Cancer Cell Spheroids Research Articles

A microfluidic platform for cultivating ovarian cancer spheroids and testing their responses to chemotherapies

There is increasing interest in utilizing in vitro cultures as patient avatars to develop personalized treatment for cancer. Typical cultures utilize Matrigel-coated plates and media to promote the proliferation of cancer cells as spheroids or tumor explants. However, standard culture conditions operate in large volumes and require a high concentration of cancer cells to initiate this process. Other limitations include variability in the ability to successfully establish a stable line and inconsistency in the dimensions of these microcancers for in vivo drug response measurements. This paper explored the utility of microfluidics in the cultivation of cancer cell spheroids. Six patient-derived xenograft (PDX) tumors of high-grade serous ovarian cancer were used as the source material to demonstrate that viability and epithelial marker expression in the microfluidic cultures was superior to that of Matrigel or large volume 3D cultures. To further demonstrate the potential for miniaturization and multiplexing, we fabricated multichamber microfluidic devices with integrated microvalves to enable serial seeding of several chambers followed by parallel testing of several drug concentrations. These valve-enabled microfluidic devices permitted the formation of spheroids and testing of seven drug concentrations with as few as 100,000 cancer cells per device. Overall, we demonstrate the feasibility of maintaining difficul-to-culture primary cancer cells and testing drugs in a microfluidic device. This microfluidic platform may be ideal for drug testing and personalized therapy when tumor material is limited, such as following the acquisition of biopsy specimens obtained by fine-needle aspiration.

Modeling Adaptive Resistance of KRAS Mutant Colorectal Cancer to MAPK Pathway Inhibitors with a Three-Dimensional Tumor Model.

Single-agent drug treatment of KRASmut colorectal cancers is often ineffective because the activation of compensatory signaling pathways leads to drug resistance. To mimic cyclic chemotherapy treatments of patients, we showed that intermittent treatments of 3D tumor spheroids of KRASmut colorectal cancer cells with inhibitors of mitogen-activated protein kinase (MAPK) signaling pathway temporarily suppressed growth of spheroids. However, the efficacy of successive single-agent treatments was significantly reduced. Molecular analysis showed compensatory activation of PI3K/AKT and STAT kinases and EGFR family proteins. To overcome the adaptation of cancer cells to MAPK pathway inhibitors, we treated tumor spheroids with a combination of MEK and EGFR inhibitors. This approach significantly blocked signaling of MAPK and PI3K/AKT pathways and prevented the growth of spheroids, but it was not effective against STAT signaling. Although the combination treatment blocked the matrix invasion of DLD1 cells, additional treatments with STAT inhibitors were necessary to prevent invasiveness of HCT116 cells. Overall, our drug resistance model elucidated the mechanisms of treatment-induced growth and invasiveness of cancer cells and allowed design-driven testing and identifying of effective treatments to suppress these phenotypes.

Potent antitumor activity of cepharanthine against triple-negative breast cancer spheroids compared with tetrandrine.

Cepharanthine (CEP) is a bis-bynzelisoquinoline alkaloid from the same class as the anticancer agent tetrandrine (TET). However, the effects of CEP against breast cancer have not been extensively studied, despite its long therapeutic history with low toxicity against other types of cancer. 3D culture systems more accurately mimic the human body and address the limitations of determining drug effectiveness compared with 2D culture systems. In the present study, the antitumor activities of TET and CEP were compared in 3D culture systems in triple-negative breast cancer (TNBC) MDA-MB-231 and estrogen receptor-positive breast cancer MCF-7 cell lines. Cell viability, apoptosis and cytotoxicity assays were performed to determine the total number of live or dead cells, the IC50 values, the number of apoptotic cells and spheroid roundness. Viability suppression of MDA-MB-231 cells was significantly greater with both TET and CEP compared with that of MCF-7 cells, and the roundness of MDA-MB-231 spheroids treated with CEP was decreased significantly compared with that of spheroid treated with TET. Cytoplasmic shrinkage in each cell line significantly increased with the treatment of TET compared with the control; however, this effect was stronger with CEP. The ratio of dead/live cells in each cell line treated with TET and CEP increased in a dose-dependent manner. Overall, the present study demonstrated that CEP had greater cell toxicity in 3D spheroids of breast cancer cells compared with TET, suggesting that CEP may have a stronger antitumor activity on TNBC spheroids compared with TET.

112 Poster - The marine natural product HB-395 selectively induces apoptosis in MDA-MB-231 triple negative breast cancer cell spheroids

112 Poster - The marine natural product HB-395 selectively induces apoptosis in MDA-MB-231 triple negative breast cancer cell spheroids

The 3D genomic landscape of differential response to EGFR/HER2 inhibition in endocrine-resistant breast cancer cells

BackgroundRecent studies suggested that crosstalk between ERα and EGFR/HER2 pathways plays a critical role in mediating endocrine therapy resistance. Several inhibitors targeting EGFR/HER2 signaling, including FDA-approved lapatinib and gefitinib as well as a novel dual tyrosine kinase inhibitor (TKI) sapitinib, showed greater therapeutic efficacies. However, how 3D chromatin landscape responds to the inhibition of EGFR/HER2 pathway remains to be elucidated. MethodsIn this study, we conducted in situ Hi-C and RNA-seq in two ERα+ breast cancer cell systems, 1) parental MCF7 cells and its associated tamoxifen-resistant MCF7TR cells; and 2) parental T47D cells and its associated tamoxifen-resistant T47DTR cells, before and after the treatment of sapitinib. ResultsWe identified differential responses in topologically associated domains (TADs), looping genes and expressed genes. Interestingly, we found that many differential TADs and looping genes are reversible after sapitinib treatment, indicating that EGFR/HER2 signaling may play a role in reshaping and rewiring the high order genome organization. We further examined and recapitulated the reversible looping genes in 3D spheroids of breast cancer cells, demonstrating that 3D cell culture spheroid of breast cancer cells could be a potential preclinical breast cancer model for studying 3D chromatin regulation. ConclusionsOur study has provided significant insights into our understanding of 3D genomic landscape changes in response to EGFR/HER2 Inhibition in endocrine-resistant breast cancer cells. Our data provides a rich resource for further evaluating chromatin structural responses to EGFR/HER2 targeted therapies in endocrine-resistant breast cancer cells. Our analyses suggest that these alterations of chromatin structures and transcriptional programs may provide new avenues for intervention or designing of patient selection for targeted endocrine treatment.

Improvement of the multi-performance biocharacteristics of cordycepin using BiloNiosome-core/chitosan-shell hybrid nanocarriers

Cordycepin, a derivative of the nucleotide adenosine, has displayed several pharmacological activities including enhanced apoptosis and cancer cells inhibition. However, oral administration of cordycepin has limited practical use due to its poor bioavailability in the intestine. Herein, we developed and demonstrated a hybrid nanocarrier system in the form of biloniosome-core/chitosan-shell hybrid nanocarriers (HNCs) in order to improve the bio-characteristics of cordycepin. In this study, HNCs were prepared by using a solvent (ethanol) injection method involving cordycepin as the biloniosome core and mucoadhesive chitosan biopolymer as a coating shell. Our results showed that the cordycepin-loaded HNCs were positively charged with enhanced mucoadhesive characteristics and highly stable in gastric fluid. The increased permeability of cordycepin-loaded HNCs compared with standard cordycepin was confirmed by in vitro intestinal permeation study across the human intestinal barrier. In addition, we demonstrated that the cordycepin-loaded HNCs are able to release their components in an active form resulting in enhanced anti-cancer activity in two-dimensional (2D) cell cultures as well as in three-dimensional (3D) multi-cellular spheroids of colon cancer cells. Further, quantitative real time PCR analysis of apoptotic gene expression revealed that cordycepin HNCs can induce apoptosis in cancer cells by negatively regulating the expression of B-cell lymphoma-extra large (BCL-XL). I Overall our results showed that the hybrid nanocarrier systems represent a promising strategy for improving the bio-characteristics of cordycepin which can be considered as a potential anti-cancer agent for colorectal cancer chemotherapy.

Hypoxia-Responsive, Polymeric Nanocarriers for Targeted Drug Delivery to Estrogen Receptor-Positive Breast Cancer Cell Spheroids.

Uncontrolled cell growth, division, and lack of enough blood supply causes low oxygen content or hypoxia in cancerous tumor microenvironments. 17β-Estradiol (E2), an estrogen receptor (ER) ligand, can be incorporated on the surface of nanocarriers for targeted drug delivery to breast cancer cells overexpressing ER. In the present study, we synthesized estradiol-conjugated hypoxia-responsive polymeric nanoparticles (polymersomes) encapsulating the anticancer drug doxorubicin (E2-Dox-HRPs) for targeted delivery into the hypoxic niches of estrogen-receptor-positive breast cancer microtumors. Estradiol-conjugated polymersomes released over 90% of their encapsulated Dox in a sustained manner within hypoxia (2% oxygen) after 12 h. However, they released about 30% of Dox in normal oxygen partial pressure (21% oxygen, normoxia) during this time. Fluorescence microscopic studies demonstrated higher cytosolic and nuclear internalization of E2-Dox-HRPs (targeted polymersomes) compared to those of Dox-HRPs (nontargeted polymersomes). Monolayer cell viability studies on ER-positive MCF7 cells showed higher cytotoxicity of targeted polymersomes in hypoxia compared to in normoxia. Cytotoxicity studies with hypoxic three-dimensional spheroid cultures of MCF7 cells treated with targeted polymersomes indicated significant differences compared to those of normoxic spheroids. The novel estradiol-conjugated hypoxia-responsive polymersomes described here have the potential for targeted drug delivery in estrogen-receptor-positive breast cancer therapy.

Abstract 980: Maligant ascites microenvironment skews M2/M1 macrophage polarization in ovarian cancer during peritoneal metastasis

Abstract Advanced ovarian cancers with peritoneal metastases are usually accompanied with metastatic recurrence and high mortality. Macrophages, as a primary cell population in the tumor microenvironment, can be active and polarized into heterogeneous tumor-associated macrophages (TAMs). Clinical evidence indicates that a high M2/M1 ratio of TAMs are commonly found in malignant ascites microenvironment (MAM), and is attributed to a poor prognosis in advanced ovarian cancers. However, the molecular mechanisms of macrophage polarization in MAM remain elusive. Here, we report that MAM could modulate Hippo and/or Wnt signaling cascades in facilitating M2 macrophage polarization. In this study, we firstly evidenced the heterogeneity macrophage populations in cancerous omental tissues and ascites cancer cell spheroids of ovarian cancers by multiplex immunohistochemical, flow cytometry and immunofluorescent analyses. Consistently, in vitro polarization model demonstrated that co-cultured monocyte (THP-1 and U937) with omentum conditioned medium (OCM) or ascites significantly led to more CD206+ M2 macrophage than CD86+ M1 macrophage or M2/M1 ratio. Quantitative RT-PCR (qPCR) analysis showed that TAM-related genes: CCL2, IL6, and IL10 genes were significantly upregulated in OCM treated macrophage. Intriguingly, OCM treated macrophage could significantly promotes ovarian cancer cell migratory capacities. Importantly, RNA-seq analysis identified Hippo and Wnt signaling cascades may play important roles in macrophage polarization in malignant OCM and ascites. Further investigations of these pathways in M2/M1 macrophage polarization in MAM are warranted. Citation Format: Huogang Wang, Mingo MH Yung, Arvin FS Chen, Hextan YS Ngan, David W. Chan. Maligant ascites microenvironment skews M2/M1 macrophage polarization in ovarian cancer during peritoneal metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 980.

Functionalized Block Co‐Polymer Pro‐Drug Nanoparticles with Anti‐Cancer Efficacy in 3D Spheroids and in an Orthotopic Triple Negative Breast Cancer Model

AbstractAmphiphilic block co‐polymers composed of poly(ethylene glycol)‐co‐poly(lactide)‐co‐poly(2‐((tert‐butoxycarbonyl)amino)‐3‐propyl carbonate) (PEG‐pLA‐pTBPC) are synthesized in monomer ratios and arrangements to enable assembly into nanoparticles with different sizes and architectures. These materials are based on components in clinical use, or known to be biodegradable, and retain the same fundamental chemistry across “AB” and “BAB” block architectures. In MCF7 and MDA‐MB‐231 breast cancer cells, nanoparticles of <100 nm are internalized most rapidly, by both clathrin‐ and caveolin‐mediated pathways. In THP‐1 cells, polymer architecture and length of the hydrophilic block is the most important factor in the rate of internalization. The organ distributions of systemically injected nanoparticles in healthy mice indicate highest accumulation of the BAB‐blocks in lungs and liver and the lowest accumulation in these organs of a methoxyPEG5000‐pLA‐pTBPC polymer. Conjugation of doxorubicin via a serum‐stable urea linker to the carbonate regions of PEG5000‐pLA‐pTBPC generates self‐assembling nanoparticles which are more cytotoxic in 2D, and penetrate further in 3D spheroids of triple negative breast cancer cells, than the free drug. In an aggressive orthotopic triple negative breast cancer mouse model, the methoxyPEG5000‐pLA‐pTBPC is of similar potency to free doxorubicin but with no evidence of adverse effects in terms of body weight.

Mass Spectrometry Imaging Combined with Metabolomics Revealing the Proliferative Effect of Environmental Pollutants on Multicellular Tumor Spheroids.

Multicellular tumor spheroids (MCTS) have gained increasing attention in cancer research because they may closely mimic some physiological characteristics of solid tumors. MCTS have been considered as a useful three-dimensional cell model for evaluating the effect of exogenous molecules on tumor progression. However, little is known about the metabolic response in MCTS after exposure to exogenous molecules. Herein, we applied metabolomics combined with MALDI-mass spectrometry imaging (MSI) to investigate the proliferation of three-dimensional MDA-MB-231 breast cancer cell spheroids treated with bisphenol S (BPS). MSI data revealed that BPS, a common environmental contaminant, penetrated MCTS in 5 min and gradually localized in the core of MCTS within 4 h. Metabolomic data demonstrated that BPS exposure induced significant changes in the levels of 28 metabolites that are involved in several pathways, including purine metabolism and the tricarboxylic acid cycle. The MSI results showed that three upregulated metabolites (ATP, ADP, and AMP) acting major roles in energy supply distributed in the proliferative zone of cell spheroids, further indicating the proliferative response of MDA-MB-231 cell spheroids caused by BPS exposure. One downregulated metabolite (xanthine) associated with reactive oxidative stress was found to localize toward the inner region of cell spheroids. These MSI results demonstrated that the increase of energy supply in the outer layer of cell spheroids might be responsible for BPS-induced proliferative response. Taken together, this integrated method might offer a more accurate and intuitive assessment for the effect of exogenous molecules on cancer progression.

Inhibition of Ovarian Cancer Cell Spheroid Formation by Synthetic Peptides Derived from Nectin-4.

The formation of 3D multicellular spheroids in the ascites fluid of ovarian cancer patients is an understudied component of the disease progression. Spheroids are less sensitive to chemotherapy, in part due to the protection afforded by their structure, but also due to their slower proliferation rate. Previous studies suggest that the cell adhesion molecule Nectin-4 plays a key role in the formation of ovarian cancer spheroids. In this study, we further examined the role of Nectin-4 at early time points in spheroid formation using real-time digital photography. Human NIH:OVCAR5 ovarian cancer cells formed aggregates within 8 h, which further contracted into compact spheroids over 24 h. In contrast, Nectin-4 knockdown cells did not form tightly compacted spheroids. Synthetic peptides derived from Nectin-4 were tested for their ability to alter spheroid formation in two ovarian cancer cell lines. Nectin-4 peptide 10 (N4-P10) had an immediate effect on disrupting ovarian cancer spheroid formation, which continued for over 24 h, while a scrambled version of the peptide had no effect. N4-P10 inhibited spheroid formation in a concentration-dependent manner and was not cytotoxic; suggesting that N4-P10 treatment could maintain the cancer cells as single cells which may be more sensitive to chemotherapy.

Autophagy in fibroblasts induced by cigarette smoke extract promotes invasion in lung cancer cells.

We investigated the effects of cigarette smoke extract (CSE) on lung fibroblasts and found that the invasiveness of lung cancer cells was facilitated by the conditioned medium from CSE-treated fibroblasts. CSE induced autophagy in fibroblasts and increased the expression of autophagy-related proteins, including optineurin and Ras-related protein Rab1B. Afterward, the fibroblasts produced high levels of interleukin-8 (IL-8), which promoted cancer cell invasion. The inhibition of either optineurin or Rab1B abrogated a rise in microtubule-associated protein 1 light chain 3 β and a decrease in p62 protein, as well as the production of IL-8, in CSE-treated fibroblasts. A three-dimensional invasion assay using cancer cell spheroids revealed that the invasion of cancer cells alone and the fibroblast-led cancer cell invasion were both enhanced by the conditioned media from CSE-treated fibroblasts. These results suggest that cigarette smoke may induce autophagy and IL-8 secretion in lung fibroblasts and modify the microenvironment to favor invasion of lung cancer cells.

Simplified low-cost methodology to establish, histologically process and analyze three-dimensional cancer cell spheroid arrays

Differently of two-dimensional cell culture, three-dimensional (3D) multicellular spheroid model allows cells to establish cell-cell/cell-matrix interactions over the entire cell surface, more closely mimicking tumor microenvironments and cellular subpopulations with specific standards of morphology, differentiation and gene expression. Thenceforth several methodologies involving or the 3D cell aggregates generation or its histological processing and analysis have emerged, but in general they are laborious, expensive and complex to set up as a routine technique. Thus, we developed a complete methodology, detailing a simple, accessible and low-cost step by step, including 1) the 3D cell aggregate generation using hanging drop technique; 2) providing a simple way to assess morphological parameters of generated spheroids; followed by 3) a multiple and organized histological processing, keeping several individual spheroids inside an agarose apparatus, maintaining a known order and position of each ones, similar to tissue microarray principle; 4) until the last step, where it is allowed a simultaneous histological composition analysis of several spheroid slices, organized side by side, in a same block section, through conventional stainings or 5) immunostaining against different molecular markers. Therefore, the present methodology aims to popularize 3D cell culture, allowing to make this a regular technique in basic cell biology research, once all steps are performed without using onerous reagents, materials or equipment. In addition to bring the agarose apparatus as a simple low cost novelty, allowing high-throughput analysis of several spheroids simultaneously in an organized manner.

Stress-induced TRAILR2 expression overcomes TRAIL resistance in cancer cell spheroids

The influence of 3D microenvironments on apoptosis susceptibility remains poorly understood. Here, we studied the susceptibility of cancer cell spheroids, grown to the size of micrometastases, to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Interestingly, pronounced, spatially coordinated response heterogeneities manifest within spheroidal microenvironments: In spheroids grown from genetically identical cells, TRAIL-resistant subpopulations enclose, and protect TRAIL-hypersensitive cells, thereby increasing overall treatment resistance. TRAIL-resistant layers form at the interface of proliferating and quiescent cells and lack both TRAILR1 and TRAILR2 protein expression. In contrast, oxygen, and nutrient deprivation promote high amounts of TRAILR2 expression in TRAIL-hypersensitive cells in inner spheroid layers. COX-II inhibitor celecoxib further enhanced TRAILR2 expression in spheroids, likely resulting from increased ER stress, and thereby re-sensitized TRAIL-resistant cell layers to treatment. Our analyses explain how TRAIL response heterogeneities manifest within well-defined multicellular environments, and how spatial barriers of TRAIL resistance can be minimized and eliminated.

Do Patient-derived Spheroid Culture Models Have Relevance in Chondrosarcoma Research?

Do Patient-derived Spheroid Culture Models Have Relevance in Chondrosarcoma Research?

Nano-vesicular formulation of propolis and cytotoxic effects in a 3D spheroid model of lung cancer.

Propolis exhibits therapeutic properties due to the presence of phenolic acids, esters, and flavonoids. The scope of this study was to develop a nano-vesicular formulation and establish a three-dimensional (3D) spheroid model in which lung cancer is recapitulated. Niosome vesicles doped with galangin-rich propolis extract were synthesized by the ether injection method using a cholesterol : surfactant mass ratio of 1 : 3 at 40 °C for 1 h. Formulated niosomes were administered to 3D lung cancer spheroid model and the cytotoxicity was compared with that of a two-dimensional (2D) setting. The galangin content was determined as 86 μg mg-1 propolis extract by ultra-performance liquid chromatography (UPLC). The particle size of loaded niosome was 151 ± 2.84 nm with a polydispersity index (PDI) of about 0.232, and an encapsulation efficiency of 70% was achieved. The decrease in cell viability and the scattering in the 3D spheroids of A549 lung cancer cells treated with propolis-loaded niosomes were notable, indicating a profound cytotoxic effect and suggesting that they can be utilized as an effective nano-vesicle. © 2020 Society of Chemical Industry.

Synthesis and Cytotoxic Activity Evaluation of New Cu(I) Complexes of Bis(pyrazol-1-yl) Acetate Ligands Functionalized with an NMDA Receptor Antagonist.

In the present article, copper(I) complexes of bis(pyrazol-1-yl) carboxylic acid (LH), bis(3,5-dimethylpyrazol-1-yl) carboxylic acid (L2H), and bis(pyrazol-1-yl) acetates conjugated with an N-methyl-d-aspartate (NMDA) receptor antagonist (LNMDA or L2NMDA) and phosphane ligands (triphenylphosphine or 1,3,5-triaza-7-phosphaadamantane) were synthesized. The selection of an NMDA antagonist for the coupling with LH and L2H was suggested by the observation that NMDA receptors are expressed and play a role in different types of cancer models. All the new complexes showed a significant antitumor activity on a panel of human tumor cell lines of different histology, with cisplatin-sensitive, cisplatin-resistant, or multi-drug-resistant phenotype. Their half maximal inhibitory concentration (IC50) values were in the low- and sub-micromolar range and, in general, significantly lower than that of cisplatin. Interestingly, the fact that all the complexes proved to be significantly more active than cisplatin even in three-dimensional (3D) spheroids of H157 and BxPC3 cancer cells increased the relevance of the in vitro results. Finally, morphological analysis revealed that the most representative complex 8 induced a massive swelling of the endoplasmic reticulum (ER) membrane, which is a clear sign of ER stress.

Pyrazine ring-based Na+/H+ exchanger (NHE) inhibitors potently inhibit cancer cell growth in 3D culture, independent of NHE1

The Na+/H+ exchanger-1 (NHE1) supports tumour growth, making NHE1 inhibitors of interest in anticancer therapy, yet their molecular effects are incompletely characterized. Here, we demonstrate that widely used pyrazinoylguanidine-type NHE1 inhibitors potently inhibit growth and survival of cancer cell spheroids, in a manner unrelated to NHE1 inhibition. Cancer and non-cancer cells were grown as 3-dimensional (3D) spheroids and treated with pyrazinoylguanidine-type (amiloride, 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), 5-(N,N-dimethyl)-amiloride (DMA), and 5-(N,N-hexamethylene)-amiloride (HMA)) or benzoylguanidine-type (eniporide, cariporide) NHE1 inhibitors for 2–7 days, followed by analyses of viability, compound accumulation, and stress- and death-associated signalling. EIPA, DMA and HMA dose-dependently reduced breast cancer spheroid viability while cariporide and eniporide had no effect. Although both compound types inhibited NHE1, the toxic effects were NHE1-independent, as inhibitor-induced viability loss was unaffected by NHE1 CRISPR/Cas9 knockout. EIPA and HMA accumulated extensively in spheroids, and this was associated with marked vacuolization, apparent autophagic arrest, ER stress, mitochondrial- and DNA damage and poly-ADP-ribose-polymerase (PARP) cleavage, indicative of severe stress and paraptosis-like cell death. Pyrazinoylguanidine-induced cell death was partially additive to that induced by conventional anticancer therapies and strongly additive to extracellular-signal-regulated-kinase (ERK) pathway inhibition. Thus, in addition to inhibiting NHE1, pyrazinoylguanidines exert potent, NHE1-independent cancer cell death, pointing to a novel relevance for these compounds in anticancer therapy.

Platinum(IV) Complexes of trans-1,2-diamino-4-cyclohexene: Prodrugs Affording an Oxaliplatin Analogue that Overcomes Cancer Resistance.

Six platinum(IV) compounds derived from an oxaliplatin analogue containing the unsaturated cyclic diamine trans-1,2-diamino-4-cyclohexene (DACHEX), in place of the 1,2-diaminocyclohexane, and a range of axial ligands, were synthesized and characterized. The derivatives with at least one axial chlorido ligand demonstrated solvent-assisted photoreduction. The electrochemical redox behavior was investigated by cyclic voltammetry; all compounds showed reduction potentials suitable for activation in vivo. X-ray photoelectron spectroscopy (XPS) data indicated an X-ray-induced surface reduction of the Pt(IV) substrates, which correlates with the reduction potentials measured by cyclic voltammetry. The cytotoxic activity was assessed in vitro on a panel of human cancer cell lines, also including oxaliplatin-resistant cancer cells, and compared with that of the reference compounds cisplatin and oxaliplatin; all IC50 values were remarkably lower than those elicited by cisplatin and somewhat lower than those of oxaliplatin. Compared to the other Pt(IV) compounds of the series, the bis-benzoate derivative was by far (5–8 times) the most cytotoxic showing that low reduction potential and high lipophilicity are essential for good cytotoxicity. Interestingly, all the complexes proved to be more active than cisplatin and oxaliplatin even in three-dimensional spheroids of A431 human cervical cancer cells.

Therapeutic potential of a cell penetrating peptide (CPP, NP1) mediated siRNA delivery: Evidence in 3D spheroids of colon cancer cells

AbstractRNA interference holds great potential for cancer therapeutics and its success is highly dependent on an effective delivery system. As most preclinical drug screening in vitro was conducted in flat monolayer cell cultures, development of more physiologically relevant models is needed to enhance testing reliability and effectiveness. Here, the aim was to develop 3D cell spheroids and evaluate the efficiency of NP1, a novel cell penetrating peptide, CPP (STR‐H16R8), developed by our group to assist siRNA delivery. NP1 elicited significant cellular uptake of siRNA and promoted great siRNA knockdown efficiency of Bcl‐2 and VEGF mRNA in 3D spheroids (53% and 51%, respectively), induced marked apoptosis after silencing HIF mRNA, and 3D spheroids displayed apoptosis resistance compared to 2D cells. Taken together, 3D spheroids provide an improved model for testing siRNA delivery and NP1 has proved to be a powerful in vitro transfection reagent.