IFNγ at the early stage induced after cryo-thermal therapy maintains CD4+ Th1-prone differentiation, leading to long-term antitumor immunity.

Introduction: Recently, more and more research illustrated the importance of inducing CD4+ T helper type (Th)-1 dominant immunity for the success of tumor immunotherapy. Our prior studies revealed the crucial role of CD4+ Th1 cells in orchestrating systemic and durable antitumor immunity, which contributes to the satisfactory outcomes of the novel cryo-thermal therapy in the B16F10 tumor model. However, the mechanism for maintaining the cryo-thermal therapy-mediated durable CD4+ Th1-dominant response remains uncovered. Additionally, cryo-thermal-induced early-stage CD4+ Th1-dominant T cell response showed a correlation with the favorable prognosis in patients with colorectal cancer liver metastasis (CRCLM). We hypothesized that CD4+ Th1-dominant differentiation induced during the early stage post cryo-thermal therapy would affect the balance of CD4+ subsets at the late phase. Methods: To understand the role of interferon (IFN)-γ, the major effector of Th1 subsets, in maintaining long-term CD4+ Th1-prone polarization, B16F10 melanoma model was established in this study and a monoclonal antibody was used at the early stage post cryo-thermal therapy for interferon (IFN)-γ signaling blockade, and the influence on the phenotypic and functional change of immune cells was evaluated. Results: IFNγ at the early stage after cryo-thermal therapy maintained long-lasting CD4+ Th1-prone immunity by directly controlling Th17, Tfh, and Tregs polarization, leading to the hyperactivation of Myeloid-derived suppressor cells (MDSCs) represented by abundant interleukin (IL)-1ß generation, and thereby further amplifying Th1 response. Discussion: Our finding emphasized the key role of early-phase IFNγ abundance post cryo-thermal therapy, which could be a biomarker for better prognosis after cryo-thermal therapy.

Combination of STING agonist with anti-vascular RGD-(KLAKLAK)2 peptide as a novel anti-tumor therapy.

Immunotherapy is one of the most promising anti-cancer treatment. It involves activating the host's own immune system to eliminate cancer cells. Activation of cGAS-STING pathway is promising therapeutic approach for cancer immunotherapy. However, in human clinical trials, targeting cGAS-STING pathway results in insufficient or unsustainable anti-tumor response. To enhance its effectiveness, combination with other anti-cancer therapies seems essential to achieve synergistic systemic anti-tumor response.The aim of this study was to evaluate whether the combination of STING agonist-cGAMP with anti-vascular RGD-(KLAKLAK)2 peptide results in a better anti-tumor response in poorly immunogenic tumors with various STING protein and αvß3 integrin status.Combination therapy inhibited growth of murine breast carcinoma more effectively than melanoma. In melanoma, the administration of STING agonist alone was sufficient to obtain a satisfactory therapeutic effect. In both tumor models we have noted stimulation of innate immune response following cGAMP administration alone or in combination. The largest population of immune cells infiltrating the TME after therapy were activated NK cells. Increased infiltration of cytotoxic CD8+ T lymphocytes within the TME was only observed in melanoma tumors. However, they also expressed the "exhaustion" PD-1 receptor. In contrast, in breast carcinoma tumors each therapy caused the drop in the number of infiltrating CD8+ T cells.The obtained results indicate an additional therapeutic benefit from combining STING agonist with an anti-vascular agent. However, this effect depends on the type of tumor, the status of its microenvironment and the expression of specific proteins such as STING and αvß3 family integrin.

Bacillus Calmette-Guérin immunotherapy induces an efficient antitumor response to control murine melanoma depending on MyD88 signaling.

Bacillus Calmette-Guérin (BCG) is the first line treatment for bladder cancer and it is also proposed for melanoma immunotherapy. BCG modulates the tumor microenvironment (TME) inducing an antitumor effective response, but the immune mechanisms involved still poorly understood. The immune profile of B16-F10 murine melanoma cells was assessed by infecting these cells with BCG or stimulating them with agonists for different innate immune pathways such as TLRs, inflammasome, cGAS-STING and type I IFN. B16-F10 did not respond to any of those stimuli, except for type I IFN agonists, contrasting with bone marrow-derived macrophages (BMDMs) that showed high production of proinflammatory cytokines. Additionally, we confirmed that BCG is able to infect B16-F10, which in turn can activate macrophages and spleen cells from mice in co-culture experiments. Furthermore, we established a subcutaneous B16-F10 melanoma model for intratumoral BCG treatment and compared wild type mice to TLR2-/-, TLR3-/-, TLR4-/-, TLR7-/-, TLR3/7/9-/-, caspase 1-/-, caspase 11-/-, IL-1R-/-, cGAS-/-, STING-/-, IFNAR-/-, MyD88-/-deficient animals. These results in vivo demonstrate that MyD88 signaling is important for BCG immunotherapy to control melanoma in mice. Also, BCG fails to induce cytokine production in the co-culture experiments using B16-F10 and BMDMs or spleen cells derived from MyD88-/- compared to wild-type (WT) animals. Immunotherapy with BCG was not able to induce the recruitment of inflammatory cells in the TME from MyD88-/- mice, impairing tumor control and IFN-γ production by T cells. In conclusion, MyD88 impacts on both innate and adaptive responses to BCG leading to an efficient antitumor response against melanoma.

Experimental evidence that carbon-ion radiotherapy utilizes cytotoxic T lymphocyte-mediated anti-tumor immunity for shrinking tumors compared to X-ray therapy.

The therapeutic efficacy of radiotherapy (RT) is primarily driven by two factors: biophysical DNA damage in cancer cells and radiation-induced anti-tumor immunity. However, Anti-tumor immune responses between X-ray RT (XRT) and carbon-ion RT (CIRT) remain unclear. In this study, we, employed mouse models to assess the immunological contribution, especially cytotoxic T-lymphocyte (CTL)-mediated immunity, to the therapeutic effectiveness of XRT and CIRT in shrinking tumors. We irradiated mouse intradermal tumors of B16F10-ovalbumin (OVA) mouse melanoma cells and 3LL-OVA mouse lung cancer cells with carbon-ion beams or X-rays in the presence or absence of CTLs. CTL removal was performed by administration of anti-CD8 monoclonal antibody (mAb) in mice. Based on tumor growth delay, we determined the tumor growth and regression curves. The enhancement ratio (ER) of the slope of regression lines in the presence of CTLs, relative to the absence of CTLs, indicates the dependency of RT on CTLs for shrinking mouse tumors, and the biological effectiveness (RBE) of CIRT relative to XRT were calculated. Tumor growth curves revealed that the elimination of CD8+ CTLs by administrating anti-CD8 mAb accelerated tumor growth compared to the presence of CTLs in both RTs. The ERs were larger in CIRT compared to XRT in the B16F10-OVA tumor models, but not in the 3LL-OVA models, suggesting a greater contribution of CTL-mediated anti-tumor immunity to tumor reduction in CIRT compared to XRT in the B16F10-OVA tumor model. In addition, the RBE values for both models were larger in the presence of CTLs compared to models without CTLs, suggesting that CIRT may utilize CTL-mediated anti-tumor immunity more than X-ray. The findings from this study suggest that although immunological contribution to therapeutic efficacy may vary depending on the type of tumor cell, CIRT utilizes CTL-mediated immunity to a greater extent compared to XRT.

Long-term combined blockade of CXCR4 and PD-L1 with in vivo reassembly for intensive tumor interference.

Negative immunoregulatory signal (PD-L1, CXCR4, et al.) and weak immunogenicity elicited immune system failing to detect and destroy cancerous cells. CXCR4 blockade promoted T cell tumor infiltration and increased tumor sensitivity to anti-PD-L1 therapy. Here, pH-responsive reassembled nanomaterials were constructed with anti-PD-L1 peptide and CXCR4 antagonists grafting (APAB), synergized with photothermal therapy for melanoma and breast tumor interference. The self-assembled APAB nanoparticles accumulated in the tumor and rapidly transformed into nanofibers in response to the acidic tumor microenvironment, leading to the exposure of grafted therapeutic agents. APAB enabling to reassemble around tumor cells and remained stable for over 96 h due to the aggregation induced retention (AIR) effect, led to long-term efficiently combined PD-L1 and CXCR4 blockade. Photothermal efficiency (ICG) induced immunogenic cell death (ICD) of tumor cells so as to effectively improve the immunogenicity. The combined therapy (ICG@APAB) could effectively inhibit the growth of primary tumor (∼83.52%) and distant tumor (∼76.24%) in melanoma-bearing mice, and significantly (p < 0.05) prolong the survival time over 42 days. The inhibition assay on tumor metastasis in 4 T1 model mice exhibited ICG@APAB almostly suppressed the occurrence of lung metastases and the expression levels of CD31, MMP-9 and VEGF in tumor decreased by 82.26%, 90.45% and 41.54%, respectively. The in vivo reassembly strategy will offer novel perspectives benefical future immunotherapies and push development of combined therapeutics into clinical settings.

Construction of lymph nodes-targeting tumor vaccines by using the principle of DNA base complementary pairing to enhance anti-tumor cellular immune response.

Tumor vaccines, a crucial immunotherapy, have gained growing interest because of their unique capability to initiate precise anti-tumor immune responses and establish enduring immune memory. Injected tumor vaccines passively diffuse to the adjacent draining lymph nodes, where the residing antigen-presenting cells capture and present tumor antigens to T cells. This process represents the initial phase of the immune response to the tumor vaccines and constitutes a pivotal determinant of their effectiveness. Nevertheless, the granularity paradox, arising from the different requirements between the passive targeting delivery of tumor vaccines to lymph nodes and the uptake by antigen-presenting cells, diminishes the efficacy of lymph node-targeting tumor vaccines. This study addressed this challenge by employing a vaccine formulation with a tunable, controlled particle size. Manganese dioxide (MnO2) nanoparticles were synthesized, loaded with ovalbumin (OVA), and modified with A50 or T20 DNA single strands to obtain MnO2/OVA/A50 and MnO2/OVA/T20, respectively. Administering the vaccines sequentially, upon reaching the lymph nodes, the two vaccines converge and simultaneously aggregate into MnO2/OVA/A50-T20 particles through base pairing. This process enhances both vaccine uptake and antigen delivery. In vitro and in vivo studies demonstrated that, the combined vaccine, comprising MnO2/OVA/A50 and MnO2/OVA/T20, exhibited robust immunization effects and remarkable anti-tumor efficacy in the melanoma animal models. The strategy of controlling tumor vaccine size and consequently improving tumor antigen presentation efficiency and vaccine efficacy via the DNA base-pairing principle, provides novel concepts for the development of efficient tumor vaccines.

Combination therapy with oncolytic virus and T cells or mRNA vaccine amplifies antitumor effects.

Antitumor therapies based on adoptively transferred T cells or oncolytic viruses have made significant progress in recent years, but the limited efficiency of their infiltration into solid tumors makes it difficult to achieve desired antitumor effects when used alone. In this study, an oncolytic virus (rVSV-LCMVG) that is not prone to induce virus-neutralizing antibodies was designed and combined with adoptively transferred T cells. By transforming the immunosuppressive tumor microenvironment into an immunosensitive one, in B16 tumor-bearing mice, combination therapy showed superior antitumor effects than monotherapy. This occurred whether the OV was administered intratumorally or intravenously. Combination therapy significantly increased cytokine and chemokine levels within tumors and recruited CD8+ T cells to the TME to trigger antitumor immune responses. Pretreatment with adoptively transferred T cells and subsequent oncolytic virotherapy sensitizes refractory tumors by boosting T-cell recruitment, down-regulating the expression of PD-1, and restoring effector T-cell function. To offer a combination therapy with greater translational value, mRNA vaccines were introduced to induce tumor-specific T cells instead of adoptively transferred T cells. The combination of OVs and mRNA vaccine also displays a significant reduction in tumor burden and prolonged survival. This study proposed a rational combination therapy of OVs with adoptive T-cell transfer or mRNA vaccines encoding tumor-associated antigens, in terms of synergistic efficacy and mechanism.

Deleting the mitochondrial respiration negative regulator MCJ enhances the efficacy of CD8+ T cell adoptive therapies in pre-clinical studies.

Mitochondrial respiration is essential for the survival and function of T cells used in adoptive cellular therapies. However, strategies that specifically enhance mitochondrial respiration to promote T cell function remain limited. Here, we investigate methylation-controlled J protein (MCJ), an endogenous negative regulator of mitochondrial complex I expressed in CD8 cells, as a target for improving the efficacy of adoptive T cell therapies. We demonstrate that MCJ inhibits mitochondrial respiration in murine CD8+ CAR-T cells and that deletion of MCJ increases their in vitro and in vivo efficacy against murine B cell leukaemia. Similarly, MCJ deletion in ovalbumin (OVA)-specific CD8+ T cells also increases their efficacy against established OVA-expressing melanoma tumors in vivo. Furthermore, we show for the first time that MCJ is expressed in human CD8 cells and that the level of MCJ expression correlates with the functional activity of CD8+ CAR-T cells. Silencing MCJ expression in human CD8 CAR-T cells increases their mitochondrial metabolism and enhances their anti-tumor activity. Thus, targeting MCJ may represent a potential therapeutic strategy to increase mitochondrial metabolism and improve the efficacy of adoptive T cell therapies.

EGC enhances tumor antigen presentation and CD8+ T cell-mediated antitumor immunity via targeting oncoprotein SND1.

The Staphylococcal nuclease and Tudor domain containing 1 (SND1) has been identified as an oncoprotein. Our previous study demonstrated that SND1 impedes the major histocompatibility complex class I (MHC-I) assembly by hijacking the nascent heavy chain of MHC-I to endoplasmic reticulum-associated degradation. Herein, we aimed to identify inhibitors to block SND1-MHC-I binding, to facilitate the MHC-I presentation and tumor immunotherapy. Our findings validated the importance of the K490-containing sites in SND1-MHC-I complex. Through structure-based virtual screening and docking analysis, (-)-Epigallocatechin (EGC) exhibited the highest docking score to prevent the binding of MHC-I to SND1 by altering the spatial conformation of SND1. Additionally, EGC treatment resulted in increased expression levels of membrane-presented MHC-I in tumor cells. The C57BL/6J murine orthotopic melanoma model validated that EGC increases infiltration and activity of CD8+ T cells in both the tumor and spleen. Furthermore, the combination of EGC with programmed death-1 (PD-1) antibody demonstrated a superior antitumor effect. In summary, we identified EGC as a novel inhibitor of SND1-MHC-I interaction, prompting MHC-I presentation to improve CD8+ T cell response within the tumor microenvironment. This discovery presents a promising immunotherapeutic candidate for tumors.

Chromosomal instability induced in cancer can enhance macrophage-initiated immune responses that include anti-tumor IgG.

Solid tumors generally exhibit chromosome copy number variation, which is typically caused by chromosomal instability (CIN) in mitosis. The resulting aneuploidy can drive evolution and associates with poor prognosis in various cancer types as well as poor response to T-cell checkpoint blockade in melanoma. Macrophages and the SIRPα-CD47 checkpoint are understudied in such contexts. Here, CIN is induced in poorly immunogenic B16F10 mouse melanoma cells using spindle assembly checkpoint MPS1 inhibitors that generate persistent micronuclei and diverse aneuploidy while skewing macrophages toward a tumoricidal 'M1-like' phenotype based on markers and short-term anti-tumor studies. Mice bearing CIN-afflicted tumors with wild-type CD47 levels succumb similar to controls, but long-term survival is maximized by SIRPα blockade on adoptively transferred myeloid cells plus anti-tumor monoclonal IgG. Such cells are the initiating effector cells, and survivors make de novo anti-cancer IgG that not only promote phagocytosis of CD47-null cells but also suppress tumor growth. CIN does not affect the IgG response, but pairing CIN with maximal macrophage anti-cancer activity increases durable cures that possess a vaccination-like response against recurrence.

Hypofractionated radiotherapy combined with lenalidomide improves systemic antitumor activity in mouse solid tumor models.

Background: Hypofractionated radiotherapy (hRT) can induce a T cell-mediated abscopal effect on non-irradiated tumor lesions, especially in combination with immune checkpoint blockade (ICB). However, clinically, this effect is still rare, and ICB-mediated adverse events are common. Lenalidomide (lena) is an anti-angiogenic and immunomodulatory drug used in the treatment of hematologic malignancies. We here investigated in solid tumor models whether lena can enhance the abscopal effect in double combination with hRT. Methods: In two syngeneic bilateral tumor models (B16-CD133 melanoma and MC38 colon carcinoma), the primary tumor was treated with hRT. Lena was given daily for 3 weeks. Besides tumor size and survival, the dependence of the antitumor effects on CD8+ cells, type-I IFN signaling, and T cell costimulation was determined with depleting or blocking antibodies. Tumor-specific CD8+ T cells were quantified, and their differentiation and effector status were characterized by multicolor flow cytometry using MHC-I tetramers and various antibodies. In addition, dendritic cell (DC)-mediated tumor antigen cross-presentation in vitro and directly ex vivo and the composition of tumor-associated vascular endothelial cells were investigated. Results: In both tumor models, the hRT/lena double combination induced a significant abscopal effect. Control of the non-irradiated secondary tumor and survival were considerably better than with the respective monotherapies. The abscopal effect was strongly dependent on CD8+ cells and associated with an increase in tumor-specific CD8+ T cells in the non-irradiated tumor and its draining lymph nodes. Additionally, we found more tumor-specific T cells with a stem-like (TCF1+ TIM3- PD1+) and a transitory (TCF1- TIM3+ CD101- PD1+) exhausted phenotype and more expressing effector molecules such as GzmB, IFNγ, and TNFα. Moreover, in the non-irradiated tumor, hRT/lena treatment also increased DCs cross-presenting a tumor model antigen. Blocking type-I IFN signaling, which is essential for cross-presentation, completely abrogated the abscopal effect. A gene expression analysis of bone marrow-derived DCs revealed that lena augmented the expression of IFN response genes and genes associated with differentiation, maturation (including CD70, CD83, and CD86), migration to lymph nodes, and T cell activation. Flow cytometry confirmed an increase in CD70+ CD83+ CD86+ DCs in both irradiated and abscopal tumors. Moreover, the hRT/lena-induced abscopal effect was diminished when these costimulatory molecules were blocked simultaneously using antibodies. In line with the enhanced infiltration by DCs and tumor-specific CD8+ T cells, including more stem-like cells, hRT/lena also increased tumor-associated high endothelial cells (TA-HECs) in the non-irradiated tumor. Conclusions: We demonstrate that lena can augment the hRT-induced abscopal effect in mouse solid tumor models in a CD8 T cell- and IFN-I-dependent manner, correlating with enhanced anti-tumor CD8 T cell immunity, DC cross-presentation, and TA-HEC numbers. Our findings may be helpful for the planning of clinical trials in (oligo)metastatic patients.

Depletion of regulatory T cells enhancing the anti-tumor effect of in situ vaccination in solid tumors.

The emergence of immune checkpoint inhibitors (ICIs) has revolutionized the clinical treatment for tumor. However, the low response rate of ICIs remains the major obstacle for curing patients and effective approaches for patients with primary or secondary resistance to ICIs remain lacking. In this study, immune stimulating agent unmethylated CG-enriched (CpG) oligodeoxynucleotide (ODN) was locally injected into the tumor to trigger a robust immune response to eradicate cancer cells, while anti-CD25 antibody was applied to remove immunosuppressive regulatory T cells, which further enhanced the host immune activity to attack tumor systematically. The combination of CpG and anti-CD25 antibody obtained notable regression in mouse melanoma model. Furthermore, rechallenge of tumor cells in the xenograft model has resulted in smaller tumor volume, which demonstrated that the combinational treatment enhanced the activity of memory T cells. Remarkably, this combinational therapy presented significant efficacy on multiple types of tumors as well and was able to prevent relapse of tumor partially. Taken together, our combinational immunotherapy provides a new avenue to enhance the clinical outcomes of patients who are insensitive or resistant to ICIs treatments.

Light-Triggered Nanozymes Remodel the Tumor Hypoxic and Immunosuppressive Microenvironment for Ferroptosis-Enhanced Antitumor Immunity.

Cancer immunotherapy holds significant promise for addressing diverse malignancies. Nevertheless, its efficacy remains constrained by the intricate tumor immunosuppressive microenvironment. Herein, a light-triggered nanozyme Fe-TCPP-R848-PEG (Fe-MOF-RP) was designed for remodeling the immunosuppressive microenvironment. The Fe-TCPP-MOFs were utilized not only as a core catalysis component against tumor destruction but also as a biocompatible delivery vector of an immunologic agonist, improving its long circulation and tumor enrichment. Concurrently, it catalyzes the decomposition of H2O2 within the tumor, yielding oxygen to augment photodynamic therapy. The induced ferroptosis, in synergy with photodynamic therapy, prompts the liberation of tumor-associated antigens from tumor cells inducing immunogenic cell death. Phototriggered on-demand release of R848 agonists stimulated the maturation of dendritic cells and reverted the tumor-promoting M2 phenotypes into adoptive M1 macrophages, which further reshaped the tumor immunosuppressive microenvironment. Notably, the nanozyme effectively restrains well-established tumors, such as B16F10 melanoma. Moreover, it demonstrates a distal tumor-inhibiting effect upon in situ light treatment. What is more, in a lung metastasis model, it elicits robust immune memory, conferring enduring protection against tumor rechallenge. Our study presents a straightforward and broadly applicable strategy for crafting nanozymes with the potential to effectively thwart cancer recurrence and metastasis.

Checkpoint inhibitor-expressing lentiviral vaccine suppresses tumor growth in preclinical cancer models.

BACKGROUND: While immunotherapy has been highly successful for the treatment of some cancers, for others, the immune response to tumor antigens is weak leading to treatment failure. The resistance of tumors to checkpoint inhibitor therapy may be caused by T cell exhaustion resulting from checkpoint activation. METHODS: In this study, lentiviral vectors that expressed T cell epitopes of an experimentally introduced tumor antigen, ovalbumin, or the endogenous tumor antigen, Trp1 were developed. The vectors coexpressed CD40 ligand (CD40L), which served to mature the dendritic cells (DCs), and a soluble programmed cell death protein 1 (PD-1) microbody to prevent checkpoint activation. Vaccination of mice bearing B16.OVA melanomas with vector-transduced DCs induced the proliferation and activation of functional, antigen-specific, cytolytic CD8 T cells. RESULTS: Vaccination induced the expansion of CD8 T cells that infiltrated the tumors to suppress tumor growth. Vector-encoded CD40L and PD-1 microbody increased the extent of tumor growth suppression. Adoptive transfer demonstrated that the effect was mediated by CD8 T cells. Direct injection of the vector, without the need for ex vivo transduction of DCs, was also effective. CONCLUSIONS: This study suggests that therapeutic vaccination that induces tumor antigen-specific CD8 T cells coupled with a vector-expressed checkpoint inhibitor can be an effective means to suppress the growth of tumors that are resistant to conventional immunotherapy.

Melanin-Ce6-loaded polydopamine nanoparticles-based enhanced phototherapy for B16 melanoma cancer cells.

Melanoma is one of the most aggressive and lethal types of cancer owing to its metastatic propensity and chemoresistance property. An alternative therapeutic option is photodynamic and photothermal therapies (PDT/PTT), which employ near-infrared (NIR) light to generate heat and reactive oxygen species (ROS). As per previous reports, Melanin (Mel), and its synthetic analogs (i.e. polydopamine nanoparticles) can induce NIR light-mediated heat energy, thereby selectively targeting and ameliorating cancer cells. Similarly, chlorin e6 (Ce6) also has high ROS generation ability and antitumor activity against various types of cancer. Based on this tenet, In the current study, we have encapsulated Mel-Ce6 in a polydopamine (PDA) nanocarrier (MCP NPs) synthesized by the oxidation polymerization method. The hydrodynamic diameter of the synthesized spherical MCP NPs was 139 ± 10 nm. The MCP NPs, upon irradiation with NIR 690 nm laser for 6 min, showed photothermal efficacy of more than 50 °C. Moreover, the red fluorescence in the MCP NPs due to Ce6 can be leveraged for diagnostic purposes. Further, the MCP NPs exhibited considerable biocompatibility with the L929 cell line and exerted nearly 70% ROS-mediated cytotoxicity on the B16 melanoma cell line after the laser irradiation. Thus, the prepared MCP NPs could be a promising theranostic agent for treating the B16 melanoma cancer.

Positron emission tomography guided synergistic treatment of melanoma using multifunctional zirconium-hematoporphyrin nanosonosensitizers.

Sonodynamic therapy (SDT) has emerged as a useful approach for tumor treatment. However, its widespread application is impeded by poor pharmacokinetics of existing sonosensitizers. Here we developed a metal-organic nanoplatform, wherein a small-molecule sonosensitizer (hematoporphyrin monomethyl ether, HMME) was ingeniously coordinated with zirconium, resulting in a multifunctional nanosonosensitizer termed Zr-HMME. Through post-synthetic modifications involving PEGylation and tumor-targeting peptide (F3) linkage, a nanoplatform capable of homing on melanoma was produced, which could elicit robust immune responses to suppress tumor lung metastasis in the host organism. Importantly, after seamless incorporation of positron-emitting 89Zr into this nanosonosensitizer, positron emission tomography (PET) could be used to monitor its in vivo pharmacokinetics. PET imaging studies revealed that this nanoplatform exhibited potent tumor accumulation and strong in vivo stability. Using intrinsic fluorescence from HMME, a dual-modal diagnostic capability (fluorescence and PET) was confirmed for this nanosonosensitizer. In addition, the mechanisms of how this nanoplatform interacted with immune system were also investigated. The collective data proved that the coordination structure between small-molecule drug cargos and metals may enhance the functions of each other while mitigating their weaknesses. This straightforward approach can expand the potential applications of suitable drug molecules.

Plasmid DNA ionisable lipid nanoparticles as non-inert carriers and potent immune activators for cancer immunotherapy.

Immunotherapy is currently a standard of care in the treatment of many malignancies. However, predictable side effects caused by systemic administration of highly immunostimulatory molecules have been a serious concern within this field. Intratumoural expression or silencing of immunogenic and immunoinhibitory molecules using nucleic acid-based approaches such as plasmid DNA (pDNA) and small interfering RNA (siRNA), respectively, could represent a next generation of cancer immunotherapy. Here, we employed lipid nanoparticles (LNPs) to deliver either non-specific pDNA and siRNA, or constructs targeting two prominent immunotherapeutic targets OX40L and indoleamine 2,3-dioxygenase-1 (IDO), to tumours in vivo. In the B16F10 mouse model, intratumoural delivery of LNP-formulated non-specific pDNA and siRNA led to strong local immune activation and tumour growth inhibition even at low doses due to the pDNA immunogenic nature. Replacement of these non-specific constructs by pOX40L and siIDO resulted in more prominent immune activation as evidenced by increased immune cell infiltration in tumours and tumour-draining lymph nodes. Consistently, pOX40L alone or in combination with siIDO could prolong overall survival, resulting in complete tumour regression and the formation of immunological memory in tumour rechallenge models. Our results suggest that intratumoural administration of LNP-formulated pDNA and siRNA offers a promising approach for cancer immunotherapy.

An IRF2-Expressing Oncolytic Virus Changes the Susceptibility of Tumor Cells to Antitumor T Cells and Promotes Tumor Clearance.

IFN regulatory factor 1 (IRF1) can promote antitumor immunity. However, we have shown previously that in the tumor cell, IRF1 can promote tumor growth, and IRF1-deficient tumor cells exhibit severely restricted tumor growth in several syngeneic mouse tumor models. Here, we investigate the potential of functionally modulating IRF1 to reduce tumor progression and prolong survival. Using inducible IRF1 expression, we established that it is possible to regulate IRF1 expression to modulate tumor progression in established B16-F10 tumors. Expression of IRF2, which is a functional antagonist of IRF1, downregulated IFNγ-induced expression of inhibitory ligands, upregulated MHC-related molecules, and slowed tumor growth and extended survival. We characterized the functional domain(s) of IRF2 needed for this antitumor activity, showing that a full-length IRF2 was required for its antitumor functions. Finally, using an oncolytic vaccinia virus as a delivery platform, we showed that IRF2-expressing vaccinia virus suppressed tumor progression and prolonged survival in multiple tumor models. These results suggest the potency of targeting IRF1 and using IRF2 to modulate immunotherapy.

Deletion of MyD88 in T Cells Improves Antitumor Activity in Melanoma.

Cytotoxic CD8+ T cells are central to the antitumor immune response by releasing cytotoxic granules that kill tumor cells. They are activated by antigen-presenting cells, which become activated by DAMPs (damage associated molecular patterns) through MyD88. However, the suppressive tumor microenvironment promotes T-cell tolerance to tumor antigens, in part by enhancing the activity of immune checkpoint molecules that prevent CD8+ T-cell activation and cytotoxicity. MyD88 limits CD4+ T-cell activation during cardiac adaptation to stress. A similar mechanism is hypothesized to exist in CD8+ T cells that could be modulated to improve antitumor immunity. Herein, adoptive transfer of MyD88-/- CD8+ T cells in melanoma-bearing T-cell-deficient mice resulted in slower tumor growth, greater intratumoral T-cell accumulation, and higher melanoma cell death compared with transfer of wild-type CD8+ T cells. These findings were also observed in T-cell-specific MyD88-/- mice compared with wild-type littermates implanted with melanoma. Mechanistically, deletion of MyD88 enhanced CD8+ T-cell activation and survival, and T-cell receptor induced degranulation of cytotoxic molecules, overall improving the killing of melanoma cells. This enhanced cytotoxicity was retained in mice bearing tumors expressing the specific antigen for which cytotoxic T-cells were restricted. This study's results demonstrate a conserved mechanism for MyD88 in modulating CD8+ T-cell activation and represent a novel target in improving cancer immunotherapy.

Neutrophils bearing adhesive polymer micropatches as a drug-free cancer immunotherapy.

Tumour-associated neutrophils can exert antitumour effects but can also assume a pro-tumoural phenotype in the immunosuppressive tumour microenvironment. Here we show that neutrophils can be polarized towards the antitumour phenotype by discoidal polymer micrometric 'patches' that adhere to the neutrophils' surfaces without being internalized. Intravenously administered micropatch-loaded neutrophils accumulated in the spleen and in tumour-draining lymph nodes, and activated splenic natural killer cells and T cells, increasing the accumulation of dendritic cells and natural killer cells. In mice bearing subcutaneous B16F10 tumours or orthotopic 4T1 tumours, intravenous injection of the micropatch-loaded neutrophils led to robust systemic immune responses, a reduction in tumour burden and improvements in survival rates. Micropatch-activated neutrophils combined with the checkpoint inhibitor anti-cytotoxic T-lymphocyte-associated protein 4 resulted in strong inhibition of the growth of B16F10 tumours, and in complete tumour regression in one-third of the treated mice. Micropatch-loaded neutrophils could provide a potent, scalable and drug-free approach for neutrophil-based cancer immunotherapy.