Ascites microenvironment conditions the peritoneal pre-metastatic niche to promote the implantation of ovarian tumor spheroids: Involvement of fibrinogen/fibrin and αV and α5ß1 integrins.

At least one-third of patients with epithelial ovarian cancer (OC) present ascites at diagnosis and almost all have ascites at recurrence especially because of the propensity of the OC cells to spread in the abdominal cavity leading to peritoneal metastasis. The influence of ascites on the development of pre-metastatic niches, and on the biological mechanisms leading to cancer cell colonization of the mesothelium, remains poorly understood. Here, we show that ascites weakens the mesothelium by affecting the morphology of mesothelial cells and by destabilizing their distribution in the cell cycle. Ascites also causes destabilization of the integrity of mesothelium by modifying the organization of cell junctions, but it does not affect the synthesis of N-cadherin and ZO-1 by mesothelial cells. Moreover, ascites induces disorganization of focal contacts and causes actin cytoskeletal reorganization potentially dependent on the activity of Rac1. Ascites allows the densification and reorganization of ECM proteins of the mesothelium, especially fibrinogen/fibrin, and indicates that it is a source of the fibrinogen and fibrin surrounding OC spheroids. The fibrin in ascites leads to the adhesion of OC spheroids to the mesothelium, and ascites promotes their disaggregation followed by the clearance of mesothelial cells. Both αV and α5ß1 integrins are involved. In conclusion ascites and its fibrinogen/fibrin composition affects the integrity of the mesothelium and promotes the integrin-dependent implantation of OC spheroids in the mesothelium.

Knockdown of CPSF4 Inhibits Bladder Cancer Cell Growth by Upregulating NRF1.

Increasing studies have shown that nuclear respiratory factor 1 (NRF1) deficiency frequently occurs in many human diseases, and its activation can protect neurons and other cells from degenerative diseases and malignant tumors. However, how NRF1 is regulated in bladder cancer remains unknown. Our research aims to reveal the role of leavage and polyadenylation-specific factor 4 (CPSF4) on the growth inhibition effect of bladder cancer and clarify its relationship with NRF1. Here, cell proliferation assay, transwell migration assay and multicellular tumor spheroids (MCTS) formation assay in the bladder cancer cell lines were carried out to measure tumor cell growth. Western bolt assay was carried out to identify the relationship between NRF1 and CPSF4. Also, subcutaneous xenograft tumors in nude mice were established to further validate the inhibition effect of CPSF4 on bladder tumor and the regulation on NRF1. The results in vitro showed that knockdown of CPSF4 strongly reduced the proliferation and migration, and inhibited MCTS formation in 5637 and HT1376 cell lines, while an additional knockdown of increased NRF1 induced by CPSF4 knockdown partially abolished these effects. The results in vivo showed that knockdown of CPSF4 strongly reduced the volume and weight of subcutaneous tumor, and decreased the expression of Ki-67 in tumor tissue, while NRF1 knockdown partially reversed these effects induced by CPSF4 knockdown. Western bolt assay demonstrated that CPSF4 could negatively regulate NRF1. Our results indicated that knock-down of CPSF4 inhibited bladder cancer cell growth by upregulating NRF1, which might provide evidence of CPSF4 as a therapeutic target for bladder cancer.

Autonomous Microlasers for Profiling Extracellular Vesicles from Cancer Spheroids.

Self-propelled micro/nanomotors are emergent intelligent sensors for analyzing extracellular biomarkers in circulating biological fluids. Conventional luminescent motors are often masked by a highly dynamic and scattered environment, creating challenges to characterize biomarkers or subtle binding dynamics. Here we introduce a strategy to amplify subtle signals by coupling strong light-matter interactions on micromotors. A smart whispering-gallery-mode microlaser that can self-propel and analyze extracellular biomarkers is demonstrated through a liquid crystal microdroplet. Lasing spectral responses induced by cavity energy transfer were employed to reflect the abundance of protein biomarkers, generating exclusive molecular labels for cellular profiling of exosomes derived from 3D multicellular cancer spheroids. Finally, a microfluidic biosystem with different tumor-derived exosomes was employed to elaborate its sensing capability in complex environments. The proposed autonomous microlaser exhibits a promising method for both fundamental biological science and applications in drug screening, phenotyping, and organ-on-chip applications.

Effects of pharmacological primary cilium disturbance in the context of in vitro 2D and 3D malignant pleura mesothelioma.

INTRODUCTION: Primary cilium (PC) is a single non-motile antenna-like organelle composed of a microtubule core axon originating from the mother centriole of the centrosome. The PC is universal in all mammalian cells and protrudes to the extracellular environment receiving mechanochemical cues that it transmits in the cell. AIM: To investigate the role of PC in mesothelial malignancy in the context of two-dimensional (2D) and three-dimensional (3D) phenotypes. MATERIALS AND METHODS: The effect of pharmacological deciliation [using ammonium sulphate (AS) or chloral hydrate (CH)] and PC elongation [using lithium chloride (LC)] on cell viability, adhesion, and migration (2D cultures) as well as in mesothelial sphere formation, spheroid invasion and collagen gel contraction (3D cultures) was investigated in benign mesothelial MeT-5A cells and in malignant pleural mesothelioma (MPM) cell lines, M14K (epithelioid) and MSTO (biphasic), and primary malignant pleural mesothelioma cells (pMPM). RESULTS: Pharmacological deciliation or elongation of the PC significantly affected cell viability, adhesion, migration, spheroid formation, spheroid invasion and collagen gel contraction in MeT-5A, M14K, MSTO cell lines and in pMPM cells compared to controls (no drug treatment). CONCLUSIONS: Our findings indicate a pivotal role of the PC in functional phenotypes of benign mesothelial cells and MPM cells.

Green synthesis of glyco-CuInS2 QDs with visible/NIR dual emission for 3D multicellular tumor spheroid and in vivo imaging.

Glyco-quantum dots (glyco-QDs) have attracted significant interest in bioimaging applications, notably in cancer imaging, because they effectively combine the glycocluster effect with the exceptional optical properties of QDs. The key challenge now lies in how to eliminate the high heavy metal toxicity originating from traditional toxic Cd-based QDs for in vivo bioimaging. Herein, we report an eco-friendly pathway to prepare nontoxic Cd-free glyco-QDs in water by the "direct" reaction of thiol-ending monosaccharides with metal salts precursors. The formation of glyco-CuInS2 QDs could be explained by a nucleation-growth mechanism following the LaMer model. As-prepared four glyco-CuInS2 QDs were water-soluble, monodispersed, spherical in shape and exhibited size range of 3.0-4.0 nm. They exhibited well-separated dual emission in the visible region (500-590 nm) and near-infrared range (~ 827 nm), which may be attributable to visible excitonic emission and near-infrared surface defect emission. Meanwhile, the cell imaging displayed the reversibly distinct dual-color (green and red) fluorescence in tumor cells (HeLa, A549, MKN-45) and excellent membrane-targeting properties of glyco-CuInS2 QDs based on their good biorecognition ability. Importantly, these QDs succeed in penetrating uniformly into the interior (the necrotic zone) of 3D multicellular tumor spheroids (MCTS) due to their high negative charge (zeta potential values ranging from - 23.9 to - 30.1 mV), which overcame the problem of poor penetration depth of existing QDs in in vitro spheroid models. So, confocal analysis confirmed their excellent ability to penetrate and label tumors. Thus, the successful application in in vivo bioimaging of these glyco-QDs verified that this design strategy is an effective, low cost and simple procedure for developing green nanoparticles as cheap and promising fluorescent bioprobes.

Molecular Mechanisms of Tumor Cell Stemness Modulation during Formation of Spheroids.

Cancer stem cells (CSCs), their properties and interaction with microenvironment are of interest in modern medicine and biology. There are many studies on the emergence of CSCs and their involvement in tumor pathogenesis. The most important property inherent to CSCs is their stemness. Stemness combines ability of the cell to maintain its pluripotency, give rise to differentiated cells, and interact with environment to maintain a balance between dormancy, proliferation, and regeneration. While adult stem cells exhibit these properties by participating in tissue homeostasis, CSCs behave as their malignant equivalents. High tumor resistance to therapy, ability to differentiate, activate angiogenesis and metastasis arise precisely due to the stemness of CSCs. These cells can be used as a target for therapy of different types of cancer. Laboratory models are needed to study cancer biology and find new therapeutic strategies. A promising direction is three-dimensional tumor models or spheroids. Such models exhibit properties resembling stemness in a natural tumor. By modifying spheroids, it becomes possible to investigate the effect of therapy on CSCs, thus contributing to the development of anti-tumor drug test systems. The review examines the niche of CSCs, the possibility of their study using three-dimensional spheroids, and existing markers for assessing stemness of CSCs.

Exploiting Vitamin D Receptor and Its Ligands to Target Squamous Cell Carcinomas of the Head and Neck.

Vitamin D (VitD) and its receptor (VDR) have been intensively investigated in many cancers. As knowledge for head and neck cancer (HNC) is limited, we investigated the (pre)clinical and therapeutic relevance of the VDR/VitD-axis. We found that VDR was differentially expressed in HNC tumors, correlating to the patients' clinical parameters. Poorly differentiated tumors showed high VDR and Ki67 expression, whereas the VDR and Ki67 levels decreased from moderate to well-differentiated tumors. The VitD serum levels were lowest in patients with poorly differentiated cancers (4.1 ± 0.5 ng/mL), increasing from moderate (7.3 ± 4.3 ng/mL) to well-differentiated (13.2 ± 3.4 ng/mL) tumors. Notably, females showed higher VitD insufficiency compared to males, correlating with poor differentiation of the tumor. To mechanistically uncover VDR/VitD's pathophysiological relevance, we demonstrated that VitD induced VDR nuclear-translocation (VitD < 100 nM) in HNC cells. RNA sequencing and heat map analysis showed that various nuclear receptors were differentially expressed in cisplatin-resistant versus sensitive HNC cells including VDR and the VDR interaction partner retinoic acid receptor (RXR). However, RXR expression was not significantly correlated with the clinical parameters, and cotreatment with its ligand, retinoic acid, did not enhance the killing by cisplatin. Moreover, the Chou-Talalay algorithm uncovered that VitD/cisplatin combinations synergistically killed tumor cells (VitD < 100 nM) and also inhibited the PI3K/Akt/mTOR pathway. Importantly, these findings were confirmed in 3D-tumor-spheroid models mimicking the patients' tumor microarchitecture. Here, VitD already affected the 3D-tumor-spheroid formation, which was not seen in the 2D-cultures. We conclude that novel VDR/VitD-targeted drug combinations and nuclear receptors should also be intensely explored for HNC. Gender-specific VDR/VitD-effects may be correlated to socioeconomic differences and need to be considered during VitD (supplementation)-therapies.

Nanoparticle-Mediated Drug Delivery of Doxorubicin Induces a Differentiated Clonogenic Inactivation in 3D Tumor Spheroids In Vitro.

Involvement of 3D tumor cell models in the in vitro biological testing of novel nanotechnology-based strategies for cancer management can provide in-depth information on the real behavior of tumor cells in complex biomimetic architectures. Here, we used polyethylene glycol-encapsulated iron oxide nanoparticles for the controlled delivery of a doxorubicin chemotherapeutic substance (IONPDOX), and to enhance cytotoxicity of photon radiation therapy. The biological effects of nanoparticles and 150 kV X-rays were evaluated on both 2D and 3D cell models of normal human keratinocytes (HaCaT) and tumor cells-human cervical adenocarcinoma (HeLa) and human squamous carcinoma (FaDu)-through cell survival. In all 2D cell models, nanoparticles were similarly internalized in a peri-nuclear pattern, but resulted in different survival capabilities following radiation treatment. IONP on normal keratinocytes showed a protective effect, but a cytotoxic effect for cancer cells. In 3D tumor cell models, IONPDOX were able to penetrate the cell spheroids towards the hypoxic areas. However, IONPDOX and 150 kV X-rays led to a dose-modifying factor DMFSF=0.1 = 1.09 ± 0.1 (200 µg/mL IONPDOX) in HeLa spheroids, but to a radioprotective effect in FaDu spheroids. Results show that the proposed treatment is promising in the management of cervical adenocarcinoma.

Applications and Advances of Multicellular Tumor Spheroids: Challenges in Their Development and Analysis.

Biomedical research requires both in vitro and in vivo studies in order to explore disease processes or drug interactions. Foundational investigations have been performed at the cellular level using two-dimensional cultures as the gold-standard method since the early 20th century. However, three-dimensional (3D) cultures have emerged as a new tool for tissue modeling over the last few years, bridging the gap between in vitro and animal model studies. Cancer has been a worldwide challenge for the biomedical community due to its high morbidity and mortality rates. Various methods have been developed to produce multicellular tumor spheroids (MCTSs), including scaffold-free and scaffold-based structures, which usually depend on the demands of the cells used and the related biological question. MCTSs are increasingly utilized in studies involving cancer cell metabolism and cell cycle defects. These studies produce massive amounts of data, which demand elaborate and complex tools for thorough analysis. In this review, we discuss the advantages and disadvantages of several up-to-date methods used to construct MCTSs. In addition, we also present advanced methods for analyzing MCTS features. As MCTSs more closely mimic the in vivo tumor environment, compared to 2D monolayers, they can evolve to be an appealing model for in vitro tumor biology studies.

Canonical Wnt Pathway Is Involved in Chemoresistance and Cell Cycle Arrest Induction in Colon Cancer Cell Line Spheroids.

The presence of cancer stem cells (CSCs) has been associated with the induction of drug resistance and disease recurrence after therapy. 5-Fluorouracil (5FU) is widely used as the first-line treatment of colorectal cancer (CRC). However, its effectiveness may be limited by the induction of drug resistance in tumor cells. The Wnt pathway plays a key role in the development and CRC progression, but it is not clearly established how it is involved in CSCs resistance to treatment. This work aimed to investigate the role played by the canonical Wnt/ß-catenin pathway in CSCs resistance to 5FU treatment. Using tumor spheroids as a model of CSCs enrichment of CRC cell lines with different Wnt/ß-catenin contexts, we found that 5FU induces in all CRC spheroids tested cell death, DNA damage, and quiescence, but in different proportions for each one: RKO spheroids were very sensitive to 5FU, while SW480 were less susceptible, and the SW620 spheroids, the metastatic derivative of SW480 cells, displayed the highest resistance to death, high clonogenic capacity, and the highest ability for regrowth after 5FU treatment. Activating the canonical Wnt pathway with Wnt3a in RKO spheroids decreased the 5FU-induced cell death. But the Wnt/ß-catenin pathway inhibition with Adavivint alone or in combination with 5FU in spheroids with aberrant activation of this pathway produced a severe cytostatic effect compromising their clonogenic capacity and diminishing the stem cell markers expression. Remarkably, this combined treatment also induced the survival of a small cell subpopulation that could exit the arrest, recover SOX2 levels, and re-grow after treatment.

Blockade of ß-Adrenergic Receptors by Nebivolol Enables Tumor Control Potential for Uveal Melanoma in 3D Tumor Spheroids and 2D Cultures.

Uveal melanoma (UM) is the most common primary cancer of the eye in adults. A new systemic therapy is needed to reduce the high metastasis and mortality rate. As ß-blockers are known to have anti-tumor effects on various cancer entities, this study focuses on investigating the effect of ß1-selective blockers atenolol, celiprolol, bisoprolol, metoprolol, esmolol, betaxolol, and in particular, nebivolol on UM. The study was performed on 3D tumor spheroids as well as 2D cell cultures, testing tumor viability, morphological changes, long-term survival, and apoptosis. Flow cytometry revealed the presence of all three ß-adrenoceptors with a dominance of ß2-receptors on cell surfaces. Among the blockers tested, solely nebivolol concentration-dependently decreased viability and altered 3D tumor spheroid structure. Nebivolol blocked the repopulation of cells spreading from 3D tumor spheroids, indicating a tumor control potential at a concentration of ≥20 µM. Mechanistically, nebivolol induced ATP depletion and caspase-3/7 activity, indicating that mitochondria-dependent signaling is involved. D-nebivolol or nebivolol combined with the ß2-antagonist ICI 118.551 displayed the highest anti-tumor effects, suggesting a contribution of both ß1- and ß2-receptors. Thus, the present study reveals the tumor control potential of nebivolol in UM, which may offer a perspective for co-adjuvant therapy to reduce recurrence or metastasis.

Cellular Effects of Selected Unsymmetrical Bisacridines on the Multicellular Tumor Spheroids of HCT116 Colon and A549 Lung Cancer Cells in Comparison to Monolayer Cultures.

Multicellular tumor spheroids are a good tool for testing new anticancer drugs, including those that may target cancer stem cells (CSCs), which are responsible for cancer progression, metastasis, and recurrence. Therefore, we applied this model in our studies of highly active antitumor unsymmetrical bisacridines (UAs). We investigated the cellular response induced by UAs in 2D and 3D cultures of HCT116 colon and A549 lung cancer cells, with an additional focus on their impact on the CSC-like population. We showed that UAs affected the viability of the studied cells, as well as their spherogenic potential in the 2D and 3D cultures. Furthermore, we proved that the most promising UAs (C-2045 and C-2053) induced apoptosis in the HCT116 and A549 spheres to a similar, or even higher, extent than what was found in monolayer conditions. Next, we identified the population of the CSC-like cells in the 2D and 3D cultures of the studied cell lines by determining the levels of CD166, CD133, CD44, and EpCAM markers. We showed that the selected UAs affected the CSC-like population in both of the cell lines, and that A549 was affected more profoundly in 3D than in 2D cultures. Thus, the UAs exhibited high antitumor properties in both the 2D and 3D conditions, which makes them promising candidates for future therapeutic applications.

Radiobiological Assessment of Targeted Radionuclide Therapy with [177Lu]Lu-PSMA-I&T in 2D vs. 3D Cell Culture Models.

In vitro therapeutic efficacy studies are commonly conducted in cell monolayers. However, three-dimensional (3D) tumor spheroids are known to better represent in vivo tumors. This study used [177Lu]Lu-PSMA-I&T, an already clinically applied radiopharmaceutical for targeted radionuclide therapy against metastatic castrate-resistant prostate cancer, to demonstrate the differences in the radiobiological response between 2D and 3D cell culture models of the prostate cancer cell lines PC-3 (PSMA negative) and LNCaP (PSMA positive). After assessing the target expression in both models via Western Blot, cell viability, reproductive ability, and growth inhibition were assessed. To investigate the geometric effects on dosimetry for the 2D vs. 3D models, Monte Carlo simulations were performed. Our results showed that PSMA expression in LNCaP spheroids was highly preserved, and target specificity was shown in both models. In monolayers of LNCaP, no short-term (48 h after treatment), but only long-term (14 days after treatment) radiobiological effects were evident, showing decreased viability and reproductive ability with the increasing activity. Further, LNCaP spheroid growth was inhibited with the increasing activity. Overall, treatment efficacy was higher in LNCaP spheroids compared to monolayers, which can be explained by the difference in the resulting dose, among others.

Tumor-penetrating peptide internalizing RGD enhances radiotherapy efficacy through reducing tumor hypoxia.

Resistance to irradiation (IR) remains a major therapeutic challenge in tumor radiotherapy. The development of novel tumor-specific radiosensitizers is crucial for effective radiotherapy against solid tumors. Here, we revealed that remodeling tumor tissue penetration via tumor-penetrating peptide internalizing arginine-glycine-aspartic acid RGD (iRGD) enhanced irradiation efficacy. The growth of 4T1 and CT26 multicellular tumor spheroids (MCTS) and tumors was delayed significantly by the treatment with IR and iRGD. Mechanistically, iRGD reduced hypoxia in MCTS and tumors, resulting in enhanced apoptosis after MCTS and tumors were treated with IR and iRGD. This is the first report that shows enhanced radiation efficacy by remodeling tumor-specific tissue penetration with iRGD, implying the potential clinical application of peptides in future tumor therapy.

Microfluidic platform for studying the anti-cancer effect of ursolic acid on tumor spheroid.

At present, the probability that a new anti-tumor drug will eventually succeed in clinical trials is extremely low. In order to make up for this shortcoming, the use of a three-dimensional (3D) cell culture model for secondary screening is often necessary. Cell spheroid is the easiest 3D model tool for drug screening. In this study, the microfluidic chip with a microwell array was manufactured, which could allow the formation of tumor spheroids with uniform size and easily retrieve cell spheroids from the chip. Cell spheroids were successfully cultured for over 15 days and the survival rate was as high as 80%. Subsequently, cellular response to the ursolic acid (UA) was observed on the chip. Compared to the monolayer culture cells in vitro, the tumor spheroids showed minor levels of epithelial-mesenchymal transition fluctuation after drug treatment. The mechanism of cell spheroid resistance to UA was further verified by detecting the expression level of upstream pathway proteins. But the invasive ability of tumor spheroids was attenuated when the duration of action of UA extended. The anti-cancer effect of UA was innovatively evaluated on breast cancer by using the microfluidic device, which could provide a basis and direction for future preclinical research on UA.

Assessment of cellular responses in three-dimensional cell cultures through chemical exchange saturation transfer and 1 H MRS.

Metabolic responses to physiological changes have been detected using chemical exchange saturation transfer (CEST) imaging in clinical settings. Similarly to other MRI techniques, the CEST technique was based originally on phantoms from buffer solutions and was then further developed through animal experiments. However, CEST imaging can capture certain dynamics of metabolism that solution phantoms cannot model. Cell culture phantoms can fill the gap between buffer phantoms and animal models. In this study, we used 1 H NMR and CEST in a B0 field of 9.4 T to investigate HEK293T cells from two-dimensional (2D) cultures, three-dimensional (3D) cultures, and 3D cultures seeded with cell spheroids. Two CEST dips were observed: the magnitude of the amine dip at 2.8 ppm increased during the incubation period, whereas the hydroxyl dip at 1.2 ppm remained approximately the same or modestly increased. We also observed a CEST dip at 2.8 ppm from the 2D culture responding dramatically to doxorubicin treatment. By cross-validating with pH values and the concentrations of amine and hydroxyl protons extracted through 1 H NMR, we observed that they did not correspond to an increase in the amine pool. We believe that the denaturation or degradation of proteins from the fetal bovine serum increased the size of the amine pool. Although 3D culture conditions can be further improved, our study suggests that 3D cultures have the potential to bridge studies of solution phantoms and those on animals.

Predicting response of micrometastases with MIRDcell V3: proof of principle with 225Ac-DOTA encapsulating liposomes that produce different activity distributions in tumor spheroids.

PURPOSE: The spatial distribution of radiopharmaceuticals within multicellular clusters is known to have a significant effect on their biological response. Most therapeutic radiopharmaceuticals distribute nonuniformly in tissues which makes predicting responses of micrometastases challenging. The work presented here analyzes published temporally dependent nonuniform activity distributions within tumor spheroids treated with actinium-225-DOTA encapsulating liposomes (225Ac-liposomes) and uses these data in MIRDcell V3.11 to calculate absorbed dose distributions and predict biological response. The predicted responses are compared with experimental responses. METHODS: Four types of liposomes were prepared having membranes with different combinations of release (R) and adhesion (A) properties. The combinations were R-A-, R-A+, R+A-, and R+A+. These afford different penetrating properties into tissue. The liposomes were loaded with either carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) or 225Ac. MDA-MB-231 spheroids were treated with the CFDA-SE-liposomes, harvested at different times, and the time-integrated CFDA-SE concentration at each radial position within the spheroid was determined. This was translated into mean 225Ac decays/cell versus radial position, uploaded to MIRDcell, and the surviving fraction of cells in spherical multicellular clusters was simulated. The MIRDcell-predicted surviving fractions were compared with experimental fractional-outgrowths of the spheroids following treatment with 225Ac-liposomes. RESULTS: The biological responses of the multicellular clusters treated with 225Ac-liposomes with physicochemical properties R+A+, R-A+, and R-A- were predicted by MIRDcell with statistically significant accuracy. The prediction for R+A- was not predicted accurately. CONCLUSION: In most instances, MIRDcell predicts responses of spheroids treated with 225Ac-liposomes that result in different tissue-penetrating profiles of the delivered radionuclides.

Hyaluronic-Acid-Tagged Cubosomes Deliver Cytotoxics Specifically to CD44-Positive Cancer Cells.

Delivery of chemotherapy drugs specifically to cancer cells raises local drug doses in tumors and therefore kills more cancer cells while reducing side effects in other tissues, thereby improving oncological and quality of life outcomes. Cubosomes, liquid crystalline lipid nanoparticles, are potential vehicles for delivery of chemotherapy drugs, presenting the advantages of biocompatibility, stable encapsulation, and high drug loading of hydrophobic or hydrophilic drugs. However, active targeting of drug-loaded cubosomes to cancer cells, as opposed to passive accumulation, remains relatively underexplored. We formulated and characterized cubosomes loaded with potential cancer drug copper acetylacetonate and functionalized their surfaces using click chemistry coupling with hyaluronic acid (HA), the ligand for the cell surface receptor CD44. CD44 is overexpressed in many cancer types including breast and colorectal. HA-tagged, copper-acetylacetonate-loaded cubosomes have an average hydrodynamic diameter of 152 nm, with an internal nanostructure based on the space group Im3m. These cubosomes were efficiently taken up by two CD44-expressing cancer cell lines (MDA-MB-231 and HT29, representing breast and colon cancer) but not by two CD44-negative cell lines (MCF-7 breast cancer and HEK-293 kidney cells). HA-tagged cubosomes caused significantly more cell death than untargeted cubosomes in the CD44-positive cells, demonstrating the value of the targeting. CD44-negative cells were equally relatively resistant to both, demonstrating the specificity of the targeting. Cell death was characterized as apoptotic. Specific targeting and cell death were evident in both 2D culture and 3D spheroids. We conclude that HA-tagged, copper-acetylacetonate-loaded cubosomes show great potential as an effective therapeutic for selective targeting of CD44-expressing tumors.

3D Printed Solutions for Spheroid Engineering and Cancer Research.

In multicellular organisms, cells are organized in a 3-dimensional framework and this is essential for organogenesis and tissue morphogenesis. Systems to recapitulate 3D cell growth are therefore vital for understanding development and cancer biology. Cells organized in 3D environments can evolve certain phenotypic traits valuable to physiologically relevant models that cannot be accessed in 2D culture. Cellular spheroids constitute an important aspect of in vitro tumor biology and they are usually prepared using the hanging drop method. Here a 3D printed approach is demonstrated to fabricate bespoke hanging drop devices for the culture of tumor cells. The design attributes of the hanging drop device take into account the need for high-throughput, high efficacy in spheroid formation, and automation. Specifically, in this study, custom-fit, modularized hanging drop devices comprising of inserts (Q-serts) were designed and fabricated using fused filament deposition (FFD). The utility of the Q-serts in the engineering of unicellular and multicellular spheroids-synthetic tumor microenvironment mimics (STEMs)-was established using human (cancer) cells. The culture of spheroids was automated using a pipetting robot and bioprinted using a custom bioink based on carboxylated agarose to simulate a tumor microenvironment (TME). The spheroids were characterized using light microscopy and histology. They showed good morphological and structural integrity and had high viability throughout the entire workflow. The systems and workflow presented here represent a user-focused 3D printing-driven spheroid culture platform which can be reliably reproduced in any research environment and scaled to- and on-demand. The standardization of spheroid preparation, handling, and culture should eliminate user-dependent variables, and have a positive impact on translational research to enable direct comparison of scientific findings.

Differential Cytotoxicity of Curcumin-Loaded Micelles on Human Tumor and Stromal Cells.

Although curcumin in the form of nanoparticles has been demonstrated as a potential anti-tumor compound, the impact of curcumin and nanocurcumin in vitro on normal cells and in vivo in animal models is largely unknown. This study evaluated the toxicity of curcumin-loaded micelles in vitro and in vivo on several tumor cell lines, primary stromal cells, and zebrafish embryos. Breast tumor cell line (MCF7) and stromal cells (human umbilical cord vein endothelial cells, human fibroblasts, and human umbilical cord-derived mesenchymal stem cells) were used in this study. A zebrafish embryotoxicity (FET) assay was conducted following the Organisation for Economic Co-operation and Development (OECD) Test 236. Compared to free curcumin, curcumin PM showed higher cytotoxicity to MCF7 cells in both monolayer culture and multicellular tumor spheroids. The curcumin-loaded micelles efficiently penetrated the MCF7 spheroids and induced apoptosis. The nanocurcumin reduced the viability and disturbed the function of stromal cells by suppressing cell migration and tube formation. The micelles demonstrated toxicity to the development of zebrafish embryos. Curcumin-loaded micelles demonstrated toxicity to both tumor and normal primary stromal cells and zebrafish embryos, indicating that the use of nanocurcumin in cancer treatment should be carefully investigated and controlled.