Tumor Tissue Microenvironment Research Articles

Clinicopathological Significance of the ET Axis in Human Oral Squamous Cell Carcinoma

The interaction between cancer cells and the surrounding microenvironment in malignant tumor tissue is known to be closely associated with cancer cell invasion and proliferation. Endothelin (ET) present in the microenvironment surrounding tumors has been reported to play a role in cancer cell invasion and proliferation by binding to receptors on the cell membrane of cancer cells. Here, we immunohistologically detected the expression of ET-1 and its receptor ETAR in oral squamous cell carcinoma (OSCC) and evaluated the association between the expression of each as well as their co-expression (ET-axis expression) and clinicopathological factors. A significant difference was observed between the invasion pattern as a parameter of cancer cell malignancy and the expressions of ET-1 and ETAR. The survival rates were significantly lower among the patients who were strongly positive for ET-1 and the ETAR-positive patients compared to negative patients. There was also a significant difference between ET-axis expression and the degree of histological differentiation and mode of invasion, and the survival rate of the positive cases was significantly lower than that of the negative cases. Our findings suggested that ET-axis assessments are important for assessing the malignancy of cancer cells and predicting the prognoses of OSCC patients.

Peptide-Induced Self-Assembly of Therapeutics into a Well-Defined Nanoshell with Tumor-Triggered Shape and Charge Switch.

Peptide-tuned self-assembly of macromolecular agents (>500 Da) such as therapeutic peptides offers a strategy to improve the properties and biofunctions of degradable nanomaterials, but the tough requirement of macromolecular therapeutics delivery and a lack of understanding of peptide-based self-assembly design present high barriers for their applications. Herein, we developed a new strategy for nanoengineering macromolecular drugs by an elaborate peptide, termed PSP (VVVVVHHRGDC), capable of directly conjugating with cargo to be a PSP-cargo monomer as building block tending to self-assemble into a well-defined nanoshell with tumor-triggered shape and charge switch. As a proof of concept, conjugation PSP to a D-peptide activator of tumor suppressor p53 termed DPMI (1492.5 Da) generated hollow spheres ~80 nm in diameter named PSP-DPMI that disintegrated only in the acidic microenvironment of tumor tissues, followed by integrin-mediated cellular uptake of PSP-DPMI monomers. Importantly, PSP-based self-assembly successfully endowed the DPMI with long circulation time and high cancer-cell-specific intracellular accumulation. PSP-DPMI nanoshells potently inhibited tumor growth in vitro and in vivo by the p53 restoration, while maintaining a highly favorable in vivo safety profile. Out of conventional encapsulation and conjugation, our study showcases a clinically viable novel method to nanoengineer macromolecular agents such as peptide for anticancer therapy and provides a hazard-free alternative strategy for the theranostics delivery.

Photodynamic Therapy Overcoming the Hypoxia Microenvironment in Tumor Tissues

Academic Journal of Polymer science (AJOP) is a Journal which publishes state-of-the-art overview articles by internationally recognized authorities in polymer science and engineering. It covers all the areas of particular interest are biomedical applications,

CpG-PEG Conjugates and their Immune Modulating Effects after Systemic Administration.

Synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs were found to be able to target cells that express Toll-like receptor 9 to modulate innate and adaptive immune reactions. But their in vivo application in immunotherapy against cancer has not been successful. We attempted in this study to examine polyethylene-glycol (PEG) conjugated CpG ODNs and investigated their mechanism of immune modulation in anti-cancer therapy. CpG-PEG conjugates with different PEG lengths were synthesized. In vitro activity as well as in vivo pharmacokinetics and pharmacodynamics properties were evaluated. CpG-PEG20Ks were found to be able to persist longer in circulation and activate various downstream effector cells. After intravenous injection, they resulted in higher levels of IL-12p70 in the circulation and lower M-MDSC infiltrates in the tumor microenvironment. Such activities were different from those of CpG ODNs without PEGylation, suggesting different PK-PD profiles systemically and locally. Our data support the development of CpG-PEGs as a new therapeutic agent that can be systemically administered to modulate immune responses and the microenvironment in tumor tissues.

Fabrication of Charge-Conversion Nanoparticles for Cancer Imaging by Flash Nanoprecipitation.

Traditional charge-conversion nanoparticles (NPs) need the breakage of acid-labile groups on the surface, which impedes the rapid response to the acidic microenvironment. Here, we developed novel rodlike charge-conversion NPs with amphiphilic dextran- b-poly(lactic- co-glycolic acid), poly(2-(dimethylamino) ethylmethylacrylate)- b-poly(ε-caprolactone), and an aggregation-induced emission-active probe through flash nanoprecipitation (FNP). These NPs exhibit reversible negative-to-positive charge transition at a slightly acidic pH relying on the rapid protonation/deprotonation of polymers. The size and the critical charge-conversion pH can be further tuned by varying the flow rate and polymer ratio. Consequently, the charge conversion endows NPs with resistance to protein adsorption at physiological pH and enhanced internalization to cancer cells under acidic conditions. Ex vivo imaging on harvest organs shows that charge-conversion NPs were predominantly distributed in tumors after intravenous administration to mice due to the robust response of NPs to the acidic microenvironment in tumor tissue, whereas control NPs or free probes were broadly accumulated in tumor, liver, kidney, and lung. These results suggest the great potential of the current FNP strategy in the facile and generic fabrication of charge-conversion NPs for tumor-targeting delivery of drugs or fluorescent probes.

WITHDRAWN: Immune imbalance of PD-1 and 1 PD-Ls in liver tissues from patients with Hepatitis B-related hepatocellular carcinoma

// Yong Liu 1,2 , Guiyang Wang 3 , Rui Huang 3 , Yingying Hao 4 , Haiyan Chang 3 , Chen Tian 3 , Yang Li 3 , Xiang-An Zhao 3 , Juan Xia 3 , Yuxin Chen 3,5 and Chao Wu 3 1 Department of Experimental Medicine, Nanjing Drum Tower Hospital, Nanjing University Medical School, Nanjing, China 2 Jiangsu Key Laboratory for Molecular Medicine, Nanjing University Medical School, Nanjing, China 3 Department of Infectious Diseases, Nanjing Drum Tower Hospital, Nanjing University Medical School, Nanjing, China 4 Department of Intensive Care Units, Nanjing Drum Tower Hospital, Nanjing University Medical School, Nanjing, China 5 Department of Laboratory Medicine, Nanjing Drum Tower Hospital, Nanjing University Medical School, Nanjing, China Correspondence to: Chao Wu, email: dr.wu@nju.edu.cn Yuxin Chen, email: yuxin_chen2015@163.com Keywords : PD-1; PD-L1; PD-L2; microenvironment; hepatitis B-related hepatocellular carcinoma Received: October 27, 2017     Accepted: December 05, 2017     Epub: January 12, 2018 Abstract 2 Dysregulated immune microenvironment, such as imbalance of programmed death-1 (PD-1) and its ligands (PD-Ls), plays a key role in hepatocarcinogenesis. Here we investigate the key immune checkpoint molecules, PD-1/PD-Ls, in liver tissues from patients with hepatitis B virus-related hepatocellular carcinoma (HBV-related HCC). We show that the characteristic of T cells in peripheral blood does not fully reflect the changes of immune microenvironment in tumor tissues. There is an imbalanced immune microenvironment in liver tissues, characterized by increased CD4/CD8 ratio and high expression of PD-1 on CD8 T cells in tumor tissues. Soluble PD-1 (sPD-1) levels in tumor tissues are increased, whereas sPD-L1 and sPD-L2 levels are decreased. Compared with control subjects, the patients with HBV-related HCC have higher serum sPD-1 and sPD-L1 levels, lower serum sPD-L2 levels. We also show that the altered immune microenvironment is not only found in tumor tissues, but also in non-tumor tissues from HBV-related HCC patients. The correlation between PD-1 and PD-L1 increases successively in hepatocellular carcinomas tissues, paracancerous tissues and adjacent non-tumor tissues. In tumor tissues, sPD-1 levels are negatively correlated with clinicopathological stages of HCC, while serum sPD-L1 levels have positive correlation with the clinicopathological stages. Combined, HBV infection alone may not affect the characteristics of T cell subpopulations, but might be related with the higher PD-1 levels in liver tissues. The immune imbalance of PD-1/PD-Ls in liver tissues may have important role in tumor microenvironment and immune escape, closely related with tumor initiation and progression.

Biomechanics of the Circulating Tumor Cell Microenvironment.

Circulating tumor cells (CTCs) exist in a microenvironment quite different from the solid tumor tissue microenvironment. They are detached from matrix and exposed to the immune system and hemodynamic forces leading to the conclusion that life as a CTC is "nasty, brutish, and short." While there is much evidence to support this assertion, the mechanisms underlying this are much less clear. In this chapter we will specifically focus on biomechanical influences on CTCs in the circulation and examine in detail the question of whether CTCs are mechanically fragile, a commonly held idea that is lacking in direct evidence. We will review multiple lines of evidence indicating, perhaps counterintuitively, that viable cancer cells are mechanically robust in the face of exposures to physiologic shear stresses that would be encountered by CTCs during their passage through the circulation. Finally, we present emerging evidence that malignant epithelial cells, as opposed to their benign counterparts, possess specific mechanisms that enable them to endure these mechanical stresses.

Targeted Therapeutic Nanoparticles: An Immense Promise to Fight against Cancer.

In nanomedicine, targeted therapeutic nanoparticle (NP) is a virtual outcome of nanotechnology taking the advantage of cancer propagation pattern. Tying up all elements such as therapeutic or imaging agent, targeting ligand, and cross-linking agent with the NPs is the key concept to deliver the payload selectively where it intends to reach. The microenvironment of tumor tissues in lymphatic vessels can also help targeted NPs to achieve their anticipated accumulation depending on the formulation objectives. This review accumulates the application of poly(lactic-co-glycolic acid) (PLGA) and polyethylene glycol (PEG) based NP systems, with a specific perspective in cancer. Nowadays, PLGA, PEG, or their combinations are the mostly used polymers to serve the purpose of targeted therapeutic NPs. Their unique physicochemical properties along with their biological activities are also discussed. Depending on the biological effects from parameters associated with existing NPs, several advantages and limitations have been explored in teaming up all the essential facts to give birth to targeted therapeutic NPs. Therefore, the current article will provide a comprehensive review of various approaches to fabricate a targeted system to achieve appropriate physicochemical properties. Based on such findings, researchers can realize the benefits and challenges for the next generation of delivery systems.

Characterization of the T-Cell Receptor Repertoire and Immune Microenvironment in Patients with Locoregionally Advanced Squamous Cell Carcinoma of the Head and Neck.

Purpose: Squamous cell carcinoma of the head and neck (SCCHN) is a lethal cancer with a suboptimal 5-year overall survival of approximately 50% with surgery and/or definitive chemoradiotherapy. Novel treatments are thus urgently awaited. Immunotherapy with checkpoint blockade has emerged as a promising option for patients with recurrent/metastatic SCCHN; however, it has not been investigated in the curative-intent setting yet. The purpose of this study was to investigate the T-cell receptor repertoire and the tumor microenvironment in tumor tissues of SCCHN patients with locoregionally advanced disease.Experimental Design: We performed T-cell receptor sequencing of tumor tissues from 44 patients with locoregionally advanced SCCHN prior to treatment with definitive chemoradiotherapy and correlated the T-cell clonality and the mRNA expression levels of immune-related genes with clinicopathologic parameters.Results: Clonal expansion of T cells was significantly higher in human papilloma virus (HPV)-negative compared with HPV-positive tumors, signifying more robust antigen presentation in HPV-negative tumors. The latter was supported by the higher percentage of HPV-negative tumors expressing HLA-A protein compared with HPV-positive tumors (P = 0.049). Higher GRZB levels correlated significantly with longer recurrence-free survival (log-rank, P = 0.003) independent of tumor size, nodal stage, and HPV status.Conclusions: Our findings support clonal expansion of T cells in SCCHN patients with locoregionally advanced disease and imply differences in the antigen presentation capacity between HPV-negative and HPV-positive tumors. Elevated GRZB mRNA levels may also serve as a favorable and independent predictor of outcome in SCCHN patients treated with chemoradiotherapy. These data provide rationale for the introduction of immunotherapeutic approaches in the curative-intent setting. Clin Cancer Res; 23(16); 4897-907. ©2017 AACR.

Abstract 3083: Prior irradiation results in elevated programmed death 1 (PD-1) in T cells

Abstract It is well known that radiation has long-lasting effects on the immune system. In this study we address the question whether these radiation-induced adverse effects on T cell activation are associated with alterations of T cell inhibitory receptors. Expression levels of inhibitory receptors on T cells were analyzed at multiple post-radiation time points ranging from one to four weeks in mice receiving whole body irradiation. The level of the checkpoint receptor, PD-1, on different T cell subpopulations was determined. Our results showed that prior irradiation resulted in significant elevated expression of PD-1 in both CD4+ and CD8+ populations. T cells with elevated PD-1 mostly were either central memory or naïve cells. In addition, the feedback induction of PD-1 expression in activated T cells declined with time after radiation. Taken together, the elevated PD-1 level observed at weeks after radiation exposure is connected to T cell dysfunction. Similar changes were seen in samples from tumor patients receiving radiotherapy. Recent preclinical and clinical studies have shown that radiation alters the immune-modulatory microenvironment in tumor tissue, and implies that a combination of radiotherapy and T cell checkpoint blockade immunotherapy including targeting the PD-L1/PD-1 axis may potentiate the antitumor response. Therefore, understanding the dynamic changes in PD-1 levels in T cells after radiation should help in the development of a more effective therapeutic strategy. Citation Format: Renxiang Chen, Deguan Li, Albert J. Fornace, Henghong Li. Prior irradiation results in elevated programmed death 1 (PD-1) in T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3083. doi:10.1158/1538-7445.AM2017-3083

Magnetic Resonance Imaging-Guided Multi-Drug Chemotherapy and Photothermal Synergistic Therapy with pH and NIR-Stimulation Release.

The combination of multidrug chemotherapy and photothermal therapy (PTT) enhances cancer therapeutic efficacy. Herein, we develop a simple and smart pH/NIR dual-stimulus-responsive degradable mesoporous CoFe2O4@PDA@ZIF-8 sandwich nanocomposite. The mesoporous CoFe2O4 core acts as T2-weighted magnetic resonance (MR) imaging probe, PTT agent, and loading platform of hydrophilic doxorubicin (DOX). A polydopamine (PDA) layer is used to avoid the premature leakage of DOX before arriving at tumor site, enhance PTT efficiency, and facilitate the integration of ZIF-8 (a kind of metal-organic framework). The ZIF-8 shell serves to encapsulate hydrophobic camptothecin (CPT) and as the switch for the pH and NIR stimulation-responsive release of the two drugs. Therefore, T2-weighted MR imaging-guided multidrug chemotherapy and PTT synergistic treatment is achieved. Two kinds of anticancer drugs, hydrophilic DOX and hydrophobic CPT, are successfully loaded in CoFe2O4 and ZIF-8, respectively, so no mutual interference between the two drugs exists. A unique two-stage stepwise release process is exhibited for CPT and DOX with an interval of 12 h to improve the anticancer efficacy under the acidic microenvironment of tumor tissue. NIR irradiation achieves the burst drug-release and PTT after laser stimulation, simultaneously. With this smart design, high drug concentration is achieved at the tumor site by quick release, especially for the therapeutic drugs that show nonlinear pharmacokinetics, and PTT is integrated efficiently. Furthermore, negligible biotoxicity and a remarkable synergic antitumor effect of the hybrid nanocomposites are validated by HepG2 cells and tumor-bearing mice as models. Our multidrug delivery-releasing composite improves tumor therapeutic efficiency significantly compared with a single-drug chemotherapy system. The simple multifunctional composite system can be applied as an effective platform for personal nanomedicine with diagnosis, smart drug delivery, and cancer treatment through its remarkable photothermal property and controllable multidrug release.

Characterization of the T-cell receptor repertoire and immune microenvironment in patients with locoregionally advanced squamous cell carcinoma of the head and neck.

3080 Background: Immunotherapy with checkpoint blockade was recently approved for patients with recurrent/metastatic SCCHN, however it has not been investigated in the curative-intent setting yet. In this study, we investigated the T-cell receptor repertoire and the immune microenvironment in tumor tissues of SCCHN patients with locoregionally advanced disease. Methods: T-cell receptor sequencing and polymerase chain reaction for immune-related genes of tumor tissues from 44 patients with locoregionally advanced SCCHN prior to treatment with definitive chemoradiotherapy were conducted. T-cell receptor clonality and the mRNA expression levels of immune-related genes were correlated with various clinicopathological parameters. Results: In patients with locoregionally advanced SCCHN, tumor infiltrating T-cells clonally expand and GRZB mRNA levels were associated significantly with longer progression-free survival (PFS) (p = 0.003) independent of HPV status, tumor and nodal stage. The TCR-β DI was significantly lower in HPV-negative compared to HPV-positive tumors (p = 0.002), signifying more clonal T-cell expansion in HPV-negative tumors. A higher percentage of HPV-negative tumors expressed HLA-A protein compared to HPV-positive tumors (p = 0.049), suggesting that the greater T-cell clonal expansion might be due to more robust antigen presentation by HPV-negative tumors. Conclusions: This study suggests the pre-existence of clonally expanded T-cells in patients with locoregionally advanced SCCHN prior to treatment, and provides rationale to introduce immunotherapy in the curative-intent setting. The association of high GRZB mRNA levels with favorable PFS independent of HPV-status, tumor and nodal stage supports that the pre-existence of an intrinsically inflamed microenvironment enhances chemoradiotherapy effects. Finally, in HPV-positive tumors, the T-cell infiltrate seemed to be more diverse which could be secondary to virally-induced defective expression of HLA class I molecules.

Tumor Microenvironment Targeting and Responsive Peptide-Based Nanoformulations for Improved Tumor Therapy.

The tumor microenvironment participates in all stages of tumor progression and has emerged as a promising therapeutic target for cancer therapy. Rapid progress in the field of molecular self-assembly using various biologic molecules has resulted in the fabrication of nanoformulations that specifically target and regulate microenvironment components to inhibit tumor growth. This inhibition process is based on differentiating between biophysicochemical cues guiding tumor and normal tissue microenvironments. Peptides and peptide derivatives, owing to their biocompatibility, chemical versatility, bioactivity, environmental sensitivity, and biologic recognition abilities, have been widely used as building blocks to construct multifunctional nanostructures for targeted drug delivery and controlled release. Several groups of peptides have been identified as having the ability to penetrate plasma membranes, regulate the essential signaling pathways of angiogenesis and immune reactions, and recognize key components in the tumor microenvironment (such as vascular systems, stromal cells, and abnormal tumor biophysicochemical features). Thus, using different modules, various functional peptides, and their derivatives can be integrated into nanoformulations specifically targeting the tumor microenvironment with increased selectivity, on-demand response, elevated cellular uptake, and improved tumor therapy. In this review, we introduce several groups of functional peptides and highlight peptide-based nanoformulations that specifically target the tumor microenvironment. We also provide our perspective on the development of smart drug-delivery systems with enhanced therapeutic efficacy.

Molecularly organic/inorganic hybrid hollow mesoporous organosilica nanocapsules with tumor-specific biodegradability and enhanced chemotherapeutic functionality.

Based on the intrinsic features of high stability and unique multifunctionality, inorganic nanoparticles have shown remarkable potentials in combating cancer, but their biodegradability and biocompatibility are still under debate. As a paradigm, this work successfully demonstrates that framework organic-inorganic hybridization can endow the inorganic mesoporous silica nanocarriers with unique tumor-sensitive biodegradability and high biocompatibility. Based on a "chemical homology" mechanism, molecularly organic-inorganic hybridized hollow mesoporous organosilica nanocapsules (HMONs) with high dispersity and sub-50nm particle dimension were constructed in mass production. A physiologically active disulfide bond (SS) was directly incorporated into the silica framework, which could break up upon contacting the reducing microenvironment of tumor tissue and biodegrade accordingly. Such a tumor-specific biodegradability is also responsible for the tumor-responsive drug releasing by the fast biodegradation and disintegration of the framework. The ultrasmall particle size of HMONs guarantees their high accumulation into tumor tissue, thus causing the high chemotherapeutic outcome. This research provides a paradigm that framework organic-inorganic hybridization can endow the inorganic nanocarrier with unique biological effects suitable for biomedical application, benefiting the development of novel nanosystems with the unique bio-functionality and performance.

Integrated analysis of somatic mutations and immune microenvironment in malignant pleural mesothelioma

ABSTRACTTo investigate the link between the genomic landscape of cancer cells and immune microenvironment in tumor tissues, we characterized somatic mutations and tumor-infiltrating lymphocytes (TILs) in malignant pleural mesothelioma (MPM), including mutation/neoantigen load, spatial heterogeneity of somatic mutations of cancer cells and TILs (T-cell receptor β (TCRβ) repertoire), and expression profiles of immune-related genes using specimens of three different tumor sites (anterior, posterior, and diaphragm) obtained from six MPM patients. Integrated analysis identified the distinct patterns of somatic mutations and the immune microenvironment signatures both intratumorally and interindividually. MPM cases showed intratumoral heterogeneity in somatic mutations with unique TCRβ clonotypes of TILs that were restricted to each tumor site, suggesting the presence of a neoantigen-related immune response. Correlation analyses showed that higher neoantigen load was significantly correlated with stronger clonal expansion of TILs (p = 0.048) and a higher expression level of an immune-associated cytolytic factor (PRF1 (p = 0.0041) in tumor tissues), suggesting that high neoantigen loads in tumor cells might promote expansion of functional tumor-specific T cells in the tumor bed. Our results collectively indicate that MPM tumors constitute a diverse heterogeneity in both the genomic landscape and immune microenvironment, and that mutation/neoantigen load may affect the immune microenvironment in MPM tissues.

Infiltration of Regulatory T Lymphocytes Impair Clinical Outcome in Ovarian Cancer Patients

Background: Ovarian carcinoma (OC) is one of the most severe and lethal cancers in women. Regulatory T (Treg) cells may participate in mediating a suppressive microenvironment in tumor tissue and contribute to tumor evasion from immune response. Specifically, some studies have indicated that Treg cell infiltration in malignant ascites and peripheral blood has been associated with poor clinical outcome. Methods: The number of CD4+, CD25+, forkhead box p3+ (Foxp3+) lymphocytes was assessed via flow cytometry in ascites and peripheral blood from 24 women with epithelial ovarian cancer (EOC). Overall survival was associated with Treg levels. Results: The study indicated an increase in the Treg cell subset from peripheral blood of EOC patients compared with those in healthy controls. Treg infiltration in ascites was higher than in blood into EOC patients. The patients with high infiltration of Treg in blood revealed less overall survival compared to a counterpart with normal levels of Treg. Regarding the association between patients with increased levels of Treg in ascites with overall survival there was no disadvantage found, compared to ascites from patients with normal levels of Treg. Conclusion: Study suggests that Treg lymphocytes were increased in ascites and peripheral blood in EOC patients and could be associated with diminished overall survival, via impairment antitumor immune response.

MnO2 Gatekeeper: An Intelligent and O2‐Evolving Shell for Preventing Premature Release of High Cargo Payload Core, Overcoming Tumor Hypoxia, and Acidic H2O2‐Sensitive MRI

Premature leakage of photosensitizer (PS) from nanocarriers significantly reduces the accumulation of PS within a tumor, thereby enhancing nonspecific accumulation in normal tissues, which inevitably leads to a limited efficacy for photodynamic therapy (PDT) and the enhanced systematic phototoxicity. Moreover, local hypoxia of the tumor tissue also seriously hinders the PDT. To overcome these limitations, an acidic H2O2‐responsive and O2‐evolving core–shell PDT nanoplatform is developed by using MnO2 shell as a switchable shield to prevent the premature release of loaded PS in core and elevate the O2 concentration within tumor tissue. The inner core SiO2‐methylene blue obtained by co‐condensation has a high PS payload and the outer MnO2 shell shields PS from leaking into blood after intravenous injection until reaching tumor tissue. Moreover, the shell MnO2 simultaneously endows the theranostic nanocomposite with redox activity toward H2O2 in the acidic microenvironment of tumor tissue to generate O2 and thus overcomes the hypoxia of cancer cells. More importantly, the Mn(ΙΙ) ion reduced from Mn(ΙV) is capable of in vivo magnetic resonance imaging selectively in response to overexpressed acidic H2O2. The facile incorporation of the switchable MnO2 shell into one multifunctional diagnostic and therapeutic nanoplatform has great potential for future clinical application.

Enhanced Cellular Internalization and On-Demand Intracellular Release of Doxorubicin by Stepwise pH-/Reduction-Responsive Nanoparticles.

The efficient delivery of antitumor agents to tumor sites faces numerous obstacles, such as poor cellular uptake and slow intracellular drug release. In this regard, smart nanoparticles (NPs) that respond to the unique microenvironment of tumor tissues have been widely used for drug delivery. In this study, novel charge-reversal and reduction-responsive histidine-grafted chitosan-lipoic acid NPs (HCSL-NPs) were selected for efficient therapy of breast cancer by enhancing cell internalization and intracellular pH- and reduction-triggered doxorubicin (DOX) release. The surface charge of HCSL-NPs presented as negative at physiological pH and reversed to positive at the extracellular and intracellular pH of the tumor. In vitro release investigation revealed that DOX/HCSL-NPs demonstrated a sustained drug release under the physiological condition, whereas rapid DOX release was triggered by both endolysosome pH and high-concentration reducing glutathione (GSH). These NPs exhibited enhanced internalization at extracellular pH, rapid intracellular drug release, and improved cytotoxicity against 4T1 cells in vitro. Excellent tumor penetrating efficacy was also found in 4T1 tumor spheroids and solid tumor slices. In vivo experiments demonstrated that HCSL-NPs exhibited excellent tumor-targeting ability in tumor tissues as well as excellent antitumor efficacy and low systemic toxicity in breast tumor-bearing BALB/c mice. These results indicated that the novel charge-reversal and reduction-responsive HCSL-NPs have great potential for targeted and efficient delivery of chemotherapeutic drugs in cancer treatments.

Abstract PR09: Antibody-mediated blockade of phosphatidylserine synergizes with immune checkpoint blockade by inhibiting multiple immune suppressive mechanisms

Abstract Background: The expression of phosphatidylserine (PS) on cell surfaces drives immunosuppressive mechanisms associated with tolerogenic cell death. In the tumor microenvironment, PS is exposed on tumor cells and tumor vascular endothelial cells treated with conventional therapies. PS signals through multiple immune cell signaling receptors where it drives the expansion of myeloid-derived suppressor cells (MDSCs), regulatory T cells, M2 macrophages, and stimulates their production of immunosuppressive cytokines (e.g., TGFβ and IL-10), rendering the tumor microenvironment non-responsive to immune activation. PS targeting antibodies have significant anti-tumor effects in multiple tumor xenografts, syngeneic tumors, and genetically engineered preclinical tumor models through mechanisms that re-program the tumor microenvironment from immunosuppressive to immune potentiating. Methods: The combination of PS-targeting and anti-PD1 antibodies was compared to single agent therapy in multiple pre-clinical mouse models, including syngeneic melanoma and breast tumors, and a genetically engineered mouse model (GEMM) of pancreatic cancer. Mice were treated weekly IP at 5 mg/kg with a PS targeting antibody, anti-PD1, or the combination for the duration of each experiment; tumor and spleen tissue microenvironments were immune profiled by FACS, ELISPOT, and immunohistochemistry. Results: In all tumor models examined, the anti-tumor effect of combination therapy was significantly superior to single agent therapy. In B16 and K1735 melanoma tumors, the combination more than doubled the anti-tumor effect of anti-PD-1 alone. Combination therapy significantly inhibited tumor growth by over 90% in E077.1 breast tumors and prolonged animal survival by 30% (n>25 each group) in the GEMM of pancreatic cancer despite the fact that anti-PD1 therapy alone was ineffective in each of these models. Analysis of immune cell subsets in the tumor microenvironment indicated that the combination of antibody-mediated blockade of PS and PD-1 significantly enhanced the effector function of tumor infiltrating CD8+ T cells and increased the ratio of T effector to T regulatory cells, as demonstrated by significant increases in TILs producing IFNγ, TNFα, IL-2, granzyme B, and Ki-67. Combination therapy re-conditioned the tumor microenvironment to favor immune potentiation, as demonstrated by significant decreases in the frequency of MDSC, the ratio of M2 to M1 macrophages, the expression of surface PD-L1, and reductions in TGFβ and IL-10. Furthermore, combination treatment induced systemic tumor specific CD8 T cell immunity, as mice demonstrating complete responses (in the K1735 and E077.1 models) rejected tumor cells upon re-challenge. Splenocytes from these mice had significantly higher numbers of tumor-specific IFNγ-producing cells in ELISPOT assay. Studies were completed without toxicity in any setting. Conclusions: Antibody-mediated PS blockade inhibits PS-mediated immune suppression and stimulates FcRγ activation. This results in immune activation that enables an otherwise non-responsive tumor microenvironment to respond to checkpoint inhibition. This abstract is also presented as Poster B08. Citation Format: Xianming Huang, Jian Gong, Michael Gray, Van Nguyen, Ryan Parks, Chris Hughes, Jeff Hutchins, Rolf Brekken, Bruce Freimark. Antibody-mediated blockade of phosphatidylserine synergizes with immune checkpoint blockade by inhibiting multiple immune suppressive mechanisms. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr PR09.

Inflammation in gastric cancer: Interplay of the COX-2/prostaglandin E2 and Toll-like receptor/MyD88 pathways.

Cyclooxygenase‐2 (COX‐2) and its downstream product prostaglandin E2 (PGE2) play a key role in generation of the inflammatory microenvironment in tumor tissues. Gastric cancer is closely associated with Helicobacter pylori infection, which stimulates innate immune responses through Toll‐like receptors (TLRs), inducing COX‐2/PGE2 pathway through nuclear factor‐κB activation. A pathway analysis of human gastric cancer shows that both the COX‐2 pathway and Wnt/β‐catenin signaling are significantly activated in tubular‐type gastric cancer, and basal levels of these pathways are also increased in other types of gastric cancer. Expression of interleukin‐11, chemokine (C‐X‐C motif) ligand 1 (CXCL1), CXCL2, and CXCL5, which play tumor‐promoting roles through a variety of mechanisms, is induced in a COX‐2/PGE2 pathway‐dependent manner in both human and mouse gastric tumors. Moreover, the COX‐2/PGE2 pathway plays an important role in the maintenance of stemness with expression of stem cell markers, including CD44, Prom1, and Sox9, which are induced in both gastritis and gastric tumors through a COX‐2/PGE2‐dependent mechanism. In contrast, disruption of Myd88 results in suppression of the inflammatory microenvironment in gastric tumors even when the COX‐2/PGE2 pathway is activated, indicating that the interplay of the COX‐2/PGE2 and TLR/MyD88 pathways is needed for inflammatory response in tumor tissues. Furthermore, TLR2/MyD88 signaling plays a role in maintenance of stemness in normal stem cells as well as gastric tumor cells. Accordingly, these results suggest that targeting the COX‐2/PGE2 pathway together with TLR/MyD88 signaling, which would suppress the inflammatory microenvironment and maintenance of stemness, could be an effective preventive or therapeutic strategy for gastric cancer.