GRP78 blockade overcomes acquired resistance to EGFR-tyrosine kinase inhibitors in non-small cell lung cancer.

AIMS: While significant upregulation of GRP78 has been documented in lung cancer patients, its association with resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) remains underexamined. Our study aimed to elucidate the functional importance of GRP78 in acquired resistance to EGFR-TKIs in non-small cell lung cancer (NSCLC) and to evaluate its potential as a therapeutic target. MAIN METHODS: Immunoblot analysis or flow cytometry was employed to assess several markers for endoplasmic reticulum (ER) stress and apoptosis. Ru(II) complex I and HA15, two known GRP78 inhibitors, were used to evaluate the functional role of GRP78. A Xenograft assay was performed to evaluate the in vivo anti-cancer effects of the GRP78 inhibitors. KEY FINDINGS: We validated a significant increase in GRP78 protein levels in HCC827-GR, H1993-GR, and H1993-ER cells. The EGFR-TKI-resistant cells overexpressing GRP78 exhibited significantly higher cell proliferation rates than did their parental counterparts. Notably, GRP78 inhibition resulted in a more profound anti-proliferative and apoptotic response via heightened ER stress and subsequent reactive oxygen species (ROS) production in EGFR-TKI-resistant cell lines compared with their parental cells. In xenograft models implanted with HCC827-GR, both Ru(II) complex I and HA15 significantly suppressed tumor growth and reduced tumor weight. Additionally, we confirmed that GRP78 plays a critical role in the proliferation of H1975, an EGFR-TKI-resistant T790M-mutant cell line, relative to other NSCLC cell lines. SIGNIFICANCE: Our findings strongly support targeting of GRP78 as a promising therapeutic strategy for NSCLC patients with acquired resistance to EGFR-TKIs.

High-Resolution In Situ High-Content Imaging of 3D-Bioprinted Single Breast Cancer Spheroids for Advanced Quantification of Benzo(a)pyrene Carcinogen-Induced Breast Cancer Stem Cells.

Cancer stem cells (CSCs), also known as tumor-initiating cells, are critically correlated with carcinogenesis and are strongly affected by the environmental factors. Environmental carcinogens, such as benzo(a)pyrene (BaP), are associated with the overproduction of CSCs in various types of cancers, including breast cancer. In this report, we present a sophisticated 3D breast cancer spheroid model for the direct identification and quantitative determination of CSCs induced by carcinogens within intact 3D spheroids. To this end, hydrogel microconstructs containing MCF-7 breast cancer cells were bioprinted within direct-made diminutive multi-well chambers, which were utilized for the mass cultivation of spheroids and in situ detection of CSCs. We found that the breast CSCs caused by BaP-induced mutations were higher in the biomimetic MCF-7 breast cancer spheroids than that in standard 2D monolayer cultures. Precisely controlled MCF-7 cancer spheroids could be generated by serially cultivating MCF-7 cells within the printed hydrogel microconstructs, which could be further utilized for high-resolution in situ high-content 3D imaging analysis to spatially identify the emergence of CSCs at the single spheroid level. Additionally, potential therapeutic agents specific to breast CSCs were successfully evaluated to verify the effectiveness of this model. This bioengineered 3D cancer spheroid system provides a novel approach to investigating the emergence of CSC induced by a carcinogen for environmental hazard assessment in a reproducible and scalable format.

Drug delivery in transarterial chemoembolization of hepatocellular carcinoma: Ex vivo evaluation using transparent tissue imaging.

In this study, we elucidated for the first time the role of anti-cancer drugs in transarterial chemoembolization (TACE) via direct visualization of the spatial distribution of drugs with respect to blood vessels in intact transparent hepatocellular carcinoma (HCC) tissues. To date, precise estimation of drug penetration into tumors using thin 3D tissue sections has been challenging. This study utilized the tissue optical clearing technique to resolve the lack of tissue clarity, thereby enabling deep tissue imaging for the quantitative assessment of drug delivery following TACE. We compared the drug delivery effect, time-dependent embolic effect, and immunogenic response following conventional TACE (cTACE), drug-eluting embolic TACE (DEE-TACE), and transarterial embolization (TAE) in a rat model of HCC. After each treatment, three-dimensional drug delivery was quantitatively evaluated via the transparent liver tumor imaging, and time-dependent tumor necrosis was analyzed by serial tumor harvesting and histological staining. The results showed that chemotherapeutic agents travel only short distances after cTACE (∼80µm) and DEE-TACE (∼110µm), whereas necrosis occurs extensively within 24 h of treatment (85.3-97.2% of tumor cells). In addition, the percentages of CD4 and IL-17+ CD4 T cells increased significantly following treatment; however, drug-loading did not appear to affect the immune response following TACE. In conclusion, transarterially delivered chemotherapeutic agents appeared to exert a limited role, owing to the rapid and overwhelming effect of embolization. STATEMENT OF SIGNIFICANCE: TACE has been widely used for the treatment of HCC, especially for unresectable intermediate and advanced HCCs. Drug use in TACE is expected to provide patients with synergistic therapeutic benefits with the effect of embolic agents; however, the role of chemotherapeutic agents in TACE remains controversial. This study quantitatively verified that chemotherapeutic agents travel only short distances after TACE, while necrosis occurs extensively within 24h, and drug loading does not significantly affect immune responses following TACE. Three-dimensional imaging of intact transparent HCC can contribute to a better understanding of drug delivery mechanisms associated with TACE and also reveal that drug use in TACE may need to be reconsidered and limited to situations when embolization is expected to be insufficient.

Integrative In Situ Photodynamic Therapy-Induced Cell Death Measurement of 3D-Bioprinted MCF-7 Tumor Spheroids.

The development of new in vitro models that closely mimic the tumor microenvironment (TME) to evaluate the efficacy of anticancer drugs has received great attention. In this study, a three-dimensional (3D) bioprinted Michigan Cancer Foundation-7 (MCF-7) cancer spheroid-embedded hydrogel model was suggested for integrative in situ determination of the half-maximal inhibitory concentration (IC50) values of photosensitizers (PSs). The MCF-7 cell-laden alginate/gelatin hydrogel was printed for the fabrication of tumor spheroids. The hydrogel was used to mimic the extracellular matrix (ECM) surrounding the cancer cells in the TME. The fluorescence intensities corresponding to photodynamic therapy (PDT)-induced death of tumor spheroids probed by the laser showed a random distribution in the hydrogel, regardless of the focus of the laser and the vertical-axis direction in which the laser was passed. These results enabled integrative in situ measurement of all tumor spheroids probed by the laser without needing to separate the tumor spheroids in the hydrogel and measure them individually. When compared with two-dimensional (2D) monolayer cultures, very large IC50 values of the PSs, chlorin e6 (Ce6) and sulfonated tetraphenyl porphyrin (sTPP), were achieved in MCF-7 spheroid-embedded hydrogels mainly due to the drug resistance of the tumor spheroids. Additionally, the heterogenic PDT response of single MCF-7 cancer cells in a single tumor spheroid was observed through 3D imaging of irregular apoptosis in a single spheroid since single tumor spheroids showed a heterogenic PDT response. Furthermore, the laser-power-dependent IC50 values of PSs were obtained using the MCF-7 spheroid-embedded hydrogel model.

3D bioprinted drug-resistant breast cancer spheroids for quantitative in situ evaluation of drug resistance.

Drug-resistant cancer spheroids were fabricated by three-dimensional (3D) bioprinting for the quantitative evaluation of drug resistance of cancer cells, which is a very important issue in cancer treatment. Cancer spheroids have received great attention as a powerful in vitro model to replace animal experiments because of their ability to mimic the tumor microenvironment. In this work, the extrusion printing of gelatin-alginate hydrogel containing MCF-7 breast cancer stem cells successfully provided 3D growth of many single drug-resistant breast cancer spheroids in a cost-effective 3D-printed mini-well dish. The drug-resistant MCF-7 breast cancer spheroids were able to maintain their drug-resistant phenotype of CD44high/CD24low/ALDH1high in the gelatin-alginate media during 3D culture and exhibited higher expression levels of drug resistance markers, such as GRP78 chaperon and ABCG2 transporter, than bulk MCF-7 breast cancer spheroids. Furthermore, the effective concentration 50 (EC50) values for apoptotic and necrotic spheroid death could be directly determined from the 3D printed-gelatin-alginate gel matrix based on in situ 3D fluorescence imaging of cancer spheroids located out of the focal point and on the focal point. The EC50 values of anti-tumor agents (camptothecin and paclitaxel) for apoptotic and necrotic drug-resistant cancer spheroid death were higher than those for bulk cancer spheroid death, indicating a greater drug resistance. STATEMENT OF SIGNIFICANCE: This study proposed a novel 3D bioprinting-based drug screening model, to quantitatively evaluate the efficacy of anticancer drugs using drug-resistant MCF-7 breast cancer spheroids formed within a 3D-printed hydrogel. Quantitative determination of anticancer drug efficacy using EC50, which is extremely important in drug discovery, was achieved by 3D printing that enables concurrent growth of many single spheroids efficiently. This study verified whether drug-resistant cancer spheroids grown within 3D-printed gelatin-alginate hydrogel could maintain and present drug resistance. Also, the EC50 values of the apoptotic and necrotic cell deaths were directly acquired in 3D-embedded spheroids based on in situ fluorescence imaging. This platform provides a single-step straightforward strategy to cultivate and characterize drug-resistant spheroids to facilitate anticancer drug screening.

A 3D cell printing-fabricated HepG2 liver spheroid model for high-content in situ quantification of drug-induced liver toxicity.

3D spheroid cultures are attractive candidates for application in in vitro drug-induced hepatotoxicity testing models to improve the reliability of biological information obtainable from a simple 2D culture model. Various 3D spheroid culture models exist for hepatotoxicity screening, but quantitative assays of spheroid response in situ are still challenging to achieve with the current 3D liver toxicity platforms. In this study, we developed a 3D printing-based HepG2 liver spheroid culture model for in situ quantitative evaluation and high-content monitoring of drug-induced hepatotoxicity. HepG2 liver spheroids grown in mini-fabricated hydrogel constructs using a 3D bioprinter were used to obtain the EC50 values and to measure the multi-parametric hepatotoxic effects, including mitochondrial permeability transition (MPT), cytosolic calcium levels, and apoptosis. Interestingly, the average fluorescence intensities of apoptotic and cell death markers, calculated for out-of-focus and in-focus spheroids, increased proportionally as a function of the drug concentration, allowing for the determination of the EC50 values. In addition, 3D HepG2 spheroids were more resistant to nefazodone-induced MPT than 2D HepG2 cells, indicating that the gelatin/alginate hydrogel culture system provides enhanced resistance to hepatotoxic drugs. The drug response of HepG2 liver spheroids was also found to be unrelated to the spheroid size. These results demonstrate that the present 3D cell-printing-based embedded HepG2 liver spheroid platform is a promising approach for screening and characterizing drug-induced hepatotoxicity.

Printing-Based Assay and Therapy of Antioxidants.

Antioxidants are essential in regulating various physiological functions and oxidative deterioration. Over the past decades, many researchers have paid attention to antioxidants and studied the screening of antioxidants from natural products and their utilization for treatments in diverse pathological conditions. Nowadays, as printing technology progresses, its influence in the field of biomedicine is growing significantly. The printing technology has many advantages. Especially, the capability of designing sophisticated platforms is useful to detect antioxidants in various samples. The high flexibility of 3D printing technology is advantageous to create geometries for customized patient treatment. Recently, there has been increasing use of antioxidant materials for this purpose. This review provides a comprehensive overview of recent advances in printing technology-based assays to detect antioxidants and 3D printing-based antioxidant therapy in the field of tissue engineering. This review is divided into two sections. The first section highlights colorimetric assays using the inkjet-printing methods and electrochemical assays using screen-printing techniques for the determination of antioxidants. Alternative screen-printing techniques, such as xurography, roller-pen writing, stamp contact printing, and laser-scribing, are described. The second section summarizes the recent literature that reports antioxidant-based therapy using 3D printing in skin therapeutics, tissue mimetic 3D cultures, and bone tissue engineering.

Multifunctional TPP-PEG-biotin self-assembled nanoparticle drug delivery-based combination therapeutic approach for co-targeting of GRP78 and lysosome.

BACKGROUND: In this study, a multifunctional tetraphenylporphyrin (TPP) conjugated polyethylene glycol with biotin (TPP-PEG-biotin) as a photo-dynamic therapy (PDT) material encapsulating a ruthenium complex 1 (Ru-1) was fabricated as self-assembled nanoparticle (Ru-1@TPP-PEG-biotin SAN) to co-target glucose-regulated protein 78 (GRP78) and the lysosome as a new anti-cancer therapeutic strategy. RESULTS: The MTT assay results reveals the enhanced anticancer activity of the Ru-1@TPP-PEG-biotin SANs due to the co-targeting of the GRP78 and lysosome. The Ru-1@TPP-PEG-biotin reduced level of GRP78 and lysosomal ceramide that contributed to the stability of the lysosomal membrane. The endoplasmic reticulum (ER) stress concomitant with the inhibition of GRP78 was clearly monitored by the phosphorylation of protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), and inositol-requiring enzyme 1 α (IRE1α) kinases to indicate the activation of the unfolded protein response (UPR) signaling using immunofluorescence assay. On the other hand, the degradation of the lysosome was observed through PDT action by the Ru-1@TPP-PEG-biotin SAN treatment. This was confirmed by the co-localization assay showing the disappearance of cathepsin D and lysosomal-associated membrane protein 1 (LAMP1) in the lysosome. CONCLUSIONS: Considering lysosome-mediated autophagy is an effective cancer cell survival mechanism, the degradation of the lysosome along with GRP78 inhibition by the Ru-1@TPP-PEG-biotin SAN combination therapy is suggested as a new co-targeting cancer treatment.

Contents of chlorogenic acids and caffeine in various coffee-related products.

Coffee is the most popular beverage in the Republic of Korea, other than Korea's traditional green tea. Coffee contains many physiologically active substances, such as chlorogenic acids (CGAs) and caffeine. Previous studies have focused on the content of CGAs and caffeine in brewed coffee. This study quantified the total amounts of CGAs and caffeine using high-performance liquid chromatography coupled with a diode array detector in 83 various coffee-related products, such as instant coffee, roasted and ground coffee sold in supermarkets, ready-to-drink coffee, and Americano coffee sold in franchise restaurants in the Republic of Korea. According to the results of this study, the coffee with the highest content of CGAs was unblended roasted and ground coffee sold in supermarkets, with a mean value of 194.1 ±â€¯67.7 mg/serving, and the most caffeine-rich coffee was Americano coffee from coffee shops, with a mean value of 166.1 ±â€¯37.5 mg/serving. The caffeine/CGA ratios were determined in various coffee beverages because they are useful parameters for estimating the human health. The lowest mean caffeine/CGAs ratio of 0.5 ±â€¯0.1 was found in unblended ground coffee sold in supermarkets, and the highest mean ratio of 2.5 ±â€¯1.4 was found in milk-added ready-to-drink coffee. Adult caffeine tolerance is defined as 400 mg a day in the Republic of Korea. However, this value highlights the importance of medicines, carbohydrate beverages, tea, chocolate, cocoa products, energy drinks and other sources of caffeine that can contribute to the total intake of caffeine.

Biotin-conjugated PEGylated porphyrin self-assembled nanoparticles co-targeting mitochondria and lysosomes for advanced chemo-photodynamic combination therapy.

The combination of chemotherapy and photodynamic therapy (chemo-PDT) has been suggested as an alternative therapy for drug-resistant cancers. In this study, biotin-conjugated PEGylated photosensitizer (PS) self-assembled nanoparticles (meso-tetraphenylporphyrin (TPP)-PEG-biotin SANs) were prepared via a self-assembly process to serve as nanocarriers for chemo-drugs as well as PSs. Electron microscopy results reveal the spherical shape of the nanoparticles (NPs). In the NPs, conjugated biotin plays a key role in selective tumor targeting. In vitro cellular experiments revealed the rapid cellular uptake of the TPP-PEG-biotin conjugates by MCF-7 cells that overexpress the biotin receptor, and verified that the conjugates were much more effective PSs than TPPS used as control in the cytotoxicity test. Interestingly, subcellular localization studies showed that the conjugates and their self-assembled NPs were localized mainly in mitochondria and partially in lysosomes, whereas TPPS was localized only in lysosomes. With the exclusive localization in mitochondria, high-content cell based assay showed that the TPP-PEG-biotin SANs induced rapid mitochondrial membrane potential transition (MPT), leading to cellular apoptosis. The chemo-drug doxorubicin (DOX) was successfully encapsulated in the TPP-PEG-biotin SANs (DOX@TPP-PEG-biotin) and had synergistic effects with enhanced cytotoxicity after PDT action. Collectively, the DOX@TPP-PEG-biotin SANs have promising potential as an effective anticancer agent in targeted combination therapy.

Determination of chlorogenic acids and caffeine in homemade brewed coffee prepared under various conditions.

Coffee, a complex mixture of more than 800 volatile compounds, is one of the most valuable commodity in the world, whereas caffeine and chlorogenic acids (CGAs) are the most common compounds. CGAs are mainly composed of caffeoylquinic acids (CQAs), dicaffeoylquinic acids (diCQAs), and feruloylquinic acids (FQAs). The major CGAs in coffee are neochlorogenic acid (3-CQA), cryptochlorogenic acid (4-CQA), and chlorogenic acid (5-CQA). Many studies have shown that it is possible to separate the isomers of FQAs by high-performance liquid chromatography (HPLC). However, some authors have shown that it is not possible to separate 4-feruloylquinic acid (4-FQA) and 5-feruloylquinic acid (5-FQA) by HPLC. Therefore, the present study was designated to investigate the chromatographic problems in the determination of CGAs (seven isomers) and caffeine using HPLC-DAD. The values of determination coefficient (R2) calculated from external-standard calibration curves were >0.998. The recovery rates conducted at 3 spiking levels ranged from 99.4% to 106.5% for the CGAs and from 98.8% to 107.1% for the caffeine. The precision values (expressed as relative standard deviations (RSDs)) were <7% and <3% for intra and interday variability, respectively. The tested procedure proved to be robust. The seven CGAs isomers except 4-FQA and 5-FQA were well distinguished and all gave good peak shapes. We have found that 4-FQA and 5-FQA could not be separated using HPLC. The method was extended to investigate the effects of different brewing conditions such as the roasting degree of green coffee bean, coffee-ground size, and numbers of boiling-water pours, on the concentration of CGAs and caffeine in homemade brewed coffee, using nine green coffee bean samples of different origins. It was reported that medium-roasted, fine-ground coffees brewed using three pours of boiling water were the healthiest coffee with fluent CGAs.