Reconciling the Cooperative-Competitive Patterns among Tumor and Immune Cells for Triple-Negative Breast Cancer Treatment Using Multimodule Nanocomplexes.

Targeting the competitive-cooperative relationships among tumor cells and various immune cells can efficiently reverse the immune-dysfunction microenvironment to boost the immunotherapies for the triple-negative breast cancer treatment. Hence, a bacterial outer membrane vesicle-based nanocomplex is designed for specifically targeting malignant cells and immune cells to reconcile the relationships based on metabolic-immune crosstalk. By uniquely utilizing the property of charge-reversal polymers to realize function separation, the nanocomplexes could synergistically regulate tumor cells and immune cells. This approach could reshape the immunosuppressive competition-cooperation pattern into one that is immune-responsive, showcasing significant potential for inducing tumor remission in TNBC models.

A New HEK293 Cell with CR2 Region of E1A Gene Deletion Prevents the Emergence of Replication-Competent Adenovirus.

PURPOSE: To eliminate the contaminants of Replication-Competent Adenovirus (RCA) during high titer recombinant oncolytic adenovirus production. METHODS: At first, we detected E1A copy numbers of different sources of 293 cells using Q-PCR, and we screened a subclone JH293-C21 of the JH293 cell line (purchased from ATCC) with lower early region 1A (E1A) copy numbers and higher adenovirus production ability. Then, we deleted the conserved region (CR)2 of the E1A gene in this subclone using the CRISPR-Cas9 system and obtained a stable cell clone JH293-C21-C14 with lower E1A expression, but the RCA formation had no significant reduction. Then, we further deleted the CR2 of JH293-C21-C14 cells with the CRISPR-Cas9 system and obtained a strain of cells named JH293-C21-C14-C28. Finally, we detected the capacity for cell proliferation, adenovirus production, and RCA formation in the production of recombinant adenovirus. RESULTS: The JH293-C21-C14-C28 cells had a similar cell proliferation ability and human adenovirus production as JH293-C21 cells. Most importantly, RCA production in JH293-C21-C14-C28 cells was lower than in JH293-C21 cells. CONCLUSION: Human adenovirus producer cell clone JH293-C21-C14-C28 with CR2 deletion can effectively prevent the RCA production of replication-competent oncolytic adenovirus; this will provide significant advantages in utility and safety in gene therapy.

Nanomaterials with dual immunomodulatory functions for synergistic therapy of breast cancer brain metastases.

A long-standing paucity of effective therapies results in the poor outcomes of triple-negative breast cancer brain metastases. Immunotherapy has made progress in the treatment of tumors, but limited by the non-immunogenicity of tumors and strong immunosuppressive environment, patients with TNBC brain metastases have not yet benefited from immunotherapy. Dual immunoregulatory strategies with enhanced immune activation and reversal of the immunosuppressive microenvironment provide new therapeutic options for patients. Here, we propose a cocktail-like therapeutic strategy of microenvironment regulation-chemotherapy-immune synergistic sensitization and construct reduction-sensitive immune microenvironment regulation nanomaterials (SIL@T). SIL@T modified with targeting peptide penetrates the BBB and is subsequently internalized into metastatic breast cancer cells, releasing silybin and oxaliplatin responsively in the cells. SIL@T preferentially accumulates at the metastatic site and can significantly prolong the survival period of model animals. Mechanistic studies have shown that SIL@T can effectively induce immunogenic cell death of metastatic cells, activate immune responses and increase infiltration of CD8+ T cells. Meanwhile, the activation of STAT3 in the metastatic foci is attenuated and the immunosuppressive microenvironment is reversed. This study demonstrates that SIL@T with dual immunomodulatory functions provides a promising immune synergistic therapy strategy for breast cancer brain metastases.

A neutrophil-biomimic platform for eradicating metastatic breast cancer stem-like cells by redox microenvironment modulation and hypoxia-triggered differentiation therapy.

Metastasis accounts for 90% of breast cancer deaths, where the lethality could be attributed to the poor drug accumulation at the metastatic loci. The tolerance to chemotherapy induced by breast cancer stem cells (BCSCs) and their particular redox microenvironment further aggravate the therapeutic dilemma. To be specific, therapy-resistant BCSCs can differentiate into heterogeneous tumor cells constantly, and simultaneously dynamic maintenance of redox homeostasis promote tumor cells to retro-differentiate into stem-like state in response to cytotoxic chemotherapy. Herein, we develop a specifically-designed biomimic platform employing neutrophil membrane as shell to inherit a neutrophil-like tumor-targeting capability, and anchored chemotherapeutic and BCSCs-differentiating reagents with nitroimidazole (NI) to yield two hypoxia-responsive prodrugs, which could be encapsulated into a polymeric nitroimidazole core. The platform can actively target the lung metastasis sites of triple negative breast cancer (TNBC), and release the escorted drugs upon being triggered by the hypoxia microenvironment. During the responsiveness, the differentiating agent could promote transferring BCSCs into non-BCSCs, and simultaneously the nitroimidazole moieties conjugated on the polymer and prodrugs could modulate the tumor microenvironment by depleting nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) and amplifying intracellular oxidative stress to prevent tumor cells retro-differentiation into BCSCs. In combination, the BCSCs differentiation and tumor microenvironment modulation synergistically could enhance the chemotherapeutic cytotoxicity, and remarkably suppress tumor growth and lung metastasis. Hopefully, this work can provide a new insight in to comprehensively treat TNBC and lung metastasis using a versatile platform.

Compound Nanoemulsion Combined with Differentiation/Cytotoxicity Drugs for Modulating Breast Cancer Stemness.

Breast cancer stem cells (BCSCs) are the culprit of triple-negative breast cancer invasiveness and are heterogeneous. It is recognized that the combination of chemotherapy and differentiation therapy for killing BCSCs and non-BCSCs simultaneously is a reliable strategy. In this study, an oil-in-water nanoemulsion was prepared by high-pressure homogenization with coencapsulation of all-trans retinoic acid (ATRA) and doxorubicin (DOX). The preparation process was simple, and the production was easy to scale up. The particle size of the nanoemulsion was 127.2 ± 2.0 nm. Cellular toxicity assay showed that the composite index of the ATRA and DOX was less than 1 and exhibited a fine combined effect. In vivo antitumor efficacy showed that the compound nanoemulsion could reduce the proportion of BCSCs to 1.18% by inhibiting the expression of Pin1. In addition, the combination of ATRA and DOX could reduce the cardiotoxicity of DOX and had higher safety. Hopefully, this work can provide a new insight into developing pharmaceutically acceptable technology for treating BCSCs.

Microenvironment-tailored micelles restrain carcinoma-astrocyte crosstalk for brain metastasis.

Breast-to-brain metastatic cells can interact with the surrounding cells, including astrocytes and microglia, to generate a pro-tumorigenic niche. Breast-to-brain metastasis can be treated using a dual strategy of eliminating metastatic tumor cells and normalizing their localized microenvironment. The effective accumulation of drugs at the action site of metastasis is crucial to realizing the above strategy, especially when dealing with the blood-brain barrier (BBB)-penetrating and tumor-targeting tactics. Here, we establish an in-situ microenvironment-tailored micelle (T-M/siRNA) to co-deliver therapeutic siRNA and paclitaxel (PTX) into the breast-to-brain metastasis. Anchored with a D-type cyclic peptide, T-M/siRNA can penetrate the BBB and subsequently target the brain metastases. Upon internalization by metastatic tumor cells, T-M/siRNA can release PTX in the high-level glutathione (GSH), resulting in killing cancer cells. Meanwhile, the micellar structure is dissociated, resulting in lowering the charge density to release the loaded siRNA that can targeted downregulate the expression of protocadherin 7 (PCDH7). Treatment of model mice revealed that T-M/siRNA can inhibit the abnormal activation of astrocytes and immunosuppressive activation of microglia, resulting in significantly enhanced synergistic anti-tumor efficacy. This study indicates that the micelle system can serve as a hopeful strategy to treat breast-to-brain metastasis.

An effective therapeutic regime for treatment of glioma using oncolytic vaccinia virus expressing IL-21 in combination with immune checkpoint inhibition.

Glioblastoma (GBM) is the most common primary malignant tumor in the brain, accounting for 51.4% of all primary brain tumors. GBM has a highly immunosuppressive tumor microenvironment (TME) and, as such, responses to immunotherapeutic strategies are poor. Vaccinia virus (VV) is an oncolytic virus that has shown tremendous therapeutic effect in various tumor types. In addition to its directly lytic effect on tumor cells, it has an ability to enhance immune cell infiltration into the TME allowing for improved immune control over the tumor. Here, we used a new generation of VV expressing the therapeutic payload interleukin-21 to treat murine GL261 glioma models. After both intratumoral and intravenous delivery, virus treatment induced remodeling of the TME to promote a robust anti-tumor immune response that resulted in control over tumor growth and long-term survival in both subcutaneous and orthotopic mouse models. Treatment efficacy was significantly improved in combination with systemic α-PD1 therapy, which is ineffective as a standalone treatment but synergizes with oncolytic VV to enhance therapeutic outcomes. Importantly, this study also revealed the upregulation of stem cell memory T cell populations after the virus treatment that exert strong and durable anti-tumor activity.

Smart hypoxia-responsive transformable and charge-reversible nanoparticles for the deep penetration and tumor microenvironment modulation of pancreatic cancer.

The compact extracellular matrix (ECM) of pancreatic ductal adenocarcinoma (PDAC) is the major physical barrier that hinders the delivery of anti-tumor drugs, leading to strong inherent chemotherapy resistance as well as establishing an immunosuppressive tumor microenvironment (TME). However, forcibly destroying the stroma barrier would break the balance of delicate signal transduction and dependence between tumor cells and matrix components. Uncontrollable growth and metastasis would occur, making PDAC more difficult to control. Hence, we design and construct an aptamer-decorated hypoxia-responsive nanoparticle s(DGL)n@Apt co-loading gemcitabine monophosphate and STAT3 inhibitor HJC0152. This nanoparticle can reverse its surficial charge in the TME, and reduce the size triggered by hypoxia. The released ultra-small DGL particles loading gemcitabine monophosphate exhibit excellent deep-tumor penetration, chemotherapy drugs endocytosis promotion, and autophagy induction ability. Meanwhile, HJC0152 inhibits overactivated STAT3 in both tumor cells and tumor stroma, softens the stroma barrier, and reeducates the TME into an immune-activated state. This smart codelivery strategy provides an inspiring opportunity in PDAC treatment.

Penetrating Micelle for Reversing Immunosuppression and Drug Resistance in Pancreatic Cancer Treatment.

Pancreatic ductal adenocarcinoma (PDAC) is on of the most lethal malignant tumors with relatively poor prognosis, characterized with insufficient drug penetration, low immune response and obvious drug resistances. The therapeutic inefficiency is multifactorially related to its specific tumor microenvironment (TME), which is representatively featured as rich stroma and immunosuppression. In this work, a versatile drug delivery system is developed that can coencapsulate two prodrugs modified from gemcitabine (GEM) and a signal transducer and activator of transcription 3 (STAT3) inhibitor (HJC0152), and the gradient pH variation is further sensed in the TME of PDAC to achieve a higher penetration by reversing its surficial charges. The escorted prodrugs can release GEM intracellularly, and respond to the hypoxic condition to yield the parental STAT3 inhibitor HJC0152, respectively. By inhibiting STAT3, the tumor immunosuppression microenvironment can be re-educated through the reversion of M2-like tumor associated macrophages (M2-TAMs), recruitment of cytotoxic T lymphocytes and downregulation of regulatory T cells (Treg s). Furthermore, cytidine deaminase (CDA) and α-smooth muscle actin (α-SMA) expression can be downregulated, plus the lipid modification of GEM, the drug resistance of GEM can be greatly relieved. Based on the above design, a synergetic therapeutic efficacy in PDAC treatment can be achieved to provide more opportunity for clinical applications.

A Micro-Environment Regulator for Filling the Clinical Treatment Gap after a Glioblastoma Operation.

The rapid postoperative recurrence and short survival time of glioblastoma (GBM) patients necessitate immediate and effective postoperative treatment. Herein, an immediate and mild postoperative local treatment strategy is developed that regulates the postoperative microenvironment and delays GBM recurrence. Briefly, an injectable hydrogel system (imGEL) loaded with Zn(II)2 -AMD3100 (AMD-Zn) and CpG oligonucleotide nanoparticles (CpG NPs) is injected into the operation cavity, with long-term function to block the recruitment of microglia/ macrophages and activate cytotoxic T cells. The finding indicated that the imGEL can regulate the immune microenvironment, inhibit GBM recurrence, and gain valuable time for subsequent adjuvant clinical chemotherapy.

Exosomes derived from immunogenically dying tumor cells as a versatile tool for vaccination against pancreatic cancer.

Despite tremendous progress achieved in immunotherapy, many critical challenges in treating pancreatic ductal adenocarcinoma (PDAC) persist. Considering the poor vascularization of PDAC, after intramuscular administration exosomes can targeted deliver "cargos" to pancreatic tumors and bypass obstructions of the intrinsic overexpressed stroma through lymphatics. Herein, we propose a strategy to derive exosomes from immunogenically dying tumor cells and exploit their properties for several purposes, including antigen presentation, adjuvant supply, and "cargo" delivery of vaccines against pancreatic cancer via intramuscular injection. To enhance the immunostimulatory effects, the MART-1 peptide is modified to the exosomes to expand T-cell-related responses. Furthermore, CCL22 siRNA is electroporated into the exosomes (referred to as spMEXO) to hinder the CCR4/CCL22 axis between DCs and Tregs, thereby suppressing Treg expansion. Both in vitro and in vivo studies demonstrate that spMEXO can serve as an effective prophylactic vaccine to delay tumor growth, whereas combining spMEXO with PDAC first-line chemotherapeutics (co-administration of gemcitabine with albumin-paclitaxel) demonstrated significantly enhanced therapeutic effects in established PANC-02 tumors. Therefore, the present work provides an effective strategy to employ cancer vaccines through intramuscular injection in PDAC and highlights the potential of exosomes derived from immunogenically dying tumor cells as a versatile tool to develop nanovaccines for immunotherapy.

Sequentially Triggered Bacterial Outer Membrane Vesicles for Macrophage Metabolism Modulation and Tumor Metastasis Suppression.

Metabolic interactions between different cell types in the tumor microenvironment (TME) often result in reprogramming of the metabolism to be totally different from their normal physiological processes in order to support tumor growth. Many studies have attempted to inhibit tumor growth and activate tumor immunity by regulating the metabolism of tumors and other cells in TME. However, metabolic inhibitors often suffer from the heterogeneity of tumors, since the favorable metabolic regulation of malignant cells and other cells in TME is often inconsistent with each other. Therefore, we reported the design of a pH-sensitive drug delivery system that targets different cells in TME successively. Outer membrane vesicles (OMVs) derived from Gram-negative bacteria were applied to coload paclitaxel (PTX) and regulated in development and DNA damage response 1 (Redd1)-siRNA and regulate tumor metabolism microenvironment and suppress tumor growth. Our siRNA@M-/PTX-CA-OMVs could first release PTX triggered by the tumor pH (pH 6.8). Then the rest of it would be taken in by M2 macrophages to increase their level of glycolysis. Great potential was observed in TAM repolarization, tumor suppression, tumor immune activation, and TME remolding in the triple-negative breast cancer model. The application of the OMV provided an insight for establishing a codelivery platform for chemical drugs and genetic medicines.

A Versatile Theranostic Platform for Colorectal Cancer Peritoneal Metastases: Real-Time Tumor-Tracking and Photothermal-Enhanced Chemotherapy.

A versatile tumor-targeting stimuli-responsive theranostic platform for peritoneal metastases of colorectal cancer is proposed in this work for tumor tracking and photothermal-enhanced chemotherapy. A quenched photosensitizer ("off" state) is developed and escorted into a tumor-targeting oxaliplatin-embedded micelle. Once reaching the tumor cell, the micelle is clasped to release free oxaliplatin, as well as the "off" photosensitizer, which is further activated ("turned-on") in the tumor reducing microenvironment to provide optical imaging and photothermal effect. The combined results from hyperthermia-enhanced chemotherapy, deep penetration, perfused O2 , and the leveraged GSH-ROS imbalance in tumor cells are achieved for improved antitumor efficacy and reduced systematic toxicity.

Macrophage-Disguised Manganese Dioxide Nanoparticles for Neuroprotection by Reducing Oxidative Stress and Modulating Inflammatory Microenvironment in Acute Ischemic Stroke.

Reperfusion injury is still a major challenge that impedes neuronal survival in ischemic stroke. However, the current clinical treatments are remained on single pathological process, which are due to lack of comprehensive neuroprotective effects. Herein, a macrophage-disguised honeycomb manganese dioxide (MnO2 ) nanosphere loaded with fingolimod (FTY) is developed to salvage the ischemic penumbra. In particular, the biomimetic nanoparticles can accumulate actively in the damaged brain via macrophage-membrane protein-mediated recognition with cell adhesion molecules that are overexpressed on the damaged vascular endothelium. MnO2 nanosphere can consume excess hydrogen peroxide (H2 O2 ) and convert it into desiderated oxygen (O2 ), and can be decomposed in acidic lysosome for cargo release, so as to reduce oxidative stress and promote the transition of M1 microglia to M2 type, eventually reversing the proinflammatory microenvironment and reinforcing the survival of damaged neuron. This biomimetic nanomedicine raises new strategy for multitargeted combined treatment of ischemic stroke.

A novel vaccinia virus enhances anti-tumor efficacy and promotes a long-term anti-tumor response in a murine model of colorectal cancer.

Colorectal cancer (CRC) is one of the leading causes of mortality and morbidity in the world, and there remains an urgent need to develop long-lasting therapies to treat CRC and prevent recurrence in patients. Oncolytic virus therapy (OVT) has demonstrated remarkable efficacy in a number of different cancer models. Here, we report a novel vaccinia virus (VV)-based OVT for treatment of CRC. The novel VV, based on the recently reported novel VVLΔTKΔN1L virus, was armed with the pleiotropic cytokine interleukin-21 (IL-21) to enhance anti-tumor immune responses stimulated after viral infection of tumor cells. Compared with an unarmed virus, VVLΔTKΔN1L-mIL-21 had a superior anti-tumor efficacy in murine CMT93 subcutaneous CRC models in vivo, mediated mainly by CD8+ T cells. Treatment resulted in development of long-term immunity against CMT93 tumor cells, as evidenced by prevention of disease recurrence. These results demonstrate that VVLΔTKΔN1L-mIL-21 is a promising therapeutic agent for treatment of CRC.

Pancreatic cancer-targeting exosomes for enhancing immunotherapy and reprogramming tumor microenvironment.

Immunotherapy has gained increasing focus in treating pancreatic ductal adenocarcinoma (PDAC), since conventional therapies like chemotherapy could not provide satisfactory improvement in overall survival outcome of PDAC patients. However, it is still not the game changing solution due to the unique tumor microenvironment and low cancer immunogenicity of PDAC. Thus, inducing more intratumoral effector immune cells as well as reversing immunosuppression is the core of PDAC treatment. Herein, we demonstrate an exosome-based dual delivery biosystem for enhancing PDAC immunotherapy as well as reversing tumor immunosuppression of M2-like tumor associated macrophages (M2-TAMs) upon disruption of galectin-9/dectin 1 axis. The deliver system is constructed from bone marrow mesenchymal stem cell (BM-MSC) exosomes, electroporation-loaded galectin-9 siRNA, and surficially modified with oxaliplatin (OXA) prodrug as an immunogenic cell death (ICD)-trigger. The use of biomaterials, BM-MSC exosomes, can significantly improve tumor targeting efficacy, thus increasing drug accumulation in the tumor site. The combined therapy (iEXO-OXA) elicits anti-tumor immunity through tumor-suppressive macrophage polarization, cytotoxic T lymphocytes recruitment and Tregs downregulation, and achieves significant therapeutic efficacy in cancer treatment.

Click-Nucleic-Acid-Containing Codelivery System Inducing Collapse of Cellular Homeostasis for Tumor Therapy through Bidirectional Regulation of Autophagy and Glycolysis.

As a rapid proliferating tissue, tumor cells have to optimize nutrient utilization to withstand harsh conditions. Several approaches have been explored to inhibit the growth and metastasis of tumor by disrupting the reprogrammed tumor metabolism. However, nutrient limitations within solid tumors may induce the metabolic flexibility of malignant cells to sustain growth and survival using one nutrient to fill metabolite pools normally supplied by the other. To overcome this predicament, a promising click-nucleic-acid-containing platform for codelivery of rapamycin, anti-PFKFB4 siRNA, and targeting ligand aptamer AS1411 was applied. PFKFB4 could act as a promising target for tumor therapy for being a molecular fulcrum that could couple glycolysis to autophagy by promoting aggressive metastatic tumors. The downregulation of PFKFB4 can help inhibit the SRC3/Akt/mTOR pathway, leading autophagy to the direction of promoting apoptosis of tumor cells, which is induced by the collapse of tumor cellular homeostasis, while low dosages of rapamycin could decrease surgery-induced immune dysfunction. Enhanced tumor autophagy, favorable in vivo antitumor efficacy, and effective systematic immune activation are observed after treatment, suggesting that autophagy and glycolysis can serve as an integrated target for tumor treatment.

Penetrable Nanoplatform for "Cold" Tumor Immune Microenvironment Reeducation.

Lack of tumor-infiltration lymphocytes (TILs) and resistances by overexpressed immunosuppressive cells (principally, myeloid-derived suppressor cells (MDSCs)) in tumor milieu are two major challenges hindering the effectiveness of immunotherapy for "immune-cold" tumors. In addition, the natural physical barrier existing in solid cancer also limits deeper delivery of drugs. Here, a tumor-targeting and light-responsive-penetrable nanoplatform (Apt/PDGs/@pMOF) is developed to elicit intratumoral infiltration of cytotoxic T cells (CTLs) and reeducate immunosuppressive microenvironment simultaneously. In particular, porphyrinic metal-organic framework (pMOF)-based photodynamic therapy (PDT) induces tumor immunogenic cell death (ICD) to promote CTLs intratumoral infiltration and hot "immune-cold" tumor. Upon being triggered by PDT, the nearly 10 nm adsorbed drug-loaded dendrimer de-shields from the nanoplatform and spreads into the deeper tumor, eliminating MDSCs and reversing immunosuppression, eventually reinforcing immune response. Meanwhile, the designed nanoplatform also has a systemic MDSC inhibition effect and moderate improvement of overall antitumor immune responses, resulting in effective suppression of distal tumors within less significant immune-related adverse effects (irAEs) induced.

Treatment and Prevention of Lung Cancer Using a Virus-Infected Reprogrammed Somatic Cell-Derived Tumor Cell Vaccination (VIReST) Regime.

Lung cancer is one of the most commonly diagnosed cancer and despite therapeutic advances, mortality remains high. The long period of clinical latency associated with lung cancer provides an ideal window of opportunity to administer vaccines to at-risk individuals that can prevent tumor progression and initiate long-term anti-tumor immune surveillance. Here we describe a personalized vaccination regime that could be applied for both therapeutic and prophylactic prevention of lung cancer, based on the derivation of lung cancer cells from induced pluripotent stem cells. Stem cells from healthy mice were modified to express Cre-dependent KRASG12D and Trp53R172H prior to differentiation to lung progenitor cells. Subsequent viral delivery of Cre caused activation of exogenous driver mutations, resulting in transformation and development of lung cancer cells. iPSC-derived lung cancer cells were highly antigenically related to lung cancer cells induced in LSL-KRASG12D/+; Trp53R172H/+ transgenic mice and were antigenically unrelated to original pluripotent stem cells or pancreatic cancer cells derived using the same technological platform. For vaccination, induced lung cancer cells were infected with oncolytic Adenovirus or Vaccinia virus, to act as vaccine adjuvants, prior to delivery of vaccines sequentially to a murine inducible transgenic model of lung cancer. Application of this Virus-Infected, Reprogrammed Somatic cell-derived Tumor cell (VIReST) regime primed tumor-specific T cell responses that significantly prolonged survival in both subcutaneous post-vaccine challenge models and induced transgenic models of lung cancer, demonstrating that stem cell-derived prophylactic vaccines may be a feasible intervention for treatment or prevention of lung cancer development in at-risk individuals.