Ultrahigh-Speed 3D DNA Walker with Dual Self-Protected DNAzymes for Ultrasensitive Fluorescence Detection and Intracellular Imaging of microRNA.

Herein, a dual self-protected DNAzyme-based 3D DNA walker (dSPD walker), composed of activated dual self-protected walking particles (ac-dSPWPs) and track particles (TPs), was constructed for ultrasensitive and ultrahigh-speed fluorescence detection and imaging of microRNAs (miRNAs) in living cells. Impressively, compared with the defect that "one" target miRNA only initiates "one" walking arm of the conventional single self-protected DNAzyme walker, the dSPD walker benefits from the secondary amplification and spatial confinement effect and could guide "one" target miRNA to generate "n" secondary targets, thereby initiating "n" nearby walking strands immediately, realizing the initial rate over one-magnitude-order faster than that of the conventional one. Moreover, in the process of relative motion between ac-dSPWPs and TPs, the ac-dSPWPs could cleave multiple substrate strands simultaneously to speed up movement and reduce the derailment rate, as well as combine with successive TPs to facilitate a large amount of continuous signal accumulation, achieving an ultrafast detection of miRNA-221 within 10 min in vitro and high sensitivity with a low detection limit of 0.84 pM. In addition, the DNA nanospheres obtained by the rolling circle amplification reaction can capture the Cy5 fluorescence dispersed in liquids, which achieves the high-contrast imaging of miRNA-221, resulting in further ultrasensitive imaging of miRNA-221 in cancer cells. The proposed strategy has made a bold innovation in the rapid and sensitive detection as well as intracellular imaging of low-abundance biomarkers, offering promising application in early diagnosis and relevant research of cancer and tumors.

Neutrophil hitchhiking for drug delivery to the bone marrow.

Pharmaceuticals have been developed for the treatment of a wide range of bone diseases and disorders, but suffer from problematic delivery to the bone marrow. Neutrophils are naturally trafficked to the bone marrow and can cross the bone marrow-blood barrier. Here we report the use of neutrophils for the targeted delivery of free drugs and drug nanoparticles to the bone marrow. We demonstrate how drug-loaded poly(lactic-co-glycolic acid) nanoparticles are taken up by neutrophils and are then transported across the bone marrow-blood barrier to boost drug concentrations in the bone marrow. We demonstrate application of this principle to two models. In a bone metastasis cancer model, neutrophil delivery is shown to deliver cabazitaxel and significantly inhibit tumour growth. In an induced osteoporosis model, neutrophil delivery of teriparatide is shown to significantly increase bone mineral density and alleviate osteoporosis indicators.

A Clinically-Achievable Injectable and Sprayable in Situ Lyotropic Liquid Crystalline Platform in Treating Hormone-Sensitive and Castration-Resistant Prostate Cancer.

When it comes to long-acting injections, lyotropic liquid crystals (LLCs) are considered as an effective and powerful drug delivery technology due to their low manufacturing and injection difficulty, consistent releasing behaviors with low burst, as well as broadly applicable drug loading capacity. However, monoolein and phytantriol, as two widely used LLC-forming materials, may give rise to tissue cytotoxicity and undesired immunological responses, which may hinder the wide application of this technology. In this study, we opted for two ingredients, phosphatidylcholine and α-tocopherol, as carriers on account of their nature-obtainable and biocompatible qualities. By changing the ratios between them, we conducted research on crystalline types, nanosized structures, viscoelastic differences, characteristics of releasing behaviors, and in vivo safety. To fully exploit this in situ LLC platform with both injectability and sprayability, we focused on the treatment of both hormone-sensitive (HSPC) and castration-resistant prostate cancer (CRPC). For HSPC, we found that spraying leuprolide and a cabazitaxel-loaded LLC platform on the tumor bed after resection greatly reduced tumor metastatic rate and prolonged the survival time. Besides, for CRPC, our results demonstrated that although leuprolide (a kind of drug for castration) alone could hardly limit the progression of CRPC with low MHC-I expression, its combination with cabazitaxel in our LLC platform achieved a significantly better tumor-inhibiting and anti-recurrent efficacy than single cabazitaxel-loaded LLC platform, owing to enhanced CD4+ T cell infiltration in tumors and immune-potentiating cytokines. In conclusion, our dual-functional and clinically achievable strategy might provide a treating solution toward both HSPC and CRPC.

Recruiting T cells and sensitizing tumors to NKG2D immune surveillance for robust antitumor immune response.

Although recruiting T cells to convert cold tumors into hot can prevent some tumors from evading immune surveillance, tumors have evolved more mechanisms to achieve immune evasion, such as downregulating major histocompatibility complex I (MHC I) molecules expression to prevent T cells from recognizing tumor-antigens, or secreting immune suppression cytokines that disable T cells. Tumor immune evasion not only promotes tumor growth, but also weakens the efficacy of existing tumor immunotherapies. Therefore, recruiting T cells while reshaping innate immunity plays an important role in preventing tumor immune escape. In this study, we constructed a long-acting in situ forming implant (ISFI) based on the Atrigel technology, co-encapsulated with G3-C12 and sulfisoxazole (SFX) as a drug depot in the tumor site (SFX + G3-C12-ISFI). First, G3-C12 could recruit T cells, and transform cold into hot tumors. Furthermore, SFX could inhibit tumor-derived exosomes secretion, reduce the shedding of NKG2D ligand (NKG2DL), repair NKG2D/NKG2DL pathway, reinvigorate natural killer (NK) cells, and evade the effects of MHC I molecules missing. In the humanized cold tumor model, our strategy showed an excellent anti-tumor effect, providing a smart strategy for solving tumor evasion immune surveillance.

Nonlysosomal Route of mRNA Delivery and Combining with Epigenetic Regulation Optimized Antitumor Immunoprophylactic Efficacy.

Currently, mRNA-based tumor therapies are in full flow because in vitro-transcribed (IVT) mRNA has the potential to express tumor antigens to initiate the adaptive immune responses. However, the efficacy of such therapy relies heavily on the delivery system. Here, a pardaxin-modified liposome loaded with tumor antigen-encoding mRNA and adjuvant (2',3'-cGAMP, (cyclic [G(2',5')pA(3',5')p])), termed P-Lipoplex-CDN is reported. Due to an nonlysosomal delivery route, the transfection efficiency on dendritic cells (DCs) is improved by reducing the lysosome disruption of cargos. The mRNA modified DCs efficiently induce tumor antigen-specific immune responses both in vitro and in vivo. As prophylactic vaccines, mRNA transfected DCs significantly delay the occurrence and development of tumors, and several immunized mice are even completely resistant to tumors. Interestingly, the efficacy depends on the major histocompatibility complex class I (MHC-I) expression level on tumor cells. Furthermore, epigenetic modification (decitabine, DAC) is applied as a combination strategy to deal with malignant tumor progression caused by deficient tumor MHC-I expression. This study highlights the close relationship between mRNA-DCs vaccine efficacy and the expression level of tumor cell MHC-I molecules. Moreover, a feasible strategy for tumor MHC-I expression deficiency is proposed, which may provide clinical guidance for the design and application of mRNA-based tumor therapies.

New advances in pharmaceutical strategies for sensitizing anti-PD-1 immunotherapy and clinical research.

Attempts have been made continuously to use nano-drug delivery system (NDDS) to improve the effect of antitumor therapy. In recent years, especially in the application of immunotherapy represented by antiprogrammed death receptor 1 (anti-PD-1), it has been vigorously developed. Nanodelivery systems are significantly superior in a number of aspects including increasing the solubility of insoluble drugs, enhancing their targeting ability, prolonging their half-life, and reducing side effects. It can not only directly improve the efficacy of anti-PD-1 immunotherapy, but also indirectly enhance the antineoplastic efficacy of immunotherapy by boosting the effectiveness of therapeutic modalities such as chemotherapy, radiotherapy, photothermal, and photodynamic therapy (PTT/PDT). Here, we summarize the studies published in recent years on the use of nanotechnology in pharmaceutics to improve the efficacy of anti-PD-1 antibodies, analyze their characteristics and shortcomings, and combine with the current clinical research on anti-PD-1 antibodies to provide a reference for the design of future nanocarriers, so as to further expand the clinical application prospects of NDDSs. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease.

Brucea javanica oil emulsion significantly improved the effect of anti-programmed cell death protein-1 immunotherapy.

BACKGROUND: Brucea javanica oil (BJO) is the active substance extracted from the dry and mature fruit of Brucea javanica. Its pharmaceutical preparation, BJO emulsion (BJOE), is one of the most widely studied traditional Chinese medicine preparations for the treatment of malignancy. However, the unrevealed anti-tumor mechanism immensely limits further development of BJOE. PURPOSE: In this study, we delved into the anti-tumor mechanism of commercial BJOE, including its influence on the tumor microenvironment (TME) and the treatment effect when combined with anti-programmed cell death protein-1 (PD-1) therapy. METHODS: The cytotoxicity of BJOE was tested in different cells in vitro, and a Förster resonance energy transfer system was also constructed to predict the release behavior of BJOE in vivo. Then, a B16 melanoma mouse model was used to explore the combination of BJOE and anti-mouse PD-1 antibody therapy. In addition, mass cytometry was used to test the impact of both drugs on the TME. RESULTS: Out data revealed that BJOE did not directly kill tumor cells in vitro. However, BJOE was mainly released at the tumor site, converting an immunosuppressive TME into an immune-activated state, and its combination with anti-PD-1 therapy significantly inhibited the growth of melanoma and prolonged the survival time of the mice due to an increase in cytotoxic T lymph (CD8+ T) and helper/inducible T lymph (CD4+ T) cells in lymph nodes and tumors. CONCLUSIONS: Our work explored the anti-tumor mechanism of commercial BJOE and the regulation of cytokines by BJOE when it was combined with anti-PD-1 therapy in vivo. The combination of these therapies could increase the numbers of CD4+ T-cells, CD8+ T-cells, and effective natural killer cells and the ratio of MI/M2 macrophages in tumor tissues, promoting inflammatory activity and enhancing the anti-tumor effect. This study provides a theoretical basis for advancing the modern development of traditional Chinese medicine preparations and stands as a reference for clinically improving the efficacy of PD-1 antibodies.

Arginine Supplementation Targeting Tumor-Killing Immune Cells Reconstructs the Tumor Microenvironment and Enhances the Antitumor Immune Response.

The tumor microenvironment (TME) is characterized by several immunosuppressive factors, of which weak acidity and l-arginine (l-arg) deficiency are two common features. A weak acidic environment threatens the survival of immune cells, and insufficient l-arg will severely restrain the effect of antitumor immune responses, both of which affect the efficiency of cancer treatments (especially immunotherapy). Meanwhile, l-arg is essential for tumor progression. Thus, two strategies, l-arg supplementation and l-arg deprivation, are developed for cancer treatment. However, these strategies have the potential risk of promoting tumor growth and impairing immune responses, which might lead to a paradoxical therapeutic effect. It is optimal to limit the l-arg availability of tumor cells from the microenvironment while supplying l-arg for immune cells. In this study, we designed a multivesicular liposome technology to continuously supply alkaline l-arg, which simultaneously changed the acidity and l-arg deficiency in the TME, and by selectively knocking down the CAT-2 transporter, l-arg starvation of tumors was maintained while tumor-killing immune cells were enriched in the TME. The results showed that our strategy promoted the infiltration and activation of CD8+ T cells in tumor, increased the proportion of M1 macrophages, inhibited melanoma growth, and prolonged survival. In combination with anti-PD-1 antibody, our strategy reversed the low tumor response to immune checkpoint blockade therapy, showing a synergistic antitumor effect. Our work provided a reference for improving the TME combined with regulating nutritional competitiveness to achieve the sensitization of immunotherapy.

A therapeutic DC vaccine with maintained immunological activity exhibits robust anti-tumor efficacy.

Dendritic cells (DCs) vaccines are a major focus of future anti-tumor immunotherapy for their pivotal role in eliciting reactive tumor-specific T-cell responses. Tumor cell-mediated DCs (TC-DC) activation and tumor antigen-mediated DCs (TA-DC) activation are two conventional modes of DC vaccine construction in clinical studies. The former physiologically mimicks the tumor identification and rejection, significantly contributing to DC-based immune recognition and migration towards the complexed tumor microenvironment (TME). However, as immunosuppressive molecules may exist in TME, these TC-DC are generally characterized with aberrant lipid accumulation and inositol-requiring kinase 1α (IRE1α)-X-box binding protein 1 (XBP1) hyperactivation, which is provoked by overwhelming oxidative stress and endoplasmic reticulum (ER) stress, resulting in TC-DC malfunction. Oppositely, without contacting immunosuppressive TME, TA-DC vaccines perform better in T-cell priming and lymph nodes (LNs) homing, but are relatively weak in TME infiltration and identification. Herein, we prepared a KIRA6-loaded α-Tocopherol nanoemulsion (KT-NE), which simultaneously ameliorated oxidative stress and ER stress in the dysfunctional lipid-laden TC-DC. The TC-DC treated by KT-NE could maintain immunological activity, simultaneously, exhibited satisfactory chemotaxis towards LNs and tumor sites in vivo, and effectively suppressed malignant progression by unleashing activated tumor-reactive T cells. This study generated a new DC-vaccine that owned puissant aptitude to identify complicated TME as well as robust immunological activity to boost T-cell initiation, which may provide some insights into the design and application of DC-vaccines for clinical application.

Dual-binding nanoparticles improve the killing effect of T cells on solid tumor.

Adoptive cell therapy (ACT) was one of the most promising anti-tumor modalities that has been confirmed to be especially effective in treating hematological malignancies. However, the clinical efficacy of ACT on solid tumor was greatly hindered by the insufficient tumor-infiltration of cytotoxic CD8 + T cells. Herein, we constructed a nanoplatform termed dual-binding magnetic nanoparticles (DBMN) that comprised PEG-maleimide (Mal), hyaluronic acid (HA) and Fe3O4 for adoptive T cell-modification and ACT-sensitization. After a simple co-incubation, DBMN was anchored onto the cell membrane (Primary linking) via Michael addition reaction between the Mal and the sulfhydryl groups on the surface of T cells, generating magnetized T cells (DBMN-T). Directed by external magnetic field and in-structure Fe3O4, DBMN-T was recruited to solid tumor where HA bond with the highly expressed CD44 on tumor cells (Secondary Linking), facilitating the recognition and effector-killing of tumor cells. Bridging adoptive T cells with host tumor cells, our DBMN effectively boosted the anti-solid tumor efficacy of ACT in a mouse model and simultaneously reduced toxic side effects.

ER-Targeting PDT Converts Tumors into In Situ Therapeutic Tumor Vaccines.

A therapeutic tumor vaccine is a promising approach to cancer treatment. One of its strategies is to treat patient-derived tumor cells in vitro and then administer them in vivo to induce an adaptive immune response and achieve cancer treatment. Here, we want to explore the possibility of converting cancer tissue into a therapeutic tumor vaccine through induced immunogenic cell death (ICD) in situ. We loaded indocyanine green (ICG) into liposomes (ICG-Lipo) and modified it with the pardaxin peptide to realize an endoplasmic reticulum (ER)-targeting function (Par-ICG-Lipo). A microfluidic technique was developed for loading ICG, a water-soluble molecule, into liposomes with a high encapsulation efficiency (greater than 90%). Under near-infrared (NIR) irradiation, ER-targeting photodynamic therapy (PDT) induced by Par-ICG-Lipo could promote the release of danger-signaling molecules (DAMPs) and tumor antigens (TAAs) in vivo, which significantly enhanced the immunogenicity in vivo and thus stimulates a strong antitumor immune response. This process would be further amplified by adopting dendritic cells. In general, our strategy transformed in situ tumor cells into therapeutic vaccines by ER-targeting PDT, which could provide a clinically applicable and effective approach for cancer treatment.

Optimized mobilization of MHC class I- and II- restricted immunity by dendritic cell vaccine potentiates cancer therapy.

Background: The participation of major histocompatibility complex (MHC) in antigen presentation shapes both the breadth and magnitude of specific T cell response. Dendritic cells (DCs) activated with nucleic acid or protein that encodes/incorporates multiple antigenic epitopes elicit MHC class I- and II- biased immunity, respectively. Studies demonstrate that an elevated MHC class I-directed CD8+ cytotoxicity T lymphocyte (CTL) response is able to provide survival benefits to patient with malignant tumor. However, a fully effective cancer therapy must elicit a diverse repertoire of both CD4+ and CD8+ T cell responses, raising demands on a multifaceted activation of the MHC system. Current therapeutic strategies usually lack an orchestrated mobilization of the MHC class I and II responses. Vaccines with little synergistic effect or unmanageable elicitation of the CD4+ and CD8+ T cell immunity usually fail to induce a potent and durable anti-tumor protection. Methods: Here, cationic nanoemulsions (CNEs) complexed with full-length tumor model antigen ovalbumin (OVA) in the form of mRNA or protein were constructed and used as two antigenic platforms to prepare DCs vaccines with tailored MHC participation (i.e., mRNA-DCs and protein-DCs). In exploring a vaccine regimen with optimal tumor suppressing effect, the mixing ratio of mRNA-DCs and protein-DCs was manipulated. Results: Therapeutic DCs vaccines involving both antigenic platforms induced better anti-tumor immunity in murine E.G7-OVA lymphoma model and B16-OVA melanoma model, which can be further augmented upon a meticulous reallocation of the MHC class I and II responses. Conclusion: This work indicated that a simultaneous and coordinated mobilization of the MHC-restricted immunity might potentiate cancer therapy.

Rational administration sequencing of immunochemotherapy elicits powerful anti-tumor effect.

As a research hotspot, immune checkpoint inhibitors (ICIs) is often combined with other therapeutics in order to exert better clinical efficacy. To date, extensive laboratory and clinical investigations into the combination of ICIs and chemotherapy have been carried out, demonstrating augmented effectiveness and broad application prospects in anti-tumor therapy. However, the administration of these two treatment modalities is usually randomized or fixed to a given chronological order. Nevertheless, the pharmacological effect of drug is closely related to its exposure behavior in vivo, which may consequently affect the synergistic outcomes of a combined therapy. In this study, we prepared a lipid nanoparticle encapsulating docetaxel (DTX-VNS), and associated it with the immune checkpoint inhibitor anti-PD-1 antibody (αPD-1) for the treatment of malignant tumors. To identify the optimum timing and sequencing for chemotherapy and immunotherapy, we designed three administration regimes, including the simultaneous delivery of DTX-VNS and αPD-1(DTX-VNS@αPD-1), DTX-VNS delivery before (DTX-VNS plus αPD-1) or post (αPD-1 plus DTX-VNS) PD-1 blockade with an interval of two days. Analysis from mass spectrometry, multi-factor detection and other techniques indicated that DTX-VNS plus αPD-1 initiated a powerful anti-tumor response in multiple tumor models, contributing to a remarkably reshaped tumor microenvironment landscape, which may attribute to the maximum therapeutic additive effects arise from a concomitant exposure of DTX-VNS and αPD-1 at the tumor site. By profiling the exposure kinetics of nanoparticles and αPD-1 in vivo, we defined the administration schedule with utmost therapeutic benefits, which may provide a valuable clinical reference for the rational administration of immunochemotherapy.

Oxygen nanocarrier broke the hypoxia trap of solid tumors and rescued transfection efficiency for gene therapy.

BACKGROUND: Gene therapy shows great promise for a broad array of diseases. However, we found that hypoxic tumor microenvironment (TME) exerted significant inhibitory effects on transfection efficiency of a variety of gene vectors (such as Lipo 2000 and PEI) in an oxygen-dependent manner. Solid tumors inevitably resulted in acute hypoxic areas due to the rapid proliferation of tumor cells and the aberrant structure of blood vessels. Thus, the hypoxic TME severely limited the efficiency and application of gene therapy. METHODS: In our previous study, we constructed endoplasmic reticulum-targeted cationic liposomes, PAR-Lipo, which could effectively deliver genes and ensure high transfection efficiency under normoxia. Unsatisfactorily, the transfection efficiency of PAR-Lipo was rather poor under hypoxia. We believed that reoxygenation was the most direct and effective means to rescue the low transfection under hypoxia. Hence, we fabricated liposomes modified with perfluorooctyl bromide (PFOB@Lipo) to load oxygen and deliver it to tumor sites, which effectively alleviated the hypoxic nature of tumor. Then PAR-Lipo were applied to mediate high-efficiency delivery of tumor suppressor gene pTP53 to inhibit tumor progression. RESULTS: The results showed that such staged strategy augmented the expression of P53 protein in tumors and extremely suppressed tumor growth. CONCLUSION: This work was the first attempt to utilize an oxygen nanocarrier to assist the therapeutic effect of gene therapy under hypoxia, providing a new reference for gene therapy in malignant tumors. GRAPHICAL ABSTARCT.

Regulating immune memory and reversing tumor thermotolerance through a step-by-step starving-photothermal therapy.

BACKGROUND: Photothermal therapy (PTT) is a highly effective treatment for solid tumors and can induce long-term immune memory worked like an in situ vaccine. Nevertheless, PTT inevitably encounters photothermal resistance of tumor cells, which hinders therapeutic effect or even leads to tumor recurrence. Naïve CD8+ T cells are mainly metabolized by oxidative phosphorylation (OXPHOS), followed by aerobic glycolysis after activation. And the differentiate of effector CD8+ T cell (CD8+ Teff) into central memory CD8+ T cell (CD8+ TCM) depends on fatty acid oxidation (FAO) to meet their metabolic requirements, which is regulated by adenosine monophosphate activated protein kinase (AMPK). In addition, the tumor microenvironment (TME) is severely immunosuppressive, conferring additional protection against the host immune response mediated by PTT. METHODS: Metformin (Met) down-regulates NADH/NADPH, promotes the FAO of CD8+ T cells by activating AMPK, increases the number of CD8+ TCM, which boosts the long-term immune memory of tumor-bearing mice treated with PTT. Here, a kind of PLGA microspheres co-encapsulated hollow gold nanoshells and Met (HAuNS-Met@MS) was constructed to inhibit the tumor progress. 2-Deoxyglucose (2DG), a glycolysis inhibitor for cancer starving therapy, can cause energy loss of tumor cells, reduce the heat stress response of tumor cell, and reverse its photothermal resistance. Moreover, 2DG prevents N-glycosylation of proteins that cause endoplasmic reticulum stress (ERS), further synergistically enhance PTT-induced tumor immunogenic cell death (ICD), and improve the effect of immunotherapy. So 2DG was also introduced and optimized here to solve the metabolic competition among tumor cells and immune cells in the TME. RESULTS: We utilized mild PTT effect of HAuNS to propose an in situ vaccine strategy based on the tumor itself. By targeting the metabolism of TME with different administration strategy of 2DG and perdurable action of Met, the thermotolerance of tumor cells was reversed, more CD8+ TCMs were produced and more effective anti-tumor was presented in this study. CONCLUSION: The Step-by-Step starving-photothermal therapy could not only reverse the tumor thermotolerance, but also enhance the ICD and produce more CD8+ TCM during the treatment.

Dual Inhibition of Endoplasmic Reticulum Stress and Oxidation Stress Manipulates the Polarization of Macrophages under Hypoxia to Sensitize Immunotherapy.

M2-tumor associated macrophages (TAMs) play an important role in tumor genesis, progression, and metastasis, and repolarizing M2-TAMs to immune-promoting M1 type is increasingly recognized as a promising strategy against the clinically intractable carcinomas. It is observed that M2 macrophages have a high tropism to the tumor hypoxic area, with their endoplasmic reticulum (ER) stress-associated IRE1-XBP1 pathway activated to inhibit cell glycolysis, promote oxidative phosphorylation (OXPHOS), and facilitate intracellular lipid accumulation, which in turn shapes the typical phenotypes of M2-TAMs, suggesting that manipulating the ER stress response of M2-TAMs might stand as a breakthrough for antitumor therapy. However, current attempts to repolarize M2 cells remain limited and are greatly challenged by the hypoxic nature of tumors. Also, the high level of reactive oxygen species (ROS) in the tumor microenvironment (TME) is favorable for the polarization of M2-TAMs. Here, we encapsulated KIRA6, an inhibitor of the IRE1-XBP1 pathway, into a reductive nanoemulsion containing α-tocopherol. Our α-T-K had dual inhibitory effects on the ER stress and oxidative stress. Both in vitro and in vivo experiments suggested that α-T-K effectively reprogrammed M2 macrophages even under hypoxia, achieved by increasing glycolysis and suppressing fatty acid oxidation (FAO). In addition, our data revealed that α-T-K not only delayed tumor growth but elevated the curative effect of PD-1 antibody. Our research demonstrated that simultaneous inhibition of ER stress and oxidative stress could effectively repolarize M2-TAMs under hypoxia, which not only filled the current gap in regulating the biological repolarization of macrophages under hypoxia but provided a meaningful reference for the clinical immunotherapy of sensitized anti-PD-1.

A clinically acceptable strategy for sensitizing anti-PD-1 treatment by hypoxia relief.

The hypoxic tumor microenvironment (TME) hinders the effectiveness of immunotherapy. Alleviating tumor hypoxia to improve the efficacy of immune checkpoint inhibitors (ICIs) represented by programmed cell death protein 1 (PD-1) antibody has become a meaningful strategy. In this study, we adopted three methods to alleviate hypoxia, including direct oxygen delivery using two different carriers and an indirect way involving HIF-1α inhibition. Both in vivo and in vitro experiments showed that liposomes modified with perfluorocarbon or hemoglobin (PFC@lipo or Hb@lipo) were able to efficiently load and release oxygen, relieving tumor hypoxia. However, the gas release behavior of PFC@lipo was uncontrollable, which might induce acute hyperoxia side effects during intravenous injection and reduce its biosafety. In contrast, whether administered locally or systemically, Hb@lipo revealed high animal tolerance, and was much safer than commercial HIF-1α inhibitor (PX-478), displaying prospects as a promising oxygen carrier for clinical practice. Pharmacodynamic experiments suggested that Hb@lipo helped PD-1 antibody break the therapeutic bottleneck and significantly inhibited the progression of 4 T1 breast cancer. But in CT26 colon cancer, the combination therapy failed to suppress tumor growth. After in-depth analysis and comparison, we found that the ratio of M1/M2 tumor associated macrophages (TAMs) between these two tumor models were dramatically different. And the lower M1/M2 ratio in CT26 tumors limited the anti-tumor effect of combination therapy. In this study, three methods for alleviating tumor hypoxia were compared from the perspectives of biosafety, efficacy and clinical applicability. Among them, Hb@lipo stood out, and its combined use with PD-1 antibody exhibit a distinct synergistic suppression effect on tumors with more M1 macrophages presented in the microenvironment. Our work provided a good reference for improving the efficacy of PD-1 antibody by alleviating tumor hypoxia.

Cocktail strategy for 'cold' tumors therapy via active recruitment of CD8+ T cells and enhancing their function.

In immunotherapy, 'cold' tumors, with low T cells infiltration, hardly benefit from the treatment of immune checkpoint inhibitors (ICIs). To address this issue, we screened two 'cold' tumor models for mice with high expression of galectin-3 (Gal-3) and designed a cocktail strategy to actively recruit CD8+ T cells into the tumor microenvironment (TME), which reversed 'cold' tumors into 'hot' and remarkably elevated their ICIs-responsiveness. Gal-3, an important driving force of tumorigenesis, inhibits T cell infiltration into tumor tissue that shapes 'cold' tumor phenotype, and promotes tumor metastasis. In this respect, Gal-3 antagonist G3-C12 peptide was chosen and further loaded into poly(lactic-co-glycolic acid) (PLGA) microspheres, with the prepared G3-C12@PLGA playing a dual role of antitumor, namely, killing two birds with one stone. Specifically, G3-C12@PLGA actively recruit T cells into 'cold' tumors by rescuing IFN-γ, and simultaneously inhibit tumor metastasis induced by Gal-3. Moreover, when combined with chemotherapeutic agent (Oxaliplatin) and anti-PD-1 peptide (APP), the immunopotentiating effect of dendritic cells (DCs) was extremely improved, with T-cell depletion dramatically reversed. In vivo experiments showed that such cocktail therapy exerted remarkable antitumor effect on 'cold' breast cancer (BC) and ovarian serous cancer (OSC). These results indicated that our strategy might be promising in treating 'cold' tumors with high expression of Gal-3, which not only enhance cancer treatment outcome, but provide a new platform for the prevention of postoperative tumor recurrence/metastasis.

Targeted regulation of lymphocytic ER stress response with an overall immunosuppression to alleviate allograft rejection.

Transplantation is the most effective, and sometimes the only resort for end-stage organ failure. However, allogeneic graft suffers greatly from lymphocyte-mediated immunorejection, which bears close relationship with a hyperactivation of endoplasmic reticulum (ER) stress response in host lymphocytes, especially in CD8+ T cells (T-8). Therefore, regulating lymphocytic ER unfolded protein response (UPR) might be a potential therapeutic breakthrough in alleviating graft rejection. Here, ER-targetable liposome is prepared via the surface modification of ER-targeting peptide (Pardaxin), which efficiently loads and directly delivers small molecule inhibitor of UPR sensor IRE1α into the ER of lymphocytes, inducing a systemic immunosuppression that facilitates tumorigenesis and metastasis in the tumor inoculation challenge in vivo. And in vitro, a stage-differential dependency of IRE1α in the phase transition of T-8 is identified. Specifically, inhibiting IRE1α at the early responding stages of T-8, especially at the activation phase, results in a shrunk proliferation, impaired effector function, and limited memory commitment, which might contribute centrally to the induced overall immunosuppression. Based on this, a classical acute rejection model, murine full-thickness trunk skin allograft that primary arises from the hyperactivity of T-lymphocyte, is used. Results suggest that lymphocytic IRE1α inactivation attenuates transplant rejection and prolongs graft survival, with a limited effector function and memory commitment of host T-8. Moreover, an even higher immunosuppressive effect is obtained when IRE1α inhibition is used in combination with immunosuppressant tacrolimus (FK506), which might owe to a synergistic regulation of inflammatory transcription factors. These findings provide a deeper insight into the biological polarization and stress response of lymphocytes, which might guide the future development of allogeneic transplantation.

A Vaccination with Boosted Cross Presentation by ER-Targeted Antigen Delivery for Anti-Tumor Immunotherapy.

Vaccination is a widely-accepted resort against the invasion or proliferation of bacteria, parasites, viruses, and even cancer, which accounts heavily on an active involvement of CD8+ T cells. As one of the pivotal strategies taken by dendritic cells (DCs) to promote the responsiveness of CD8+ T cells to exogenous antigens, cross presentation culminates in an elevated overall host defense against cancer or infection. However, the precise mechanisms regulating such a process remains elusive, and current attempts to fuel cross presentation usually fail to exert efficiency. Here, model antigen OVA-loaded, endoplasmic reticulum (ER)-targeting cationic liposome (OVA@lipoT) is developed and characterized with a booster effect on the activation and maturation of DCs. Moreover, OVA@lipoT pulsed DCs exhibit overwhelming superiority in triggering cytotoxic T lymphocyte response both in vivo and in vitro. Data reveal that lipoT alters the intracellular trafficking and presenting pathway of antigen, which promotes cross presentation and bears close relationship to the ER-associated degradation (ERAD). These results may drop a hint about the interconnectivity between cross presentation and ER-targeted antigen delivery, provide extra information to the understanding of ERAD-mediated cross priming, and even shed new light on the design and optimization of vaccines against currently intractable cancers or virus-infection.