Evaluation of the effect of hyperthermia and electron radiation on prostate cancer stem cells

The aim of this study was to investigate the effect of hyperthermia, 6 MeV electron radiation and combination of these treatments on cancer cell line DU145 in both monolayer culture and spheroids enriched for prostate cancer stem cells (CSCs). Flowcytometric analysis of the expression of molecular markers CD133⁺/CD44⁺ was carried out to determine the prostate CSCs in cell line DU145 grown as spheroids in serum-free medium. Following monolayer and spheroid culture, DU145 cells were treated with different doses of hyperthermia, electron beam and combination of them. The survival and self-renewing of the cells were evaluated by colony formation assay (CFA) and spheroid formation assay (SFA). Flowcytometry results indicated that the percentage of CD133⁺/CD44⁺ cells in spheroid culture was 13.9-fold higher than in the monolayer culture. The SFA showed significant difference between monolayer and spheroid culture for radiation treatment (6 Gy) and hyperthermia (60 and 90 min). The CFA showed significantly enhanced radiosensitivity in DU145 cells grown as monolayer as compared to spheroids, but no effect of hyperthermia. In contrast, for the combination of radiation and hyperthermia the results of CFA and SFA showed a reduced survival fraction in both cultures, with larger effects in monolayer than in spheroid culture. Thus, hyperthermia may be a promising approach in prostate cancer treatment that enhances the cytotoxic effect of electron radiation. Furthermore, determination and characterization of radioresistance and thermoresistance of CSCs in the prostate tumor is the key to develop more efficient therapeutic strategies.     

A 3D Fibrous Scaffold Inducing Tumoroids: A Platform for Anticancer Drug Development

The development of a suitable three dimensional (3D) culture system for anticancer drug development remains an unmet need. Despite progress, a simple, rapid, scalable and inexpensive 3D-tumor model that recapitulates in vivo tumorigenesis is lacking. Herein, we report on the development and characterization of a 3D nanofibrous scaffold produced by electrospinning a mixture of poly(lactic-co-glycolic acid) (PLGA) and a block copolymer of polylactic acid (PLA) and mono-methoxypolyethylene glycol (mPEG) designated as 3P. Cancer cells cultured on the 3P scaffold formed tight irregular aggregates similar to in vivo tumors, referred to as tumoroids that depended on the topography and net charge of the scaffold. 3P scaffolds induced tumor cells to undergo the epithelial-to-mesenchymal transition (EMT) as demonstrated by up-regulation of vimentin and loss of E-cadherin expression. 3P tumoroids showed higher resistance to anticancer drugs than the same tumor cells grown as monolayers. Inhibition of ERK and PI3K signal pathways prevented EMT and reduced tumoroid formation, diameter and number. Fine needle aspirates, collected from tumor cells implanted in mice when cultured on 3P scaffolds formed tumoroids, but showed decreased sensitivity to anticancer drugs, compared to tumoroids formed by direct seeding. These results show that 3P scaffolds provide an excellent platform for producing tumoroids from tumor cell lines and from biopsies and that the platform can be used to culture patient biopsies, test for anticancer compounds and tailor a personalized cancer treatment.     

Fabrication of 3D calcium-alginate scaffolds for human glioblastoma modeling and anticancer drug response evaluation

The three-dimensional (3D) cell culture model has been increasingly used to study cancer biology and screen for anticancer agents due to its close mimicry to in vivo tumor biopsies. In this study, 3D calcium(Ca)-alginate scaffolds were developed for human glioblastoma cell culture and an investigation of the responses to two anticancer agents, doxorubicin and cordycepin. Compared to the 2D monolayer culture, glioblastoma cells cultured on these 3D Ca-alginate scaffolds showed reduced cell proliferation, increased tumor spheroid formation, enhanced expression of cancer stem cell genes (CD133, SOX2, Nestin, and Musashi-1), and improved expression of differentiation potential-associated genes (GFAP and β-tubulin III). Additionally, the vascularization potential of the 3D glioblastoma cells was increased, as indicated by a higher expression of tumor angiogenesis biomarker (VEGF) than in the cells in 2D culture. To highlight the application of Ca-alginate scaffolds, the 3D glioblastomas were treated with anticancer agents, including doxorubicin and cordycepin. The results demonstrated that the 3D glioblastomas presented a greater resistance to the tested anticancer agents than that of the cells in 2D culture. In summary, the 3D Ca-alginate scaffolds for glioblastoma cells that were developed in this study offer a promising platform for anticancer agent screening and the discovery of drug-resistant mechanisms of cancer.     

Development and characterization of cancer stem cell-based tumoroids as an osteosarcoma model

Three-dimensional (3D) cancer tumor models are becoming vital approaches for high-throughput drug screening, drug targeting, development of novel theranostic systems, and personalized medicine. Yet, it is becoming more evident that the tumor progression and metastasis is fueled by a subpopulation of stem-like cells within the tumor that are also called cancer stem cells (CSCs). This study aimed to develop a tumoroid model using CSCs. For this purpose CD133⁺ cells were isolated from SaOS-2 osteosarcoma cell line with magnetic-activated cell sorting. To evaluate tumoroid formation ability, the cells were incubated in different cell numbers in agar gels produced by 3D Petri Dish® method. Subsequently, CD133⁺ cells and CD133⁻ cells were co-cultured to investigate CD133⁺ cell localization in tumoroids. The characterization of tumoroids was performed using Live&Dead staining, immunohistochemistry, and quantitative polymerase chain reaction analysis. The results showed that, CD133⁺, CD133⁻ and SaOS-2 cells were all able to form 3D tumoroids regardless of the initial cell number, but, while 72 hr were needed for CD133⁺ cells to self-assemble, 24 hr were enough for CD133⁻ and SaOS-2 cells. CD133⁺ cells were located within tumoroids randomly with high cell viability. Finally, when compared to two-dimensional (2D) cultures, there were 5.88, 4.14, 6.95, and 1.68-fold higher messenger RNA expressions for Sox2, OCT3/4, Nanog, and Nestin, respectively, in CD133⁺ cells that were cultured within 3D tumoroids, showing longer maintenance of stem cell phenotype in 3D, that can allow more relevant screening and targeting efficiency in pharmaceutical testing. It was concluded that CSC-based tumoroids are propitious as 3D tumor models to fill the gap between conventional 2D in vitro culture and in vivo animal experiments for cancer research.     

Induction of cancer cell stemness by chemotherapy

Recent studies indicate that cancer stem cells (CSCs) exist in most hematological and solid tumors. CSCs are characterized by their ability to self-renew and their capacity to differentiate into the multitude of cells that comprise the tumor mass. Moreover, these cells have been shown to be intrinsically resistant to conventional anticancer therapies. Despite their fundamental role in cancer pathogenesis, the cellular origin of CSCs remains highly controversial. The aim of this study was to examine whether heterogeneous cancer cells can acquire stem cell-like properties in response to chemotherapy. We demonstrate that carboplatin can induce the self-renewal (spherogenesis) and pluripotency (Sox2 and Oct3/4 expression) of hepatocellular carcinoma (HCC) cells grown under stem cell culture conditions. Moreover, we show that non-CSC cells, obtained by side population flow cytometric sorting using Hoechst 33342, can acquire stem-like properties after exposure to carboplatin. Finally, we show that knockdown of Sox2 and Oct3/4 gene expression in HCC cells can reduce carboplatin-mediated increases in sphere formation and increase cellular sensitivity to chemotherapy. Taken together, our data indicate that bulk cancer cells may be an important source of CSCs during tumor development, and that targeting Sox2 and/or Oct3/4 may be a promising approach for targeting CSCs in clinical cancer treatment.     

以胶原膜为支架的心肌细胞体外三维培养

将从新生乳鼠心室肌组织获取的心肌细胞接种于鼠尾胶原膜三维支架和组织培养板,以细胞形态、细胞搏动、葡萄糖比消耗率(q_glu)、乳酸比产率(q_lac)、乳酸转化率(Y_lac/glu)、肌酸激酶及乳酸脱氢酶的活力为观察指标,比较心肌细胞在鼠尾胶原膜中三维(3D)培养和组织培养板中二维(2D)培养的差异。培养于鼠尾胶原膜的乳鼠心肌细胞在第5天形成闰盘连接,形成面积约为80mm³、肉眼可见自律性同步收缩的心肌细胞3D培养物。3D培养体系中乳鼠心肌细胞的q_glu、q_lac和Y_lac/glu的均值分别为7.37 μmol/10.6cells/d、2.92 μmol/106cells/d和0.38 μmol/μmol;2D培养体系中乳鼠心肌细胞的q_glu、q_lac和Y_lac/glu的均值分别为7.59 μmol/10.6cells/d、3.83 μmol/10.6cells/d和 0.51 μmol/μmol。两种培养体系中乳鼠心肌细胞的肌酸激酶及乳酸脱氢酶的活力无明显差别。实验结果表明：培养于鼠尾胶原膜的心肌细胞保持正常心肌细胞的代谢活力和收缩功能。     

Doxorubicin and 5-fluorouracil resistant hepatic cancer cells demonstrate stem-like properties

The efficacy of hepatocellular carcinoma (HCC) treatment is very low because of the high percentage of recurrence and resistance to anticancer agents. Hepatic cancer stem cells (HCSCs) are considered the origin of such recurrence and resistance. Our aim was to evaluate the stemness of doxorubicin and 5-fluorouracil resistant hepatic cancer cells and establish the new method to isolate the HCSCs from primary cultured HCC tumors. HCC biopsies were used to establish primary cultures. Then, primary cells were selected for HCSCs by culture in medium supplemented with doxorubicin (0, 0.1, 0.25, 0.5 or 1 μg/mL), 5-fluorouracil (0, 0.1, 0.25, 0.5 or 1 μg/mL) or their combination. Selection was confirmed by detection of HCSC markers such as CD133, CD13, CD90, and the side population was identified by rhodamine 123 efflux. The cell population with the strongest expression of these markers was used to evaluate the cell cycle, gene expression profile, tumor sphere formation, marker protein expression, and in vivo tumorigenesis. Selective culture of primary cells in medium supplemented with 0.5 μg/mL doxorubicin and 1 μg/mL 5-fluorouracil selected cancer cells with the highest stemness properties. Selected cells strongly expressed CD13, CD133, CD90, and CD326, efflux rhodamine 123 and formed tumor spheres in suspension. Moreover, selected cells were induced to differentiate into cells with high expression of CD19 and AFP (alpha-fetoprotein), and importantly, could form tumors in NOD/SCID mice upon injection of 1 × 10⁵ cells/mouse. Selective culture with doxorubicin and 5-fluorouracil will enrich HCSCs, is an easy method to obtain HCSCs that can be used to develop better therapeutic strategies for patients with HCC, and particularly HCSC-targeting therapy.     

Three-dimensional polymeric systems for cancer cell studies

Three-dimensional (3-D) culture of cancer cells and of normal mammalian cells in a polymeric matrix is generally a better alternate model for understanding the regulation of cancer cell proliferation and for evaluation of different anticancer drugs. A substantial amount of evidence demonstrates important differences in the behavior of cells grown in monolayer, i.e., two-dimensional (2-D), and in 3-D cultures. Cancer cells grown in 3-D culture are more resistant to cytotoxic agents than cells in 2-D culture; growth of cells in vitro in 3-D requires a suitable polymer that provides a structural scaffold for cell adhesion and growth. Many naturally derived polymers as well as synthetic polymers have been investigated as scaffolds. The aim of this review is to overview the polymeric materials of natural and synthetic origin that are of specific interest to 3-D cell cultures, and discuss the development of new polymers that should be specifically designed for 3-D culture applications.     

Evidence that aberrant expression of tissue transglutaminase promotes stem cell characteristics in mammary epithelial cells

Cancer stem cells (CSCs) or tumor initiating cells (TICs) make up only a small fraction of total tumor cell population, but recent evidence suggests that they are responsible for tumor initiation and the maintenance of tumor growth. Whether CSCs/TICs originate from normal stem cells or result from the dedifferentiation of terminally differentiated cells remains unknown. Here we provide evidence that sustained expression of the proinflammatory protein tissue transglutaminase (TG2) confers stem cell like properties in non-transformed and transformed mammary epithelial cells. Sustained expression of TG2 was associated with increase in CD44(high)/CD24(low/-) subpopulation, increased ability of cells to form mammospheres, and acquisition of self-renewal ability. Mammospheres derived from TG2-transfected mammary epithelial cells (MCF10A) differentiated into complex secondary structures when grown in Matrigel cultures. Cells in these secondary structures differentiated into Muc1-positive (luminal marker) and integrin α6-positive (basal marker) cells in response to prolactin treatment. Highly aggressive MDA-231 and drug-resistant MCF-7/RT breast cancer cells, which express high basal levels of TG2, shared many traits with TG2-transfected MCF10A stem cells but unlike MCF10A-derived stem cells they failed to form the secondary structures and to differentiate into Muc1-positive luminal cells when grown in Matrigel culture. Downregulation of TG2 attenuated stem cell properties in both non-transformed and transformed mammary epithelial cells. Taken together, these results suggested a new function for TG2 and revealed a novel mechanism responsible for promoting the stem cell characteristics in adult mammary epithelial cells.     

miR-130b Promotes CD133(+) liver tumor-initiating cell growth and self-renewal via tumor protein 53-induced nuclear protein 1

A novel paradigm in tumor biology suggests that cancer growth is driven by stem-like cells within a tumor, called tumor-initiating cells (TICs) or cancer stem cells (CSCs). Here we describe the identification and characterization of such cells from hepatocellular carcinoma (HCC) using the marker CD133. CD133 accounts for approximately 1.3%-13.6% of the cells in the bulk tumor of human primary HCC samples. When compared with their CD133? counterparts, CD133(+) cells not only possess the preferential ability to form undifferentiated tumor spheroids in vitro but also express an enhanced level of stem cell-associated genes, have a greater ability to form tumors when implanted orthotopically in immunodeficient mice, and can be serially passaged into secondary animal recipients. Xenografts resemble the original human tumor and maintain a similar percentage of tumorigenic CD133(+) cells. Quantitative PCR analysis of 41 separate HCC tissue specimens with follow-up data found that CD133(+) tumor cells were frequently detected at low quantities in HCC, and their presence was also associated with worse overall survival and higher recurrence rates. Subsequent differential microRNA expression profiling of CD133(+) and CD133? cells from human HCC clinical specimens and cell lines identified an overexpression of miR-130b in CD133(+) TICs. Functional studies on miR-130b lentiviral-transduced CD133? cells demonstrated superior resistance to chemotherapeutic agents, enhanced tumorigenicity in vivo, and a greater potential for self renewal. Conversely, antagonizing miR-130b in CD133(+) TICs yielded an opposing effect. The increased miR-130b paralleled the reduced TP53INP1, a known miR-130b target. Silencing TP53INP1 in CD133? cells enhanced both self renewal and tumorigenicity in vivo. Collectively, miR-130b regulates CD133(+) liver TICs, in part, via silencing TP53INP1.     

Turning Hepatic Cancer Stem Cells Inside Out – A Deeper Understanding through Multiple Perspectives

Hepatocellular carcinoma (HCC), a highly malignant disease and the third leading cause of all cancer mortalities worldwide, often responses poorly to current treatments and results in dismal outcomes due to frequent chemoresistance and tumor relapse. The heterogeneity of HCC is an important attribute of the disease. It is the outcome of many factors, including the cross-talk between tumor cells within the tumor microenvironment and the acquisition and accumulation of genetic and epigenetic alterations in tumor cells. In addition, there is accumulating evidence in recent years to show that the malignancy of HCC can be attributed partly to the presence of cancer stem cell (CSC). CSCs are capable to self-renew, differentiate and initiate tumor formation. The regulation of the stem cell-like properties by several important signaling pathways have been found to endow the tumor cells with an increased level of tumorigenicity, chemoresistance, and metastatic ability. In this review, we will discuss the recent findings on hepatic CSCs, with special emphasis on their putative origins, relationship with hepatitis viruses, regulatory signaling networks, tumor microenvironment, and how these factors control the stemness of hepatic CSCs. We will also discuss some novel therapeutic strategies targeted at hepatic CSCs for combating HCC and perspectives of future investigation.     

26S proteasome activity is down-regulated in lung cancer stem-like cells propagated in vitro

Cancer stem cells (CSCs) are a small subset of cancer cells capable of self-renewal and tumor maintenance. Eradicating cancer stem cells, the root of tumor origin and recurrence, has emerged as one promising approach to improve lung cancer survival. Cancer stem cells are reported to reside in the side population (SP) of cultured lung cancer cells. We report here the coexistence of a distinct population of non-SP (NSP) cells that have equivalent self-renewal capacity compared to SP cells in a lung tumor sphere assay. Compared with the corresponding cells in monolayer cultures, lung tumor spheres, formed from human non-small cell lung carcinoma cell lines A549 or H1299, showed marked morphologic differences and increased expression of the stem cell markers CD133 and OCT3/4. Lung tumor spheres also exhibited increased tumorigenic potential as only 10,000 lung tumor sphere cells were required to produce xenografts tumors in nude mice, whereas the same number of monolayer cells failed to induce tumors. We also demonstrate that lung tumor spheres showed decreased 26S proteasome activity compared to monolayer. By using the ZsGreen-cODC (C-terminal sequence that directs degradation of Ornithine Decarboxylase) reporter assay in NSCLC cell lines, only less than 1% monolayer cultures were ZsGreen positive indicating low 26S proteasome, whereas lung tumor sphere showed increased numbers of ZsGreen-positive cells, suggesting the enrichment of CSCs in sphere cultures.     

Osteogenic differentiation is inhibited and angiogenic expression is enhanced in MC3T3-E1 cells cultured on three-dimensional scaffolds

Osteogenic differentiation of osteoprogenitor cells in three-dimensional (3D) in vitro culture remains poorly understood. Using quantitative real-time RT-PCR techniques, we examined mRNA expression of alkaline phosphatase, osteocalcin, and vascular endothelial growth factor (VEGF) in murine preosteoblastic MC3T3-E1 cells cultured for 48 h and 14 days on conventional two-dimensional (2D) poly(L-lactide-co-glycolide) (PLGA) films and 3D PLGA scaffolds. Differences in VEGF secretion and function between 2D and 3D culture systems were examined using Western blots and an in vitro Matrigel-based angiogenesis assay. Expression of both alkaline phosphatase and osteocalcin in cells cultured on 3D scaffolds was significantly downregulated relative to 2D controls in 48 h and 14 day cultures. In contrast, elevated levels of VEGF expression in 3D culture were noted at every time point in short- and long-term culture. VEGF protein secretion in 3D cultures was triple the amount of secretion observed in 2D controls. Conditioned medium from 3D cultures induced an enhanced level of angiogenic activity, as evidenced by increases in branch points observed in in vitro angiogenesis assays. These results collectively indicate that MC3T3-E1 cells commit to osteogenic differentiation at a slower rate when cultured on 3D PLGA scaffolds and that VEGF is preferentially expressed by these cells when they are cultured in three dimensions. gene expression; osteogenesis; angiogenesis     

Stem cell-derived extracellular matrix enables survival and multilineage differentiation within superporous hydrogels

Hydrophilic poly(ethylene glycol) diacrylate (PEGDA) hydrogel surfaces resist protein adsorption and are generally thought to be unsuitable for anchorage-dependent cells to adhere. Intriguingly, our previous findings revealed that PEGDA superporous hydrogel scaffolds (SPHs) allow anchorage of bone marrow derived human mesenchymal stem cells (hMSCs) and support their long-term survival. Therefore, we hypothesized that the physicochemical characteristics of the scaffold impart properties that could foster cellular responses. We examined if hMSCs alter their microenvironment to allow cell attachment by synthesizing their own extracellular matrix (ECM) proteins. Immunofluorescence staining revealed extensive expression of collagen type I, collagen type IV, laminin, and fibronectin within hMSC-seeded SPHs by the end of the third week. Whether cultured in serum-free or serum-supplemented medium, hMSC ECM protein gene expression patterns exhibited no substantial changes. The presence of serum proteins is required for initial anchorage of hMSCs within the SPHs but not for the hMSC survival after 24 h. In contrast to 2D expansion on tissue culture plastic (TCP), hMSCs cultured within SPHs proliferate similarly in the presence or absence of serum. To test whether hMSCs retain their undifferentiated state within the SPHs, cell-seeded constructs were cultured for 3 weeks in stem cell maintenance medium and the expression of hMSC-specific cell surface markers were evaluated by flow cytometry. CD105, CD90, CD73, and CD44 were present to a similar extent in the SPH and in 2D monolayer culture. We further demonstrated multilineage potential of hMSCs grown in the PEGDA SPHs, whereby differentiation into osteoblasts, chondrocytes, and adipocytes could be induced. The present study demonstrates the potential of hMSCs to alter the "blank" PEGDA environment to a milieu conducive to cell growth and multilineage differentiation by secreting adhesive ECM proteins within the porous network of the SPH scaffolds.     

Transient mTOR inhibition facilitates continuous growth of liver tumors by modulating the maintenance of CD133+ cell populations

The mammalian target of the rapamycin (mTOR) pathway, which drives cell proliferation, is frequently hyperactivated in a variety of malignancies. Therefore, the inhibition of the mTOR pathway has been considered as an appropriate approach for cancer therapy. In this study, we examined the roles of mTOR in the maintenance and differentiation of cancer stem-like cells (CSCs), the conversion of conventional cancer cells to CSCs and continuous tumor growth in vivo. In H-Ras-transformed mouse liver tumor cells, we found that pharmacological inhibition of mTOR with rapamycin greatly increased not only the CD133+ populations both in vitro and in vivo but also the expression of stem cell-like genes. Enhancing mTOR activity by over-expressing Rheb significantly decreased CD133 expression, whereas knockdown of the mTOR yielded an opposite effect. In addition, mTOR inhibition severely blocked the differentiation of CD133+ to CD133- liver tumor cells. Strikingly, single-cell culture experiments revealed that CD133- liver tumor cells were capable of converting to CD133+ cells and the inhibition of mTOR signaling substantially promoted this conversion. In serial implantation of tumor xenografts in nude BALB/c mice, the residual tumor cells that were exposed to rapamycin in vivo displayed higher CD133 expression and had increased secondary tumorigenicity compared with the control group. Moreover, rapamycin treatment also enhanced the level of stem cell-associated genes and CD133 expression in certain human liver tumor cell lines, such as Huh7, PLC/PRC/7 and Hep3B. The mTOR pathway is significantly involved in the generation and the differentiation of tumorigenic liver CSCs. These results may be valuable for the design of more rational strategies to control clinical malignant HCC using mTOR inhibitors.     

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 µM cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.     

Multifunctionalized iron oxide nanoparticles for selective targeting of pancreatic cancer cells

Nanomedicine nowadays offers novel solutions in cancer therapy by introducing multimodal treatments in one single formulation. In addition, nanoparticles act as nanocarriers changing the solubility, biodistribution and efficiency of the therapeutic molecules, thus generating more efficient treatments and reducing their side effects. To apply these novel therapeutic approaches, efforts are focused on the multi-functionalization of the nanoparticles and will open up new avenues to advanced combinational therapies. Pancreatic ductal adenocarcinoma (PDAC) is a cancer with unmet medical needs. Abundant expression of the anti-phagocytosis signal CD47 has also been observed on pancreatic cancer cells, in particular a subset of cancer stem cells (CSCs) responsible for resistance to standard therapy and metastatic potential. CD47 receptor is found on pancreatic cancer and highly expressed on CSCs, but not on normal pancreas. Inhibiting CD47 using monoclonal antibodies has been shown as an effective strategy to treat PDAC in vivo. However, CD47 inhibition effectively slowed tumor growth only in combination with Gemcitabine or Abraxane. In this work, we present the generation of multifunctionalized iron oxide magnetic nanoparticles (MNPs) that include the anti-CD47 antibody and the chemotherapeutic drug Gemcitabine in a single formulation. We demonstrate the in vitro efficacy of the formulation against CD47-positive pancreatic cancer cells. This article is part of a Special Issue entitled "Recent Advances in Bionanomaterials" Guest Editor: Dr. Marie-Louise Saboungi and Dr. Samuel D. Bader.     

Immune modulation of effector CD4+ and regulatory T cell function by sorafenib in patients with hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a difficult to treat cancer characterized by poor tumor immunity with only one approved systemic drug, sorafenib. If novel combination treatments are to be developed with immunological agents, the effects of sorafenib on tumor immunity are important to understand. In this study, we investigate the impact of sorafenib on the CD4+CD25? effector T cells (Teff) and CD4+CD25+ regulatory T cells (Tregs) from patients with HCC. We isolated Teff and Treg from peripheral mononuclear cells of HCC patients to determine immune reactivity by thymidine incorporation, ELISA and flow cytometry. Teff cultured alone or with Treg were supplemented with different concentrations of sorafenib. The effects of sorafenib on Teff responses were dose-dependent. Pharmacologic doses of sorafenib decreased Teff activation by down regulating CD25 surface expression. In contrast, sub-pharmacologic concentrations of sorafenib resulted in Teff activation. These low doses of sorafenib in the Teff cultures led to a significant increase in Teff proliferation, IL2 secretion and up-regulation of CD25 expression on the cell surface. In addition, low doses of sorafenib in the suppression Teff/Treg cocultures restored Teff responses by eliminating Treg suppression. The loss of Treg suppressive function correlated with an increase in IL2 and IL6 secretion. Our findings show that sub-pharmacologic doses of sorafenib impact subsets of T cells differently, selectively increasing Teff activation while blocking Treg function. In conclusion, this study describes novel immune activating properties of low doses of sorafenib by promoting immune responsiveness in patients with HCC.     

An EpCAM/CD3 bispecific antibody efficiently eliminates hepatocellular carcinoma cells with limited galectin-1 expression

There have been several studies suggesting that cancer stem cells (CSCs) contribute to the high rates of recurrence and resistance to therapies observed in hepatocellular carcinoma (HCC). Epithelial cell adhesion molecule (EpCAM) has been demonstrated to be a biomarker of CSCs and a potential therapeutic target in HCC. Here, we prepared two anti-EpCAM monoclonal antibodies (1H8 and 2F2) and an anti-EpCAM bispecific T cell engager (BiTE) 1H8/CD3, which was derived from 1H8, and used them to treat HCC in vitro and in vivo. The results demonstrated that all of the developed anti-EpCAM antibodies specifically bound to EpCAM. Neither anti-EpCAM monoclonal antibody had obvious anti-HCC activities in vitro or in vivo. However, anti-EpCAM BiTE 1H8/CD3 induced strong peripheral blood mononuclear cell-dependent cellular cytotoxicity in Huh-7 and Hep3B cells but not EpCAM-negative SK-Hep-1 cells. Notably, 1H8/CD3 completely inhibited the growth of Huh-7 and Hep3B xenografts in vivo. Treatment of the Huh-7 HCC xenografts with 1H8/CD3 significantly suppressed tumor proliferation and reduced the expression of most CSC biomarkers. Intriguingly, galectin-1 (Gal-1) overexpression inhibited 1H8/CD3-induced lymphocytotoxicity in HCCs while knockdown of Gal-1 increased the lymphocytotoxicity. Collectively, these results indicate that anti-EpCAM BiTE 1H8/CD3 is a promising therapeutic agent for HCC treatment. Gal-1 may contribute to the resistance of HCC cells to 1H8/CD3-induced lysis.     

Three‐dimensional cell culture model utilization in cancer stem cell research

Three‐dimensional (3D) cell culture models are becoming increasingly popular in contemporary cancer research and drug resistance studies. Recently, scientists have begun incorporating cancer stem cells (CSCs) into 3D models and modifying culture components in order to mimic in vivo conditions better. Currently, the global cell culture market is primarily focused on either 3D cancer cell cultures or stem cell cultures, with less focus on CSCs. This is evident in the low product availability officially indicated for 3D CSC model research. This review discusses the currently available commercial products for CSC 3D culture model research. Additionally, we discuss different culture media and components that result in higher levels of stem cell subpopulations while better recreating the tumor microenvironment. In summary, although progress has been made applying 3D technology to CSC research, this technology could be further utilized and a greater number of 3D kits dedicated specifically to CSCs should be implemented.