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Simple Summary: Recently, CMTM6 has been found to stabilize PD-L1 on the plasma membrane of some tumor cells, favoring tumor immune system evasion. Until the present, it was unknown whether this protein could be present in a soluble form in the plasma of patients with cervical cancer (CC) or, additionally, if it could be found in atypical subcellular compartments in cell lines derived from CC. The results of our present research confirm that CMTM6 is increased in the plasma of patients with CC and associated, but not exclusively so, with exosomes, and that exosomal CMTM6 levels are correlated with exosomal PD-L1 levels. In addition, in CC cell lines, CMTM6 was found to be secreted and present both on the membrane and intracellularly. CMTM6 derived from CC in soluble, exosomal membrane and intracellular forms could have an impact on the biology of tumor cells by affecting the expression of molecules associated with tumor development and progression, such as PD-L1. CMTM6 is a membrane protein that acts as a regulator of PD-L1, maintaining its expression on the cell surface, and can prevent its lysosome-mediated degradation. It is unknown if CMTM6 is present in the plasma of patients with cervical cancer, and if it has non-canonical subcellular localizations in cell lines derived from cervical cancer. Our objective was to determine whether CMTM6 is found in plasma derived from cervical cancer patients and its subcellular localization in cell lines. Patient plasma was separated into exosome-enriched, exosome-free, and total plasma fractions. The levels of CMTM6 in each fraction were determined using ELISA and Western blot. Finally, for the cellular model, HeLa, SiHa, CaSki, and HaCaT were used; the subcellular locations of CMTM6 were determined using immunofluorescence and flow cytometry. Soluble CMTM6 was found to be elevated in plasma from patients with cervical cancer, with a nearly three-fold increase in patients (966.27 pg/mL in patients vs. 363.54 pg/mL in controls). CMTM6 was preferentially, but not exclusively, found in the exosome-enriched plasma fraction, and was positively correlated with exosomal PD-L1; CMTM6 was identified in the membrane, intracellular compartments, and culture supernatant of the cell lines. These results highlight that CMTM6, in its various presentations, may play an important role in the biology of tumor cells and in immune system evasion. [ABSTRACT FROM AUTHOR]

Simple Summary: Breast cancer is one of the most prevalent forms of cancer diagnosed in women worldwide. Since the estradiol receptor (ER) is overexpressed in 75% of breast tumors, it is a reasonable target for tumor diagnosis and therapy. This study focuses on the development and preclinical evaluation of readily synthesized 18F-labeled estradiol derivatives with different lipophilicity. The least hydrophilic derivative, 18F-TA-Glyco-EE, showed the highest cellular uptake in ER-positive breast cancer cells. The in vivo PET imaging of breast tumor-bearing mice demonstrated the desired rapid clearance of the tracer from the excretory organ through the liver. The in vitro autoradiography of ER-positive tumor sections confirmed the high specific binding of 18F-TA-Glyco-EE. In conclusion, 18F-TA-Glyco-EE may be a promising candidate for imaging of ER-positive breast cancer. About 75% of breast tumors show an overexpression of the estradiol receptor (ER), making it a valuable target for tumor diagnosis and therapy. To date, 16α-[18F]fluoroestradiol (FES) is the only FDA-approved imaging probe for the positron emission tomography (PET) imaging of ER-positive (ER+) breast cancer. However, FES has the drawback of a high retention in the liver. Therefore, the aim of this study was the development and preclinical evaluation of estradiol (E2) derivatives with different lipophilicity. Three 18F-labeled prosthetic groups (two glycosyl and one PEG azide) were chosen for conjugation with ethinyl estradiol (EE) by 18F-CuAAC (Cu-catalyzed azide-alkyne cycloaddition). The cellular uptake in ER+ MCF-7 tumor cells was highest for the less hydrophilic derivative (18F-TA-Glyco-EE). In nude mice bearing different breast tumors (ER+ MCF-7 and T47D versus ER− MDA-MB-231), 18F-TA-Glyco-EE revealed a high uptake in the liver (13%ID/g, 30 min p.i.), which decreased over 90 min to 1.2%ID/g, indicating fast hepatobiliary clearance. The statistically significant difference of 18F-TA-Glyco-EE uptake in T47D compared to MDA-MB-231 tumors at 60–90 min p.i. indicated ER-specific uptake, whereas in vivo PET imaging did not provide evidence for specific uptake of 18F-TA-Glyco-EE in MCF-7 tumors, probably due to ER occupation by E2 after E2-dependent MCF-7 tumor growth in mice. However, in vitro autoradiography revealed a high specific binding of 18F-TA-Glyco-EE to ER+ tumor slices. We conclude that 18F-TA-Glyco-EE, with its increased hydrophilicity after deacetylation in the blood and thus rapid washout from non-target tissues, may be a viable alternative to FES for the PET imaging of breast cancer. [ABSTRACT FROM AUTHOR]

Introduction: Cancers in general, and specifically lung cancer, continue to have low patient survival rates when the patient is at an advanced stage when diagnosed. It appears that the local environment, especially fibroblasts and their signaling molecules, tends to induce metastasis, increase cancer cell resistance to treatment, and aid in tumor growth rates. Since 3-D models quickly become too complex and/or expensive and therefore rarely leave the lab they are developed in, it is interesting to develop a 2-D model that more closely mimics clustered tumor formation and bulk interaction with a surrounding fibroblast environment. Methods: In the present study, we utilize an off-the-shelf stereolithography 3-D printer, standard use well plates, magnets, and metallic tubes to create a customizable 2-D co-culture system capable of being analyzed quantitatively with staining and qualitatively with standard fluorescent/brightfield microscopy to determine cancer-fibroblast interactions while also being able to test chemotherapeutic drugs in a high-throughput manner with standard 96-well plates. Results: Comparisons from monoculture and co-culture growth rates show that the presence of fibroblasts allows for significantly increased growth rates for H460 cancer. Additionally, the viability of cancer cells can be quantified with simple cell staining methods, and morphology and cell-cell interactions can be observed and studied. Discussion: The high throughput model demonstrates that boundary condition changes can be observed between cancer cells and fibroblasts based upon the different chemotherapeutics that have been administered. [ABSTRACT FROM AUTHOR]

Objective: To investigate the in vivo immunomodulatory and anti-tumor mechanisms of the combined treatment of novel Four-Herb formula (4HF) and doxorubicin in triple-negative breast cancer (TNBC). Methods: Murine-derived triple-negative mammary carcinoma cell line, 4T1 cells, was cultured and inoculated into mouse mammary glands. Sixty-six mice were randomly assigned into 6 groups (n=11 in ench): naïve, control, LD 4HF (low dose 4HF), HD 4HF (high dose 4HF), LD 4HF + D (low dose and doxorubicin), and D (doxorubicin). Apart from the naïve group, each mouse received subcutaneous inoculation with 5 × 105 4T1 cells resuspended in 100 µL of normal saline in the mammary fat pads. Starting from the day of tumor cell inoculation, tumors were grown for 6 days. The LD and HD groups received daily oral gavage of 658 and 2,630 mg/kg 4HF, respectively. The LD 4HF+D group received daily oral gavage of 658 mg/kg 4HF and weekly intraperitoneal injection of doxorubicin (5 mg/kg). The D group received weekly intraperitoneal injections of doxorubicin (5 mg/kg). The treatment naïve mice received daily oral gavage of 0.2 mL double distilled water and 0.1 mL normal saline via intraperitoneal injection once a week. The control group received daily oral gavage of 0.2 mL double-distilled water. The treatment period was 30 days. At the end of treatment, mice organs were harvested to analyze immunological activities via immunophenotyping, gene and multiplex analysis, histological staining, and gut microbiota analysis. Results: Mice treated with the combination of 4HF and doxorubicin resulted in significantly reduced tumor and spleen burdens (P<0.05), altered the hypoxia and overall immune lymphocyte landscape, and manipulated gut microbiota to favor the anti-tumor immunological activities. Moreover, immunosuppressive genes, cytokines, and chemokines such as C-C motif chemokine 2 and interleukin-10 of tumors were significantly downregulated (P<0.05). 4HF-doxorubicin combination treatment demonstrated synergetic activities and was most effective in activating the anti-tumor immune response (P<0.05). Conclusion: The above results provide evidence for evaluating the immune regulating mechanisms of 4HF in breast cancer and support its clinical significance in its potential as an adjunctive therapeutic agent or immune supplement. [ABSTRACT FROM AUTHOR]

Simple Summary: We have previously demonstrated that the interaction between cancer cells and bystander cells in the tumor microenvironment can promote the development of tumor cell heterogeneity. This heterogeneity is the basis for cancer progression, therapeutic resistance, and distant metastasis. In this study, we co-cultured prostate cancer cells with stromal cells to study the process of cancer–bystander cell interaction. Compared to primary stromal cells, spontaneously transformed stromal cells had significantly enhanced activity in inducing heterogeneity in cancer cells. By tracking the individual cancer–stromal interactions, we found that the transformed stromal sublines had a high tendency to fuse with cancer cells, forming cancer–stromal fusion hybrids that had the ability to proliferate. Hybridization between individual cancer cells and stromal cells resulted in genotypic and phenotypic diversification. This study indicates that the pathophysiologic status of the bystander cell compartment is crucial to the progression of tumor cell heterogeneity. Tumor cells gain advantages in growth and survival by acquiring genotypic and phenotypic heterogeneity. Interactions with bystander cells in the tumor microenvironment contribute to the progression of heterogeneity. We have shown that fusion between tumor and bystander cells is one form of interaction, and that tumor–bystander cell fusion has contrasting effects. By trapping fusion hybrids in the heterokaryon or synkaryon state, tumor–bystander cell fusion prevents the progression of heterogeneity. However, if trapping fails, fusion hybrids will resume replication to form derivative clones with diverse genomic makeups and behavioral phenotypes. To determine the characteristics of bystander cells that influence the fate of fusion hybrids, we co-cultured prostate mesenchymal stromal cell lines and their spontaneously transformed sublines with LNCaP as well as HPE-15 prostate cancer cells. Subclones derived from cancer–stromal fusion hybrids were examined for genotypic and phenotypic diversifications. Both stromal cell lines were capable of fusing with cancer cells, but only fusion hybrids with the transformed stromal subline generated large numbers of derivative subclones. Each subclone had distinct cell morphologies and growth behaviors and was detected with complete genomic hybridization. The health conditions of the bystander cell compartment play a crucial role in the progression of tumor cell heterogeneity. [ABSTRACT FROM AUTHOR]

Simple Summary: Platelets express the C-type lectin-like receptor 2 (CLEC-2) that enables binding to podoplanin (PDPN)-expressing tumor cells and, thereby, promote metastatic spread. An increased level of soluble CLEC-2 was reported in patients with thromboinflammatory and malignant disease, presumably released from activated platelets. In our study, we show that in vitro platelet activation leads to a significant decrease in CLEC-2 on platelets and in the plasma. We also found decreased levels of soluble CLEC-2 in plasma samples of patients with colorectal carcinoma (stages I to IV), breast cancer or melanoma (stages I to III) compared to healthy donors. Interestingly, in patients with glioblastoma, the plasma level of soluble CLEC-2 was significantly higher. We concluded that an increased plasma level of soluble CLEC-2 is not a suitable biomarker of platelet activation and tumor progression in most types of cancer. The C-type lectin-like receptor 2 (CLEC-2) is expressed on platelets and mediates binding to podoplanin (PDPN) on various cell types. The binding to circulating tumor cells (CTCs) leads to platelet activation and promotes metastatic spread. An increased level of soluble CLEC-2 (sCLEC-2), presumably released from activated platelets, was shown in patients with thromboinflammatory and malignant disease. However, the functional role of sCLEC-2 and the mechanism of sCLEC-2 release are not known. In this study, we focused on the effect of platelet activation on CLEC-2 expression and the sCLEC-2 plasma level in patients with cancer. First, citrated blood from healthy volunteer donors (n = 20) was used to measure the effect of platelet stimulation by classical agonists and PDPN on aggregation, CLEC-2 expression on platelets with flow cytometry, sCLEC-2 release to the plasma with ELISA and total CLEC-2 expression with Western blot analysis. Second, sCLEC-2 was determined in plasma samples from healthy donors (285) and patients with colorectal carcinoma (CRC; 194), melanoma (160), breast cancer (BC; 99) or glioblastoma (49). PDPN caused a significant increase in the aggregation response induced by classical agonists. ADP or PDPN stimulation of platelets caused a significant decrease in CLEC-2 on platelets and sCLEC-2 in the plasma, whereas total CLEC-2 in platelet lysates remained the same. Thus, the increased plasma level of sCLEC-2 is not a suitable biomarker of platelet activation. In patients with CRC (median 0.9 ng/mL), melanoma (0.9 ng/mL) or BC (0.7 ng/mL), we found significantly lower sCLEC-2 levels (p < 0.0001), whereas patients with glioblastoma displayed higher levels (2.6 ng/mL; p = 0.0233) compared to healthy controls (2.1 ng/mL). The low sCLEC-2 plasma level observed in most of the tumor entities of our study presumably results from the internalization of sCLEC-2 by activated platelets or binding of sCLEC-2 to CTCs. [ABSTRACT FROM AUTHOR]

Simple Summary: In this study, X-ray Photoelectron Spectroscopy (XPS) is used as a fast technique to characterize the composition of nanosized small extracellular vesicles (EVs), with the aim of evaluating its possible application in cancer screening and diagnosis. This spectroscopic technique is an ideal approach to characterize membranes due to its sensitivity and its penetration depth, in the nanometric range, meaning that the results will mainly correspond to the membrane composition, with little interference from the biomolecules in the EV´s lumen. We propose the use of XPS to analyze the membranes of small extracellular vesicles isolated from bodily fluids of cancer patients and compared them to those of healthy cells and donors. Notably, we have focused our analysis on the N chemical environment of EV membranes, as this can be related to previous complex chemical analyses of lipid and protein contents in EVs. We provide preliminary results on a fast and non-invasive novel strategy, as a method or tool for research-based EV investigations with potential application in cancer screening and monitoring. Small extracellular vesicle (EV) membranes display characteristic protein-lipidic composition features that are related to their cell of origin, providing valuable clues regarding their parental cell composition and real-time state. This could be especially interesting in the case of cancer cell-derived EVs, as their membranes could serve as valuable tools in liquid biopsy applications and to detect changes in the tumor malignancy. X-Ray Photoelectron Spectroscopy (XPS) is a powerful surface analysis technique able to detect every chemical element present, being also sensitive to their chemical environment. Here we explore the use of XPS as a fast technique to characterize EV membrane composition, with possible application in cancer research. Notably, we have focused on the nitrogen environment as an indicator of the relative abundance of pyridine-type bonding, primary, secondary and tertiary amines. Specifically, we have analyzed how tumoral and healthy cells have different nitrogen chemical environments that can indicate the presence or absence of malignancy. In addition, a collection of human serum samples from cancer patients and healthy donors was also analyzed. The differential XPS analysis of EVs collected from patients confirmed that the patterns of amine evolution could be related to markers of cancer disease, opening the possibility of their use as a non-invasive blood biomarker. [ABSTRACT FROM AUTHOR]

Three-dimensional cell culture methods are able to confer new predictive relevance to in vitro tumor models. In particular, the 3D multicellular tumor spheroids model is considered to better resemble tumor complexity associated with drug resistance compared to the 2D monolayer model. Recent advances in 3D printing techniques and suitable biomaterials have offered new promises in developing 3D tissue cultures at increased reproducibility and with high-throughput characteristics. In our study, we compared the sensitivity to dasatinib treatment in two different cancer cell lines, prostate cancer cells DU145 and glioblastoma cells U87, cultured in the 3D spheroids model and in the 3D bioprinting model. DU145 and U87 cells were able to proliferate in 3D alginate/gelatin bioprinted structures for two weeks, forming spheroid aggregates. The treatment with dasatinib demonstrated that bioprinted cells were considerably more resistant to drug toxicity than corresponding cells cultured in monolayer, in a way that was comparable to behavior observed in the 3D spheroids model. Recovery and analysis of cells from 3D bioprinted structures led us to hypothesize that dasatinib resistance was dependent on a scarce penetrance of the drug, a phenomenon commonly reported also in spheroids. In conclusion, the 3D bioprinted model utilizing alginate/gelatin hydrogel was demonstrated to be a suitable model in drug screening when spheroid growth is required, offering advantages in feasibility, reproducibility, and scalability compared to the classical 3D spheroids model. [ABSTRACT FROM AUTHOR]

Background: Oral malignancies have a significant effect on the quality of life among the affected patients. Curcumin is an antioxidant with a low bioavailability in the target tissue. Niosomes are carriers of increasing the therapeutic effects of drugs and reducing their side effects. This study aimed to determine the effective dose of curcumin niosome in the culture and then to compare its prophylactic effect in the form of mouthwash with that of its injectable form on oral cancer in rats. Methods: This was an in-vitro and in-vivo study. Firstly, KB oral cancer cells and human umbilical vein endothelial cells (HUVEC) were treated in separate groups with free curcumin, curcumin-loaded niosomes, and the unloaded niosomes at four doses of 4, 8, 16, and 32 μg. The study rats were then divided into the following four groups: 1) no intervention, 2) only carcinogenic substance, 3) carcinogenic substance with curcumin-loaded niosome injection, and 4) carcinogenic substance with a mouthwash containing niosome. Results: At the cellular level, a dose of 16 μg after 24 h was selected as an effective dose. In the animal phase, the use of injectable curcumin niosome was observed to significantly prevent the development of severe forms of dysplasia. Conclusions: In this in-vitro and in-vivo study, curcumin-loaded niosome was effective in preventing the development of severe forms of dysplasia and the inhibition of the growth of cancer cells. [ABSTRACT FROM AUTHOR]

Background: The treatment of pancreatic cancer (PDAC) remains clinically challenging, and neoadjuvant therapy (NAT) offers down staging and improved surgical resectability. Abundant fibrous stroma is involved in malignant characteristic of PDAC. We aimed to investigate tissue remodelling, particularly the alteration of the collagen architecture of the PDAC microenvironment by NAT.Methods: We analysed the alteration of collagen and gene expression profiles in PDAC tissues after NAT. Additionally, we examined the biological role of Ephrin-A5 using primary cultured cancer-associated fibroblasts (CAFs).Results: The expression of type I, III, IV, and V collagen was reduced in PDAC tissues after effective NAT. The bioinformatics approach provided comprehensive insights into NAT-induced matrix remodelling, which showed Ephrin-A signalling as a likely pathway and Ephrin-A5 (encoded by EFNA5) as a crucial ligand. Effective NAT reduced the number of Ephrin-A5+ cells, which were mainly CAFs; this inversely correlated with the clinical tumour shrinkage rate. Experimental exposure to radiation and chemotherapeutic agents suppressed proliferation, EFNA5 expression, and collagen synthesis in CAFs. Forced EFNA5 expression altered CAF collagen gene profiles similar to those found in PDAC tissues after NAT.Conclusion: These results suggest that effective NAT changes the extracellular matrix with collagen profiles through CAFs and their Ephrin-A5 expression. [ABSTRACT FROM AUTHOR]

Context: Anaplastic thyroid cancer (ATC) is a rare, aggressive, and deadly disease. Robust preclinical thyroid cancer models are needed to adequately develop and study novel therapeutic agents. Patient-derived xenograft (PDX) models may resemble patient tumors by recapitulating key genetic alterations and gene expression patterns, making them excellent preclinical models for drug response evaluation.Objective: We developed distinct ATC PDX models concurrently with cell lines and characterized them in vitro and in vivo.Methods: Fresh thyroid tumor from patients with a preoperative diagnosis of ATC was surgically collected and divided for concurrent cell line and PDX model development. Cell lines were created by generating single cells through enzymatic digestion. PDX models were developed following direct subcutaneous implantation of fresh tumor on the flank of immune compromised/athymic mice.Results: Six ATC PDX models and 4 cell lines were developed with distinct genetic profiles. Mutational characterization showed one BRAF/TP53/CDKN2A, one BRAF/CDKN2A, one BRAF/TP53, one TP53 only, one TERT-promoter/HRAS, and one TERT-promoter/KRAS/TP53/NF2/NFE2L2 mutated phenotype. Hematoxylin-eosin staining comparing the PDX models to the original patient surgical specimens show remarkable resemblance, while immunohistochemistry stains for important biomarkers were in full concordance (cytokeratin, TTF-1, PAX8, BRAF). Short tandem repeats DNA fingerprinting analysis of all PDX models and cell lines showed strong concordance with the original tumor. PDX successful establishment rate was 32%.Conclusion: We have developed and characterized 6 novel ATC PDX models with 4 matching cell lines. Each PDX model harbors a distinct genetic profile, making them excellent tools for preclinical therapeutic trials. [ABSTRACT FROM AUTHOR]

Objective: To determine the expression and functional roles of indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO2) in leiomyoma.Design: Experimental study.Setting: Academic research laboratory.Patient(s): Women undergoing hysterectomy for leiomyoma.Intervention(s): Blockade of IDO1 and TDO2.Main Outcome Measure(s): Expression of IDO1 and TDO2 in leiomyoma and the effects of their inhibitors on the extracellular matrix.Result(s): Leiomyoma expressed significantly higher levels of IDO1 and TDO2 messenger ribonucleic acid (mRNA; 60.3%, 35/58 pairs and 98.3%, 57/58 pairs, respectively) and protein (54%, 27/50 pairs and 92%, 46/50 pairs, respectively) as well as the enzyme activity marker kynurenine (78.3%, 36/46 pairs for IDO1/TDO2) compared with levels in matched myometrium. The expression of TDO2 but not IDO1 mRNA was significantly higher in fibroids from African American compared with that in Caucasian and Hispanic patients. The TDO2 but not the IDO1 protein and mRNA levels were more abundant in fibroids bearing the MED12 mutation compared with results in wild-type leiomyomas. Treatment of leiomyoma smooth muscle cell and myometrial smooth muscle cell spheroids with the TDO2 inhibitor 680C91 but not the IDO1 inhibitor epacadostat significantly repressed cell proliferation and the expression of collagen type I (COL1A1) and type III (COL3A1) in a dose-dependent manner; these effects were more pronounced in leiomyoma smooth muscle cells compared with myometrial smooth muscle cell spheroids.Conclusion(s): These results underscore the physiological significance of the tryptophan degradation pathway in the pathogenesis of leiomyomas and the potential utility of anti-TDO2 drugs for treatment of leiomyomas. [ABSTRACT FROM AUTHOR]

Rationale: Lymphangioleiomyomatosis (LAM) is a multisystem disease that causes lung cysts and respiratory failure. Loss of TSC (tuberous sclerosis complex) gene function results in a clone of "LAM cells" with dysregulated mTOR (mechanistic target of rapamycin) activity. LAM cells and fibroblasts form lung nodules that also contain mast cells, although their significance is unknown. Objectives: To understand the mechanism of mast-cell accumulation and the role of mast cells in the pathogenesis of LAM. Methods: Gene expression was examined using transcriptional profiling and qRT-PCR. Mast cell/LAM nodule interactions were examined in vitro using spheroid TSC2-null cell/fibroblast cocultures and in vivo using an immunocompetent Tsc2-null murine homograft model. Measurements and Main Results: LAM-derived cell/fibroblast cocultures induced multiple CXC chemokines in fibroblasts. LAM lungs had increased tryptase-positive mast cells expressing CXCRs (CXC chemokine receptors) (P < 0.05). Mast cells located around the periphery of LAM nodules were positively associated with the rate of lung function loss (P = 0.016). LAM spheroids attracted mast cells, and this process was inhibited by pharmacologic and CRISPR/cas9 inhibition of CXCR1 and CXCR2. LAM spheroids caused mast-cell tryptase release, which induced fibroblast proliferation and increased LAM-spheroid size (1.36 ± 0.24-fold; P = 0.0019). The tryptase inhibitor APC366 and sodium cromoglycate (SCG) inhibited mast cell-induced spheroid growth. In vivo, SCG reduced mast-cell activation and Tsc2-null lung tumor burden (vehicle: 32.5.3% ± 23.6%; SCG: 5.5% ± 4.3%; P = 0.0035). Conclusions: LAM-cell/fibroblast interactions attract mast cells where tryptase release contributes to disease progression. Repurposing SCG for use in LAM should be studied as an alternative or adjunct to mTOR inhibitor therapy. [ABSTRACT FROM AUTHOR]

Objective: Natural compounds have gained considerable attention in recent years due to disadvantages and properties of current chemotherapy drugs in cancer therapy. In addition, the impact of these compounds is specific for each type and/or subtypes of cancer due to different treatment response. Rutaecarpine, an alkaloid obtained from Evodia Rutaecarpa Chinese herb, has anticancer activity by inhibiting topoisomerase and/or cyclo-oxygenase-2 levels. However, the effectiveness of rutaecarpine has not been well known in breast cancer in terms of subtype. Therefore, we investigated the potential therapeutic effects of rutaecarpine on two different subtypes of breast cancer cells.Materials and Methods: The cytotoxic and apoptotic effects of rutaecarpine on MCF-7 and MDA-MB-231 cells were analyzed by WST-1, Annexin V, cell cycle, and acridine orange staining.Results: WST-1 results indicated that rutaecarpine significantly inhibited the growth of both cancer cells for 48 h (P < 0.05). In addition, rutaecarpine treatment caused apoptotic cell death through chromatin condensation and nuclear blebbing and G0/G1 arrest in both breast cancer cells. However, the efficacy of rutaecarpine was more profound in MCF-7 cells than MDA-MB-231 cells.Conclusions: Consequently, rutaecarpine has a potential therapeutic effect on breast cancer. However, the effectiveness of rutaecarpine is dependent on the subtype of breast cancer. [ABSTRACT FROM AUTHOR]

Objective In multiple myeloma (MM), stimulation of osteoclasts and bone marrow (BM) lesions lead to hypercalcemia, renal failure, and anemia. Co-culture of the myeloma cells in both hypocalcemia and hypercalcemia concentrations with bone marrow-mesenchymal stem cells were evaluated. Materials and Methods Viability and survival of myeloma cells were assessed by microculture tetrazolium test and flow cytometric assays. Mesenchymal stem cells (MSCs) were extracted from normal and myeloma patients and were co-cultured with myeloma cells. Results Myeloma cells showed less survival in both hypocalcaemia and hypercalcemia conditions (P  <.01). The paracrine and juxtacrine conditions of demineralized bone matrix-induced hypercalcemia increased the proliferation and survival of the cells (P  <.05). Unlike myeloma MSCs, normal MSCs reduced the survival of and induced apoptosis in myeloma cells (P  <.1). Conclusion Normal healthy-MSCs do not protect myeloma cells, but inhibit them. However, increasing the ratio of myeloma cells to MSCs reduces their inhibitory effects of MSCs and leads to their myelomatous transformation. [ABSTRACT FROM AUTHOR]

Purpose: Vitreoretinal lymphoma (VRL) is a potentially fatal intraocular malignancy. Diagnosis is hampered by poor preservation of morphology and DNA/RNA integrity, which precludes adjunctive molecular analysis. We aimed to determine the optimum fixative protocol for VRL biopsies that permits cytology, IHC/flow cytometry and molecular analyses.Methods: Six fixatives were compared on cultured Pfeiffer cells used as a cellular model. Cells were fixed and evaluated on cellular morphology, antibody staining, DNA/RNA amount and integrity. VRL clinical cases were used as validation and proof-of-concept.Results: PreservCyt was the best fixative for preserving cellular morphology and high-quality RNA/DNA from vitreous fluid biopsies. Cells from clinical VRL cases fixed with PreservCyt showed adequate cellular morphology and IHC positivity. Sufficient DNA was obtained for IgH clonality and MYD88 mutation detection using remnant cytological fluid.Conclusions: PreservCyt maintains good morphology and RNA/DNA integrity suggesting that it is a suitable fixative for VRL diagnosis and molecular analysis. [ABSTRACT FROM AUTHOR]

Background: MPNST is a rare soft-tissue sarcoma that can arise from patients with NF1. Existing chemotherapeutic and targeted agents have been unsuccessful in MPNST treatment, and recent findings implicate STAT3 and HIF1-α in driving MPNST. The DNA-binding and transcriptional activity of both STAT3 and HIF1-α is regulated by Redox factor-1 (Ref-1) redox function. A first-generation Ref-1 inhibitor, APX3330, is being tested in cancer clinical trials and could be applied to MPNST.Methods: We characterised Ref-1 and p-STAT3 expression in various MPNST models. Tumour growth, as well as biomarkers of apoptosis and signalling pathways, were measured by qPCR and western blot following treatment with inhibitors of Ref-1 or STAT3.Results: MPNSTs from Nf1-Arfflox/floxPostnCre mice exhibit significantly increased positivity of p-STAT3 and Ref-1 expression when malignant transformation occurs. Inhibition of Ref-1 or STAT3 impairs MPNST growth in vitro and in vivo and induces apoptosis. Genes highly expressed in MPNST patients are downregulated following inhibition of Ref-1 or STAT3. Several biomarkers downstream of Ref-1 or STAT3 were also downregulated following Ref-1 or STAT3 inhibition.Conclusions: Our findings implicate a unique therapeutic approach to target important MPNST signalling nodes in sarcomas using new first-in-class small molecules for potential translation to the clinic. [ABSTRACT FROM AUTHOR]

BACKGROUND Pancreatic cancer is considered as the most deadly tumor among gastrointestinal cancers because of its poor prognosis. The frequently deregulated pathway in the cancer cell is associated with an increased expression of various genes, including the synthesis of fatty acids. We aimed to evaluate the level of serum fatty acid synthase (FASN) as a diagnostic marker for early diagnosis of pancreatic cancer. METHODS Serum FASN levels were measured by ELISA in 92 patients with pancreatic adenocarcinomas and in 92 healthy controls. Logistic regression analysis was used to identify independent predictors of certain diagnostic categories. RESULTS Serum FASN levels were significantly higher in patients with pancreatic cancer than in healthy controls (1.35 [0.98-2.3] ng/mL vs 1.04 [0.19-1.34] ng/mL, p < 0.001) and in smokers compared to non-smokers (1.41 [0.79-2.52] ng/mL vs 1.07 [0.21-1.74] ng/mL, p < 0.001). FASN levels and smoking were associated with increased risk of PC (1.54 [1.1- 2.14] ng/mL, p = 0.011 and 5.69 [2.68-12.09] ng/mL, p < 0.001, respectively). CONCLUSION Elevated serum FASN levels in patients with pancreatic cancer indicate the need for the production of large numbers of lipids for the survival and proliferation of human cancer cells and the diagnostic value of FASN as a new diagnostic biomarker. [ABSTRACT FROM AUTHOR]

Background: Human epidermal growth factor 2 (HER2/ERBB2) is frequently amplified/mutated in cancer. The tyrosine kinase inhibitors (TKIs) lapatinib, neratinib, and tucatinib are FDA-approved for the treatment of HER2-positive breast cancer. Direct comparisons of the preclinical efficacy of the TKIs have been limited to small-scale studies. Novel biomarkers are required to define beneficial patient populations.Methods: In this study, the anti-proliferative effects of the three TKIs were directly compared using a 115 cancer cell line panel. Novel TKI response/resistance markers were identified through cross-analysis of drug response profiles with mutation, gene copy number and expression data.Results: All three TKIs were effective against HER2-amplified breast cancer models; neratinib showing the most potent activity, followed by tucatinib then lapatinib. Neratinib displayed the greatest activity in HER2-mutant and EGFR-mutant cells. High expression of HER2, VTCN1, CDK12, and RAC1 correlated with response to all three TKIs. DNA damage repair genes were associated with TKI resistance. BRCA2 mutations were correlated with neratinib and tucatinib response, and high expression of ATM, BRCA2, and BRCA1 were associated with neratinib resistance.Conclusions: Neratinib was the most effective HER2-targeted TKI against HER2-amplified, -mutant, and EGFR-mutant cell lines. This analysis revealed novel resistance mechanisms that may be exploited using combinatorial strategies. [ABSTRACT FROM AUTHOR]

Background: Eukaryotic protein translation elongation factor 1α2 (EEF1A2) is an oncogene that promotes the progression of breast and pancreatic cancer. In this study, we aimed to elucidate the oncogenic function of EEF1A2 in the metastasis of lung adenocarcinoma (LUAD).Methods: Immunohistochemistry and western blot were used to study EEF1A2 expression levels in LUAD tissues and cells, respectively. The role of EEF1A2 in LUAD progression were investigated in vitro and in vivo. We identified potential EEF1A2-binding proteins by liquid chromatography-electrospray mass spectrometry (LC-MS)/MS. Protein-protein interactions were determined by immunofluorescence and co-immunoprecipitation (Co-IP).Results: In this study, we report that EEF1A2 mediates the epithelial-mesenchymal transformation (EMT), to promote the metastasis of LUAD cells in vitro and in vivo. Moreover, EEF1A2 interacts with HSP90AB1 to increase TGFβ Receptor (TβR)-I, and TβRII expression, followed by enhanced SMAD3 and pSMAD3 expression and nuclear localisation, which promotes the EMT of LUAD cells. Overexpression of EEF1A2 in cancer tissues is associated with poor prognosis and short survival of patients with LUAD.Conclusions: These findings underscore the molecular functions of EEF1A2 in LUAD metastasis and indicate that EEF1A2 represents a promising target in the treatment of aggressive LUAD. [ABSTRACT FROM AUTHOR]

Background: Anti-programmed cell death 1 (PD-1)/programmed death-ligand 1 (PD-L1) monoclonal antibodies (mAbs) show remarkable clinical anti-tumour efficacy. However, rational combinations are needed to extend the clinical benefit to primary resistant tumours. The design of such combinations requires the identification of the kinetics of critical immune cell populations in the tumour microenvironment.Methods: In this study, we compared the kinetics of immune cells in the tumour microenvironment upon treatment with immunotherapy combinations with different anti-tumour efficacies in the non-inflamed tumour model TC-1/A9. Tumour-bearing C57BL/6J mice were treated with all possible combinations of a human papillomavirus (HPV) E7 long peptide, polyinosinic-polycytidylic acid (PIC) and anti-PD-1 mAb. Tumour growth and kinetics of the relevant immune cell populations were assessed over time. The involvement of key immune cells was confirmed by depletion with mAbs and immunophenotyping with multiparametric flow cytometry.Results: The maximum anti-tumour efficacy was achieved after intratumoural administration of HPV E7 long peptide and PIC combined with the systemic administration of anti-PD-1 mAb. The intratumoural immune cell kinetics of this combination was characterised by a biphasic immune response. An initial upsurge of proinflammatory myeloid cells led to a further rise in effector CD8+ T lymphocytes at day 8. Depletion of either myeloid cells or CD8+ T lymphocytes diminished the anti-tumour efficacy of the combination.Conclusions: The anti-tumour efficacy of a successful immunotherapy combination in a non-inflamed tumour model relies on an early inflammatory process that remodels the myeloid cell compartment. [ABSTRACT FROM AUTHOR]

Background: Poly(ADP-ribose) polymerase inhibitors (PARPi) target tumours defective in homologous recombination (HR). Most BRCA-wild-type (WT) HR-proficient breast cancers are intrinsically resistant to PARP inhibitors, e.g., talazoparib. We evaluated the role of autophagy in this de novo resistance and determined the underlying mechanism to overcome this.Methods: Autophagosome formation and autophagic flux were assessed by evaluating endogenous LC3-II levels and ectopic expression of EGFP-LC3 and mRFP-EGFP-LC3 in breast cancer cells. Autophagy-defective cells were generated by genetic depletion of BECN1, ATG5, p62/SQSTM1 and LAMP1 by using CRISPR-Cas9 double nickase system. The response of PARPi was evaluated in autophagy-proficient and -defective breast cancer cells and in xenograft SCID-mice model.Results: Pro-survival autophagy was significantly enhanced upon talazoparib treatment in BRCA-WT breast cancer cell lines. Autophagy-deficient cells were hypersensitive to talazoparib. Targeting autophagy synergistically enhanced the therapeutic efficacy of talazoparib in BRCA1-WT breast cancer cells in vitro and in vivo xenograft tumour mouse model. Mechanistically, autophagy inhibition by chloroquine promoted deleterious NHEJ mediated DSB-repair, leading to extensive genomic instability and mitotic catastrophe.Conclusions: Autophagy confers de novo resistance to PARP inhibitor, talazoparib. Autophagy inhibition improves the therapeutic outcome of PARPi treatment in preclinical mice model, bearing HR-proficient breast tumours, warranting its usage in the clinical settings. [ABSTRACT FROM AUTHOR]

Background: Triple negative breast cancers (TNBC) have poor prognoses despite aggressive treatment with cytotoxic chemotherapy. Cancer-associated fibroblasts (CAFs) are prominent in tumour stroma. Our hypothesis was that CAFs modulate chemotherapy sensitivity.Methods: TNBC cells and breast fibroblasts were cultured; survival after chemotherapeutics was assessed using luciferase or clonogenic assays. Signalling was investigated using transcriptomics, reporters, recombinant proteins and blocking antibodies. Clinical relevance was investigated using immunohistochemistry.Results: Breast CAFs dose-dependently protected TNBC cell lines MDA-MB-231 and MDA-MB-157, but not MDA-MB-468s, from chemotherapy. CAF-induced protection was associated with interferon (IFN) activation. CAFs were induced to express IFNβ1 by chemotherapy and TNBC co-culture, leading to paracrine activation in cancer cells. Recombinant IFNs were sufficient to protect MDA-MB-231 and MDA-MB-157 but not MDA-MB-468 cells. In TNBC patients, IFNβ1 expression in CAFs correlated with cancer cell expression of MX1, a marker of activated IFN signalling. High expression of IFNβ1 (CAFs) or MX1 (tumour cells) correlated with reduced survival after chemotherapy, especially in claudin-low tumours (which MDA-MB-231 and MDA-MB-157 cells represent). Antibodies that block IFN receptors reduced CAF-dependent chemoprotection.Conclusions: CAF-induced activation of IFN signalling in claudin-low TNBCs results in chemoresistance. Inhibition of this pathway represents a novel method to improve breast cancer outcomes. [ABSTRACT FROM AUTHOR]

Background: The investigation of underlying mechanism and the exploitation of novel therapies for metastatic prostate cancer (PCa) are still urgently needed. miR-199b-5p has been suggested to function as tumour suppressor in various human cancers. However, the clinical significance and role of miR-199b-5p in PCa remain unclear.Methods: The current study sought to investigate the expression status of miR-199b-5p in PCa and the involved molecular mechanisms in PCa metastasis by using bioinformatics analyses, loss-and gain-of-functions and rescue experiments in vitro and in vivo.Results: We demonstrated that miR-199b-5p was significantly downregulated in metastatic PCa tissues and cells when compared with the normal prostate tissue, the localised disease, the weakly metastatic and androgen-dependent PCa cell and the normal prostate epithelial cell. We also found that miR-199b-5p drastically suppressed PCa cell proliferation, migration and invasion in vitro and inhibited xenografts tumour growth and metastasis in vivo. Mechanistically, our results showed that miR-199b-5p could inhibit discoidin domain receptor tyrosine kinase 1 (DDR1) expression by directly targeting its 3'-UTR, thereby hindering epithelial-mesenchymal transition (EMT)-associated traits, which were induced by DDR1 activating ERK signalling pathway. Moreover, PCa patients with low miR-199b-5p expression level had a remarkably shorter overall survival than those with high miR-199b-5p level, indicating an association of miR-199b-5p loss with poor prognosis in patients with PCa. Furthermore, DDR1 was upregulated in PCa, and significantly correlated with high Gleason score, advanced pathological stage, tumour metastasis and shorter overall survival.Conclusions: Our study, for the first time, provide evidence of a tumour-suppressive function of miR-199b-5p in the invasion and metastasis of PCa, supporting the translational exploitation of miR-199b-5p-based therapeutic approaches for PCa metastases. Also, the miR-199b-5p-DDR1-ERK signalling axis identified in this study represents a novel mechanism of regulating EMT in PCa metastases. [ABSTRACT FROM AUTHOR]

The perforation of plasma membrane by ultrasound-driven microbubbles (i.e., sonoporation) provides a temporary window for transporting macromolecules into the cytoplasm that is promising with respect to drug delivery and gene therapy. To improve the efficacy of delivery while ensuring biosafety, membrane resealing and cell recovery are required to help sonoporated cells defy membrane injury and regain their normal function. Blebs are found to accompany the recovery of sonoporated cells. However, the spatiotemporal characteristics of blebs and the underlying mechanisms remain unclear. With a customized platform for ultrasound exposure and 2-D/3-D live single-cell imaging, localized membrane perforation was induced with ultrasound-driven microbubbles, and the cellular responses were monitored using multiple fluorescent probes. The results indicated that localized blebs undergoing four phases (nucleation, expansion, pausing and retraction) on a time scale of tens of seconds to minutes were specifically involved in the reversibly sonoporated cells. The blebs spatially correlated with the membrane perforation site and temporally lagged (about tens of seconds to minutes) the resealing of perforated membrane. Their diameter (about several microns) and lifetime (about tens of seconds to minutes) positively correlated with the degree of sonoporation. Further studies revealed that intracellular calcium transients might be an upstream signal for triggering blebbing nucleation; exocytotic lysosomes not only contributed to resealing of the perforated membrane, but also to the increasing bleb volume during expansion; and actin components accumulation facilitated bleb retraction. These results provide new insight into the short-term strategies that the sonoporated cell employs to recover on membrane perforation and to remodel membrane structure and a biophysical foundation for sonoporation-based therapy. [ABSTRACT FROM AUTHOR]

Glioblastoma (GBM) is an incurable brain tumor with inevitable recurrence. This is in part due to a highly malignant cancer stem cell (CSC) subpopulation of tumor cells that is particularly resistant to conventional treatments, including radiotherapy. Here we show that CBL0137, a small molecule anti-cancer agent, sensitizes GBM CSCs to radiotherapy. CBL0137 sequesters the FACT (facilitates chromatin transcription) complex to chromatin, resulting in cytotoxicity preferentially within tumor cells. We show that when combined with radiotherapy, CBL0137 inhibited GBM CSC growth and resulted in more DNA damage in the CSCs compared to irradiation or drug alone. Using an in vivo subcutaneous model, we showed that the frequency of GBM CSCs was reduced when tumors were pretreated with CBL0137 and then exposed to irradiation. Survival studies with orthotopic GBM models resulted in significantly extended survival for mice treated with combinatorial therapy. As GBM CSCs contribute to the inevitable recurrence in patients, targeting them is imperative. This work establishes a new treatment paradigm for GBM that sensitizes CSCs to irradiation and may ultimately reduce tumor recurrence. [ABSTRACT FROM AUTHOR]

Human kinome/phosphatome screen identified CAMK2N1 genes suppressing the development of human hepatocellular carcinoma (HCC). CAMK2N1 downregulation was found in 47% HCCs and associated with poor prognosis. The downregulation was mainly attributed to its genome deletion (28.4%) and DNA hypermethylation of its promoter (12.5%). Silencing and ectopic expression of CAMK2N1 respectively enhanced and suppressed cell proliferation, colony formation, and xenograft tumor growth in nude mice. Comparative proteomics revealed that CAMK2N1 silencing transcriptionally deregulated the genes regulated by E2F1 (89 out of the 114 E2F-signaling targets, P = 8.8E-240). The promoter assays revealed that CAMK2N1 suppressed E2F1-mediated transcriptional activities. CAMK2N1 silencing induced cyclins D/E expression, whereas its ectopic expression induced P27/KIP1 expression and suppressed the cell cycle. CAMK2N1 was translocated from the nuclei to the cytoplasm when cell proliferation reached the stationary phase, where its functions as an endogenous inhibitor of CAMK2. In conclusion, CAMK2NA is a novel 1p36 tumor suppressor gene that inhibits E2F1 transcriptional activities and induces P27/KIP1 expression. CAMK2N1-CAMK2 signaling forms a mechanism that restricts the cell cycle progression. Its deregulation could lead to tumorigenesis and might serve as promising therapeutic targets. [ABSTRACT FROM AUTHOR]

Arsenic, a human carcinogen, causes various human cancers, including those of the skin, lung, and liver. Hepatocellular carcinomas (HCCs), which have high mortality, are common malignancies worldwide. Tumor-associated macrophages (TAMs), which are considered to be similar to M2-polarized macrophages, promote tumor invasion and progression. Small non-coding RNAs (miRNAs) regulate expression of genes involved in progression of various malignancies. Extracellular vesicles (EVs), as mediators of cell communication, pass specific miRNAs directly from TAMs to tumor cells, promoting tumor pathogenesis and metastasis. In HCCs, large tumor suppressor kinase 1 (LATS1), functions as a tumor suppressor. However, the molecular mechanism by which miRNA modulates LATS1 expression in HCCs remains unclear. The results show that exposure to arsenite, increased miR-15b levels and induced M2 polarization of THP-1 cells. Elevated levels of miR-15b were transferred from arsenite-treated-THP-1 (As-THP-1) cells to HCC cells via miR-15b in EVs inhibited activation of the Hippo pathway by targeting LATS1, and was involved in promoting the proliferation, migration, and invasion of HCC cells. In conclusion, miR-15b in EVs from As-THP-1 cells is transferred to HCC cells, in which it targets and downregulates LATS1 expression and promotes the proliferation, migration, and invasion of HCC cells. [ABSTRACT FROM AUTHOR]

Adrenocortical carcinomas (ACCs) overexpress insulin-like growth factor 2 (IGF2), that drives a proliferative autocrine loop by binding to IGF1R and IR, but IGF1R/IR-targeted therapies failed in ACC patients. The cytoskeleton actin-binding protein filamin A (FLNA) impairs IR signalling in melanoma cells. Aims of this study were to test FLNA involvement in regulating IGF1R and IR responsiveness to both IGF2 and inhibitors in ACC. In ACC cells H295R and SW13 and primary cultures (1ACC, 4 adenomas) we found that IGF1R and IR interacted with FLNA, and FLNA silencing increased IGF1R and reduced IR expression, with a downstream effect of increased cell proliferation and ERK phosphorylation. In addition, FLNA knockdown potentiated antiproliferative effects of IGF1R/IR inhibitor Linsitinib and IGF1R inhibitor NVP-ADW742 in H295R. Finally, Western blot showed lower FLNA expression in ACCs (n = 10) than in ACAs (n = 10) and an inverse correlation of FLNA/IGF1R ratio with ERK phosphorylation in ACCs only. In conclusion, we demonstrated that low FLNA levels enhance both IGF2 proliferative effects and IGF1R/IR inhibitors efficacy in ACC cells, suggesting FLNA as a new factor influencing tumor clinical behavior and the response to the therapy with IGF1R/IR-targeted drugs. [ABSTRACT FROM AUTHOR]

Surgery is the only curative treatment option for pancreatic cancer, but patients often develop postoperative recurrence. Surgical invasiveness might be involved in the mechanism of recurrence. The associations among inflammation caused by surgery, neutrophils, and cancer metastasis were investigated. At first, neutrophil extracellular traps (NETs) were examined in clinical specimens, and NETs were observed around metastatic tumors. To explore how NETs were induced, neutrophils were cultured with pancreatic cancer or in cancer-conditioned medium. Neutrophils formed NETs when they were cultured with pancreatic cancer or even its conditioned medium. The effects of NETs on cancer cells were further investigated in vitro and in vivo. NETs induced the epithelial to mesenchymal transition in cancer cells and thereby promoted their migration and invasion. HMGB1 derived from NETs appeared to potentiate the malignancy of cancer cells. In a mouse model of liver metastasis with inflammation, NETs participated in the metastatic process by enhancing extravasation. Interestingly, thrombomodulin degraded HMGB1 and consequently inhibited the induction of NETs, thereby preventing pancreatic cancer metastasis to the liver. In conclusion, NETs interact reciprocally with pancreatic cancer cells, which play a pivotal role in inflammation-associated metastasis. Targeting NETs with thrombomodulin can be a novel strategy to improve the surgical outcome of pancreatic cancer patients. [ABSTRACT FROM AUTHOR]

Solid tumors are composed of tumor cells and stromal cells including lymphatic endothelial cells (LEC), which are mainly viewed as cells forming lymphatic vessels involved in the transport of metastatic and immune cells. We here reveal a new mechanism by which tumor exposed-LEC (teLEC) exert mitogenic effects on tumor cells. Our conclusions are supported by morphological and molecular changes induced in teLEC that in turn enhance cancer cell invasion in 3D cultures and tumor cell proliferation in vivo. The characterization of teLEC secretome by RNA-Sequencing and cytokine array revealed that interleukine-6 (IL6) is one of the most modulated molecules in teLEC, whose production was negligible in unexposed LEC. Notably, neutralizing anti-human IL6 antibody abrogated teLEC-mediated mitogenic effects in vivo, when LEC were mixed with tumor cells in the ear sponge assay. We here assign a novel function to teLEC that is beyond their role of lymphatic vessel formation. This work highlights a new paradigm, in which teLEC exert "fibroblast-like properties", contribute in a paracrine manner to the control of tumor cell properties and are worth considering as key stromal determinant in future studies. [ABSTRACT FROM AUTHOR]

Cancer cells can communicate with the tumor microenvironment and contribute to tumor progression. However, the effects of drug-resistant tumor cells on angiogenesis are unclear. Current anti-angiogenic strategies also have limitations and it would be useful to develop novel targets and treatment strategies. Here, our study showed that the conditioned medium and exosomes from 5-FU-resistant colon cancer cells promoted angiogenesis, and we observed that exosomal dipeptidyl peptidase IV (DPP4) was a potent inducer of this angiogenesis. DPP4-enriched exosomes increased periostin (POSTN) expression in human umbilical vein endothelial cells via Twist1 nuclear translocation or activating Smad signaling pathway, while silencing or inhibition of DPP4 neutralized those effects. The in vivo and clinical data indicated that high DPP4 expression was related to tumor progression. These findings indicate that DPP4 may be a target for inhibiting angiogenesis in 5-FU-resistant colon cancer. Furthermore, exosomal DPP4 concentrations may be a useful prognostic marker for colon cancer. [ABSTRACT FROM AUTHOR]

Human papillomavirus (HPV) is the etiological agent of cervical cancer; however, the mechanisms underlying HPV-mediated carcinogenesis remain poorly understood. Here, we showed that nuclear receptor related-1 protein (Nurr1) was upregulated in primary cervical cancer tissue-derived spheroid cells and HPV-positive cell lines, and Nurr1 upregulation was correlated with cancer grade. Nurr1 promoted cell proliferation, migration, invasion, and anchorage-independent cell growth. In addition to its effect on cancer aggressiveness, Nurr1 enhanced the self-renewal ability of cells in vitro and in vivo, underscoring the importance of Nurr1 in maintaining the stemness of cancer stem-like cells (CSLCs). Mechanistically, Nurr1 independently activated the MEK/ERK and PI3K/Akt/mTOR signaling cascades. The MEK inhibitor trametinib (GSK) and PI3K/mTOR dual inhibitor dactolisib (BEZ) were shown to abrogate Nurr1-augmented tumorigenesis by upregulating p21 and p27 expression and by suppressing MMP9 and KLF4 expression. We provided further evidence that BEZ, but not GSK, could abolish Nurr1-enhanced radioresistance, suggesting its potential value for radiosensitizing CSLCs in the clinical setting. This study highlights the unprecedented roles of Nurr1 and elucidates mechanisms by which Nurr1 promotes tumor progression and radioresistance, providing a novel therapeutic strategy for cervical cancer treatment. [ABSTRACT FROM AUTHOR]

Most nucleoside anticancer drugs show a primary resistance to p53-deficient or p53-mutated cancer cells and are limited in the clinic to the treatment of hematological malignancies. However, 2'-fluoro-4'-seleno-ara-C (F-Se-Ara-C), a new generation of cytarabine (Ara-C) analogs, exhibited potent antitumor activity against the p53-deficient prostate cancer cell line PC-3. The distinct activity of F-Se-Ara-C was achieved by targeting the synthetic lethal interaction between p53 and mitogen-activated protein kinase-activated protein kinase-2 (MK2). MK2 is a checkpoint effector for DNA damage responses to drive cell cycle arrest and DNA repair in p53-deficient cancer cells. Therefore, targeting MK2 may be an effective therapeutic strategy that induces apoptosis for cancers deficient in p53. F-Se-Ara-C effectively induced anti-prostate cancer activity in vitro and in vivo by inhibition of MK2 activation in p53-deficient prostate cancer cells. Moreover, combining F-Se-Ara-C with cabozantinib, an anticancer drug currently in clinical use, induced synergistic antitumor activity in p53-deficient prostate cancer cells. Taken together, these data show that F-Se-Ara-C may become great anticancer drug candidate with its unique mechanism of action for overcoming the apoptotic resistance of p53-deficient cells by targeting the synthetic lethal interaction. [ABSTRACT FROM AUTHOR]

In cancer genomics, recurrence of mutations in gene families that share homologous domains has recently emerged as a reliable indicator of functional impact and can be exploited to reveal the pro-oncogenic effect of previously uncharacterized variants. Pan-cancer analyses of mutation hotspots in the catalytic domain of a subset of tyrosine kinase receptors revealed that two infrequent mutations of VEGFR2 (R1051Q and D1052N) recur in analogous proteins and correlate with reduced patient survival. Functional validation showed that both R1051Q and D1052N mutations increase the enzymatic activity of VEGFR2. The expression of VEGFR2R1051Q potentiates the PI3K/Akt signaling axis in cancer cells, increasing their tumorigenic potential in vitro and in vivo. In addition, it confers to cancer cells an increased sensitivity to the VEGFR2-targeted tyrosine kinase inhibitor Linifanib. In the context of an efficacious application of anti-cancer targeted therapies, these findings indicate that the screening for uncharacterized mutations, like VEGFR2R1051Q, may help to predict patient prognosis and drug response, with significant clinical implications. [ABSTRACT FROM AUTHOR]

An increasing number of studies have shown that circular RNAs (circRNAs) play important roles in malignant tumor initiation and progression; however, many circRNAs are yet unidentified, and the role of circRNAs in nasopharyngeal carcinoma (NPC) is unclear. Using RNA sequencing, we discovered a novel circRNA, termed circARHGAP12, that was processed from the pre-mRNA of the ARHGAP12 gene. CircARHGAP12 was significantly upregulated in NPC tissues and cell lines and promoted NPC cell migration and invasion. Overexpression or knockdown experiments revealed that circARHGAP12 regulates the expression of cytoskeletal remodeling-related proteins EZR, TPM3, and RhoA. CircARHGAP12 was found to bind directly to the 3' UTR of EZR mRNA and promote its stability; moreover, EZR protein interacted with TPM3 and RhoA and formed a complex to promote NPC cell invasion and metastasis. This study identified the novel circRNA circARHGAP12, characterized its biological function and mechanism, and increased our understanding of circRNAs in NPC pathogenesis. In particular, circARHGAP12 was found to promote the malignant biological phenotype of NPC via cytoskeletal remodeling, thus providing a clue for targeted therapy of NPC. [ABSTRACT FROM AUTHOR]

Context: Cushing disease (CD) is a life-threatening disorder. Therapeutic goals include symptom relief, biochemical control, and tumor growth inhibition. Current medical therapies for CD by and large exert no action on tumor growth.Objective: To identify drugs that inhibit corticotroph tumor adrenocorticotropic hormone (ACTH) secretion and growth.Design: High throughput screen employing a novel "gain of signal" ACTH AlphaLISA assay.Setting: Academic medical center.Patients: Corticotroph tumor tissues from patients with CD.Interventions: None.Main Outcome Measures: Potent inhibitors of corticotroph tumor ACTH secretion and growth.Results: From a kinase inhibitor library, we identified the dual PI3K/HDAC inhibitor CUDC-907 as a potent inhibitor of murine and human corticotroph tumor ACTH secretion (median effective concentration 1-5 nM), and cell proliferation (median inhibitory concentration 5 nM). In an in vivo murine corticotroph tumor xenograft model, orally administered CUDC-907 (300 mg/kg) reduced corticotroph tumor volume (TV [cm3], control 0.17 ± 0.05 vs CUDC-907 0.07 ± 0.02, P < .05) by 65% and suppressed plasma ACTH (ACTH [pg/mL] control 206 ± 27 vs CUDC-907 47 ± 7, P < .05) and corticosterone (corticosterone [ng/mL] control 180 ± 87 vs CUDC-907 27 ± 5, P < .05) levels by 77% and 85% respectively compared with controls. We also demonstrated that CUDC-907 acts through HDAC1/2 inhibition at the proopiomelanocortin transcriptional level combined with its PI3K-mediated inhibition of corticotroph cell viability to reduce ACTH secretion.Conclusions: Given its potent efficacy in in vitro and in vivo models of CD, combined with proven safety and tolerance in clinical trials, we propose CUDC-907 may be a promising therapy for CD. [ABSTRACT FROM AUTHOR]

Purpose: The study of breast cancer nearly always involves patients close to menopause or older. Therefore, young patients are mostly underrepresented. Our aim in this study was to demonstrate biological differences in breast cancer of young people using as a model available cell lines derived from people with breast cancer younger than 35 years. Methods: Global miRNA expression was analyzed in breast cancer cells from young (HCC1500, HCC1937) and old patients (MCF-7, MDA-MB-231, HCC1806, and MDA-MB-468). In addition, it was compared with same type of results from patients. Results: We observed a differential profile for 155 miRNAs between young and older cell lines. We identified a set of 24 miRNA associated with aggressiveness that were regulating pluripotency of stem cell-related pathways. Combining the miRNA expression data from cell lines and breast cancer patients, 132 miRNAs were differently expressed between young and old samples, most of them previously found in cell lines. MiR-23a-downregulation was also associated with poor survival in young patients. Conclusions: Our results suggest that HCC1500 and HCC1937 cell lines could be suitable cellular models for breast cancer affecting young women. The miR-23a-downregulation could have a potential role as a poor prognosis biomarker in this age group. [ABSTRACT FROM AUTHOR]

Background: Stearoyl-CoA desaturase-1 (SCD1) is reported to play essential roles in cancer stemness among several cancers. Our previous research revealed significant overexpression of SCD1 in primary gastric cancer stem cells (GCSCs), with its functional role still unknown.Methods: We stably established three primary GCSCs by sphere-forming assays and flow cytometry. Protein quantification and bioinformatics analysis were performed to reveal the differential protein pattern. Lentivirus-based small-interfering RNA (siRNA) knockdown and pharmacological inhibition approaches were used to characterise the function and molecular mechanism role of SCD1 in the regulation of GC stemness and tumour metastasis capacity both in vitro and in vivo.Results: SCD1 was found to increase the population of GCSCs, whereas its suppression by an SCD1 inhibitor or knockdown by siRNA attenuated the stemness of GCSCs, including chemotherapy resistance and sphere-forming ability. Furthermore, SCD1 suppression reversed epithelial-to-mesenchymal transition and reduced the GC metastasis probability both in vitro and in vivo. Downregulation of SCD1 in GCSCs was associated with the expression of Yes-associated protein (YAP), a key protein in the Hippo pathway, and nuclear YAP translocation was also blocked by the SCD1 decrease.Conclusions: SCD1 promotes GCSC stemness through the Hippo/YAP pathway. Targeting SCD1 might be a novel therapeutic strategy, especially to suppress GC metastasis and sensitise chemotherapy. [ABSTRACT FROM AUTHOR]

Background and Purpose: Breast cancer is the leading cause of death in women worldwide, with resistance to current therapeutic strategies, including tamoxifen, causing major clinical challenges and leading to more aggressive and metastatic disease. To address this, novel strategies that can inhibit the mechanisms responsible for tamoxifen resistance need to be assessed.Experimental Approach: We examined the effect of the novel, clinically-trialled, thiosemicarbazone anti-cancer agent, DpC, and its potential as a combination therapy with the clinically used estrogen receptor (ER) antagonist, tamoxifen, using both tamoxifen-resistant and -sensitive, human breast cancer cells (MDA-MB-453, MDA-MB-231 and MCF-7) in 2D and 3D cell-culture. Synergy was assessed using the Chou-Talalay method. The molecular and anti-proliferative effects of these agents and their combination was examined via Western blot, immunofluorescence and colony formation assays.Key Results: Combinations of tamoxifen with DpC were highly synergistic, leading to potent inhibition of cell proliferation, colony formation, and ER-α transcriptional activity. The combination also more efficiently reduced major molecular drivers of proliferation of tamoxifen-resistant cells, including c-Myc, cyclin D1, and p-AKT, while up-regulating the cell cycle inhibitor, p27, and inhibiting oncogenic phosphorylation of ER-α at Ser167. Assessing these effects using 3D cell culture further confirmed the greater effects of DpC combined with tamoxifen in reducing ER-α expression, and that of the proliferation marker, Ki-67, in both tamoxifen-sensitive and -resistant MCF-7 spheroids.Conclusions and Implications: These studies demonstrate that the synergistic combination of DpC with tamoxifen could be a promising new therapeutic strategy to overcome tamoxifen resistance in ER-positive breast cancer. [ABSTRACT FROM AUTHOR]

Background: Urothelial carcinomas (UCs) are highly prevalent in patients with end-stage renal disease. Chronic kidney disease (CKD) is the predecessor of end-stage renal disease, and it is also associated with UC. However, the interplay between CKD and UC lacks solid evidence. Acrolein is produced by polyamines and has been suggested to be the uremic "toxin." The level of acrolein correlates well with chronic renal failure. We recently found that acrolein-induced DNA damage and inhibited DNA repair in urothelial cells, which contribute to bladder cancer. Therefore, we hypothesize that acrolein is involved in the formation of UC in patients with CKD.Materials and Methods: A total of 62 UC patients and 43 healthy control subjects were recruited. Acrolein-DNA (Acr-dG) adducts and p53 gene mutations in UC tissues, plasma acrolein-protein conjugates (Acr-PC) and S-(3-hydroxypropyl)-N-acetylcysteine levels, and urinary Acr metabolites were analyzed in these patients.Results: Acr-dG levels were statistically correlated with CKD stages in UC patients (P < 0.01). Most p53 mutations were G to A and G to T mutations in these patients, and 50% of mutations at G:C pairs occurred in CpG sites, which is similar to the mutational spectra induced by Acr-dG adducts. Acr-PC levels in the plasma of UC patients with CKD were significantly higher than those of control subjects (P < 0.001). Altered urinary S-(3-hydroxypropyl)-N-acetylcysteine was also found in UC patients with CKD compared to control subjects (P < 0.005).Conclusion: These results indicate that acrolein acts as an endogenous uremic toxin and contributes to UC formation in patients with CKD. [ABSTRACT FROM AUTHOR]

Approximately 15% of patients with multiple myeloma (MM) harbour the t(4;14) chromosomal translocation, leading to the overexpression of the histone methyltransferase NSD2. Patients with this translocation display increased tumour dissemination, accelerated disease progression and rapid relapse. Using publicly available gene expression profile data from NSD2high (n = 135) and NSD2low (n = 878) MM patients, we identified 39 epithelial-mesenchymal transition (EMT)-associated genes which are overexpressed in NSD2high MM plasma cells. In addition, our analyses identified Twist-1 as a key transcription factor upregulated in NSD2high MM patients and t(4;14)-positive cell lines. Overexpression and knockdown studies confirmed that Twist-1 is involved in driving the expression of EMT-associated genes in the human MM cell line KMS11 and promoted the migration of myeloma cell lines in vitro. Notably, Twist-1 overexpression in the mouse MM cell line 5TGM1 significantly increased tumour dissemination in an intratibial tumour model. These findings demonstrate that Twist-1, downstream of NSD2, contributes to the induction of an EMT-like signature in t(4;14)-positive MM and enhances the dissemination of MM plasma cells in vivo, which may, in part, explain the aggressive disease features associated with t(4;14)-positive MM. [ABSTRACT FROM AUTHOR]

Clinical data analysis reveals that the expression of the EphB4 receptor tyrosine kinase is significantly elevated in HER2-positive breast cancer and high levels of EphB4 strongly correlate with poor disease prognosis. However, the impact of EphB4 activation on HER2-positive breast cancer cells and the potential of EphB4 as a therapeutic target remain to be explored. Here, we show that EphB4 overexpression confers gain-of-function activities to HER2-positive breast cancer cells, rendering resistance to a HER2/EGFR inhibitor Lapatinib. Furthermore, using integrated transcriptomic and tyrosine phosphoproteomic analyses, followed by biochemical confirmation, we establish that EphB4 activation engages the SHP2/GAB1-MEK signaling cascade and downstream c-MYC activation, and thereby limits the overall drug responses to Lapatinib. Finally, we demonstrate that, in HER2-positive breast tumors, inhibition of EphB4 combined with Lapatinib is more effective than either alone. These findings provide new insights into the signaling networks dictating therapeutic response to Lapatinib as well as a rationale for co-targeting EphB4 in HER2-positive breast cancer. [ABSTRACT FROM AUTHOR]

Tumor-associated macrophages (TAMs) are important immunocytes associated with cancer metastasis. However, whether TAMs play a dominant role in mediating CXCL12/CXCR4-induced liver metastasis of colorectal cancer (CRC) remains unexplored. Herein, we found that CD206+ TAMs, which infiltrated at the invasive front, were correlated with CXCR4 expression and liver metastasis of CRC in clinical specimens. Several miRNAs (miR-25-3p, miR-130b-3p, miR-425-5p), upregulated in CRC cells by activation of the CXCL12/CXCR4 axis, could be transferred to macrophages via exosomes. These exosomal miRNAs induced M2 polarization of macrophages by regulating PTEN through activation of PI3K/Akt signaling pathway. In turn, M2 polarized macrophages promoted cancer metastasis by enhancing epithelial-mesenchymal transition (EMT) and secreting vascular endothelial growth factor (VEGF). Co-culture of CRC cells with macrophages transfected with these miRNAs or treated with exosomes enhanced their metastatic capacity both in vitro and in vivo. Clinically, the serum levels of exosomal miR-25-3p, miR-130b-3p and miR-425-5p were correlated with progression and metastasis of CRC. In conclusion, these results reveal a crucial role of exosomal miRNAs in mediating the crosstalk between CXCR4 overexpressing cancer cells and TAMs, providing potential therapeutic targets for circumventing liver metastasis of CRC. [ABSTRACT FROM AUTHOR]

Nuclear factor erythroid-2-related factor 2 (Nrf2), a transcription factor, participates in protecting cells from electrophilic or oxidative stresses through regulating expression of cytoprotective and antioxidant genes. It has become one of the emerging targets for cancer chemosensitization, and small molecule inhibitors of Nrf2 can enhance the efficacy of chemotherapeutic drugs. Here, we found that flumethasone, a glucocorticoid, inhibited Nrf2 signaling in A549 and H460 cells by promoting Nrf2 protein degradation. Moreover, flumethasone significantly increased the sensitivity of A549 and H460 cells to chemotherapeutic drugs including cisplatin, doxorubicin and 5-FU. In mice bearing A549-shControl cells-derived xenografts, the size and weight of xenografts in the flumethasone and cisplatin combination group had a significant reduction compared with those in the cisplatin group, while in mice bearing A549-shNrf2 cells-derived xenografts, the size and weight of the xenografts in the combination group had no significant difference compared with those in the cisplatin group, demonstrating that chemosensitization effect of flumethasone is Nrf2-dependent. This work suggests that flumethasone can potentially be used as an adjuvant sensitizer to enhance the efficacy of chemotherapeutic drugs in lung cancer. [ABSTRACT FROM AUTHOR]

Background: PLEKHA5 has previously been identified as a novel molecule implicated in melanoma brain metastasis, a disease that continues to portend a poor prognosis. The aim of this study was to further investigate the functional role of PLEKHA5 in disseminated melanoma.Methods: The impact of PLEKHA5 on proliferation and tumor growth was examined in vitro and in melanoma xenograft models, including brain-tropic melanomas (melanomas tending to disseminate to the brain). In vitro loss- and gain-of-function studies were used to explore the underlying mechanisms of PLEKHA5-mediated tumor growth and the crosstalk between PLEKHA5 and PI3K/AKT/mTOR or MAPK/ERK signaling. The clinical relevance of PLEKHA5 dysregulation was further investigated in a cohort of matched cranial and extracranial melanoma metastases.Results: PLEKHA5 stable knockdown negatively regulated cell proliferation by inhibiting the G1 -to-S cell cycle transition, which coincided with upregulation of the cell cycle regulator PDCD4. Conversely, ectopic PLEKHA5 expression exhibited the inverse effect. PLEKHA5 knockdown significantly inhibited tumor growth, whereas its overexpression upregulated the growth of tumors, which was induced by cranial and subcutaneous inoculation of cells in nude mice. PLEKHA5 modulation affected PDCD4 protein stability and was coupled with changes in PI3K/AKT/mTOR pathway signaling. High PDCD4 expression in cerebral specimens was associated with better overall survival.Conclusions: This study further supports the role of PLEKHA5 as a regulator of melanoma growth at distant sites, including the brain. Furthermore, the results highlight the significance of PDCD4 dysregulation in disseminated melanoma and implicate PDCD4 as a possible causal link between PLEKHA5 and cell proliferation and growth. [ABSTRACT FROM AUTHOR]

Purpose: Pyruvate kinase M2 (PKM2) catalyzes the final step in glycolysis, the key process of tumor metabolism. PKM2 is found in high levels in glioblastoma (GBM) cells with marginal expression within healthy brain tissue, rendering it a key biomarker of GBM metabolic re-programming. Our group has reported the development of a novel radiotracer, 1-((2-fluoro- 6-[18F]fluorophenyl)sulfonyl)-4-((4-methoxyphenyl)sulfonyl)piperazine ([18F]DASA- 23), to non-invasively detect PKM2 levels with positron emission tomography (PET).Procedure: U87 human GBM cells were treated with the IC50 concentration of various agents used in the treatment of GBM, including alkylating agents (temozolomide, carmustine, lomustine, procarbazine), inhibitor of topoisomerase I (irinotecan), vascular endothelial and epidermal growth factor receptor inhibitors (cediranib and erlotinib, respectively) anti-metabolite (5-fluorouracil), microtubule inhibitor (vincristine), and metabolic agents (dichloroacetate and IDH1 inhibitor ivosidenib). Following drug exposure for three or 6 days (n = 6 replicates per condition), the radiotracer uptake of [18F]DASA-23 and 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) was assessed. Changes in PKM2 protein levels were determined via Western blot and correlated to radiotracer uptake.Results: Significant interactions were found between the treatment agent (n = 12 conditions total comprised 11 drugs and vehicle) and the duration of treatment (3- or 6-day exposure to each drug) on the cellular uptake of [18F]DASA-23 (p = 0.0001). The greatest change in the cellular uptake of [18F]DASA-23 was found after exposure to alkylating agents (p < 0. 0001) followed by irinotecan (p = 0. 0012), erlotinib (p = 0. 02), and 5-fluorouracil (p = 0. 005). Correlation of PKM2 protein levels and [18F]DASA-23 cellular uptake revealed a moderate correlation (r = 0.44, p = 0.15).Conclusions: These proof of principle studies emphasize the superiority of [18F]DASA-23 to [18F]FDG in detecting the glycolytic response of GBM to multiple classes of anti-neoplastic drugs in cell culture. A clinical trial evaluating the diagnostic utility of [18F]DASA-23 PET in GBM patients (NCT03539731) is ongoing. [ABSTRACT FROM AUTHOR]

Background: We aimed at clarifying the role of lipocalin-2 (LCN-2) in clear-cell renal cell carcinoma (ccRCC). Since LCN-2 was recently identified as a novel iron transporter, we explored its iron load as a decisive factor in conferring its biological function.Methods: LCN-2 expression was analysed at the mRNA and protein level by using immunohistochemistry, RNAscope® and qRT-PCR in patients diagnosed with clear-cell renal cell carcinoma compared with adjacent healthy tissue. We measured LCN-2-bound iron by atomic absorption spectrometry from patient-derived samples and applied functional assays by using ccRCC cell lines, primary cells, and 3D tumour spheroids to verify the role of the LCN-2 iron load in tumour progression.Results: LCN-2 was associated with poor patient survival and LCN-2 mRNA clustered in high- and low-expressing ccRCC patients. LCN-2 protein was found overexpressed in tumour compared with adjacent healthy tissue, whereby LCN-2 was iron loaded. In vitro, the iron load determines the biological function of LCN-2. Iron-loaded LCN-2 showed pro-tumour functions, whereas iron-free LCN-2 produced adverse effects.Conclusions: We provide new insights into the pro-tumour function of LCN-2. LCN-2 donates iron to cells to promote migration and matrix adhesion. Since the iron load of LCN-2 determines its pro-tumour characteristics, targeting either its iron load or its receptor interaction might represent new therapeutic options. [ABSTRACT FROM AUTHOR]

The PIK3 CA gene encodes the p110α protein subunit and is one of the most efficient cancer genes in solid and hematological tumors including hepatocellular carcinoma (HCC). There are currently ongoing therapies against tumors based on PIK3 CA inhibition. Because microRNAs (miRNAs) play an important role in post-transcriptional regulation and are also involved in the inhibition of PIK3 CA expression to suppress cancer cell proliferation, overexpression of tumor-suppressive miRNA is a promising therapeutic approach for HCC therapy. The successful and localized delivery of miRNA overexpression vectors (pre-miRNA plasmids) is very important in improving the therapeutic efficacy of this miRNA therapy strategy. In the study described here, submicron acoustic phase-shifted nanodroplets were used to efficiently deliver pre-miRNA plasmid in vitro and in vivo for HCC therapy under focused ultrasound (US) activation. Briefly, six miRNAs, inhibiting PIK3 CA and downregulated in HCC, were selected through summary and analysis of the currently existing literature data. Quantitative real-time polymerase chain reaction (qRT-PCR), Western blot and cell apoptosis assay revealed that pre-miR-139, -203a, -378a and -422a plasmids among the six miRNA overexpression vectors could suppress growth of the hepatoma cell line SMMC-7721. These four pre-miRNA plasmids were then electrostatically adhered to positively charged lipid-shelled nanodroplets to obtain plasmid-loaded nanodroplets (PLNDs). The PLND-generated microbubbles oscillated and even collapsed under US exposure to release the loaded pre-miRNA plasmids and enhance their cellular uptake through consequent sonoporation, that is, formation of small pores on the cell membrane induced by the mechanical effects of PLND cavitation. Fluorescence microscopy results revealed that PLNDs could effectively deliver the aforementioned four pre-miRNA plasmids into SMMC-7721 cells in vitro under 1.2-MHz 60-cycle sinusoid US exposure with a peak negative pressure >5.5 MPa at a 40-Hz pulse repetition frequency. Plasmid delivery efficiency and cell viability positively correlated with the inertial cavitation dose that was determined mainly by peak negative pressure. Furthermore, PLNDs combined with US were evaluated in vivo to deliver these four pre-miRNAs plasmids and verify their therapeutic efficacy in subcutaneous tumor of the mouse xenograft HCC model. The results revealed that the PLNDs loaded with pre-miR-139 and -378a plasmids could effectively suppress tumor growth after US treatment. Thus, combination of pre-miRNA PLNDs with US activation seems to constitute a potential strategy for HCC therapy. [ABSTRACT FROM AUTHOR]

Purpose: Triple-negative breast cancer (TNBC) is an aggressive, lethal, and heterogeneous subtype of breast cancers, tending to have lower 5-year survival rates than other BC subtypes in response to conventional chemotherapies. This study's aim was to identify advanced regimens to effectively control TNBC tumor development.Methods: We investigated the combination of the DNA synthesis inhibitor gemcitabine, the DNA-damaging agent cisplatin, and the histone deacetylase inhibitor romidepsin to control a variety of breast cells in vitro. We studied the toxicity of drug doses and administration schedules to determine tolerable combination regimens in immune-deficient nude and -competent BALB/c mice. We then studied the efficacy of tolerable regimens in controlling TNBC cell-derived xenograft development in nude mice. By reducing clinically equivalent doses of each agent in combination, we formulated tolerable regimens in animals. We verified that the tolerable triple combination gemcitabine plus romidepsin + cisplatin regimen more efficacious than double combination regimens in controlling xenograft tumor development in nude mice.Results: A triple combination of gemcitabine + romidepsin + cisplatin synergistically induced death of the TNBC M.D. Anderson-Metastatic Breast cancer (MDA-MB) 231 and MDA-MB468, as well as Michigan Cancer Foundation (MCF) 7, MCF10A, and MCF10A-Ras cells. Cell death induced by gemcitabine + romidepsin + cisplatin was in a reactive oxygen species-dependent manner.Conclusion: Considering the high costs for developing a new anticancer agent, we used the FDA-approved drugs gemcitabine, romidepsin (is approved for T-cell lymphoma and is under clinical trial for TNBC), and cisplatin to economically formulate an efficacious and safe combination regimen. The highly efficacious gemcitabine plus romidepsin + cisplatin regimen should be poised for efficient translation into clinical trials, ultimately contributing to reduced mortality and improved quality of life for TNBC patients. [ABSTRACT FROM AUTHOR]