Laser-Ignited Lipid Peroxidation Nanoamplifiers for Strengthening Tumor Photodynamic Therapy Through Aggravating Ferroptotic Propagation and Sustainable High Immunogenicity.

Photodynamic therapy (PDT) is extensively investigated for tumor therapy in the clinic. However, the efficacy of PDT is severely limited by the tissue penetrability of light, short effective half-life and radius of reactive oxygen species (ROS), and the weak immunostimulatory effect. In this study, a glutathione (GSH)-activatable nano-photosensitizer is developed to load with arachidonic acid (AA) and camouflage by erythrocyte membrane, which serves as a laser-ignited lipid peroxidation nanoamplifier (MAR). The photosensitive effect of MAR is recovered accompanied by the degradation in the tumor microenvironment and triggers the peroxidation of AA upon laser excitation. Interestingly, it aggravates the propagation of ferroptosis among cancer cells by driving the continuous lipid peroxidation chain reactions with the participation of the degradation products, ferrous ions (Fe2+ ), and AA. Consequently, even the deep-seated tumor cells without illumination also undergo ferroptosis owing to the propagation of ferroptotic signal. Moreover, the residual tumor cells undergoing ferroptosis still maintain high immunogenicity after PDT, thus continuously triggering sufficient tumor-associated antigens (TAAs) release to remarkably promote the anti-tumor immune response. Therefore, this study will provide a novel "all-in-one" nano-photosensitizer that not only amplifies the damaging effect and expands the effective range of PDT but also improves the immunostimulatory effect after PDT.

Nanoplatforms Potentiated Ablation-Immune Synergistic Therapy through Improving Local Control and Suppressing Recurrent Metastasis.

Minimally invasive ablation has been widely applied for treatment of various solid tumors, including hepatocellular carcinoma, renal cell carcinoma, breast carcinomas, etc. In addition to removing the primary tumor lesion, ablative techniques are also capable of improving the anti-tumor immune response by inducing immunogenic tumor cell death and modulating the tumor immune microenvironment, which may be of great benefit to inhibit the recurrent metastasis of residual tumor. However, the short-acting activated anti-tumor immunity of post-ablation will rapidly reverse into an immunosuppressive state, and the recurrent metastasis owing to incomplete ablation is closely associated with a dismal prognosis for the patients. In recent years, numerous nanoplatforms have been developed to improve the local ablative effect through enhancing the targeting delivery and combining it with chemotherapy. Particularly, amplifying the anti-tumor immune stimulus signal, modulating the immunosuppressive microenvironment, and improving the anti-tumor immune response with the versatile nanoplatforms have heralded great application prospects for improving the local control and preventing tumor recurrence and distant metastasis. This review discusses recent advances in nanoplatform-potentiated ablation-immune synergistic tumor therapy, focusing on common ablation techniques including radiofrequency, microwave, laser, and high-intensity focused ultrasound ablation, cryoablation, and magnetic hyperthermia ablation, etc. We discuss the advantages and challenges of the corresponding therapies and propose possible directions for future research, which is expected to provide references for improving the traditional ablation efficacy.

Thermoacoustic Imaging-Guided Thermo-Chemotherapy for Hepatocellular Carcinoma Sensitized by a Microwave-Responsive Nitric Oxide Nanogenerator.

Imaging-guided percutaneous microwave thermotherapy has been regarded as an important alternative nonsurgical therapeutic strategy for hepatocellular carcinoma (HCC) that provides excellent local tumor control and favorable survival benefit. However, providing a high-resolution, real-time, and noninvasive imaging technique for intraoperative guidance and controlling postoperative residual tumor recurrence are urgent needs for the clinical setting. In this study, a cisplatin (CDDP)-loaded nanocapsule (NPs@CDDP) with microwave responsive property was prepared to simultaneously serve as a contrast agent of emerging thermoacoustic imaging and a sensitizing agent of microwave thermo-chemotherapy. Accompanying the enzymolysis in the tumor microenvironment, the NPs@CDDP responsively release l-arginine (l-Arg) and CDDP. l-Arg with excellent microwave-absorbing property allowed it to serve as a thermoacoustic imaging contrast agent for accurately delineating the tumor and remarkably increasing tumor temperature under ultralow power microwave irradiation. Apart from the chemotherapeutic effect, CDDP elevated the intracellular H2O2 level through cascade reactions and further accelerated the continuous transformation of l-Arg to nitric oxide (NO), which endowed the NPs@CDDP with NO-generation capability. Notably, the high concentration of intracellular NO was proved to aggravate lipid peroxidation and greatly improved the efficacy of microwave thermo-chemotherapy. Thereby, NPs@CDDP was expected to serve as a theranostic agent integrating the functions of tumor microenvironment-responsive drug delivery system, contrast agent of thermoacoustic imaging, thermal sensitizing agent, and NO nanogenerator, which was promising to provide a potential imaging-guided therapeutic strategy for HCC.

Hydrogen sulfide activatable metal-organic frameworks for Fluorescence Imaging-Guided Photodynamic Therapy of colorectal cancer.

Photodynamic therapy (PDT) is a promising alternative and palliative therapeutic strategy for colorectal cancer (CRC). A novel photosensitizer with higher selectivity for CRC and fewer side effects is vital for clinical application. Given that the overexpression of hydrogen sulfide (H2S) in CRC, it is expected to provide a selective stimulus for activatable photosensitizers that in respond to the specific microenvironment. Herein, we report a novel development of metal-organic frameworks (MOFs) composed of meso-Tetra (4-carboxyphenyl) porphine (TCPP) and ferric ion (Fe3+) through a facile one-pot process. Experiments both in vitro and in vivo reveal that the MOF is capable of depredating in response to the high content of H2S in tumor microenvironment of CRC. Accompanying with the degradation and release of TCPP, the fluorescence and photosensitivity effect is switched from "off" to "on", enabling the MOF to serve as a H2S activatable nano-photosensitizer for real-time fluorescence imaging-guided and targeted PDT of CRC.

Multifunctional Nanosystems Powered Photodynamic Immunotherapy.

Photodynamic Therapy (PDT) with the intrinsic advantages including non-invasiveness, spatiotemporal selectivity, low side-effects, and immune activation ability has been clinically approved for the treatment of head and neck cancer, esophageal cancer, pancreatic cancer, prostate cancer, and esophageal squamous cell carcinoma. Nevertheless, the PDT is only a strategy for local control of primary tumor, that it is hard to remove the residual tumor cells and inhibit the tumor metastasis. Recently, various smart nanomedicine-based strategies are developed to overcome the barriers of traditional PDT including the drawbacks of traditional photosensitizers, limited tissue penetrability of light, inefficient induction of tumor cell death and tumor resistance to the therapy. More notably, a growing number of studies have focused on improving the therapeutic efficiency by eliciting host immune system with versatile nanoplatforms, which heralds a broader clinical application prospect of PDT in the future. Herein, the pathways of PDT induced-tumor destruction, especially the host immune response is summarized, and focusing on the recent progress of nanosystems-enhanced PDT through eliciting innate immunity and adaptive immunity. We expect it will provide some insights for conquering the drawbacks current PDT and expand the range of clinical application through this review.

Tumor Microenvironment-Responsive Nanodrug for Clear-Cell Renal Cell Carcinoma Therapy via Triggering Waterfall-Like Cascade Ferroptosis.

The most common type of kidney tumor, clear-cell renal cell carcinoma (ccRCC) with relatively insidious development and easily metastatic characteristics is generally insensitive to cytotoxic chemotherapy. The abundant polyunsaturated fatty acids (PUFAs) content in advanced ccRCC allows it to be intrinsically vulnerable to ferroptosis-based therapeutic strategies. Nevertheless, the strategy to cause the "iron overload" by administration with iron-based nanomaterials has limited therapeutic efficacy. And the classic ferroptosis agonist (RSL3) with low specificity for tumors, short half-life in the blood, poor water solubility and deficient accumulation at the tumor site prevents its reliable application in vivo. In this study, iron-based metal-organic framework nanoparticles (MIL-101(Fe) NPs) delivered RSL3 to ccRCC tumors, and then released the iron ions and RSL3 accompanied by the degradation of MIL-101(Fe) NPs in the acidic tumor microenvironment. The MIL-101(Fe)@RSL3 as a pH-responsive nanodrug causes cellular iron overload and promotes the hydroxyl radical (•OH) generation by Fenton reaction to attack PUFAs, leading to the aberrant accumulation of lipid peroxides (L-OOH). Additionally, RSL3 directly inhibits glutathione peroxidase 4 (GPX4) to detoxify L-OOH, and ferrous ions further catalyze the irreversible conversion of highly reactive lipid alkoxyl radicals (L-O•) from L-OOH to triggering waterfall-like cascade ferroptosis. In contrast to the limited antitumor efficiency of free RSL3, MIL-101(Fe)@RSL3 with high encapsulation efficiency (88.7%) shows a significant ccRCC-specific antitumor effect and negligible side effects. Taken together, MIL-101(Fe)@RSL3 could aggravate ferroptosis and be expected to be a promising nanodrug for ccRCC systemic therapy due to the targeted delivery and responsive release of RSL3 and iron ions.

Doxorubicin-Loaded Metal-Organic Framework Nanoparticles as Acid-Activatable Hydroxyl Radical Nanogenerators for Enhanced Chemo/Chemodynamic Synergistic Therapy.

Doxorubicin (DOX) is a widely used first-line antitumor agent; however, acquired drug resistance and side effects have become the main challenges to effective cancer therapy. Herein, DOX is loaded into iron-rich metal-organic framework/tannic acid (TA) nanocomplex to form a tumor-targeting and acid-activatable drug delivery system (MOF/TA-DOX, MTD). Under the acidic tumor microenvironment, MTD simultaneously releases DOX and ferrous ion (Fe2+) accompanied by degradation. Apart from the chemotherapeutic effect, DOX elevates the intracellular H2O2 levels through cascade reactions, which will be beneficial to the Fenton reaction between the Fe2+ and H2O2, to persistently produce hydroxyl radicals (•OH). Thus, MTD efficiently mediates chemodynamic therapy (CDT) and remarkably enhances the sensitivity of chemotherapy. More encouragingly, the cancer cell killing efficiency of MTD is up to ~86% even at the ultralow equivalent concentration of DOX (2.26 µg/mL), while the viability of normal cells remained >88% at the same concentration of MTD. Taken together, MTD is expected to serve as drug-delivery nanoplatforms and •OH nanogenerators for improving chemo/chemodynamic synergistic therapy and reducing the toxic side effects.

Roles of the ERK1/2 and PI3K/PKB signaling pathways in regulating the expression of extracellular matrix genes in rat pulmonary artery smooth muscle cells.

PURPOSE:: To investigate the mechanisms by which PD98059 and LY294002 interfere with the abnormal deposition of extracellular matrix regulated by connective tissue growth factor (CTGF) of rat pulmonary artery smooth muscle cells (PASMCs). METHODS:: Rat PASMCs were cultured and separated into a control group. Real-time fluorescence quantitative PCR was performed to detect the expression of collagen III and fibronectin mRNA. Immunohistochemistry and western blot analyses were performed to detect the expression of collagen III protein. RESULTS:: The expression of collagen III and fibronectin mRNA was greater in PASMCs stimulated with CTGF for 48 h, than in the control group. After 72h of stimulation, the expression of collagen III protein in the PASMCs was greater than in the control. The equivalent gene and protein expression of the CPL group were much more significant. CONCLUSIONS:: CTGF can stimulate the gene expression of collagen III and fibronectin in PASMCs, which may be one of the factors that promote pulmonary vascular remodeling (PVR) under the conditions of pulmonary arterial hypertension (PAH). PD98059 and LY294002 can inhibit the ERK1/2 and PI3K/PKB signaling pathways, respectively, thus interfering with the biological effects of CTGF. This may be a new way to reduce PAH-PVR.

Roles of the ERK1/2 and PI3K/PKB signaling pathways in regulating the expression of extracellular matrix genes in rat pulmonary artery smooth muscle cells

Abstract Purpose: To investigate the mechanisms by which PD98059 and LY294002 interfere with the abnormal deposition of extracellular matrix regulated by connective tissue growth factor (CTGF) of rat pulmonary artery smooth muscle cells (PASMCs). Methods: Rat PASMCs were cultured and separated into a control group. Real-time fluorescence quantitative PCR was performed to detect the expression of collagen III and fibronectin mRNA. Immunohistochemistry and western blot analyses were performed to detect the expression of collagen III protein. Results: The expression of collagen III and fibronectin mRNA was greater in PASMCs stimulated with CTGF for 48 h, than in the control group. After 72h of stimulation, the expression of collagen III protein in the PASMCs was greater than in the control. The equivalent gene and protein expression of the CPL group were much more significant. Conclusions: CTGF can stimulate the gene expression of collagen III and fibronectin in PASMCs, which may be one of the factors that promote pulmonary vascular remodeling (PVR) under the conditions of pulmonary arterial hypertension (PAH). PD98059 and LY294002 can inhibit the ERK1/2 and PI3K/PKB signaling pathways, respectively, thus interfering with the biological effects of CTGF. This may be a new way to reduce PAH-PVR.

DNA Duplex-Based Photodynamic Molecular Beacon for Targeted Killing of Retinoblastoma Cell.

PURPOSES: Retinoblastoma (RB) is the most common primary intraocular malignancy of infancy. An alternative RB treatment protocol is proposed and tested. It is based on a photodynamic therapy (PDT) with a designed molecular beacon that specifically targets the murine double minute x (MDMX) high-expressed RB cells. METHODS: A MDMX mRNA triggered photodynamic molecular beacon is designed by binding a photosensitizer molecule (pyropheophorbide-a, or PPa) and a black hole quencher-3 (BHQ3) through a complementary oligonucleotide sequence. Cells with and without MDMX high-expression are incubated with the beacon and then irradiated with a laser. The fluorescence and reactive oxygen species are detected in solution to verify the specific activation of PPa by the perfectly matched DNA targets. The cell viabilities are evaluated with CCK-8 and flow cytometry assay. RESULTS: The fluorescence and photo-cytoxicity of PPa is recovered and significantly higher in the MDMX high-expressed Y79 and WERI-Rb1 cells, compared to that with the MDMX low-expressed cells. CONCLUSIONS: The synthesized beacon exhibits high PDT efficiency toward MDMX high-expressed RB cells. The data suggest that the designed beacon may provide a potential alternative for RB therapy and secures the ground for future investigation.

Photo-enhancement of macrophage phagocytic activity via Rac1-mediated signaling pathway: Implications for bacterial infection.

Phagocytosis and the subsequent destruction of invading pathogens by macrophages are indispensable steps in host immune responses to microbial infections. Low-power laser irradiation (LPLI) has been found to exert photobiological effects on immune responses, but the signaling mechanisms underlying this photobiomodulation of phagocytosis remains largely unknown. Here, we demonstrated for the first time that LPLI enhanced the phagocytic activity of macrophages by stimulating the activation of Rac1. The overexpression of constitutively activated Rac1 clearly enhanced LPLI-induced phagocytosis, whereas the overexpression of dominant negative Rac1 exerted the opposite effect. The phosphorylation of cofilin was involved in the effects of LPLI on phagocytosis, which was regulated by the membrane translocation and activation of Rac1. Furthermore, the photoactivation of Rac1 was dependent on the Src/PI3K/Vav1 pathway. The inhibition of the Src/PI3K pathway significantly suppressed LPLI-induced actin polymerization and phagocytosis enhancement. Additionally, LPLI-treated mice exhibited increased survival and a decreased organ bacterial load when challenged with Listeria monocytogenes, indicating that LPLI enhanced macrophage phagocytosis in vivo. These findings highlight the important roles of the Src/PI3K/Vav1/Rac1/cofilin pathway in regulating macrophage phagocytosis and provide a potential strategy for treating phagocytic deficiency via LPLI.

Phototherapy-Induced Antitumor Immunity: Long-Term Tumor Suppression Effects via Photoinactivation of Respiratory Chain Oxidase-Triggered Superoxide Anion Burst.

AIMS: Our previous studies have demonstrated that as a mitochondria-targeting cancer phototherapy, high-fluence, low-power laser irradiation (HF-LPLI) results in oxidative damage that induces tumor cell apoptosis. In this study, we focused on the immunological effects of HF-LPLI phototherapy and explored its antitumor immune regulatory mechanism. RESULTS: We found not only that HF-LPLI treatment induced tumor cell apoptosis but also that HF-LPLI-treated apoptotic tumor cells activated macrophages. Due to mitochondrial superoxide anion burst after HF-LPLI treatment, tumor cells displayed a high level of phosphatidylserine oxidation, which mediated the recognition and uptake by macrophages with the subsequent secretion of cytokines and generation of cytotoxic T lymphocytes. In addition, in vivo results showed that HF-LPLI treatment caused leukocyte infiltration into the tumor and efficaciously inhibited tumor growth in an EMT6 tumor model. These phenomena were absent in the respiration-deficient EMT6 tumor model, implying that the HF-LPLI-elicited immunological effects were dependent on the mitochondrial superoxide anion burst. INNOVATION: In this study, for the first time, we show that HF-LPLI mediates tumor-killing effects via targeting photoinactivation of respiratory chain oxidase to trigger a superoxide anion burst, leading to a high level of oxidatively modified moieties, which contributes to the phenotypic changes in macrophages and mediates the antitumor immune response. CONCLUSION: Our results suggest that HF-LPLI may be an effective cancer treatment modality that both eradicates the treated primary tumors and induces an antitumor immune response via photoinactivation of respiratory chain oxidase to trigger superoxide anion burst.

[Integrin ß3 pathway mediated connective tissue growth factor-induced proliferation, migration and extracellular matrix deposition of pulmonary arterial smooth muscle cells].

OBJECTIVE: To explore the effects of integrin ß3 pathway on the proliferation, migration and extracellular matrix deposition of pulmonary arterial smooth muscle cells (PASMCs) induced by connective tissue growth factor (CTGF). METHODS: PASMCs of SD Rats were cultured in M199 culture system in vitro and the 3rd-7th passages of PASMCs were used in the experiments. The cells were randomly divided into three groups: (1) CONTROL GROUP: culture system contained no any stimulation factor; (2) CTGF group: culture system was added into 50 ng/ml CTGF; (3) CTGF+ anti-integrin ß3 antibody group:culture system was added with 50 ng/ml CTGF and 10 mg/L anti-integrin ß3 antibody. The PASMCs were cultured with 50 ng/ml CTGF and anti-integrin ß3 antibody (0, 5, 10, 15, 20 mg/L) for 24, 48, 72 and 96 h, the proliferation of PASMCs was detected by WST-1 Cell Proliferation Assay Kit. The migration of PASMCs was observed by Transwell cell test under the phase contrast microscope. RT-PCR assay was applied to detect the mRNA expression of collagenI-α1, collagen III-α1 and fibronectin-1 gene of PASMCs. The expression of fibronectin protein was examined by Western blotting and immunohistochemistry. RESULTS: The results of WST-1 test showed that the anti-integrin ß3 antibody inhibited significantly the proliferation of PASMCs induced by CTGF (P < 0.05), among which the inhibition rate of anti-integrin ß3 antibody (10 mg/L) was the most significant. Transwell test results showed that CTGF group of PASMCs migration numbers (25 ± 1.57) were higher than that of the control group (11 ± 2.08, P < 0.01); PASMCs migration numbers of CTGF+ integrin ß3 antibody group (17 ± 4.16) were less than that of the CTGF group (P < 0.05). Compared with the control group, the mRNA expression of collagen typeI-α1 (4.28 ± 0.33), collagen typeIII-α1 (4.41 ± 0.35), fibronectin-1 (4.05 ± 0.33) of PASMCs was increased in CTGF group, with a time-dependence (P < 0.01); Compared with the CTGF group, the mRNA expression of collagen typeI-α1 (3.38 ± 0.30), collagen typeIII-α1 (3.40 ± 0.30), fibronectin-1 (3.12 ± 0.29) of PASMCs was reduced in CTGF+ anti-integrin ß3 antibody group (P < 0.05), which was higher than that of the control group (P < 0.05); Western blot and immunohistochemical tests showed that compared with the control group, CTGF group could stimulate the expression of fibronectin protein of PASMCs (P < 0.01); the anti-integrin ß3 antibody could inhibit the expression of fibronectin protein induced by CTGF(P < 0.01), which was more remarkable than that in the control group (P < 0.01). CONCLUSION: Integrin ß3 pathway can mediate CTGF-induced proliferation, migration and extracellular matrix deposition of PASMCs, CTGF-integrin ß3 signaling pathway may play an important role in pulmonary vascular remodeling.

Loss expression of active fragile sites genes associated with the severity of breast epithelial abnormalities.

BACKGROUND: WWOX and FHIT are two candidate tumor suppressor genes located in active fragile sites, the damage of which has been associated with the development of breast cancer. The association of the expression of these genes and the development of breast cancer has not been fully explored. We evaluated mRNA and protein expression of WWOX and FHIT in breast tissue with normal histological appearances, atypical ductal hyperplasia, ductal carcinoma in situ, and invasive cancer to see if a progressive decline in expression was present. METHODS: Reverse transcription-polymerase chain reaction and Western blotting were used to evaluate the specimens for mRNA and protein expression, including 28 specimens with normal tissue, 28 specimens with atypical ductal hyperplasia, 33 specimens with ductal carcinoma in situ, and 51 specimens with invasive ductal carcinoma. RESULTS: Compared with in situ and invasive cancer specimens, both normal and atypical hyperplasia specimens had greater rates of detectable mRNA (WWOX rate ratio = 2.95, 95% CI 1.24 - 7.08; FHIT rate ratio = 4.58, 95% CI 1.82 - 11.81) and Western blotting detectable protein (WWOX rate ratio = 4.12, 95% CI 1.63 - 10.73; FHIT rate ratio = 3.76, 95% CI 1.44 - 10.06). For both proteins, differences between normal and atypical hyperplasia specimens and between in situ and invasive carcinoma specimens were explainable by chance (P > 0.05 for each analysis). Within each histological category, differences among fractions of specimens showed that FHIT and WWOX mRNA and protein expression were explainable by chance (P > 0.05 for each analysis). CONCLUSION: Expression of FHIT and WWOX decreases along with breast tissue progress from a normal histological appearance to atypical ductal hyperplasia, in situ cancer, and the final invasive cancer.