Accurately Controlled Tumor Temperature with Silica-Coated Gold Nanorods for Optimal Immune Checkpoint Blockade Therapy.

Photothermal therapy (PTT) at mild temperatures ranging from 44 to 45 °C holds tremendous promise as a strategy for inducing potent immunogenic cell death (ICD) within tumor tissues, which can reverse the immunosuppressive tumor microenvironment (ITM) into an immune-responsive milieu. However, accurately and precisely controlling the tumor temperature remains a formidable challenge. Here, we report the precision photothermal immunotherapy by using silica-coated gold nanorods (AuNR@SiO2), and investigating the optimal administration routes and treatment protocols, which enabled to achieve the sustained and controlled mild heating within the tumor tissues. First, the highest photothermal performance of AuNR@SiO2 with 20-nm silica shell thickness than 5 or 40 nm was confirmed in vitro and in vivo. Then, the optimal conditions for precision immunotherapy were further investigated to produce mild temperature (44 to 45 °C) accurately in tumor tissues. The optimal conditions with AuNR@SiO2 result in a distinct cell death with high early/late apoptosis and low necrosis, leading to very efficient ICD compared to lower or higher temperatures. In colon tumor-bearing mice, intratumorally injected AuNR@SiO2 efficiently promotes a mild temperature within the tumor tissues by local irradiation of near-infrared (NIR) laser. This mild PTT substantially increases the population of mature dendritic cells (DCs) and cytotoxic T cells (CTLs) within tumor tissues, ultimately reversing the ITM into an immune-responsive milieu. Furthermore, we found that the combination mild PTT with AuNR@SiO2 and anti-PD-L1 therapy could lead to the 100% complete regression of primary tumors and immunological memory to prevent tumor recurrence. Collectively, this study demonstrates that AuNR@SiO2 with a robust methodology capable of continuously inducing mild temperature accurately within the ITM holds promise as an approach to achieve the precision photothermal immunotherapy.

Cancer cell-specific and pro-apoptotic SMAC peptide-doxorubicin conjugated prodrug encapsulated aposomes for synergistic cancer immunotherapy.

BACKGROUND: Immunogenic cell death (ICD) is a crucial approach to turn immunosuppressive tumor microenvironment (ITM) into immune-responsive milieu and improve the response rate of immune checkpoint blockade (ICB) therapy. However, cancer cells show resistance to ICD-inducing chemotherapeutic drugs, and non-specific toxicity of those drugs against immune cells reduce the immunotherapy efficiency. METHODS: Herein, we propose cancer cell-specific and pro-apoptotic liposomes (Aposomes) encapsulating second mitochondria-derived activator of caspases mimetic peptide (SMAC-P)-doxorubicin (DOX) conjugated prodrug to potentiate combinational ICB therapy with ICD. The SMAC-P (AVPIAQ) with cathepsin B-cleavable peptide (FRRG) was directly conjugated to DOX, and the resulting SMAC-P-FRRG-DOX prodrug was encapsulated into PEGylated liposomes. RESULTS: The SMAC-P-FRRG-DOX encapsulated PEGylated liposomes (Aposomes) form a stable nanostructure with an average diameter of 109.1 ± 5.14 nm and promote the apoptotic cell death mainly in cathepsin B-overexpressed cancer cells. Therefore, Aposomes induce a potent ICD in targeted cancer cells in synergy of SMAC-P with DOX in cultured cells. In colon tumor models, Aposomes efficiently accumulate in targeted tumor tissues via enhanced permeability and retention (EPR) effect and release the encapsulated prodrug of SMAC-P-FRRG-DOX, which is subsequently cleaved to SMAC-P and DOX in cancer cells. Importantly, the synergistic activity of inhibitors of apoptosis proteins (IAPs)-inhibitory SMAC-P sensitizing the effects of DOX induces a potent ICD in the cancer cells to promote dendritic cell (DC) maturation and stimulate T cell proliferation and activation, turning ITM into immune-responsive milieu. CONCLUSIONS: Eventually, the combination of Aposomes with anti-PD-L1 antibody results in a high rate of complete tumor regression (CR: 80%) and also prevent the tumor recurrence by immunological memory established during treatments.

Micro-syringe chip-guided intratumoral administration of lipid nanoparticles for targeted anticancer therapy.

BACKGROUND: Nano-sized drug delivery system has been widely studied as a potential technique to promote tumor-specific delivery of anticancer drugs due to its passive targeting property, but resulting in very restricted improvements in its systemic administration so far. There is a requirement for a different approach that dramatically increases the targeting efficiency of therapeutic agents at targeted tumor tissues. METHODS: To improve the tumor-specific accumulation of anticancer drugs and minimize their undesirable toxicity to normal tissues, a tumor-implantable micro-syringe chip (MSC) with a drug reservoir is fabricated. As a clinically established delivery system, six liposome nanoparticles (LNPs) with different compositions and surface chemistry are prepared and their physicochemical properties and cellular uptake are examined in vitro. Subsequently, MSC-guided intratumoral administration is studied to identify the most appropriate for the higher tumor targeting efficacy with a uniform intratumoral distribution. For efficient cancer treatment, pro-apoptotic anticancer prodrugs (SMAC-P-FRRG-DOX) are encapsulated to the optimal LNPs (SMAC-P-FRRG-DOX encapsulating LNPs; ApoLNPs), then the ApoLNPs are loaded into the 1 µL-volume drug reservoir of MSC to be delivered intratumorally for 9 h. The tumor accumulation and therapeutic effect of ApoLNPs administered via MSC guidance are evaluated and compared to those of intravenous and intratumoral administration of ApoLNP in 4T1 tumor-bearing mice. RESULTS: MSC is precisely fabricated to have a 0.5 × 4.5 mm needle and 1 µL-volume drug reservoir to achieve the uniform intratumoral distribution of LNPs in targeted tumor tissues. Six liposome nanoparticles with different compositions of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (PC), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (PS), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)2000] (PEG2000-DSPE) are prepared with average sizes of 100-120 nm and loaded into the 1 µL-volume drug reservoir in MSC. Importantly negatively charged 10 mol% of PS-containing LNPs are very slowly infused into the tumor tissue through the micro-syringe of the MSC over 6 h. The intratumoral targeting efficiency of MSC guidance is 93.5%, effectively assisting the homogeneous diffusion of LNPs throughout the tumor tissue at 3.8- and 2.7-fold higher concentrations compared to the intravenous and intratumoral administrations of LNPs, respectively. Among the six LNP candidates 10 mol% of PS-containing LNPs are finally selected for preparing pro-apoptotic SMAC-P-FRRG-DOX anticancer prodrug-encapsulated LNPs (ApoLNPs) due to their moderate endocytosis rate high tumor accumulation and homogenous intratumoral distribution. The ApoLNPs show a high therapeutic effect specifically to cathepsin B-overexpressing cancer cells with 6.6 µM of IC50 value while its IC50 against normal cells is 230.7 µM. The MSC-guided administration of ApoLNPs efficiently inhibits tumor growth wherein the size of the tumor is 4.7- and 2.2-fold smaller than those treated with saline and intratumoral ApoLNP without MSC, respectively. Moreover, the ApoLNPs remarkably reduce the inhibitor of apoptosis proteins (IAPs) level in tumor tissues confirming their efficacy even in cancers with high drug resistance. CONCLUSION: The MSC-guided administration of LNPs greatly enhances the therapeutic efficiency of anticancer drugs via the slow diffusion mechanism through micro-syringe to tumor tissues for 6 h, whereas they bypass most hurdles of systemic delivery including hepatic metabolism, rapid renal clearance, and interaction with blood components or other normal tissues, resulting in the minimum toxicity to normal tissues. The negatively charged ApoLNPs with cancer cell-specific pro-apoptotic prodrug (SMAC-P-FRRG-DOX) show the highest tumor-targeting efficacy when they are treated with the MSC guidance, compared to their intravenous or intratumoral administration in 4T1 tumor-bearing mice. The MSC-guided administration of anticancer drug-encapsulated LNPs is expected to be a potent platform system that facilitates overcoming the limitations of systemic drug administration with low delivery efficiency and serious side effects.

Recent Studies and Progress in the Intratumoral Administration of Nano-Sized Drug Delivery Systems.

Over the last 30 years, diverse types of nano-sized drug delivery systems (nanoDDSs) have been intensively explored for cancer therapy, exploiting their passive tumor targetability with an enhanced permeability and retention effect. However, their systemic administration has aroused some unavoidable complications, including insufficient tumor-targeting efficiency, side effects due to their undesirable biodistribution, and carrier-associated toxicity. In this review, the recent studies and advancements in intratumoral nanoDDS administration are generally summarized. After identifying the factors to be considered to enhance the therapeutic efficacy of intratumoral nanoDDS administration, the experimental results on the application of intratumoral nanoDDS administration to various types of cancer therapies are discussed. Subsequently, the reports on clinical studies of intratumoral nanoDDS administration are addressed in short. Intratumoral nanoDDS administration is proven with its versatility to enhance the tumor-specific accumulation and retention of therapeutic agents for various therapeutic modalities. Specifically, it can improve the efficacy of therapeutic agents with poor bioavailability by increasing their intratumoral concentration, while minimizing the side effect of highly toxic agents by restricting their delivery to normal tissues. Intratumoral administration of nanoDDS is considered to expand its application area due to its potent ability to improve therapeutic effects and relieve the systemic toxicities of nanoDDSs.

Prediction of IGS RTS Orbit Correction Using LSTM Network at the Time of IOD Change.

The international GNSS service (IGS) real-time service (RTS) provides orbit and clock corrections for the global navigation satellite system (GNSS) via the internet. RTS is widely used for real-time, precise positioning and its data is transmitted via the internet. Intermittent data loss can occur and cause positioning accuracy degradation. RTS data has a discontinuity when the issue of data (IOD) changes every two hours. If the signal loss occurs immediately after the IOD change, then the performance of the RTS prediction degrades significantly. We propose an adjustment method to make the RTS data across the IOD change, which makes it possible to use long RTS data for building a prediction model. The proposed adjustment method is combined with a long-short-term memory (LSTM) network to improve long-period prediction accuracy. Experiments with GPS and RTS were performed to evaluate the RTS orbit prediction accuracy. The LSTM with the IOD adjustment outperforms other polynomial prediction methods, and the positioning accuracy with the predicted RTS orbit correction shows a significant improvement.

Implantable micro-scale LED device guided photodynamic therapy to potentiate antitumor immunity with mild visible light.

BACKGROUND: Photodynamic therapy (PDT) is a promising strategy to promote antitumor immunity by inducing immunogenic cell death (ICD) in tumor cells. However, practical PDT uses an intense visible light owing to the shallow penetration depth of the light, resulting in immunosuppression at the tumor tissues. METHODS: Herein, we propose an implantable micro-scale light-emitting diode device (micro-LED) guided PDT that enables the on-demand light activation of photosensitizers deep in the body to potentiate antitumor immunity with mild visible light. RESULTS: The micro-LED is prepared by stacking one to four micro-scale LEDs (100 µm) on a needle-shape photonic device, which can be directly implanted into the core part of the tumor tissue. The photonic device with four LEDs efficiently elicits sufficient light output powers without thermal degradation and promotes reactive oxygen species (ROS) from a photosensitizer (verteporfin; VPF). After the intravenous injection of VPF in colon tumor-bearing mice, the tumor tissues are irradiated with optimal light intensity using an implanted micro-LED. While tumor tissues under intense visible light causes immunosuppression by severe inflammatory responses and regulatory T cell activation, mild visible light elicits potent ICD in tumor cells, which promotes dendritic cell (DC) maturation and T cell activation. The enhanced therapeutic efficacy and antitumor immunity by micro-LED guided PDT with mild visible light are assessed in colon tumor models. Finally, micro-LED guided PDT in combination with immune checkpoint blockade leads to 100% complete tumor regression and also establishes systemic immunological memory to prevent the recurrence of tumors. CONCLUSION: Collectively, this study demonstrates that micro-LED guided PDT with mild visible light is a promising strategy for cancer immunotherapy.

Tumor-Specific Monomethyl Auristatin E (MMAE) Prodrug Nanoparticles for Safe and Effective Chemotherapy.

A prodrug is bioreversible medication that is specifically converted to the active drugs by enzymes overexpressed in the tumor microenvironment, which can considerably reduce the chemotherapy-induced side effects. However, prodrug strategies usually have low antitumor efficacy compared to free drugs by delayed drug release. This is because they need time to be activated by enzymatic cleavage and they also cannot be fully recovered to the active drugs. Therefore, highly potent anticancer drug should be considered to expect a sufficient antitumor efficacy. Herein, we propose tumor-specific monomethyl auristatin E (MMAE) prodrug nanoparticles for safe and effective chemotherapy. The cathepsin B-specific cleavable FRRG peptide and MMAE are chemically conjugated via one-step simple synthetic chemistry. The resulting FRRG-MMAE molecules form stable nanoparticles without any additional carrier materials by hydrophobic interaction-derived aggregations. The FRRG-MMAE nanoparticles efficiently accumulate within the tumor tissues owing to the enhanced permeability and retention (EPR) effect and inhibit the tubulin polymerization by releasing free MMAE in the cathepsin B-overexpressed tumor cells. In contrast, FRRG-MMAE nanoparticles maintain a non-toxic inactive state in the normal tissues owing to innately low cathepsin B expression, thereby reducing MMAE-related severe toxicity. Collectively, this study provides a promising approach for safe and effective chemotherapy via MMAE-based prodrug nanoparticles, which may open new avenues for advanced drug design for translational nanomedicine.

Comparative study of cathepsin B-cleavable linkers for the optimal design of cathepsin B-specific doxorubicin prodrug nanoparticles for targeted cancer therapy.

A carrier-free prodrug nanoparticle has emerged as a potential approach to cancer therapy. It plays a vital role in enhancing the tumor targeting and therapeutic efficacy of the anticancer agent at sites of intention wherein the prodrug nanoparticle is potentially activated. Herein, five derivatives of cathepsin B-cleavable prodrugs are synthesized via chemically conjugating different cathepsin B-cleavable peptides (Phe-Arg-Arg-Gly, Phe-Arg-Arg-Leu, Phe-Arg-Arg-Leu-Gly, Phe-Leu-Arg-Arg-Gly) to doxorubicin (DOX). The peptide-DOX prodrugs can spontaneously assemble into nanoparticles via their intermolecular hydrophobic and π-π stacking interactions. The resulting cathepsin B-cleavable prodrugs nanoparticles formed different nanoparticle structures according to the amphiphilicity and flexibility of different peptides and their particle stability and cellular uptake mechanism are carefully evaluated in vitro. Among five prodrug nanoparticles, the Phe-Arg-Arg-Leu-DOX (FRRL-DOX) nanoparticle was formed to a size of 167.5 ± 12.4 nm and stably maintains its nanoparticle structure in saline media for 3 days. The FRRL-DOX nanoparticle is well taken up by tumoral nuclei and effectively induces cancer cell death with minimal toxicity to normal cells. In addition, the FRRL-DOX nanoparticle shows 2.3-16.3-fold greater tumor-specific accumulation in vivo than other prodrug nanoparticles and free DOX. The therapeutic effect of FRRL-DOX is finally examined, demonstrating 2.1-fold better anticancer efficacy compared to that of free DOX. Notably, the FRRL-DOX nanoparticle does not exert serious toxicity in its repeated intravenous administration at a high dose of up to 10 mg/kg (equiv. to DOX). In conclusion, the peptide sequence for cathepsin B-cleavable prodrug nanoparticle is determined to be successfully optimized in a way of increasing its tumor selectivity and lowering toxicity to normal tissues.

Injectable Hydrogel-Based Combination Cancer Immunotherapy for Overcoming Localized Therapeutic Efficacy.

Various immunotherapeutic agents that can elicit antitumor immune responses have recently been developed with the potential for improved efficacy in treating cancer. However, insufficient delivery efficiency at the tumor site, along with severe side effects after systemic administration of these anticancer agents, have hindered their therapeutic application in cancer immunotherapy. Hydrogels that can be directly injected into tumor sites have been developed to help modulate or elicit antitumor responses. Based on the biocompatibility, degradability, and controllable mechanochemical properties of these injectable hydrogels, various types of immunotherapeutic agents, such as hydrophobic anticancer drugs, cytokines, antigens, and adjuvants, have been easily and effectively encapsulated, resulting in the successful elicitation of antitumor immune responses and the retention of long-term immunotherapeutic efficacy following administration. This review summarizes recent advances in combination immunotherapy involving injectable hydrogel-based chemoimmunotherapy, photoimmunotherapy, and radioimmunotherapy. Finally, we briefly discuss the current limitations and future perspectives on injectable hydrogels for the effective combination immunotherapy of tumors.

Sustained and Long-Term Release of Doxorubicin from PLGA Nanoparticles for Eliciting Anti-Tumor Immune Responses.

Immunogenic cell death (ICD) is a powerful trigger eliciting strong immune responses against tumors. However, traditional chemoimmunotherapy (CIT) does not last long enough to induce sufficient ICD, and also does not guarantee the safety of chemotherapeutics. To overcome the disadvantages of the conventional approach, we used doxorubicin (DOX) as an ICD inducer, and poly(lactic-co-glycolic acid) (PLGA)-based nanomedicine platform for controlled release of DOX. The diameter of 138.7 nm of DOX-loaded PLGA nanoparticles (DP-NPs) were stable for 14 days in phosphate-buffered saline (PBS, pH 7.4) at 37 °C. Furthermore, DOX was continuously released for 14 days, successfully inducing ICD and reducing cell viability in vitro. Directly injected DP-NPs enabled the remaining of DOX in the tumor site for 14 days. In addition, repeated local treatment of DP-NPs actually lasted long enough to maintain the enhanced antitumor immunity, leading to increased tumor growth inhibition with minimal toxicities. Notably, DP-NPs treated tumor tissues showed significantly increased maturated dendritic cells (DCs) and cytotoxic T lymphocytes (CTLs) population, showing enhanced antitumor immune responses. Finally, the therapeutic efficacy of DP-NPs was maximized in combination with an anti-programmed death-ligand 1 (PD-L1) antibody (Ab). Therefore, we expect therapeutic efficacies of cancer CIT can be maximized by the combination of DP-NPs with immune checkpoint blockade (ICB) by achieving proper therapeutic window and continuously inducing ICD, with minimal toxicities.

The safe and effective intraperitoneal chemotherapy with cathepsin B-specific doxorubicin prodrug nanoparticles in ovarian cancer with peritoneal carcinomatosis.

Intraperitoneal (IP) chemotherapy has shown promising efficacy in ovarian cancer with peritoneal carcinomatosis (PC), but in vivo rapid clearance and severe toxicity of free anticancer drugs hinder the effective treatment. Herein, we propose the safe and effective IP chemotherapy with cathepsin B-specific doxorubicin prodrug nanoparticles (PNPs) in ovarian cancer with PC. The PNPs are prepared by self-assembling cathepsin B-specific cleavable peptide (FRRG) and doxorubicin (DOX) conjugates, which are further formulated with pluronic F68. The PNPs exhibit stable spherical structure and cytotoxic DOX is specifically released from PNPs via sequential enzymatic degradation by cathepsin B and intracellular proteases. The PNPs induce cytotoxicity in cathepsin B-overexpressing ovarian (SKOV-3 and HeyA8) and colon (MC38 and CT26) cancer cells, but not in cathepsin B-deficient normal cells in cultured condition. With enhanced cancer-specificity and in vivo residence time, IP injected PNPs efficiently accumulate within PC through two targeting mechanisms of direct penetration (circulation independent) and systemic blood vessel-associated accumulation (circulation dependent). As a result, IP chemotherapy with PNPs efficiently inhibit tumor progression with minimal side effects in peritoneal human ovarian tumor-bearing xenograft (POX) and patient derived xenograft (PDX) models. These results demonstrate that PNPs effectively inhibit progression of ovarian cancer with peritoneal carcinomatosis with minimal local and systemic toxicities by high cancer-specificity and favorable in vivo PK/PD profiles enhancing PC accumulation.

Deep Learning-Based Prediction of Material Properties Using Chemical Compositions and Diffraction Patterns as Experimentally Accessible Inputs.

We report a deep learning (DL) model that predicts various material properties while accepting directly accessible inputs from routine experimental platforms: chemical compositions and diffraction data, which can be obtained from the X-ray or electron-beam diffraction and energy-dispersive spectroscopy, respectively. These heterogeneous forms of inputs are treated simultaneously in our DL model, where the novel chemical composition vector is proposed by developing element embedding with the normalized composition matrix. With 1524 binary samples available in the Materials Project database, the model predicts formation energies and band gaps with mean absolute errors of 0.29 eV/atom and 0.66 eV, respectively. According to the weighing test between these two inputs, the properties tend to be more influenced by the chemical composition than the crystal structure. This work intentionally avoids using inputs that are not directly accessible (e.g., atomic coordinates) in experimental platforms, and thus is expected to substantially improve the practical use of DL models.

Visible-Light-Triggered Prodrug Nanoparticles Combine Chemotherapy and Photodynamic Therapy to Potentiate Checkpoint Blockade Cancer Immunotherapy.

Immune checkpoint blockade is a promising approach for cancer immunotherapy, but many patients do not respond due to the immunosuppressive tumor microenvironment (ITM). Herein, we propose visible-light-triggered prodrug nanoparticles (LT-NPs) for reversing ITM into high immunogenic tumors to potentiate checkpoint blockade immunotherapy. The photosensitizer (verteporfin; VPF), cathepin B-specific cleavable peptide (FRRG), and doxorubicin (DOX) conjugates are self-assembled into LT-NPs without any additional carrier material. The LT-NPs are specifically cleaved to VPF and DOX in cathepsin B-overexpressing cancer cells, thereby inducing cancer-specific cytotoxicity and immunogenic cell death (ICD) upon visible light irradiation. In tumor models, LT-NPs highly accumulate within tumors via the enhanced permeability and retention effect, and photochemotherapy of VPF and DOX induces effective ICD and maturation of dendritic cells to stimulate cross-presentation of cancer-antigens to T cells. Furthermore, LT-NPs with PD-L1 blockade greatly inhibit tumor growth, tumor recurrence, and lung metastasis by initiating a strong antitumor immune response. The photochemotherapy by LT-NPs provides a promising strategy for effective checkpoint blockade immunotherapy.

Cancer-activated doxorubicin prodrug nanoparticles induce preferential immune response with minimal doxorubicin-related toxicity.

The effective chemotherapeutic drug, doxorubicin (DOX), elicits immunogenic cell death (ICD) and additional anticancer immune responses during chemotherapy. However, it also induces severe side effects and systemic immunosuppression, hampering its wide clinical application. Herein, we constructed cancer-activated DOX prodrug by conjugating the cathepsin B-cleavable peptide (Phe-Arg-Arg-Gly, FRRG) to a doxorubicin (DOX), resulting in FRRG-DOX that self-assembled into cancer-activated DOX prodrug nanoparticles (CAP-NPs). The resulting CAP-NPs were further stabilized with the FDA-approved compound, Pluronic F68. CAP-NPs formed stable prodrug nanoparticles and they were specifically cleaved to cytotoxic DOX molecules only in cathepsin B-overexpressing cancer cells, inducing a cancer cell-specific cytotoxicity. In particular, the CAP-NPs induced ICD through cathepsin B-cleavage mechanism only in targeted cancer cells in vitro. In colon tumor-bearing mice, selectively accumulated CAP-NPs at tumors enhanced antitumor immunity without DOX-related severe toxicity, inflammatory response and systemic immunosuppression. Moreover, cytotoxicity against immune cells infiltrated into tumor microenvironment was significantly reduced compared to free DOX, leading to increased response to checkpoint inhibitor immunotherapy. The combinatorial treatment of CAP-NPs with anti-PD-L1 exhibited high rate of complete tumor regression (50%) compared to free DOX with anti-PD-L1. Concurrently, DOX-related side effects were greatly reduced during chemoimmunotherapy. Collectively, our results suggest that cancer-activated DOX prodrug nanoparticles provide a promising approach to increase clinical benefit by inducing an immune response preferentially only to targeted cancer cells, not to normal cells and immune cells, and potentiates checkpoint inhibitor immunotherapy.

PARsylated transcription factor EB (TFEB) regulates the expression of a subset of Wnt target genes by forming a complex with ß-catenin-TCF/LEF1.

Wnt signaling is mainly transduced by ß-catenin via regulation of the ß-catenin destruction complex containing Axin, APC, and GSK3ß. Transcription factor EB (TFEB) is a well-known master regulator of autophagy and lysosomal biogenesis processes. TFEB's nuclear localization and transcriptional activity are also regulated by various upstream signals. In this study, we found that Wnt signaling induces the nuclear localization of TFEB and the expression of Wnt target genes is regulated by TFEB-ß-catenin-TCF/LEF1 as well as ß-catenin-TCF/LEF1 complexes. Our biochemical data revealed that TFEB is a part of the ß-catenin destruction complex, and destabilization of the destruction complex by knockdown of either Axin or APC causes nuclear localization of TFEB. Interestingly, RNA-sequencing analysis revealed that about 27% of Wnt3a-induced genes were TFEB dependent. However, these "TFEB mediated Wnt target genes" were different from TFEB target genes involved in autophagy and lysosomal biogenesis processes. Mechanistically, we found that Tankyrase (TNKS) PARsylates TFEB with Wnt ON signaling, and the nuclear localized PARsylated TFEB forms a complex with ß-catenin-TCF/LEF1 to induce the "TFEB mediated Wnt target genes". Finally, we found that in various types of cancer, the levels of TFEB mediated Wnt target genes exhibit strong correlations with the level of Axin2, which represents the activity of Wnt signaling. Overall, our data suggest that Wnt signaling induces the expression of a subset of genes that are distinct from previously known genes regulated by the ß-catenin-TCF/LEF1 complex or TFEB, by forming a transcription factor complex consisting of PARsylated TFEB and ß-catenin-TCF/LEF1.

Combination of cancer-specific prodrug nanoparticle with Bcl-2 inhibitor to overcome acquired drug resistance.

Multiple combination therapies with chemotherapeutic drugs and inhibitors of drug resistance have been effective in the clinical cases, but concerns have been raised about the severe toxicity of these chemotherapeutic drugs. Herein, we report a potent and safe combination strategy of cancer-specific doxorubicin (DOX) prodrug nanoparticles (PNPs) and B-cell lymphoma-2 (Bcl-2) anti-apoptotic inhibitor, Navitoclax, to overcome acquired drug resistance during chemotherapy. The cancer-specific PNPs were constructed by conjugating cathepsin B-specific cleavable peptide (Phe-Arg-Arg-Gly; FRRG) to DOX, resulting in FRRG-DOX that self-assembled into nanoparticles and the FRRG-DOX nanoparticles were further stabilized with the FDA-approved pharmaceutical excipient, Pluronic F68. The resulting PNPs are specifically cleaved and metabolized to free DOX in cathepsin B-overexpressing cancer cells, but they exhibited minimal cytotoxicity in cathepsin B-deficient normal cells. As expected, free DOX and PNPs induced overexpression of Bcl-2 in MDA-MB-231 cells, due to acquired drug resistance in a cell culture system. However, combination therapy with PNPs and Navitoclax showed the outstanding synergetic cytotoxicity by decreasing the expression level of Bcl-2. In MDA-MB231 breast tumor-bearing mice, intravenously injected PNPs efficiently accumulated in targeted tumor tissues via enhanced permeability and retention (EPR) effect. When combined with orally administered Navitoclax, PNPs exhibited more potent therapeutic efficacy in aquired drug resistant models than free DOX plus Navitoclax, whereas PNPs greatly reduced systemic toxic side effects in normal organs. Our cancer-specific PNP-based combination therapy with Bcl-2 inhibitor may provide a promising approach for the potent and safe treatment of acquired drug-resistant cancers.

Doxorubicin-Loaded PLGA Nanoparticles for Cancer Therapy: Molecular Weight Effect of PLGA in Doxorubicin Release for Controlling Immunogenic Cell Death.

Direct local delivery of immunogenic cell death (ICD) inducers to a tumor site is an attractive approach for leading ICD effectively, due to enabling the concentrated delivery of ICD inducers to the tumor site. Herein, we prepared doxorubicin (DOX)-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) using different molecular weight PLGA (7000 g/mol and 12,000 g/mol), showing different drug release kinetics. The different release kinetics of DOX might differently stimulate a tumor cell-specific immune response by releasing damage-associated molecular patterns (DAMPs), resulting in showing a different antitumor response in the living body. DOX-PLGA7K NPs showed faster DOX release kinetics than DOX-PLGA12K NPs in the physiological condition. DOX-PLGA7K NPs and DOX-PLGA12K NPs were successfully taken up by the CT-26 tumor cells, subsequently showing different DOX localization times at the nucleus. Released DOX successfully lead to cytotoxicity and HMGB1 release in vitro. Although the DOX-PLGA7K NPs and DOX-PLGA12K NPs showed different sustained DOX release kinetics in vitro, tumor growth of the CT-26 tumor was similarly inhibited for 28 days post-direct tumor injection. Furthermore, the immunological memory effect was successfully established by the ICD-based tumor-specific immune responses, including DC maturation and tumor infiltration of cytotoxic T lymphocytes (CTLs). We expect that the controlled release of ICD-inducible chemotherapeutic agents, using different types of nanomedicines, can provide potential in precision cancer immunotherapy by controlling the tumor-specific immune responses, thus improving the therapeutic efficacy.

Stage-Dependent Gene Expression Profiling in Colorectal Cancer.

Temporal gene expression profiles have been widely considered to uncover the mechanism of cancer development and progression. Gene expression patterns, however, have been analyzed for limited stages with small samples, without proper data pre-processing, in many cases. With those approaches, it is difficult to unveil the mechanism of cancer development over time. In this study, we analyzed gene expression profiles of two independent colorectal cancer sample datasets, each of which contained 556 and 566 samples, respectively. To find specific gene expression changes according to cancer stage, we applied the linear mixed-effect regression model (LMER) that controls other clinical variables. Based on this methodology, we found two types of gene expression patterns: continuously increasing and decreasing genes as cancer develops. We found that continuously increasing genes are related to the nervous and developmental system, whereas the others are related to the cell cycle and metabolic processes. We further analyzed connected sub-networks related to the two types of genes. From these results, we suggest that the gene expression profile analysis can be used to understand underlying the mechanisms of cancer development such as cancer growth and metastasis. Furthermore, our approach can provide a good guideline for advancing our understanding of cancer developmental processes.

Pja2 Inhibits Wnt/ß-catenin Signaling by Reducing the Level of TCF/LEF1.

Ubiquitination of proteins plays an essential role in various cellular processes, including protein degradation, DNA repair, and cell signaling pathways. Previous studies have shown that protein ubiquitination is implicated in regulating pluripotency as well as fate determination of stem cells. To identify how protein ubiquitination affects differentiation of embryonic stem cells, we analyzed microarray data, which are available in the public domain, of E3 ligases and deubiquitinases whose levels changed during stem cell differentiation. Expression of pja2, a member of the RING-type E3 ligase family, was up-regulated during differentiation of stem cells. Wnt/ß-catenin signaling is one of the most important signaling pathways for regulation of the self-renewal and differentiation of embryonic stem cells. Pja2 was shown to bind to TCF/LEF1, which are transcriptional factors for Wnt/ß-catenin signaling, and regulate protein levels by ubiquitination, leading to down-regulation of Wnt signaling activity. Based on these results, we suggest that E3 ligase Pja2 regulates stem cell differentiation by controlling the level of TCF/LEF1 by ubiquitination.

Identification and characterization of the leaf specific networks of inner and rosette leaves in Brassica rapa.

Inner and rosette leaves of Chinese cabbage (Brassica rapa) have different characteristics in terms of nutritional value, appearance, taste, color and texture. Many researchers have utilized differentially expressed genes for exploring the difference between inner and rosette leaves of Brassica rapa. The functional characteristics of a gene, however, is determined by complex interactions between genes. Hence, a noble network approach is required for elucidating such functional difference that is not captured by gene expression profiles alone. In this study, we measured gene expression in the standard cabbage genome by RNA-Sequencing and constructed rosette and inner leaf networks based on the gene expression profiles. Furthermore, we compared the topological and functional characteristics of these networks. We found significant functional difference between the rosette and inner leaf networks. Specifically, we found that the genes in the rosette leaf network were associated with homeostasis and response to external stimuli whereas the genes in the inner leaf network were mainly related to the glutamine biosynthesis processes and developmental processes with hormones. Overall, the network approach provides an insight into the functional difference of the two leaves.