Organoid forming potential as complementary parameter for accurate evaluation of breast cancer neoadjuvant therapeutic efficacy.

BACKGROUND: 13-15% of breast cancer/BC patients diagnosed as pathological complete response/pCR after neoadjuvant systemic therapy/NST suffer from recurrence. This study aims to estimate the rationality of organoid forming potential/OFP for more accurate evaluation of NST efficacy. METHODS: OFPs of post-NST residual disease/RD were checked and compared with clinical approaches to estimate the recurrence risk. The phenotypes of organoids were classified via HE staining and ER, PR, HER2, Ki67 and CD133 immuno-labeling. The active growing organoids were subjected to drug sensitivity tests. RESULTS: Of 62 post-NST BC specimens, 24 were classified as OFP-I with long-term active organoid growth, 19 as OFP-II with stable organoid growth within 3 weeks, and 19 as OFP-III without organoid formation. Residual tumors were overall correlated with OFP grades (P < 0.001), while 3 of the 18 patients (16.67%) pathologically diagnosed as tumor-free (ypT0N0M0) showed tumor derived-organoid formation. The disease-free survival/DFS of OFP-I cases was worse than other two groups (Log-rank P < 0.05). Organoids of OFP-I/-II groups well maintained the biological features of their parental tumors and were resistant to the drugs used in NST. CONCLUSIONS: The OFP would be a complementary parameter to improve the evaluation accuracy of NST efficacy of breast cancers.

Dual Inhibition of BRAF-MAPK and STAT3 Signaling Pathways in Resveratrol-Suppressed Anaplastic Thyroid Cancer Cells with BRAF Mutations.

Anaplastic thyroid cancer is an extremely lethal malignancy without reliable treatment. BRAFV600E point mutation is common in ATCs, which leads to MAPK signaling activation and is regarded as a therapeutic target. Resveratrol inhibits ATC cell growth, while its impact on BRAF-MAPK signaling remains unknown. This study aims to address this issue by elucidating the statuses of BRAF-MAPK and STAT3 signaling activities in resveratrol-treated THJ-11T, THJ-16T, and THJ-21T ATC cells and Nthyori 3-1 thyroid epithelial cells. RT-PCR and Sanger sequencing revealed MKRN1-BRAF fusion mutation in THJ-16T, BRAF V600E point mutation in THJ-21T, and wild-type BRAF genes in THJ-11T and Nthyori 3-1 cells. Western blotting and immunocytochemical staining showed elevated pBRAF, pMEK, and pERK levels in THJ-16T and THJ-21T, but not in THJ-11T or Nthyori 3-1 cells. Calcein/PI, EdU, and TUNEL assays showed that compared with docetaxel and doxorubicin and MAPK-targeting dabrafenib and trametinib, resveratrol exerted more powerful inhibitory effects on mutant BRAF-harboring THJ-16T and THJ-21T cells, accompanied by reduced levels of MAPK pathway-associated proteins and pSTAT3. Trametinib- and dabrafenib-enhanced STAT3 activation was efficiently suppressed by resveratrol. In conclusion, resveratrol acts as dual BRAF-MAPK and STAT3 signaling inhibitor and a promising agent against ATCs with BRAF mutation.

Frequently expressed glypican-3 as a promising novel therapeutic target for osteosarcomas.

Osteosarcoma (OS) is the most common bone malignancy without a reliable therapeutic target. Glypican-3 (GPC3) mutation and upregulation have been detected in multidrug resistant OS, and anti-GPC3 immunotherapy can effectively suppress the growth of organoids. Further profiling of GPC3 mutations and expression patterns in OS is of clinical significance. To address these issues, fresh OS specimens were collected from 24 patients for cancer-targeted next-generation sequencing (NGS) and three-dimensional patient-derived organoid (PDO) culture. A tumor microarray was prepared using 37 archived OS specimens. Immunohistochemical (IHC) staining was performed on OS specimens and microarrays to profile GPC3 and CD133 expression as well as intratumoral distribution patterns. RT-PCR was conducted to semiquantify GPC3 and CD133 expression levels in the OS tissues. Anti-GPC3 immunotherapy was performed on OS organoids with or without GPC3 expression and its efficacy was analyzed using multiple experimental approaches. No OS cases with GPC3 mutations were found, except for the positive control (OS-08). IHC staining revealed GPC3 expression in 73.77% (45/61) of OSs in weak (+; 29/45), moderate (++; 8/45), and strong (+++; 8/45) immunolabeling densities. The intratumoral distribution of GPC3-positive cells was variable in the focal (+; 10%-30%; 8/45), partial (++; 31%-70%; 22/45), and the most positive patterns (+++; >71%; 15/45), which coincided with CD133 immunolabeling (P = 9.89 × 10-10 ). The anti-GPC3 antibody efficiently inhibits Wnt/ß-catenin signaling and induces apoptosis in GPC3-positive PDOs and PDXs, as opposed to GPC3-negative PDOs and PDXs. The high frequency of GPC3 and CD133 co-expression and the effectiveness of anti-wild-type GPC3-Ab therapy in GPC3-positive OS models suggest that GPC3 is a novel prognostic parameter and a promising therapeutic target for osteosarcoma.

Higher efficacy of resveratrol against advanced breast cancer organoids: A comparison with that of clinically relevant drugs.

The lack of reliable drugs is a therapeutic challenge of advanced breast cancers (ABCs). Resveratrol (Res) exerts inhibitory effects on breast cancer cell lines and animal models, while its efficacy against individual breast cancer cases remains unknown. This study aims to use ABC-derived organoids (ABCOs) as the ex vivo therapeutic platform to clarify the effectiveness of resveratrol against different ABC subtypes. Immunohistochemical staining confirmed that the ABCOs maintained their original tumors' ER, PR, HER2, and Ki67 expression patterns. ABCO proliferation and viability tests showed >50% cell death rates in 79.2% (19/24) of Res-treated, 28.6% (2/7) fulvestrant-treated, 66.7% (4/6) paclitaxel-treated, and 66.7% (6/9) gemcitabine-treated ABCOs. pSTAT3 nuclear translocation was more frequent in Res-sensitive (17/19; 89.47%) than that (1/5; 20%) of Res-insensitive ABCOs, which were suppressed upon Res treatment. Statistical analysis revealed a close correlation of STAT3 activation with the efficacy of Res, but not related to tumor receptor expression patterns (ER, PR, HER2) and pathological classification. We demonstrate for the first time the higher efficacy and broader spectrum of Res against different subtypes of ABCOs in comparison with that of conventional antibreast cancer drugs, providing an alternative approach for better management of ABCs.

Construction of IL-13 Receptor α2-Targeting Resveratrol Nanoparticles against Glioblastoma Cells: Therapeutic Efficacy and Molecular Effects.

Glioblastoma multiforme (GBM) is the most common lethal primary brain malignancy without reliable therapeutic drugs. IL-13Rα2 is frequently expressed in GBMs as a molecular marker. Resveratrol (Res) effectively inhibits GBM cell growth but has not been applied in vivo because of its low brain bioavailability when administered systemically. A sustained-release and GBM-targeting resveratrol form may overcome this therapeutic dilemma. To achieve this goal, encapsulated Res 30 ± 4.8 nm IL-13Rα2-targeting nanoparticles (Pep-PP@Res) were constructed. Ultraviolet spectrophotometry revealed prolonged Res release (about 25%) from Pep-PP@Res in 48 h and fluorescent confocal microscopy showed the prolonged intracellular Res retention time of Pep-PP@Res (>24 h) in comparison with that of free Res (<4 h) and PP@Res (<4 h). MTT and EdU cell proliferation assays showed stronger suppressive effects of Pep-PP@Res on rat C6 GBM cells than that of PP@Res (p = 0.024) and Res (p = 0.009) when used twice for 4 h/day. Pep-PP@Res had little toxic effect on normal rat brain cells. The in vivo anti-glioblastoma effects of Res can be distinctly improved in the form of Pep-PP@Res nanoparticles via activating JNK signaling, upregulating proapoptosis gene expression and, finally, resulting in extensive apoptosis. Pep-PP@Res with sustained release and GBM-targeting properties would be suitable for in vivo management of GBMs.

Identification of GPC3 mutation and upregulation in a multidrug resistant osteosarcoma and its spheroids as therapeutic target.

BACKGROUND: Drug resistance and the lack of molecular therapeutic target are the main challenges in the management of osteosarcomas (OSs). Identification of novel genetic alteration(s) related with OS recurrence and chemotherapeutic resistance would be of scientific and clinical significance. METHODS: To identify potential genetic alterations related with OS recurrence and chemotherapeutic resistance, the biopsies of a 20-year-old male osteosarcoma patient were collected at primary site (p-OS) and from its metastatic tumor (m-OS) formed after 5 months of adjuvant chemotherapy. Both OS specimens were subjected to cancer-targeted next generation sequencing (NGS) and their cell suspensions were cultured under three-dimensional condition to establish spheroid therapeutic model. Transcript-oriented Sanger sequencing for GPC3, the detected mutated gene, was performed on RNA samples of p-OS and m-OS tissues and spheroids. The effects of anti-GPC3 antibody and its combination with cisplatin on m-OS spheroids were elucidated. RESULTS: NGS revealed 4 mutations (GPC3, SOX10, MDM4 and MAPK8) and 6 amplifications (MDM2, CDK4, CCND3, RUNX2, GLI1 and FRS2) in p-OS, and 3 mutations (GPC3, SOX10 and EGF) and 10 amplifications (CDK4, CCND3, MDM2, RUNX2, GLI1, FRS2, CARD11, RAC1, SLC16A7 and PMS2) in m-OS. Among those alterations, the mutation abundance of GPC3 was the highest (56.49%) in p-OS and showed 1.54 times increase in m-OS. GPC3 transcript-oriented Sanger sequencing confirmed the mutation at 1046 in Exon 4, and immunohistochemical staining showed increased GPC3 production in m-OS tissues and its spheroids. EdU cell proliferation and Calcein/PI cell viability assays revealed that of the anti-OS first line drugs (doxorubicin, cisplatin, methotrexate, ifosfamide and carboplatin), 10 µM carboplatin exerted the best inhibitory effects on the p-OS but not the m-OS spheroids. 2 µg/mL anti-GPC3 antibody effectively committed m-OS spheroids to death by itself (76.43%) or in combination with cisplatin (92.93%). CONCLUSION: This study demonstrates increased abundance and up-regulated expression of mutant GPC3 in metastatic osteosarcoma and its spheroids with multidrug resistance. As GPC3-targeting therapy has been used to treat hepatocellular carcinomas and it is also effective to OS PDSs, GPC3 would be a novel prognostic parameter and therapeutic target of osteosarcomas.

Resveratrol and its Nanoparticle suppress Doxorubicin/Docetaxel-resistant anaplastic Thyroid Cancer Cells in vitro and in vivo.

Background: Docetaxel and doxorubicin combination has been widely used in anaplastic thyroid cancer/ATC treatment but often results in serious adverse effects and drug resistance. Resveratrol effectively inhibits ATC cell proliferation in vitro without affecting the corresponding normal cells, while its in vivo anti-ATC effects especially on the ones with docetaxel/doxorubicin-resistance have not been reported due to its low bioavailability. Nanoparticles with sustained-release and cancer-targeting features may overcome this therapeutic bottleneck. Methods: The resveratrol nanoparticles with sustained-release and IL-13Rα2-targeting capacities (Pep-1-PEG3.5k-PCL4k@Res) were prepared to improve the in vivo resveratrol bioavailability. Human THJ-16T ATC cell line was employed to establish nude mice subcutaneous transplantation model. The tumor-bearing mice were divided into four groups as Group-1, without treatment, Group-2, treated by 30 mg/kg free resveratrol, Group-3, treated by 30 mg/kg Pep-1-PEG3.5k-PCL4k@Res and Group-4, treated by 5 mg/kg docetaxel/5 mg/kg doxorubicin combination. TUNEL staining was used to detect the apoptotic cells in the tumor tissues. Docetaxel/doxorubicin resistant xenografts named as THJ-16T/R were isolated and subjected to 2D and 3D culture. The docetaxel/doxorubicin and resveratrol sensitivities of the original THJ-16T and THJ-16T/R cells were analyzed by multiple methods. Results: Docetaxel/doxorubicin and Pep-1-PEG3.5k-PCL4k@Res but not free resveratrol significantly delayed tumor growth (P < 0.01) and caused extensive apoptosis. The mice in docetaxel/doxorubicin-treated group suffered from weight loss (> 10%) and 2/3 of them died within 3 times of treatment and the chemotherapy was stop to avoid further animal loss. One week after drug withdrawal, the subcutaneous tumors regrew and the tumor volume increased 55.28% within 14 days. The cells isolated from the regrowing tumors (THJ-16T/R) were successfully cultured under 2D and 3D condition and underwent drug treatments. Compared with THJ-16T, the death rate of docetaxel/doxorubicin-treated THJ-16T/R population was lower (39.3% vs 18.0%), which remained almost unchanged in resveratrol-treated group (45.3% vs 49.3%). Conclusion: Resveratrol sustained-release targeting nanoparticles effectively inhibit in vivo ATC growth. Docetaxel/doxorubicin suppresses ATC xenografts but causes obvious side effects and secondary drug resistance that can be overcome by resveratrol.

Biological implications of PTEN upregulation and altered sodium/iodide symporter intracellular distribution in resveratrol-suppressed anaplastic thyroid cancer cells.

Objective: Anaplastic thyroid cancer/ATC is a highly aggressive malignancy with extremely poor prognosis. Resveratrol/Res promotes re-differentiation of cancer cells and exerts inhibitory effects on ATC cells. Sodium/iodide symporter/NIS and phosphate and tension homology deleted on chromsome ten/PTEN levels are positively correlated with the grade of thyroid cancer differentiation, while the impact of Res on them remain unknown. Materials and Methods: The patterns of NIS and PTEN expression and intracellular distribution in THJ-16T and THJ-21T ATC and Nthy-ori 3-1 normal thyroid cells and their relevance with Res-caused ATC suppression were investigated via multiple experimental methods. E-cadherin was cited as a re-differentiation biomarker of ATC cells. Results: MTT and EdU cell proliferation assays showed distinct growth suppression in ATC cells after Res treatment. TUNEL staining revealed extensive apoptosis of Res-treated THJ-16T and THJ-21T rather than Nthy-ori 3-1 cells. Western blotting, immunocytochemical/ICC and double-labeled immunofluorescent/IF staining showed increased PTEN levels accompanied with distinct NIS and PTEN nuclear co-translocation in Res-treated THJ-16T and THJ-21T cells. E-cadherin but not NIS appeared on the outer membrane. Conclusion: PTEN upregulation and the concurrent NIS and PTEN nuclear translocation in Res-suppressed ATC cells may indicate the better therapeutic outcome and would be a group of beneficial prognostic factors of ATCs.

Roles of Non-Coding RNAs in Normal Human Brain Development, Brain Tumor, and Neuropsychiatric Disorders.

One of modern biology's great surprises is that the human genome encodes only ~20,000 protein-coding genes, which represents less than 2% of the total genome sequence, and the majority of them are transcribed into non-coding RNAs (ncRNAs). Increasing evidence has shown that ncRNAs, including miRNAs, long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs), play important roles in regulating a wide range of biological processes of the human brain. They not only regulate the pathogenesis of brain tumors, but also the development of neuropsychiatric diseases. This review provides an integrated overview of the roles of ncRNAs in normal human brain function, brain tumor development, and neuropsychiatric disease. We discussed the functions and molecular mechanisms of miRNAs, lncRNAs, and circRNAs in normal brain function and glioma, respectively, including those in exosome vesicles that can act as a molecular information carrier. We also discussed the regulatory roles of ncRNAs in the development of neuropsychiatric diseases. Lastly, we summarized the currently available platforms and tools that can be used for ncRNA identification and functional exploration in human diseases. This study will provide comprehensive insights for the roles of ncRNAs in human brain function and disease.

Differential Exosomic Proteomic Patterns and Their Influence in Resveratrol Sensitivities of Glioblastoma Cells.

Glioblastoma multiforme (GBM) is the commonest primary brain malignancy with extremely poor prognosis. Resveratrol posseses anti-cancer effects, while GBM cells respond differently to it due to certain unknown reason(s). Because the tumor-derived exosomes are supposed to influence chemosensitivity, the exosomic proteins released from resveratrol-sensitive U251 and resveratrol-resistant glioblastoma LN428 cells are profiled before (N/Exo) and after drug treatment (Res/Exo) by label-free liquid chromatography-mass spectrometry (LC-MS). The therapeutic implications of the proteomic findings are estimated by gene ontology enrichment analysis (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG)-based bioinformatic analyses and further elucidated by exosome co-incubating. The results reveal that U251/N/Exo but not U251/Res/Exo enhances resveratrol sensitivity of resveratrol-resistant LN428 cells. The resveratrol sensitive properties of U251 cells are not altered by either LN428/N/Exo or LN428/Res/Exo. U251/N/Exo contains higher levels of chromatin silencing and epidermis development proteins, while U251/Res/Exo has more oxygen transport and G protein-coupled receptor. Both of LN428/N/Exo and LN428/Res/Exo are rich in the proteins related with nucleosome assembly, microtubule-based process and chromatin silencing. In conclusion, U251/N/Exo sensitizes LN428 cells to resveratrol via delivering drug sensitizing signals, suggesting the presence of additional factor(s) that may determine the resveratrol sensitivities of glioblastoma cells.

Correlation of ARHI upregulation with growth suppression and STAT3 inactivation in resveratrol-treated ovarian cancer cells.

BACKGROUND: Aplysia ras homology member I/ARHI is known as ovarian cancer suppressor gene and potential inhibitor of signal transducer and activator of transcription 3/STAT3 signaling. Resveratrol suppresses growth and STAT3 activation of ovarian cancer cells, while its influence in ARHI expression remains unknown. OBJECTIVE: The current study aims to elucidate the status of ARHI expression and its relevance with growth suppression and STAT3 inactivation of resveratrol-treated cells. METHODS: ARHI expression patterns of three ovarian cancer cell lines (human CAOV-3, OVCAR-3 and rat NUTU-19) without and with 100 µM resveratrol treatment were checked by immunocytochemical staining, Western blotting and RT-PCR. The involvement of ARHI in the growth inhibition and STAT3 inactivation of resveratrol-treated OVCAR-3 cells was investigated by transfection of ARHI-specific siRNA. RESULTS: ARHI is expressed in low levels in three ovarian cancer cell lines, which is upregulated upon resveratrol treatment accompanied with growth arrest, extensive apoptosis, increased autophagic activity and inactivated STAT3 signaling. Specific siRNA transfection efficiently knocked down ARHI expression in resveratrol-treated CAOV-3 and OVCAR-3 cells and increased the total cell number in limited extents (P> 0.05) in comparison with that of resveratrol-treated ovarian cancer cells without any transfection or transfected with mock oligonucleotides. ARHI knockdown failed to prevent resveratrol-caused STAT3 inactivation and cell crisis. CONCLUSION: ARHI upregulation is another molecular event caused by resveratrol and one of the elements related with resveratrol's anti-ovarian cancer efficacy. Resveratrol may inactivate STAT3 signaling of ovarian cancer cells in ARHI unrelated pattern(s).

Changes in synaptic plasticity and expression of glutamate receptor subunits in the CA1 and CA3 areas of the hippocampus after transient global ischemia.

Excess glutamate release from the presynaptic membrane has been thought to be the major cause of ischemic neuronal death. Although both CA1 and CA3 pyramidal neurons receive presynaptic glutamate input, transient cerebral ischemia induces CA1 neurons to die while CA3 neurons remain relatively intact. This suggests that changes in the properties of pyramidal cells may be the main cause related to ischemic neuronal death. Our previous studies have shown that the densities of dendritic spines and asymmetric synapses in the CA1 area are increased at 12h and 24h after ischemia. In the present study, we investigated changes in synaptic structures in the CA3 area and compared the expression of glutamate receptors in the CA1 and CA3 hippocampal regions of rats after ischemia. Our results demonstrated that the NR2B/NR2A ratio became larger after ischemia although the expression of both the NR2B subunit (activation of apoptotic pathway) and NR2A subunit (activation of survival pathway) decreased in the CA1 area from 6h to 48h after reperfusion. Furthermore, expression of the GluR2 subunit (calcium impermeable) of the AMPA receptor class significantly decreased while the GluR1 subunit (calcium permeable) remained unchanged at the same examined reperfusion times, which subsequently caused an increase in the GluR1/GluR2 ratio. Despite these notable differences in subunit expression, there were no obvious changes in the density of synapses or expression of NMDAR and AMPAR subunits in the CA3 area after ischemia. These results suggest that delayed CA1 neuronal death may be related to the dramatic fluctuation in the synaptic structure and relative upregulation of NR2B and GluR1 subunits induced by transient global ischemia.