, suggesting targeting efficiency enhanced by T cells. After 48 h tracing, the NIR signals of DOX-A has accumulated in the metabolic organ (Fig. 3b), such as kidney and liver while the NIR signals of DOX-A@T cell remained in tumor site, complying with
the fact that DOX-A@T cells actively infiltrate into tumor microenvironment as endogenous counterpart. Given above observations, DOX-A@T cells actively recruited into tumor microenvironment. After infiltrating into the tumor tissue, DOX-A conjugated would be departed from T cells under acidic tumor microenvironment, mediating its penetration and endocytosis through albumin receptor, containing SPARC and gp60 [27]. The tumor-bearing mice were intravenously injected with Bodipy-labeled DOX-A and DOX-A@T cell. After 48 h treatment, the frozen tumor slices were obtained and stained with SPARC primary antibody and fluorescent secondary antibody, which were observed under confocal microscope. As captured in Fig. 3c, SPARC overexpressed (green signal) in tumor tissues could enhance the accumulation of either vehicle. Colocalization of SPARC and either vehicle was also captured (yellow signal), suggesting that the binding between SPARC and albumin from DOX-A or DOX-A@T cell in tumor microenvironment was of great importance for the substantial accumulation. More importantly, co-localization of DOX-A@T cells and SPARC confirmed that pH responsive-dependent dissociation of DOX-A from T cells and regulation of drug release in the tumor microenvironment. In our previous work, we noted that mice that received low dose platinum drug could not only rescue the tumor growth but also induce the exposure of ICD related signal [17]. Thus, we envisioned that DOX-A@T cell could not only perform as a conventional chemotherapeutic but also regulate the suppressive tumor immune microenvironment. After receiving 4 doses at a dosage of 2.5 mg DOX/kg every other day via tail-vein intravenously injection, tumor volume and body weight were recorded at determined
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Fig. 4. Immune responses generated after DOX-A@T cell treatment. Observation of ICD effect and T lymphocyte infiltration after treatment with different formulations. Release of Hsp70 and HMGB-1 in the whole tumor tissue lysates were determined using western blot, and -actin was selected as the control protein (a); cell membrane surface CRT exposure using by two-photon laser microscopy. CRT-positive tissues immunofluorescence with antibodies against CRT, followed by FITC-conjugated secondary antibodies (green) and DAPI (blue) staining nucleus. Scale bar: 100 m (b); DC maturation effect determined by CD80+ and CD86+ DC distributed in tumor draining lymph node. Data are presented as means ± SD (n = 3) (c); representative flow cytometry data of CD45+ CD4− CD8+ and CD45+ CD4+ CD8− effector T lymphocytes infiltration in tumors. Data are presented as means ± SD (n = 3) (d); the amount of helper T cells determined by CD4+ CD25− T lymphocytes and regulatory T cells determined by CD4+ CD25+ T lymphocytes distributed in tumors. Data are presented as means ± SD (n = 3) (e); CD8+ T lymphocyte infiltration into tumor microenvironment determined by immunofluorescence with CD8-FITC antibody (green). Scale bar: 100 m (f); IFN-␥ levels intra-tumor were analyzed by ELISA method. Data are presented as mean ± SD (n = 4) (g). Significance is defined as ns, no significance, *p < 0.05, **p < 0.01, ***p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
time points to evaluate general antitumor effects (Fig. 3d and e). The tumor sections from mice in each group were detected from tissue-level apoptosis staining method (Fig. 3f). After all, DOX-A@T cell manifested the strongest antitumor efficacy proven by the steadiest curve of growth rate and tumor size change, as well as could induce the strongest extent of tumor apoptosis. In contrast, when administered in the form of DOX-A@T cells, no detectable abnormality was observed in the major organs, suggesting the biocompatibility of formulation (Figure S16). As a result, this chemical approach for engineered DOX-A@T cells complements the safe delivery of doxorubicin and controllable release profiling in the tumor microenvironment, which would further contribute to ICD signals exposure and awaken anti-tumor immunity. Given that the ability of doxorubicin to induce immunogenic cell death of tumor cells in vitro, it is reasonable to predict that doxorubicin treatment might give rise to the subsequent modulation of immune response in tumor microenvironment. To prove that doxorubicin has the ability to induce ICD in 4T1 cells bearing mice, at first Hsp70 and HMGB-1 were detected by western blot assay, which showed that DOX-A@T cell could increase the expression of these two proteins (Fig. 4a). It has been reported that Hsp70 and HMGB-1 protein were closely relevant to the extent of ICD induced by chemotherapeutics [24]. Furthermore, CRT exposure after each treatment was observed through immunofluorescence staining under confocal microcopy. Consistent with the result of in vitro assay, DOX-A@T cell could induce more CRT translocation
from cytoplasm to membrane (Fig. 4b). Besides, T cell infiltration as a prerequisite for activating immune response, CD3+ T lymphocyte was then monitored by immunofluorescence staining to investigate that infiltration level was dependent on the transformation of tumor immune microenvironment. As imaged (Figure S17), T lymphocyte infiltration has been restored after DOX-A@T cell treatment, suggesting that ICD signals exposure after DOX-A@T cells treatment in tumor-bearing mice could be processed by immune cells to further alleviate the tumor immune microenvironment. Tumor development is not only triggered by activation of oncogenes or inactivation of suppressor genes, but also by the composition in the tumor immune microenvironment, indicating by the categories and density of infiltrated immune cells [28]. An effective antitumor immune response is often driven by a combination of effector lymphocytes and a subset of dendritic cells. Accumulating evidence suggests that activation of immune response is critical for improving prognosis and patient survival, which could be attained by using agents that induce ICD in tumor cells. Among, ICD defines that dying cancer cells by ICD inducers (conventional chemotherapeutics) would operate as tumor vaccines that stimulate the immune response. Therefore, it is crucial to explore the efficacy of ICD inducer contributing to restore immune response inside tumor immune microenvironment. After treatment with different groups, mice were first sacrificed to dissect their lymph nodes and tumor tissues, and subsequently collected to evaluate the levels of flow cytometry analysis. It was found that DOX-A@T
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cells could promote robust DC maturation among other injections, which means that the sufficient ICD cell debris generated from DOX-A@T cells group could be recognized as tumor associated antigen for DC processing and presentation (Fig. 4c). Next, the differentiation levels of infiltrated T lymphocyte were determined and evaluated by classical biomarkers. Tumor tissues were processed to stain with fluorescent biomarkers after treatment to distinguish the tumor-infiltrating effector T cells (CD45+ CD4− CD8+ ), helper T cells (CD45+ CD4+ CD25− ) and regulatory T cells (CD45+ CD4+ CD25+ ) by flow cytometry (Fig. 4d and e). Cross-priming of CD8+ T lymphocyte was triggered by mature DCs and antigen presentation. The percentage of effector T cells was obviously increased (by ∼1.5 fold compared to the saline control) after treatment with DOX-A@T cell, suggesting the antitumor immunological response generating from ICD inducer against immunosuppressive microenvironment. The helper T cells could also be classified into effector lymphocytes which could interact with cytotoxic T lymphocyte by secreting relative cytokines. The higher percentage of helper T cells emerged after DOX-A@T cell (by ∼2 fold compared to the saline control) could also contribute to potent immune response. Meanwhile, regulatory T cells are regarded as suppressive immune cells, playing an important role in tumor evasion mechanism [29]. DOX-A@T cell could effectively lower down the infiltration level of regulatory T cells. Furthermore, DOX-A@T cell could facilitate CD8+ T lymphocyte infiltrating into dense magnificent tissues (Fig. 4f). Beyond that, the secretion level of IFN-␥ was positively upregulated in DOX-A@T cell (Fig. 4g). Accumulating results indicated that the DOX delivery by engineered T cells is not solely attributed to tumor regression, but also the restoration of immune response. In summary, we have rationally exploited tumor infiltrating lymphocyte as the delivery platform of ICD inducer doxorubicin aiming at maximizing the combined effects of chemotherapeutic efficacy and immunotherapy. To overcome the limitation of cell-based carrier, we have selected albumin to conjugate more drug and tethered trigger responsive linker anchored the surface of T lymphocyte. In addition to the chemotherapeutic effect of DOX, DOX-A@T cell take advantage of DOX-induced ICD to directly destroy tumor cells and to stimulate immune responses by alternating the maturation of DCs and subsequently inducing infiltrating T lymphocyte differentiation into more effector T cells, ultimately awakening the immune response. In present work, DOX-A@T cell has been developed as maximizing the ICD effect on tumor cells to indirectly alleviate the suppressive TIME. To the best of our knowledge, the interplay of immunotherapy and chemotherapy has been harnessed in several tumor models, holding great potential in cancer treatment. Our strategy may open a new direction to precise combination therapy tailored to different cancer types. Acknowledgements We acknowledge the financial funding from National Science Fund for Distinguished Young Scholars (Grant 81425023), Program of Shanghai Academic Research Leader (No. 18XD1400500) and special Funding for Aging and Medicine of Fudan University (No. IDF301110). Appendix A. Supplementary data
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Applied Materials Today journal homepage: www.elsevier.com/locate/apmt
Short communication
Nano-engineered lymphocytes for alleviating suppressive tumor immune microenvironment Lisha Liu 1 , Qinjun Chen 1 , Chunhui Ruan, Xinli Chen, Xi He, Yu Zhang, Yujie Zhang, Yifei Lu, Qin Guo, Wenxi Zhou, Chao Li, Tao Sun, Chen Jiang ∗ Key Laboratory of Smart Drug Delivery, Ministry of Education, State Key Laboratory of Medical Neurobiology, Department of Pharmaceutics, School of Pharmacy, Fudan University, Shanghai 201203, China
a r t i c l e
i n f o
Article history: Received 30 April 2019 Received in revised form 20 June 2019 Accepted 21 June 2019 Keywords: Tumor-infiltrating lymphocytes Doxorubicin Albumin conjugate Tumor immune microenvironment Drug delivery
a b s t r a c t The intrinsic trafficking ability of host cells to infiltrate various disease sites and act as living chaperones for chemotherapeutics alone or chemotherapeutics-loaded nanoparticles to actively transmigrate the endothelial barrier within solid tumor and achieve efficient tumor accumulation is being explored in a quantity of contexts. Here, we leveraged tumor-infiltrating T lymphocytes as delivery vehicle to increase the tumor apoptosis and immunogenic cell death (ICD) of doxorubicin to efficiently boost immune response and eliminate the tumor growth. First we decorated the redox responsive doxorubicin-albumin conjugates (DOX-A) onto the surface of T lymphocytes (DOX-A@T cell) via a pH sensitive bond, which allows for stable retention of DOX-A on the surface of T lymphocytes in blood circulation but the responsive release of DOX-A within the weak acid tumor microenvironment. The released DOX-A exerted deep tumor penetration, efficient tumor cell uptake and rapid intracellular drug release via redox responsive cleavage. We showed that DOX-A can be stably conjugated to the surface of T lymphocytes for at least 6 h without any detectable toxicity or interference with intrinsic cell functions. DOX-A@T cell had the strong tumor infiltration and penetration both in vitro and in vivo. More significantly, this DOX-A@T cell actively efficiently augmented the DOX-induced ICD efficacy through activation of dendritic cells (DCs) and consequent activation of specific T cell response, thus potentiating immune response in tumor microenvironment. © 2019 Published by Elsevier Ltd.
Although immunotherapy and chemotherapy fight against cancer via targeting different module of tumor-cell survival and their combined effects, the fact is that potent chemotherapy is well known to blunt immune systems long perplexes the successful application of this combination [1]. However, over the past few years accumulating evidence indicates the positive immunologic effects of chemotherapeutic agents [2]. Among, immunogenic cell death (ICD) is a unique modality of cell death in response to some antineoplastic agents such as anthracyclines and oxaliplatin. Unlike normal apoptosis mechanism, ICD involves changes in the composition of the cell surface as well as releases a large number of immune-stimulatory signals, including calreticulin (CRT) exposure, high mobility group box 1 (HMGB1) translocation and heat shock protein (HSP) expression, into tumor immune microenvironment (TIME), then induce an effective antitumor immune
∗ Corresponding author. E-mail address: [email protected] (C. Jiang). 1 These authors contributed equally to this paper. https://doi.org/10.1016/j.apmt.2019.06.009 2352-9407/© 2019 Published by Elsevier Ltd.
response through activation of dendritic cells (DCs) and consequent activation of specific T cell response [3,4]. Recently the increasing preclinical evidences have demonstrated that ICD induced by chemotherapeutics efficiently broke down the immune tolerance and arrested tumor growth in animals [5,6]. However, effective dose of ICD drug, which is required to break immune tolerance to the tumor meanwhile avoid undesired host autoimmune outcomes, is usually a tremendous challenge to most of active targeting nanoparticles, in view of insufficient tumor accumulation and inferior penetration within the tumor microenvironment [7]. Therefore, how to design a smart drug delivery system, which can actively transmigrate the endothelial barrier of many solid tumors with higher densities of blood vessels, efficiently accumulate in tumor tissues and improve the deep penetration of nanoparticle into deep tumor region, still encounter a major challenge. Cell engineering strategies showed their promising application for targeted drug delivery, cancer immunotherapy and blood glucose regulation [8,9]. Among T lymphocytes play a central role in immune protection against cancer. Their infiltration in tumor site is usually associated with a favorable prognosis and may predict
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outcome of therapies with drugs that block immune inhibitory receptors. A recent meta-analysis reported that tumor-infiltrating lymphocytes (TIL) can be identified in 48% of all breast cancers. Interestingly, triple-negative breast cancers show the highest incidence of lymphocyte predominance [10]. The intrinsic trafficking ability of T lymphocytes to infiltrate tumor sites and act as living chaperones for therapeutics has been explored in a number of contexts [11–16]. Those outcomes demonstrated that leveraging T lymphocytes as drug carriers greatly enhanced the amount of drug that can be delivered to tumor, achieving the quantity in the tumor that are orders of magnitude greater than that which can be delivered by nanoparticles alone or even most of active targeted nanoparticles [11,14]. In those previous works, there were two methods for T lymphocytes to load therapeutic cargoes, such as indirect way and direct way. The indirect way conjugated the surface of drug-loaded nanoparticles with an antibody against antigen of T lymphocytes, which showed specific T lymphocytes binding and then effective receptor-mediated internalization by T lymphocytes [15]. The direct way used complementary chemistry-based approaches to conjugate drug-loaded nanoparticles directly to the plasma membrane of T lymphocytes, where nanoparticles were not internalized and slowly released their cargo compounds. This retention of nanoparticles on the surfaces of chaperon cells was shown to avoid the premature degradation of nanoparticles carrier or cargo owing to internalization into degradative intracellular compartments [12–14,16]. However, both internalized drug cargos (indirect T lymphocyte drug loading) and local permeation of drug cargos released from nanoparticles conjugated on the surface of T lymphocyte through the plasma membrane to the cytosol (direct T lymphocyte drug loading) might lead to some adverse effects and dampen the intrinsic functions of T lymphocytes. In addition, the lack of control on drug release from nanoparticles might lead to some premature prior to target sites or insufficient release of drug cargos within target cells, which affect the drug accumulation in the target sites or drug concentration-dependent antitumor efficacy. To avoid the internalization of drug-loaded nanoparticles and control the drug release from nanoparticles, here a pH sensitive covalent bond was used to decorate the surface of T lymphocytes by novel ICD drug-albumin conjugates containing a redox cleavable bond. We expected that drug-albumin conjugates would stably reside on the surface of T lymphocytes and then autologous T lymphocytes would act as “Trojan horses” to efficiently deliver doxorubicin-albumin conjugates to tumor tissue, which efficiently accumulated in peripheral cells of the tumor mass but not tumor core region. Under the more acidic (pH 6.5) tumor microenvironment, the doxorubicin-albumin conjugates would be automatically dislodged from T lymphocytes by the pH sensitive covalent bond. In comparison to the T lymphocytes drug carriers, the ultra-smaller size enables doxorubicin-albumin conjugates to reach the tumor core region and achieve the deep tumor penetration. We also expect that our doxorubicin-albumin conjugates exhibit avid uptake by tumor cells owing to the natural fast-growing demand. Once being internalized into the cytoplasm, the redox cleavable bond between the doxorubicin-albumin conjugate trigger the efficient doxorubicin release under the action of abundant intracellular redox species. This high concentration of doxorubicin within tumor cells exerts potent therapeutic benefits with absence of systemic toxicity via both concentration-dependent chemotherapy and immunogenic cell death. In order to fulfill our hypothesis, we first devised a hyperbranched self-immolative polymers with dendrimer like structure (termed as Dendron), which has multiple chemotherapeutics coupling points on the branch end and a disulfide bond on the capping end. Next, we used a bi-functional polyethylene glycol space to couple four doxorubicin (DOX) molecules onto one
Dendron molecule and then hooked the Dendron-DOX conjugate to the amine group of albumin to form the DOX-albumin conjugate (DOX-A). We then synthesized another PEG derivative consisting of a pH sensitive bond and a maleimide end group as the linkage to hook DOX-A to the free thiol groups T lymphocytes plasma membrane, which is termed as DOX-A@T cell. Herein, we investigated DOX-A@T cell as an innovative drug delivery platform substantially implementation in chemotherapy-mediated tumor immune microenvironment (TIME) modulation and antitumor efficacy. In summary, we developed here a novel approach for active targeting of therapeutics (drug-albumin conjugates) to tumor sites, using autologous T lymphocytes as “Trojan horses” (Scheme 1). As designed, DOX-A@T cells consist of two sections, DOX-A conjugate synthesis and engineered DOX-A@ T cells construction (Fig. 1a). As for the conjugate part, we noted that dendrimerbased nanomaterials possessed higher drug loading efficiency and smaller particle size, which endowed with remarkable enrichment in tumor tissues, in the light of our previous work [17,18]. Unfortunately, there’s still unmet criteria primarily in controllable drug release pattern. Considering the unique biological condition in tumor microenvironment, stimuli-responsive trigger could be applied into the design of nanocarriers. A smart self-immolative polymers with branched structure (termed as Dendron) were initially synthesized step by step (Scheme S1–S2, Figure S1–S12). Then DOX could be conjugated with hydroxyl group of Dendron (four branches) while the disulfide linker was introduced into the head group of Dendron. Hence, drug disassembled from Dendron could follow like domino fashion, in other words, like a self-immolative reaction resulting in controllable release [19]. Dendron-DOX derivative was water insoluble due to its structure without any hydrophilic block adding. To increase the hydrophilicity of Dendron-DOX derivative and construct the bridge linking to the T lymphocytes, albumin was chosen to act as the bridge protein because of the following merits [20]: albumin is the most naturally abundant plasma protein in blood and has been success® fully approved as drug delivery vehicle like Abraxane ; albumin conjugation could increase the in vivo stability and facilitate the tumor transportation of small chemotherapeutics. Albumin could be simply bridged with Dendron-DOX via a bi-functional PEG linker, termed as DOX-albumin conjugate (termed as DOX-A). The morphology of DOX-A conjugate showed near spheroid particle under normal condition while collapsed immediately in the media supplied with 10 mM DTT solution, where the disulfide bond was cleaved by reductive reagent, such as DTT or GSH, thus leading to a complete break up in Dendron molecule. Dynamic light scattering (DLS) analysis further validated the structure transformation under the redox condition (Fig. 1b). Consistent with those expectations, DTT as reducing reagent apparently accelerated the release of DOX from DOX-A in a manner dependent on the cleavable disulfide bond, while DOX-A remains intact in pH 7.4 PBS buffer (Fig. 1c). We postulated that this redox-responsive release could occur in tumor cells. To further construct the engineered DOX-A@ T cells, T lymphocytes were freshly isolated from mouse spleen and stimulated via anti-CD3/anti-CD28 activation kit [21]. Activated T lymphocyte could be sorted out through detecting the CD3 expression for the subsequent nanoparticle labeling (Figure S13). Driven by the phenomenon that activated T lymphocyte have elevated levels of free thiol groups on cell membrane [12], we attempted to link DOX-A to carrier cell through insertion of maleimide-activated linkers into the DOX-A structure, for coupling the thiol groups on cell surface. More importantly, to achieve the deep penetration of therapeutics into hypoperfusion area in tumors, we synthesized a novel bi-functional PEG linker containing pH sensitive bond which could be cleaved synthesized regarding the weakly acidic tumor microenvironment (Scheme S3). Thus, DOX-A was backpacked to
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Scheme 1. Construction of DOX-A@T cells for bolstering up ICD within tumors.
Fig. 1. Design of DOX-A@T cell. Construction of engineered DOX-A@T cells include DOX-A conjugate synthesis and T cells labeling strategy (a); TEM images and size distribution of HDD in the absence and presence of 10 mM DTT, scale bar = 200 nm (b); in vitro DOX release from DOX-A triggered by 10 mM DTT or not in PBS 7.4. Data were presented as mean ± SD (n = 3) (c); representative dot plots of CD3+ T lymphocyte after activation (upper panel) and IFN-␥ production from CD8+ T lymphocyte (lower panel), in comparison between endogenous T lymphocyte and engineered DOX-A@T cell (d); colocalization of DOX-A and T cells after conjugate labeling, membrane stained with WGA (green) and Bodipy labeled DOX-A (red) were imaged by confocal microscopy after 1 h and 6 h, respectively. Scale bar = 5 m (e). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
the plasma membrane of T lymphocyte through this PEG linker, constructing DOX-A@T cell. Next we scrutinized the total CD3 expression level of T cells, occupation of CD8+ effector T cells and secreted level of IFN-␥ from endogenous T cells and engineered
DOX-A@T cells, respectively (Figure S15). There were no obvious difference in endogenous T lymphocyte and DOX-A@T cell, suggesting that this chemical conjugation exhibited no toxicity or interference with intrinsic cell functions (Fig. 1d). DOX-A should
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Fig. 2. In vitro study of DOX-A@T cell. In vitro penetration of Bodipy labeled DOX-A@T cell on 4T1 tumor spheroids with 2 h incubation under pH 6.5 acidic environment. Scale bar 100 m, z-axis depth 20 m (a); in vitro killing of 4T1 cells by free DOX, DOX-A conjugate and DOX-A@T cells, unconjugated T cells as control. Data were presented as means ± SD (n = 4) (c and d); (c). Cell apoptosis of 4T1 cells induced by free DOX, DOX-A, DOX-A@T cells and unconjugated T cells were determined by FACS analysis after 12 h treatment (d); observation of immunogenic cell death induced by free DOX, DOX-A, DOX-A@T cell and unconjugated T cells after 12 h treatment, cell membrane surface CRT exposure were detected by immunofluorescence with antibodies against CRT, followed by FITC-conjugated secondary antibodies (green) and DAPI (blue) staining nucleus under two-photon laser microscopy. Scale bar: 10 m (e). Release of Hsp70 in the whole cell lysate was measured using western blot, and -actin was selected as the control protein. Data are presented as means ± SD (n = 3) (f). Significance is defined as ns, no significance, *p < 0.05, **p < 0.01, ***p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
localize on the membrane of T cells instead of internalization. Thus, the presence of Bodipy-labeled DOX-A at the surface of T lymphocyte was visualized by confocal microscopy. The images captured in Fig. 1e showed that the red signal of Bodipy co-localized with the green signal T cell membrane indicating that the DOX-A were predominantly retained on the T cell membrane through thiol ether bond without endocytosis into T cells for at least 6 h (Fig. 1e), which was in consistent with L. Tang’s work [13]. In addition, covalent coupling the hyper-branched Dendron-DOX to the albumin particle also maximized the loading efficiency per cell, we also confirmed the drug loading efficiency of this complex through cell lysis approach, which approximately calculated as 6.5 g DOX/106 cells. Given that the in vivo microenvironment of many solid tumors is slightly acidic (pH 6.5) [22,23], we anticipated that incubating DOX-A@T cell with tumor spheroid under culture medium with different pH values could mimic the tumor extracellular environment in vitro. At the meantime, a pH insensitive PEG linker was synthesized as control. Compared with DOX-A@T cells without pH sensitive linker, DOX-A@T cells we fabricated could be able to cleave under weakly acidic (pH 6.8) media with more Bodipy signal distributed the inner core of tumor spheroid, enabling the deep penetration behavior of DOX-A (Fig. 2a). Besides, the microsize of DOX-A@T cell impeded penetration under physiological condition, suggesting the stability of DOX-A conjugate conserved with the “Trojan horse” role of T cells (Figure S15). Next we adopted several assays to investigate the killing efficacy of DOX-A@T cells complex in vitro. To begin with, the inhibitory role of DOX-A@T cells on the viability of tumor cells was detected using MTT assay. As shown in Fig. 2b, the inhibition of tumor cell viability was concentration-dependent. There were no obvious difference among the three DOX formulations, respectively (Table S2). Previous results showed that endogenous T cells origin from mouse
spleen with very subtle CD8+ T cells population. We next conversed the drug concentration of DOX-A@T cells into different T cells/4T1 cell ratio comparing with the same ratio under endogenous T cells incubation. The killing efficacy of plain T cells was quite marginal, compared with DOX-A@T cells, suggesting that endogenous T cells possess the inferior CD8 and less interferon gamma secretion primarily attributed to the devoid stimulation of peculiar tumor antigen. Low dose of doxorubicin, similar to platinum-based drug, reckons on their capacity as ICD inducer to promote apoptosis of tumor cells [4]. Key events in this pathway include early translocation to the tumor cell surface of calreticulin protein (CRT), which could be recognized as an “eat-me” signal for DCs phagocytosis and tumor antigen presentation [24]. Meanwhile, molecular chaperones, such as Hsp70 appear on the tumor cell surface, could stimulate DC maturation [25]. Additionally, the post-apoptotic release signal like the nuclear chromatin binding protein HMGB1 released into the extracellular matrix, might contribute to the antigen presentation by DCs as well as activation of CD8+ T cells [4]. To investigate the ICD inducing apoptosis in vitro, cell apoptosis assay via Annexin V/PI staining is the most intuitive way. In agreement with previous antitumor efficacy in vitro, endogenous T lymphocytes merely generate anti-tumor efficacy probably due to the undifferentiated state isolated from mouse spleen. DOX-A and DOX-A@T cell induced significantly apoptosis than their free compound counterparts in virtue of the high concentration of intracellular reducing agents, such as glutathione (GSH) accelerating the drug release from DOX-A or DOX-A@T cell (Fig. 2d). Of note, the detachment of DOX-A@T cell should be conducted artificially under weakly acidic condition to ensure the subsequent uptake from tumor cells and the similar responsiveness with DOX-A conjugate.
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Fig. 3. In vivo study of DOX-A@T cell. In vivo fluorescence imaging of mice post injection of DOX-A and DOX-A@T cell (a); ex vivo fluorescence images of normal organs and tumors collected at 48 h after i.v. administration (b); colocalization between SPARC protein (green) and Bodipy labeled DOX-A or DOX-A@T cell after 36 h injection. SPARC protein were detected by immunofluorescence method with antibodies against SPARC, followed by FITC conjugated secondary antibodies (green) and DAPI (blue) staining nucleus. Scale bar: 100 m (c); antitumor efficacy of saline, free DOX, DOX-A and DOX-A@T cell evaluated on body weight and tumor volume. Data are presented as means ± SD (n = 6) (d and e); representative histological images of 4T1 tumor xenografts of different DOX-based treated groups using the TUNEL assay. Green: apoptosis cells. Green, apoptosis cells; original magnification = 100×. Scale bar: 100 m (d). Significance is defined as *p < 0.05, **p < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
As described previously [26], the exposure of some ICD signals is essential for ICD mediated anti-tumor efficacy and rescue effect on suppressive TIME (Fig. 2e and f). To analyze the membrane translocation of CRT protein, post-treatment cells were labeled with anti-CRT antibodies and fluorescent secondary antibodies for observation by confocal microscopy. As expected, the non-ICD inducer, unconjugated T lymphocyte had extremely weak effects on triggering CRT exposure. Treatment with DOX, DOX-A and DOXA@T cell could increase CRT exposure on the cell membrane, in which exhibiting obvious aggregation and shrinkage of nucleus. Furthermore, the expression of Hsp70 was enhanced after free DOX, DOX-A and DOX-A@T cell treatment due to the ICD effect, suggesting the possibility of DC maturation and effector T cell infiltration in tumor immune microenvironment. Inspired by above findings in vitro, we next investigated the effect of DOX-A@T cells on cancer treatment in vivo, via establishing xenograft mouse model of breast cancer. We anticipated that the homing feature of T cells could enhance the tumor targeting of DOX-A@T cells. Bodipy as fluorescent probe was labeled into DOX-A and DOX-A@T cells, respectively. The biodistribution in vivo was monitored at different time after treatment with either of two vehicles. As illustrated in Fig. 3a, the mice injected with DOX-A@T cells exhibited stronger near-infrared (NIR) signal at tumor sites (indicated with the red circle) than the one injected with DOXA conjugate, suggesting targeting efficiency enhanced by T cells. After 48 h tracing, the NIR signals of DOX-A has accumulated in the metabolic organ (Fig. 3b), such as kidney and liver while the NIR signals of DOX-A@T cell remained in tumor site, complying with
the fact that DOX-A@T cells actively infiltrate into tumor microenvironment as endogenous counterpart. Given above observations, DOX-A@T cells actively recruited into tumor microenvironment. After infiltrating into the tumor tissue, DOX-A conjugated would be departed from T cells under acidic tumor microenvironment, mediating its penetration and endocytosis through albumin receptor, containing SPARC and gp60 [27]. The tumor-bearing mice were intravenously injected with Bodipy-labeled DOX-A and DOX-A@T cell. After 48 h treatment, the frozen tumor slices were obtained and stained with SPARC primary antibody and fluorescent secondary antibody, which were observed under confocal microscope. As captured in Fig. 3c, SPARC overexpressed (green signal) in tumor tissues could enhance the accumulation of either vehicle. Colocalization of SPARC and either vehicle was also captured (yellow signal), suggesting that the binding between SPARC and albumin from DOX-A or DOX-A@T cell in tumor microenvironment was of great importance for the substantial accumulation. More importantly, co-localization of DOX-A@T cells and SPARC confirmed that pH responsive-dependent dissociation of DOX-A from T cells and regulation of drug release in the tumor microenvironment. In our previous work, we noted that mice that received low dose platinum drug could not only rescue the tumor growth but also induce the exposure of ICD related signal [17]. Thus, we envisioned that DOX-A@T cell could not only perform as a conventional chemotherapeutic but also regulate the suppressive tumor immune microenvironment. After receiving 4 doses at a dosage of 2.5 mg DOX/kg every other day via tail-vein intravenously injection, tumor volume and body weight were recorded at determined
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Fig. 4. Immune responses generated after DOX-A@T cell treatment. Observation of ICD effect and T lymphocyte infiltration after treatment with different formulations. Release of Hsp70 and HMGB-1 in the whole tumor tissue lysates were determined using western blot, and -actin was selected as the control protein (a); cell membrane surface CRT exposure using by two-photon laser microscopy. CRT-positive tissues immunofluorescence with antibodies against CRT, followed by FITC-conjugated secondary antibodies (green) and DAPI (blue) staining nucleus. Scale bar: 100 m (b); DC maturation effect determined by CD80+ and CD86+ DC distributed in tumor draining lymph node. Data are presented as means ± SD (n = 3) (c); representative flow cytometry data of CD45+ CD4− CD8+ and CD45+ CD4+ CD8− effector T lymphocytes infiltration in tumors. Data are presented as means ± SD (n = 3) (d); the amount of helper T cells determined by CD4+ CD25− T lymphocytes and regulatory T cells determined by CD4+ CD25+ T lymphocytes distributed in tumors. Data are presented as means ± SD (n = 3) (e); CD8+ T lymphocyte infiltration into tumor microenvironment determined by immunofluorescence with CD8-FITC antibody (green). Scale bar: 100 m (f); IFN-␥ levels intra-tumor were analyzed by ELISA method. Data are presented as mean ± SD (n = 4) (g). Significance is defined as ns, no significance, *p < 0.05, **p < 0.01, ***p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
time points to evaluate general antitumor effects (Fig. 3d and e). The tumor sections from mice in each group were detected from tissue-level apoptosis staining method (Fig. 3f). After all, DOX-A@T cell manifested the strongest antitumor efficacy proven by the steadiest curve of growth rate and tumor size change, as well as could induce the strongest extent of tumor apoptosis. In contrast, when administered in the form of DOX-A@T cells, no detectable abnormality was observed in the major organs, suggesting the biocompatibility of formulation (Figure S16). As a result, this chemical approach for engineered DOX-A@T cells complements the safe delivery of doxorubicin and controllable release profiling in the tumor microenvironment, which would further contribute to ICD signals exposure and awaken anti-tumor immunity. Given that the ability of doxorubicin to induce immunogenic cell death of tumor cells in vitro, it is reasonable to predict that doxorubicin treatment might give rise to the subsequent modulation of immune response in tumor microenvironment. To prove that doxorubicin has the ability to induce ICD in 4T1 cells bearing mice, at first Hsp70 and HMGB-1 were detected by western blot assay, which showed that DOX-A@T cell could increase the expression of these two proteins (Fig. 4a). It has been reported that Hsp70 and HMGB-1 protein were closely relevant to the extent of ICD induced by chemotherapeutics [24]. Furthermore, CRT exposure after each treatment was observed through immunofluorescence staining under confocal microcopy. Consistent with the result of in vitro assay, DOX-A@T cell could induce more CRT translocation
from cytoplasm to membrane (Fig. 4b). Besides, T cell infiltration as a prerequisite for activating immune response, CD3+ T lymphocyte was then monitored by immunofluorescence staining to investigate that infiltration level was dependent on the transformation of tumor immune microenvironment. As imaged (Figure S17), T lymphocyte infiltration has been restored after DOX-A@T cell treatment, suggesting that ICD signals exposure after DOX-A@T cells treatment in tumor-bearing mice could be processed by immune cells to further alleviate the tumor immune microenvironment. Tumor development is not only triggered by activation of oncogenes or inactivation of suppressor genes, but also by the composition in the tumor immune microenvironment, indicating by the categories and density of infiltrated immune cells [28]. An effective antitumor immune response is often driven by a combination of effector lymphocytes and a subset of dendritic cells. Accumulating evidence suggests that activation of immune response is critical for improving prognosis and patient survival, which could be attained by using agents that induce ICD in tumor cells. Among, ICD defines that dying cancer cells by ICD inducers (conventional chemotherapeutics) would operate as tumor vaccines that stimulate the immune response. Therefore, it is crucial to explore the efficacy of ICD inducer contributing to restore immune response inside tumor immune microenvironment. After treatment with different groups, mice were first sacrificed to dissect their lymph nodes and tumor tissues, and subsequently collected to evaluate the levels of flow cytometry analysis. It was found that DOX-A@T
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cells could promote robust DC maturation among other injections, which means that the sufficient ICD cell debris generated from DOX-A@T cells group could be recognized as tumor associated antigen for DC processing and presentation (Fig. 4c). Next, the differentiation levels of infiltrated T lymphocyte were determined and evaluated by classical biomarkers. Tumor tissues were processed to stain with fluorescent biomarkers after treatment to distinguish the tumor-infiltrating effector T cells (CD45+ CD4− CD8+ ), helper T cells (CD45+ CD4+ CD25− ) and regulatory T cells (CD45+ CD4+ CD25+ ) by flow cytometry (Fig. 4d and e). Cross-priming of CD8+ T lymphocyte was triggered by mature DCs and antigen presentation. The percentage of effector T cells was obviously increased (by ∼1.5 fold compared to the saline control) after treatment with DOX-A@T cell, suggesting the antitumor immunological response generating from ICD inducer against immunosuppressive microenvironment. The helper T cells could also be classified into effector lymphocytes which could interact with cytotoxic T lymphocyte by secreting relative cytokines. The higher percentage of helper T cells emerged after DOX-A@T cell (by ∼2 fold compared to the saline control) could also contribute to potent immune response. Meanwhile, regulatory T cells are regarded as suppressive immune cells, playing an important role in tumor evasion mechanism [29]. DOX-A@T cell could effectively lower down the infiltration level of regulatory T cells. Furthermore, DOX-A@T cell could facilitate CD8+ T lymphocyte infiltrating into dense magnificent tissues (Fig. 4f). Beyond that, the secretion level of IFN-␥ was positively upregulated in DOX-A@T cell (Fig. 4g). Accumulating results indicated that the DOX delivery by engineered T cells is not solely attributed to tumor regression, but also the restoration of immune response. In summary, we have rationally exploited tumor infiltrating lymphocyte as the delivery platform of ICD inducer doxorubicin aiming at maximizing the combined effects of chemotherapeutic efficacy and immunotherapy. To overcome the limitation of cell-based carrier, we have selected albumin to conjugate more drug and tethered trigger responsive linker anchored the surface of T lymphocyte. In addition to the chemotherapeutic effect of DOX, DOX-A@T cell take advantage of DOX-induced ICD to directly destroy tumor cells and to stimulate immune responses by alternating the maturation of DCs and subsequently inducing infiltrating T lymphocyte differentiation into more effector T cells, ultimately awakening the immune response. In present work, DOX-A@T cell has been developed as maximizing the ICD effect on tumor cells to indirectly alleviate the suppressive TIME. To the best of our knowledge, the interplay of immunotherapy and chemotherapy has been harnessed in several tumor models, holding great potential in cancer treatment. Our strategy may open a new direction to precise combination therapy tailored to different cancer types. Acknowledgements We acknowledge the financial funding from National Science Fund for Distinguished Young Scholars (Grant 81425023), Program of Shanghai Academic Research Leader (No. 18XD1400500) and special Funding for Aging and Medicine of Fudan University (No. IDF301110). Appendix A. Supplementary data
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