Cancer Cell Spheroids Research Articles

Visualizing and Quantifying mRNA Localization at the Invasive Front of 3D Cancer Spheroids.

Localization of mRNAs at the front of migrating cells is a widely used mechanism that functionally supports efficient cell movement. It is observed in single cells on two-dimensional surfaces, as well as in multicellular three-dimensional (3D) structures and in tissue in vivo. 3D multicellular cultures can reveal how the topology of the extracellular matrix and cell-cell contacts influence subcellular mRNA distributions. Here we describe a method for mRNA imaging in an inducible system of collective cancer cell invasion. MDA-MB-231 cancer cell spheroids are embedded in Matrigel, induced to invade, and processed to image mRNAs with single-molecule sensitivity. An analysis algorithm is used to quantify and compare mRNA distributions at the front of invasive leader cells. The approach can be easily adapted and applied to analyze RNA distributions in additional settings where cells polarize along a linear axis.

Palmitoyl Carnitine-Anchored Nanoliposomes for Neovasculature-Specific Delivery of Gemcitabine Elaidate to Treat Pancreatic Cancer

Being the fourth most fatal malignancy worldwide, pancreatic cancer is on track to become the second leading cause of cancer-related deaths in the United States by 2030. Gemcitabine is a first-line chemotherapeutic agent for pancreatic ductal adenocarcinoma (PDAC). Gemcitabine Elaidate (Gem Elaidate) is a lipophilic derivative which allows hENT1-independent intracellular delivery of gemcitabine and better pharmacokinetics and entrapment in a nanocarrier. Cancer cells and neovasculature are negatively charged compared to healthy cells. Palmitoyl-DL-carnitine chloride (PC) is a Protein kinase C (PKC) inhibitor which also provides a cationic surface charge to nanoliposomes for targeting tumor neovasculature and augmented anticancer potency. The objectives of our study are: (a) to develop and characterize a PKC inhibitor-anchored Gem Elaidate-loaded PEGylated nanoliposome (PGPLs) and (b) to investigate the anticancer activity of Gem Elaidate and PGPLs in 2D and 3D models of pancreatic cancer. The optimized PGPLs resulted in a particle size of 80 ± 2.31 nm, a polydispersity index of 0.15 ± 0.05 and a ζ-potential of +31.6 ± 3.54 mV, with a 93.25% encapsulation efficiency of Gem Elaidate in PGPLs. Our results demonstrate higher cellular uptake, inhibition in migration, as well as angiogenesis potential and significant apoptosis induced by PGPLs in 3D multicellular tumor spheroids of pancreatic cancer cells. Hence, PGPLs could be an effective and novel nanoformulation for the neovasculature-specific delivery of Gemcitabine Elaidate to treat PDAC.

EGFR-targeted bacteriophage lambda penetrates model stromal and colorectal carcinoma tissues, is taken up into carcinoma cells, and interferes with 3-dimensional tumor formation.

Colorectal cancer and other adult solid cancers pose a significant challenge for successful treatment because the tumor microenvironment both hinders the action of conventional therapeutics and suppresses the immune activities of infiltrating leukocytes. The immune suppression is largely the effect of enhanced local mediators such as purine nucleosides and eicosanoids. Genetic approaches have the promise of interfering with these mechanisms of local immunosuppression to allow both intrinsic and therapeutic immunological anticancer processes. Bacterial phages offer a novel means of enabling access into tissues for therapeutic genetic manipulations. We generated spheroids of fibroblastic and CRC cancer cells to model the 3-dimensional stromal and parenchymal components of colorectal tumours. We used these to examine the access and effects of both wildtype (WT) and epidermal growth factor (EGF)-presenting bacteriophage λ (WT- λ and EGF-λ) as a means of delivery of targeted genetic interventions in solid cancers. We used both confocal microscopy of spheroids exposed to AF488-tagged phages, and the recovery of viable phages as measured by plaque-forming assays to evaluate access; and measures of mitochondrial enzyme activity and cellular ATP to evaluate the outcome on the constituent cells. Using flourescence-tagged derivatives of these bacteriophages (AF488-WT-λ and AF488-EGF-λ) we showed that phage entry into these tumour microenvironments was possible and that the EGF ligand enabled efficient and persistent uptake into the cancer cell mass. EGF-λ became localized in the intracellular portion of cancer cells and was subjected to subsequent cellular processing. The targeted λ phage had no independent effect upon mature tumour spheroids, but interfered with the early formation and growth of cancer tissues without the need for addition of a toxic payload, suggesting that it might have beneficial effects by itself in addition to any genetic intervention delivered to the tumour. Interference with spheroid formation persisted over the duration of culture. We conclude that targeted phage technology is a feasible strategy to facilitate delivery into colorectal cancer tumour tissue (and by extension other solid carcinomas) and provides an appropriate delivery vehicle for a gene therapeutic that can reduce local immunosuppression and/or deliver an additional direct anticancer activity.

Aquaporin water channels affect the response of conventional anticancer therapies of 3D grown breast cancer cells

Aquaporin (AQP) water channels facilitate water transport across cellular membranes and are essential in regulation of body water balance. Moreover, several AQPs are overexpressed or ectopically expressed in breast cancer. Interestingly, several in vitro studies have suggested that AQPs can affect the response to conventional anticancer chemotherapies. Therefore, we took a systematic approach to test how AQP1, AQP3 and AQP5, which are often over-/ectopically expressed in breast cancer, affect total viability of 3-dimensional (3D) breast cancer cell spheroids when treated with the conventional anticancer chemotherapies Cisplatin, 5-Fluorouracil (5-FU) and Doxorubicin, a Combination of the three drugs as well as the Combination plus the Ras inhibitor Salirasib. Total viability of spheroids overexpressing AQP1 were decreased by all treatments except for 5-FU, which increased total viability by 20% compared to DMSO treated controls. All treatments reduced viability of spheroids overexpressing AQP3. In contrast, only Doxorubicin, Combination and Combination + Salirasib reduced total viability of spheroids overexpressing AQP5. Thus, this study supports a significant role of AQPs in the response to conventional chemotherapies. Evaluating the role of individual proteins that contribute to resistance to chemotherapies is essential in advancing personalized medicine in breast carcinomas.

Cell-Taxi: Mesenchymal Cells Carry and Transport Clusters of Cancer Cells.

Cell clusters that collectively migrate from primary tumors appear to be far more potent in forming distant metastases than single cancer cells. A better understanding of the collective cell migration phenomenon and the involvement of various cell types during this process is needed. Here, an in vitro platform based on inverted-pyramidal microwells to follow and quantify the collective migration of hundreds of tumor cell clusters at once is developed. These results indicate that mesenchymal stromal cells (MSCs) or cancer-associated fibroblasts (CAFs) in the heterotypic tumor cell clusters may facilitate metastatic dissemination by transporting low-motile cancer cells in a Rac-dependent manner and that extracellular vesicles secreted by mesenchymal cells only play a minor role in this process. Furthermore, in vivo studies show that cancer cell spheroids containing MSCs or CAFs have faster spreading rates. These findings highlight the active role of co-traveling stromal cells in the collective migration of tumor cell clusters and may help in developing better-targeted therapies.

Harvestable tumour spheroids initiated in a gelatin-carboxymethyl cellulose hydrogel for cancer targeting and imaging with fluorescent gold nanoclusters

Cancer cell spheroids are the simplest 3D in vitro cancer models and have been extensively used for cancer research. More recently, models have been becoming complex, with the introduction of a matrix and non-cancer cell types to mimic specific tumour aspects. However, applying drugs or agents in matrix-embedded cancer spheroids can be problematic. Most matrices can impede and also bind drugs or visualizing agents non-specifically, in the vicinity of the embedded spheroids. This may interfere with imaging or further analysis without breaking apart the 3D model into its constituents. Here, we developed a combined gelatin-carboxymethyl cellulose (G-CMC) hydrogel for initiating cancer spheroids that enabled intact harvesting pre/post treatment for further investigation, such as targeting and imaging. We combined CMC (1.25%) and gelatin (2.5%) at 25 °C and initiated polymerisation after autoclaving (121 °C) to obtain a mechanical strength (sheer stress) of 38 Pas versus 1.28 Pas for CMC alone. These matrix conditions facilitated separation of the spheroids from the G-CMC, using low centrifugation (100 g). We described growth of colorectal and breast cancer spheroids within the G-CMC matrix (with average diameters of 220 mm and 180 μm for representative cell lines HT29 and MCF7 at 10 days, respectively). As the cancer cells express the surface biomarker calreticulin (CRT), we manufactured anti-calreticulin IgG (anti-CRT) conjugated to fluorescent gold nanoclusters (anti-CRT-AuNC) as a probe. We harvested cancer spheroids and incubated live with the nanoclusters. Imaging demonstrated strong binding of CRT-targeted AuNCs compared to control AuNCs. This novel model preserves cancer spheroid integrity upon isolation and is well suited for targeted imaging and drug delivery of cancer in 3D.

Three-Dimensional Mass Spectrometry Imaging Reveals Distributions of Lipids and the Drug Metabolite Associated with the Enhanced Growth of Colon Cancer Cell Spheroids Treated with Triclosan.

The application of mass spectrometry imaging (MSI) to explore the responses of cancer cell spheroids (CCS) after treatment of exogenous molecules has attracted growing attention. Increasing studies have utilized MSI to image the two-dimensional distributions of exogenous and endogenous molecules in planar CCS sections. However, because CCS are volumetric and heterogenous, maintaining their three-dimensional (3D) information is essential for acquiring a better understanding of the tumor microenvironment and mechanisms of action of exogenous molecules. Here, an established method of 3D MSI was applied to distinguish the distributions of triclosan sulfate and endogenous lipids in three microregions of colon CCS with an enhanced growth induced by the treatment of triclosan, a common antimicrobial agent. The results of 3D MSI showed that triclosan sulfate gradually accumulated from the periphery to the entire structure of CCS and finally localized in the core region. Spatial lipidomics analysis revealed that the upregulated phosphatidylethanolamine (fold change (FD) = 1.26, p = 0.0021), phosphatidylinositol (FD = 1.17, p = 0.0180), and phosphatidylcholine (FD = 1.22, p = 0.0178) species mainly distributed in the outer proliferative region, while the upregulated sphingomyelin (FD = 1.18, p = 0.024) species tended to distribute in the inner necrotic region. Our results suggest that a competitive mechanism between inhibiting and promoting CCS growth might be responsible for the proliferation of CCS treated with triclosan.

Artemisia argyi extract induces apoptosis in human gemcitabine-resistant lung cancer cells via the PI3K/MAPK signaling pathway

Ethnopharmacological relevance: Artemisia argyi H. Lév. & Vaniot (Asteraceae), also called “Chinese mugwort”, is frequently used as a herbal medicine in China, Japan, Korea, and eastern parts of Russia. It is known as “ai ye” in China and “Gaiyou” in Japan. In ancient China, the buds and leaves of A. argyi were commonly consumed before and after Tomb-sweeping Day. It is used to treat malaria, hepatitis, cancer, inflammatory diseases, asthma, irregular menstrual cycle, sinusitis, and pathologic conditions of the kidney and liver. Although A. argyi extract (AAE) has shown anti-tumor activity against various cancers, the therapeutic effect and molecular mechanism of AAE remains to be further studied in lung cancer.Aim of the study: This study aimed to demonstrate the anti-tumor effect of AAE and its associated biological mechanisms in CL1-0 parent and gemcitabine-resistant (CL1-0-GR) lung cancer cells.Experimental procedure: Human lung cancer cells CL1-0 and CL1-0-GR cells were treated with AAE. Cell viability was assessed using the MTT, colony, and spheroid formation assays. Migration, invasion, and immunofluorescence staining were used to determine the extent of epithelial– mesenchymal transition (EMT). JC-1 and MitoSOX fluorescent assays were performed to investigate the effect of AAE on mitochondria. Apoptosis was detected using the TUNEL assay and flow cytometry with Annexin V staining.Result: We found that A. argyi significantly decreased cell viability and induced apoptosis, accompanied by mitochondrial membrane depolarization and increased ROS levels in both parent cells (CL1-0) and gemcitabine-resistant lung cancer cells (CL1-0-GR). AAE-induced apoptosis is regulated via the PI3K/AKT and MAPK signaling pathways. It also prevents CL1-0 and CL1-0-GR cancer cell invasion, migration, EMT, colony formation, and spheroid formation. In addition, AAE acts cooperative with commercial chemotherapy drugs to enhance tumor spheroid shrinkage.Conclusion: Our study provides the first evidence that A. argyi treatment suppresses both parent and gemcitabine-resistant lung cancer cells by inducing ROS, mitochondrial membrane depolarization, and apoptosis, and reducing EMT. Our finding provides insights into the anti-cancer activity of A. argyi and suggests that A. argyi may serve as a chemotherapy adjuvant that potentiates the efficacy of chemotherapeutic agents.

Therapeutic Targeting of Cancer Stem Cells Prevents Resistance of Colorectal Cancer Cells to MEK Inhibition.

Drug resistance is a leading cause for the failure of cancer treatments. Plasticity of cancer cells to acquire stem cell-like properties enables them to escape drug toxicity through different adaptive mechanisms. Eliminating cancer stem cells (CSCs) can potentially improve treatment outcomes for patients. To determine the role of CSCs in resistance of colorectal cancer cells to targeted therapies and identify treatment strategies, we treated spheroids of BRAFmut and KRASmut colorectal cancer cells with inhibitors of the mitogen-activated protein kinase pathway and studied resistance mechanisms through gene and protein expression analyses. We found that treatments activated several oncogenic pathways and expression of CSC markers CD166 and ALDH1A3. We identified a specific combination treatment using trametinib and mithramycin A to simultaneously inhibit the CSC phenotype and activities of several pathways in cancer cells. This study demonstrates the feasibility of therapeutic targeting of CSCs as a strategy to block tumorigenic activities of cancer cells.

A Biomimetic High Throughput Model of Cancer Cell Spheroid Dissemination onto Aligned Fibrillar Collagen.

Cell dissemination during tumor development is a characteristic of cancer metastasis. Dissemination from three-dimensional spheroid models on extracellular matrices designed to mimic tissue-specific physiological microenvironments may allow us to better elucidate the mechanism behind cancer metastasis and the response to therapeutic agents. The orientation of fibrillar collagen plays a key role in cellular processes and mediates metastasis through contact-guidance. Understanding how cells migrate on aligned collagen fibrils requires in vitro assays with reproducible and standardized orientation of collagen fibrils on the macro-to-nanoscale. Herein, we implement a spheroid-based migration assay, integrated with a fibrillar type I collagen matrix, in a manner compatible with high throughput image acquisition and quantitative analysis. The migration of highly proliferating U2OS osteosarcoma cell spheroids onto an aligned fibrillar type I collagen matrix was quantified. Cell dissemination from the spheroid was polarized with increased invasion in the direction of fibril alignment. The resulting area of cell dissemination had an aspect ratio of 1.2 ± 0.1 and an angle of maximum invasion distance of 5° ± 44° relative to the direction of collagen fibril alignment. The assay described here can be applied to a fully automated imaging and analysis pipeline for the assessment of tumor cell migration with high throughput screening.

13C metabolic flux analysis clarifies distinct metabolic phenotypes of cancer cell spheroid mimicking tumor hypoxia

Cancer cells adapt their intracellular energy metabolism to the oxygen-deprived tumor microenvironment (TME) to ensure tumor progression. This adaptive mechanism has focused attention on the metabolic phenotypes of tumor cells under hypoxic TME for developing novel cancer therapies. Although widely used monolayer (2D) culture does not fully reflect in vivo hypoxic TME, spheroid (3D) culture can produce a milieu similar to the TME in vivo. However, how different metabolic phenotypes are expressed in 3D cultures mimicking tumor hypoxia compared with 2D cultures under hypoxia remains unclear. To address this issue, we investigated the metabolic phenotypes of 2D- and 3D-cultured cancer cells by 13C-metabolic flux analysis (13C-MFA). Principal component analysis of 13C mass isotopomer distributions clearly demonstrated distinct metabolic phenotypes of 3D-cultured cells. 13C-MFA clarified that 3D culture significantly upregulated pyruvate carboxylase flux in line with the pyruvate carboxylase protein expression level. On the other hand, 3D culture downregulated glutaminolytic flux. Consistent with our findings, 3D-cultured cells are more resistant to a glutaminase inhibitor than 2D-cultured cells. This study suggests the importance of considering the metabolic characteristics of the particular in vitro model used for research on cancer metabolism.

The intriguing role of collagen on the rheology of cancer cell spheroids

Spheroids are multicellular systems with an interesting rheology giving rise to elasto-visco-plastic properties. They are good tumor models, but the role of the extracellular matrix (ECM) is not fully understood. ECM is an important link between cells and may have a significant impact on tissue organization. Here we determine viscoelastic properties of spheroids including different collagen I amounts using AFM and predict new frequency–dependent properties leading to soft glassy rheology behavior. A unified model – similar to single cell behavior – is proposed and discussed, while complementary confocal experiments reveal the microstructure of spheroids, with collagen I fibers serving as a skeleton for cells, thus reinforcing the spheroid viscoelastic behavior.

Gossypol Induces Apoptosis of Human Pancreatic Cancer Cells via CHOP/Endoplasmic Reticulum Stress Signaling Pathway.

Gossypol, a natural phenolic aldehyde present in cotton plants, was originally used as a means of contraception, but is currently being studied for its anti-proliferative and anti-metastatic effects on various cancers. However, the intracellular mechanism of action regarding the effects of gossypol on pancreatic cancer cells remains unclear. Here, we investigated the anti-cancer effects of gossypol on human pancreatic cancer cells (BxPC-3 and MIA PaCa-2). Cell counting kit-8 assays, annexin V/propidium iodide staining assays, and transmission electron microscopy showed that gossypol induced apoptotic cell death and apoptotic body formation in both cell lines. RNA sequencing analysis also showed that gossypol increased the mRNA levels of CCAAT/enhancer-binding protein homologous protein (CHOP) and activating transcription factor 3 (ATF3) in pancreatic cancer cell lines. In addition, gossypol facilitated the cleavage of caspase-3 via protein kinase RNA-like ER kinase (PERK), CHOP, and Bax/Bcl-2 upregulation in both cells, whereas the upregulation of ATF was limited to BxPC-3 cells. Finally, a three-dimensional culture experiment confirmed the successful suppression of cancer cell spheroids via gossypol treatment. Taken together, our data suggest that gossypol may trigger apoptosis in pancreatic cancer cells via the PERK-CHOP signaling pathway. These findings propose a promising therapeutic approach to pancreatic cancer treatment using gossypol.

Cytotoxic effect of metformin on butyrate-resistant PMF-K014 colorectal cancer spheroid cells

Three-dimensional (3D) cell culture models are used in cancer research because they mimic physiological responses in vivo compared with two-dimensional (2D) culture systems. Recently, cross-resistance of butyrate-resistant (BR) cells and chemoresistance in colorectal cancer (CRC) cells have been reported; however, effective treatments for BR cells have not been identified. In this study, we investigated the cytotoxicity of metformin (MET), an anti-diabetic drug, on BR CRC cells in a 3D spheroid culture model. The results demonstrate that MET decreases spheroid size, migration, and spheroid viability, while it increases spheroid death. The molecular mechanism revealed that AMP-activated protein kinase (AMPK) and Akt serine/threonine kinase 1(Akt) were significantly upregulated, whereas the acetyl-CoA-carboxylase (ACC) and mammalian target of rapamycin (mTOR) were downregulated, which led to caspase activation and apoptosis. Our findings show the potential cytotoxicity of MET on CRC-BR cells. The combination of MET and conventional chemotherapeutic drugs should be addressed in further studies to reduce the side effects of standard chemotherapy for CRC.

Ordered micropattern arrays fabricated by lung-derived dECM hydrogels for chemotherapeutic drug screening.

AimsThis study aims to evaluate ECM-coated micropattern arrays derived from decellularization of native porcine lungs as a novel three-dimensional cell culture platform.MethodsECM derived from decellularization of native porcine lungs was exploited to prepare hydrogels. Then, dECM-coated micropattern arrays were fabricated at four different diameters (50, 100, 150 and 200 ​μm) using polydimethylsiloxane (PDMS). Two lung cancer cell lines, A549 and H1299, were tested on a dECM-coated micropattern array as a novel culture platform for cell adhesion, distribution, proliferation, viability, phenotype expression, and drug screening to evaluate the cytotoxicity of paclitaxel, doxorubicin and cisplatin.ResultsThe ECM derived from decellularization of native porcine lungs supported cell adhesion, distribution, viability and proliferation better than collagen I and Matrigel as the coated matrix on the surface. Moreover, the optimal diameter of the micropattern arrays was 100–150 ​μm, as determined by measuring the morphology, viability, proliferation and phenotype of the cancer cell spheroids. Cell spheroids of A549 and H1299 on dECM-coated micropattern arrays showed chemoresistance to anticancer drugs compared to that of the monolayer. The different distributions of HIF-1α, MCL-1 (in the center) and Ki-67 and MRP2 (in the periphery) of the spheroids demonstrated the good establishment of basal-lateral polarity and explained the chemoresistance phenomenon of spheroids.ConclusionsThis novel three-dimensional cell culture platform is stable and reliable for anticancer drug testing. Drug screening in dECM-coated micropattern arrays provides a powerful alternative to existing methods for drug testing and metabolic profiling in the drug discovery process.

Intrinsic Differences in Spatiotemporal Organization and Stromal Cell Interactions Between Isogenic Lung Cancer Cells of Epithelial and Mesenchymal Phenotypes Revealed by High-Dimensional Single-Cell Analysis of Heterotypic 3D Spheroid Models.

The lack of inadequate preclinical models remains a limitation for cancer drug development and is a primary contributor to anti-cancer drug failures in clinical trials. Heterotypic multicellular spheroids are three-dimensional (3D) spherical structures generated by self-assembly from aggregates of two or more cell types. Compared to traditional monolayer cell culture models, the organization of cells into a 3D tissue-like structure favors relevant physiological conditions with chemical and physical gradients as well as cell-cell and cell-extracellular matrix (ECM) interactions that recapitulate many of the hallmarks of cancer in situ. Epidermal growth factor receptor (EGFR) mutations are prevalent in non-small cell lung cancer (NSCLC), yet various mechanisms of acquired resistance, including epithelial-to-mesenchymal transition (EMT), limit the clinical benefit of EGFR tyrosine kinase inhibitors (EGFRi). Improved preclinical models that incorporate the complexity induced by epithelial-to-mesenchymal plasticity (EMP) are urgently needed to advance new therapeutics for clinical NSCLC management. This study was designed to provide a thorough characterization of multicellular spheroids of isogenic cancer cells of various phenotypes and demonstrate proof-of-principle for the applicability of the presented spheroid model to evaluate the impact of cancer cell phenotype in drug screening experiments through high-dimensional and spatially resolved imaging mass cytometry (IMC) analyses. First, we developed and characterized 3D homotypic and heterotypic spheroid models comprising EGFRi-sensitive or EGFRi-resistant NSCLC cells. We observed that the degree of EMT correlated with the spheroid generation efficiency in monocultures. In-depth characterization of the multicellular heterotypic spheroids using immunohistochemistry and high-dimensional single-cell analyses by IMC revealed intrinsic differences between epithelial and mesenchymal-like cancer cells with respect to self-sorting, spatiotemporal organization, and stromal cell interactions when co-cultured with fibroblasts. While the carcinoma cells harboring an epithelial phenotype self-organized into a barrier sheet surrounding the fibroblasts, mesenchymal-like carcinoma cells localized to the central hypoxic and collagen-rich areas of the compact heterotypic spheroids. Further, deep-learning-based single-cell segmentation of IMC images and application of dimensionality reduction algorithms allowed a detailed visualization and multiparametric analysis of marker expression across the different cell subsets. We observed a high level of heterogeneity in the expression of EMT markers in both the carcinoma cell populations and the fibroblasts. Our study supports further application of these models in pre-clinical drug testing combined with complementary high-dimensional single-cell analyses, which in turn can advance our understanding of the impact of cancer-stroma interactions and epithelial phenotypic plasticity on innate and acquired therapy resistance in NSCLC.

Glycolytic oscillations in HeLa cervical cancer cell spheroids.

Previous studies have unravelled glycolytic oscillations in cancer cells, such as HeLa cervical and DU145 prostate cancer cells, using a monolayer culture system. Here, we demonstrate glycolytic oscillations in HeLa cervical cancer cell spheroids. Experiments revealed that a small number of HeLa cells in spheroids exhibited heterogeneous oscillations with a higher frequency than those in monolayers. Model analyses and our previous experiments indicated that the higher frequencies of oscillations in spheroids were mostly due to the increase in glycolytic enzyme activity in the cells, and to the decrease in glucose concentration by diffusional transport of glucose from the surface to inside the spheroids, as well as the increase in cell density through spheroid formation. These results and our previous studies imply that more malignant cancer cells tend to exhibit glycolytic oscillations with higher frequencies than less malignant cells. Adjacent cells in spheroids oscillated within a 10% difference in frequency, but did not synchronize with each other. This suggests that weak cell-to-cell interactions might exist among HeLa cells connected with cadherins in the spheroid microenvironment; however, the interactions were not strong enough to induce synchronization of glycolytic oscillations.

Modified Bovine Milk Exosomes for Doxorubicin Delivery to Triple-Negative Breast Cancer Cells.

Biological nanoparticles, such as exosomes, offer an approach to drug delivery because of their innate ability to transport biomolecules. Exosomes are derived from cells and an integral component of cellular communication. However, the cellular cargo of human exosomes could negatively impact their use as a safe drug carrier. Additionally, exosomes have the intrinsic yet enigmatic, targeting characteristics of complex cellular communication. Hence, harnessing the natural transport abilities of exosomes for drug delivery requires predictably targeting these biological nanoparticles. This manuscript describes the use of two chemical modifications, incorporating a neuropilin receptor agonist peptide (iRGD) and a hypoxia-responsive lipid for targeting and release of an encapsulated drug from bovine milk exosomes to triple-negative breast cancer cells. Triple-negative breast cancer is a very aggressive and deadly form of malignancy with limited treatment options. Incorporation of both the iRGD peptide and hypoxia-responsive lipid into the lipid bilayer of bovine milk exosomes and encapsulation of the anticancer drug, doxorubicin, created the peptide targeted, hypoxia-responsive bovine milk exosomes, iDHRX. Initial studies confirmed the presence of iRGD peptide and the exosomes' ability to target the αvβ3 integrin, overexpressed on triple-negative breast cancer cells' surface. These modified exosomes were stable under normoxic conditions but fragmented in the reducing microenvironment created by 10 mM glutathione. In vitro cellular internalization studies in monolayer and three-dimensional (3D) spheroids of triple-negative breast cancer cells confirmed the cell-killing ability of iDHRX. Cell viability of 50% was reached at 10 μM iDHRX in the 3D spheroid models using four different triple-negative breast cancer cell lines. Overall, the tumor penetrating, hypoxia-responsive exosomes encapsulating doxorubicin would be effective in reducing triple-negative breast cancer cells' survival.

New synthetic phenylquinazoline derivatives induce apoptosis by targeting the pro-survival members of the BCL-2 family

Chemo-resistant cancer cells acquire robust growth potential through cell signaling mechanisms such as the down-regulation of tumor suppressors and the up-regulation of pro-survival proteins, respectively. To overcome chemo-resistance of cancer, small molecule drugs that interact with the cell signaling proteins to enhance sensitization of cancer cells toward cancer therapies are likely to be effective for the treatment of chemo-drug resistant cancer. To identify high potency small molecules, a series of ten novel phenylquinazoline derivatives were synthesized to determine their cellular effects in MCF-7 and MCF-7- cisplatin-resistant (CR) human breast cancer cells which led to the identification of two bioactive compounds, SMS-IV-20 and SMS-IV-40, that exhibited an elevated level of cytotoxicity against the human breast cancer cells and spheroid cells. In addition, both compounds enhanced chemo-sensitization of the human breast cancer cells that were genetically engineered to express the tumor suppressor and pro-apoptotic proteins, MOAP-1, Bax, and RASSF1a (MBR), suggesting that the compounds interact with the MBR signaling pathway. Furthermore, when MCF-7-CR cells were treated with SMS-IV-20 and SMS-IV-40 in the presence of ABT-737, a BCL-XL and BCL-2 inhibitor, enhanced chemo-sensitization was observed, suggesting SMS-IV-20 and SMS-IV-40 exert antagonistic activity to regulate the functional activity of BCL-2 and BCL-XL. Western blot analysis showed that both SMS-IV-20 and SMS-IV-40 induced down-regulation of BCL-2 or both BCl-2 and BCL-XL expression, respectively while promoting the release of mitochondrial Cytochrome C. Taken together, the data showed that SMS-IV-20 and SMS-IV-40 are potent activators of apoptosis that enhance chemo-sensitization through their antagonistic actions on the pro-survival activity of the BCl-2 family in human cancer cells.

Transglutaminase 2 enhances hepatocyte growth factor signaling to drive the mesothelioma cancer cell phenotype.

Transglutaminase 2 (TG2) is an important mesothelioma cancer cell survival protein. However, the mechanism whereby TG2 maintains mesothelioma cell survival is not well understood. We present studies showing that TG2 drives hepatocyte growth factor (HGF)-dependent MET receptor signaling to maintain the aggressive mesothelioma cancer phenotype. TG2 increases HGF and MET messenger RNA and protein levels to enhance MET signaling. TG2 inactivation reduces MET tyrosine kinase activity to reduce cancer cell spheroid formation, invasion and migration. We also confirm that HGF/MET signaling is a biologically important mediator of TG2 action. Reducing MET level using genetic methods or treatment with MET inhibitors reduces spheroid formation, invasion and migration and this is associated with reduced MEK1/2 and ERK1/2. In addition, MEK1/2 and ERK1/2 inhibitors suppress the cancer phenotype. Moreover, MET knockout mesothelioma cells form 10-fold smaller tumors compared to wild-type cells and these tumors display reduced MET, MEK1/2, and ERK1/2 activity. These findings suggest that TG2 maintains HGF and MET levels in cultured mesothelioma cells and tumors to drive HGF/MET, MEK1/2, and ERK1/2 signaling to maintain the aggressive mesothelioma cancer phenotype.