HCT116 Spheroids Research Articles

The Role of Mechanical Properties and Structure of Type I Collagen Hydrogels on Colorectal Cancer Cell Migration.

Mechanical interactions between cells and their microenvironment play an important role in determining cell fate, which is particularly relevant in metastasis, a process where cells invade tissue matrices with different mechanical properties. In vitro, type I collagen hydrogels have been commonly used for modeling the microenvironment due to its ubiquity in the human body. In this work, the combined influence of the stiffness of these hydrogels and their ultrastructure on the migration patterns of HCT-116 and HT-29 spheroids are analyzed. For this, six different types of pure type I collagen hydrogels by changing the collagen concentration and the gelation temperature are prepared. The stiffness of each sample is measured and its ultrastructure is characterized. Cell migration studies are then performed by seeding the spheroids in three different spatial conditions. It is shown that changes in the aforementioned parameters lead to differences in the mechanical stiffness of the matrices as well as the ultrastructure. These differences, in turn, lead to distinct cell migration patterns of HCT-116 and HT-29 spheroids in either of the spatial conditions tested. Based on these results, it is concluded that the stiffness and the ultrastructural organization of the matrix can actively modulate cell migration behavior in colorectal cancer spheroids.

Anti-Cancer Effects of Artesunate in Human 3D Tumor Models of Different Complexity.

The anti-malaria drug Artesunate (ART) shows strong anti-cancer effects in vitro; however, it shows only marginal treatment results in clinical cancer studies. In this study, ART was tested in preclinical 3D cancer models of increasing complexity using clinically relevant peak plasma concentrations to obtain further information for translation into clinical use. ART reduced cell viability in HCT-116 and HT-29 derived cancer spheroids (p < 0.001). HCT-116 spheroids responded dose-dependently, while HT-29 spheroids were affected more strongly by ART than by cytostatics (p < 0.001). HCT-116 spheroids were chemo-sensitized by ART (p < 0.001). In patient-derived cancer spheroids (PDCS), ART led to inhibition of cell viability in 84.62% of the 39 samples tested, with a mean inhibitory effect of 13.87%. Viability reduction of ART was 2-fold weaker than cytostatic monotherapies (p = 0.028). Meanwhile, tumor-stimulation of up to 16.30% was observed in six (15.38%) PDCS-models. In 15 PDCS samples, ART modulated chemotherapies in combined testing, eight of which showed chemo-stimulation (maximum of 36.90%) and seven chemo-inhibition (up to 16.95%). These results demonstrate that ART's anti-cancer efficacy depends on the complexity of the tumor model used. This emphasizes that cancer treatment with ART should be evaluated before treatment of the individual patient to ensure its benefits and prevent unwanted effects.

Anti-colon cancer effects of Spirulina polysaccharide and its mechanism based on 3D models

Spirulina polysaccharides (PSP) possess significant biological properties. However, it is still a lack of investigation on the anti-colorectal cancer effect and mechanism. In this study, PSP showed significant effects on LoVo cell spheroids with an IC50 value of 0.1943 mg/mL. The analysis of transcriptomics and metabolomics indicated the impact of PSP on LoVo spheroid cells through involvement in the two pathways of “glycine, serine, and threonine metabolism” and “ABC transporters”. And, the q-PCR data further verified the pointed mechanism of PSP on colon cancer (CC) by regulating the expression levels of relevant genes in the synthesis pathways of serine and glycine in tumor cells. Furthermore, the anti-colon cancer effects of PSP were verified via other human colon cancer cell lines HCT116 and HT29 spheroids (IC50 = 0.0646 mg/mL and 0.2213 mg/mL, respectively), and three patient-derived organoids (PDOs) with IC50 values ranging from 3.807 to 7.788 mg/mL. In addition, this study found that a mild concentration of PSP cannot enhance the anti-tumor effect of 5-Fu. And a significant inhibition was found of PSP in 5-Fu resistance organoids. These results illustrated that PSP could be a treatment or supplement for 5-Fu resistant colorectal cancer (CRC).

The Effect of Dynamic, In Vivo-like Oxaliplatin on HCT116 Spheroids in a Cancer-on-Chip Model Is Representative of the Response in Xenografts.

The cancer xenograft model in which human cancer cells are implanted in a mouse is one of the most used preclinical models to test the efficacy of novel cancer drugs. However, the model is imperfect; animal models are ethically burdened, and the imperfect efficacy predictions contribute to high clinical attrition of novel drugs. If microfluidic cancer-on-chip models could recapitulate key elements of the xenograft model, then these models could substitute the xenograft model and subsequently surpass the xenograft model by reducing variation, increasing sensitivity and scale, and adding human factors. Here, we exposed HCT116 colorectal cancer spheroids to dynamic, in vivo-like, concentrations of oxaliplatin, including a 5 day drug-free period, on-chip. Growth inhibition on-chip was comparable to existing xenograft studies. Furthermore, immunohistochemistry showed a similar response in proliferation and apoptosis markers. While small volume changes in xenografts are hard to detect, in the chip-system, we could observe a temporary growth delay. Lastly, histopathology and a pharmacodynamic model showed that the cancer spheroid-on-chip was representative of the proliferating outer part of a HCT116 xenograft, thereby capturing the major driver of the drug response of the xenograft. Hence, the cancer-on-chip model recapitulated the response of HCT116 xenografts to oxaliplatin and provided additional drug efficacy information.

A Screening of Native (Poly)phenols and Gut-Related Metabolites on 3D HCT116 Spheroids Reveals Gut Health Benefits of a Flavan-3-ol Metabolite.

Epidemiological evidence suggests that a reduced risk of colorectal cancer (CRC) is correlated with high consumption of fruits and vegetables, which are major sources of fiber and phytochemicals, such as flavan-3-ols. However, it remains unknown how these phytochemicals and their specific gut-related metabolites may alter cancer cell behavior. A focused screening using native (poly)phenols and gut microbial metabolites (GMMs) on 3D HCT116 spheroids is carried out using a high-throughput imaging approach. Dose-responses, IC50 , and long-term exposure are calculated for the most promising native (poly)phenols and GMMs. As a result, this research shows that (poly)phenol catabolites may play a key role in preventing cancer propagation. Indeed, µM concentration levels of (4R)-5-(3',4'-dihydroxyphenyl)-γ-valerolactone significantly decrease spheroid size at early stages of spheroid aggregation and gene expression of matrix metalloproteinases. A chronic exposure to (4R)-5-(3',4'-dihydroxyphenyl)-γ-valerolactone may lead to a reduced CRC risk. Daily intake of monomeric, oligomeric, and polymeric flavan-3-ols may increase the colonic concentrations of this metabolite, and, in turn, this compound may act locally interacting with intestinal epithelial cells, precancerous and cancer cells.

Phototoxicity of two positive-charged diaryl porphyrins in multicellular tumor spheroids.

Photodynamic therapy (PDT) is a clinically approved cancer treatment in which reactive oxygen species are formed only when three harmless components, a photosensitizer (PS), light and molecular oxygen, are present at the same time, leading to cell death. Most of the PSs were tested on monolayer cells, but differences between 2D cells and solid tumors significantly limit the value of in vitro PDT studies, whereas the use of 3D spheroid might be more suitable for drug development and preclinical drug testing for PDT. In a previous work we have shown that two positive-charged diaryl porphyrins (2 and 4) were more potent than the corresponding neutral molecules (1 and 3) on a panel of 2D-cultured cancer cell lines. In the present study the photodynamic effects of these molecules have been evaluated on HCT116 and MCF7 spheroids. Induction of apoptotic and necrotic cell death, and generation of reactive oxygen species (ROS) have been also evaluated, along with accumulation and localization of PSs into spheroids. Our findings indicate that 2 and 4 retained their phototoxic effects also in 3D spheroids; furthermore, they were more potent than 1 and 3 and as potent as Foscan (m-THPC), the most successful PS approved for clinical PDT of cancer, used as reference. Although further aspects of their mechanisms of action need to be addressed, our results strongly suggest a potential in vivo photodynamic application of 2 and 4, considering that spheroids represent a more realistic indicator of in vivo therapeutic efficacy than 2D cell lines.

The Influence of Antitumor Unsymmetrical Bisacridines on 3D Cancer Spheroids Growth and Viability.

The culture of 3D spheroids is a promising tool in drug development and testing. Recently, we synthesized a new group of compounds, unsymmetrical bisacridines (UAs), which exhibit high cytotoxicity against various human cell lines and antitumor potency against several xenografts. Here, we describe the ability of four UAs—C-2028, C-2041, C-2045, and C-2053—to influence the growth of HCT116 and H460 spheres and the viability of HCT116 cells in 3D culture compared with that in 2D standard monolayer culture. Spheroids were generated using ultra-low-attachment plates. The morphology and diameters of the obtained spheroids and those treated with UAs were observed and measured under the microscope. The viability of cells exposed to UAs at different concentrations and for different incubation times in 2D and 3D cultures was assessed using 7-AAD staining. All UAs managed to significantly inhibit the growth of HCT116 and H460 spheroids. C-2045 and C-2053 caused the death of the largest population of HCT116 spheroid cells. Although C-2041 seemed to be the most effective in the 2D monolayer experiments, in 3D conditions, it turned out to be the weakest compound. The 3D spheroid culture seems to be a suitable method to examine the efficiency of new antitumor compounds, such as unsymmetrical bisacridines.

Encapsulation of a cationic antimicrobial peptide into self-assembled polyion complex nano-objects enhances its antitumor properties

Antimicrobial peptides, a large class of molecules synthetized by various organisms as an innate defense against pathogens are more and more used for their anticancer properties as well. In order to overcome some of their limitations and to enhance their therapeutic efficiency, the use of delivery systems was taken into consideration. In this study we describe an original delivery system for antimicrobial peptides based on its physico-chemical properties, namely the self-assembled polyion complexes (PIC) based on electrostatic interactions of cationic antimicrobial peptide P6 with negatively charged double hydrophilic block copolymers, the poly(ethylene oxide)-poly(acrylic acid) (PEO-PAA) in our study. The drug delivery system was tested on 3D human tumor HCT-116 spheroids. The spheroid evolution and cell viability were monitored at 24 and 48 h after the treatment was applied. Our study demonstrates for the first time the feasibility of forming a polyion complex PIC with an antimicrobial peptide and that this self-assembled organization provides added value in terms of anti-tumor therapeutic efficacy compared to the free form of the antimicrobial peptide.

Engineering the Escherichia coli Nitroreductase NfsA to Create a Flexible Enzyme-Prodrug Activation System.

Bacterial nitroreductase enzymes that can efficiently convert nitroaromatic prodrugs to a cytotoxic form have numerous applications in targeted cellular ablation. For example, the generation of cytotoxic metabolites that have low bystander potential (i.e., are largely confined to the activating cell) has been exploited for precise ablation of specific cell types in animal and cell-culture models; while enzyme-prodrug combinations that generate high levels of bystander cell killing are useful for anti-cancer strategies such as gene-directed enzyme-prodrug therapy (GDEPT). Despite receiving substantial attention for such applications, the canonical nitroreductase NfsB from Escherichia coli has flaws that limit its utility, in particular a low efficiency of conversion of most prodrugs. Here, we sought to engineer a superior broad-range nitroreductase, E. coli NfsA, for improved activity with three therapeutically-relevant prodrugs: the duocarmycin analogue nitro-CBI-DEI, the dinitrobenzamide aziridine CB1954 and the 5-nitroimidazole metronidazole. The former two prodrugs have applications in GDEPT, while the latter has been employed for targeted ablation experiments and as a precise ‘off-switch’ in GDEPT models to eliminate nitroreductase-expressing cells. Our lead engineered NfsA (variant 11_78, with the residue substitutions S41Y, L103M, K222E and R225A) generated reduced metabolites of CB1954 and nitro-CBI-DEI that exhibited high bystander efficiencies in both bacterial and 2D HEK-293 cell culture models, while no cell-to-cell transfer was evident for the reduced metronidazole metabolite. We showed that the high bystander efficiency for CB1954 could be attributed to near-exclusive generation of the 2-hydroxylamine reduction product, which has been shown in 3D cell culture to cause significantly greater bystander killing than the 4-hydroxylamine species that is also produced by NfsB. We similarly observed a high bystander effect for nitro-CBI-DEI in HCT-116 tumor spheroids in which only a small proportion of cells were expressing variant 11_78. Collectively, our data identify variant 11_78 as a broadly improved prodrug-activating nitroreductase that offers advantages for both targeted cellular ablation and suicide gene therapy applications.

Time- and Dose-Dependent Toxicity Studies in 3D Cultures Using a Luminescent Lactate Dehydrogenase Assay.

Three-dimensional (3D) in vitro systems closely resemble tissue microenvironments and provide predictive models for studying cytotoxic drug responses. The ability to capture the kinetic profiles of such responses in a dynamic and noninvasive way can further advance the utility of 3D cell cultures. Here, we describe the use of a luminescent lactate dehydrogenase (LDH) toxicity assay for monitoring time- and dose-dependent effects of drug treatment in 3D cancer spheroids. HCT116 spheroids formed in 96-well ultralow attachment plates were treated with increasing drug concentrations. Medium samples were collected at different timepoints, frozen, stored, and analyzed at the end of experiments using the luminescent LDH-Glo™ Assay. High assay sensitivity and low volume sampling enabled drug-induced toxicity profiling in a time- and dose-dependent manner.

Expression of Epithelial Cell Adhesion Molecule (EpCAM) in Tumor Spheroids of Human Colorectal Adenocarcinoma Cells.

We studied the formation of spheroids by Caco-2, SW480, and HCT116 human colorectal adenocarcinoma cell lines under low-adhesion culturing conditions. Of these three cell lines, only HCT116 formed stable tumor spheroids. Flow cytometry analysis of 19 surface markers in monolayer HCT116 culture and spheroids formed by these cells revealed considerable similarity of the expression profiles in these two culturing modes. The only exception was EpCAM molecule: its expression in spheroids was 3-fold higher than in the monolayer culture. Scanning confocal laser microscopy showed equal EpCAM distribution in the inner mass of the spheroids.

MK615 Suppresses Hypoxia Tolerance by Up-regulation of E-cadherin in Colorectal Cancer Cells With Mutant KRAS.

The Japanese apricot "Prunus mume" is a traditional Japanese medicine. MK615, a compound extract from Prunus mume has been reported to have anti-tumor effects. Herein, we used 3D floating (3DF) culture to evaluate the anticancer effects of MK615 against human colorectal cancer (CRC) cells that contain mutant (mt) KRAS. HKe3 cells exogenously expressing mtKRAS (HKe3-mtKRAS) were treated with MK615 in 3DF cultures. The protein levels of hypoxia-inducible factor 1 (HIF-1) and E-cadherin were quantified by western blotting. MtKRAS enhanced hypoxia tolerance via up-regulation of HIF-1. The expression of HIF-1 protein was suppressed by constitutive overexpression of E-cadherin in CRC HCT116 spheroids. MK615 increased the expression of E-cadherin and decreased the expression of HIF-1 in HKe3-mtKRAS. These results suggest that MK615 suppresses hypoxia tolerance by up-regulation of E-cadherin in CRC cells with mtKRAS. MK615 exhibits properties useful for the potential treatment of CRC patients with mtKRAS.

Enhancing the therapeutic effects of in vitro targeted radionuclide therapy of 3D multicellular tumor spheroids using the novel stapled MDM2/X-p53 antagonist PM2

BackgroundPrecision therapeutics continuously make advances in cancer therapy, and a field of growing interest is the combination of targeted radionuclide therapy (TRNT) with potential radiosensitizing agents. This study evaluated whether the effects of in vitro TRNT, using the 177Lu-labeled anti-CD44v6 antibody AbN44v6, were potentiated by the novel stapled MDM2/X-p53 antagonist PM2.Materials and methodsTwo wt p53 cell lines, HCT116 (colorectal carcinoma) and UM-SCC-74B (head and neck squamous cell carcinoma), expressing different levels of the target antigen, CD44v6, were used. Antigen-specific binding of 177Lu-AbN44v6 was initially verified in a 2D cell assay, after which the potential effects of unlabeled AbN44v6 on downstream phosphorylation of Erk1/2 were evaluated by western blotting. Further, the therapeutic effects of unlabeled AbN44v6, 177Lu-AbN44v6, PM2, or a combination (labeled/unlabeled AbN44v6 +/− PM2) were assessed in 3D multicellular tumor spheroid assays.ResultsRadiolabeled antibody bound specifically to CD44v6 on both cell lines. Unlabeled AbN44v6 binding did not induce downstream phosphorylation of Erk1/2 at any of the concentrations tested, and repeated treatments with the unlabeled antibody did not result in any spheroid growth inhibition. 177Lu-AbN44v6 impaired spheroid growth in a dose-dependent and antigen-dependent manner. A single modality treatment with 20 μM of PM2 significantly impaired spheroid growth in both spheroid models. Furthermore, the combination of TRNT and PM2-based therapy proved significantly more potent than either monotherapy. In HCT116 spheroids, this resulted in a two- and threefold spheroid growth rate decrease for the combination of PM2 and 100 kBq 177Lu-AbN44v6 compared to monotherapies 14-day post treatment. In UM-SCC-74B spheroids, the combination therapy resulted in a reduction in spheroid size compared to the initial spheroid size 10-day post treatment.ConclusionTRNT using 177Lu-AbN44v6 proved efficient in stalling spheroid growth in a dose-dependent and antigen-dependent manner, and PM2 treatment demonstrated a growth inhibitory effect as a monotherapy. Moreover, by combining TRNT with PM2-based therapy, therapeutic effects of TRNT were potentiated in a 3D multicellular tumor spheroid model. This proof-of-concept study exemplifies the strength and possibility of combining TRNT targeting CD44v6 with PM2-based therapy.

Effective Targeting of Colon Cancer Cells with Piperine Natural Anticancer Prodrug Using Functionalized Clusters of Hydroxyapatite Nanoparticles.

Targeted drug delivery offers great opportunities for treating cancer. Here, we developed a novel anticancer targeted delivery system for piperine (Pip), an alkaloid prodrug derived from black pepper that exhibits anticancer effects. The tailored delivery system comprises aggregated hydroxyapatite nanoparticles (HAPs) functionalized with phosphonate groups (HAP-Ps). Pip was loaded into HAPs and HAP-Ps at pH 7.2 and 9.3 to obtain nanoformulations. The nanoformulations were characterized using several techniques and the release kinetics and anticancer effects investigated in vitro. The Pip loading capacity was >20%. Prolonged release was observed with kinetics dependent on pH, surface modification, and coating. The nanoformulations fully inhibited monolayer HCT116 colon cancer cells compared to Caco2 colon cancer and MCF7 breast cancer cells after 72 h, whereas free Pip had a weaker effect. The nanoformulations inhibited ~60% in HCT116 spheroids compared to free Pip. The Pip-loaded nanoparticles were also coated with gum Arabic and functionalized with folic acid as a targeting ligand. These functionalized nanoformulations had the lowest cytotoxicity towards normal WI-38 fibroblast cells. These preliminary findings suggest that the targeted delivery system comprising HAP aggregates loaded with Pip, coated with gum Arabic, and functionalized with folic acid are a potentially efficient agent against colon cancer.

Abstract C031: Ras processing by RRSP protease as a strategy to inhibit Ras-driven tumors

Abstract RAS genes are the most frequently mutated oncogenes in human cancers encoding four highly-related proteins that are critical molecular hubs for cancer cell proliferation and survival. Although promising results have been recently reported in early trials for KRas G12C inhibitors, there are no FDA-approved drugs that are effective against Ras-driven cancers, and Ras is still considered one of the most elusive targets in cancer research. We discovered the Ras-Rap1-Specific Protease (RRSP) from bacterium Vibrio vulnificus. RRSP is a naturally occurring endopeptidase that site-specifically cleaves the major Ras isoforms (H, N and K) and the most common mutated Ras oncoproteins (G12V, G13D and Q61R) within the Switch I region. In order to bring RRSP into cancer cells, RRSP was fused to diphtheria toxin binding subunit B (DTB). DTB delivers RRSP into the cytosol of targeted cells via binding to its receptor hHB-EGF (human heparin-binding epidermal growth factor-like growth factor). KRAS mutations occur in up to 50% of colorectal cancer (CRC) and in approximately 2% of triple-negative breast cancer (TNBC). However, 50% of TNBC show Ras upregulation via mutation-independent activation mechanisms. We investigated the anticancer potential of RRSP in CRC HCT-116 KRAS G13D as well as TNBC MDA-MB-231 KRAS G13D and MDA-MB-436 KRAS WT cell lines. RRSP was successfully delivered via DTB into CRC and TNBC cancer cell lines as indicated by Ras cleavage and pERK1/2 dephosphorylation at concentrations as low as 10 pM. As a consequence of Ras processing, a strong decrease in cell viability and proliferation was observed in all cell lines tested as per crystal violet staining and CellTiter Glo assay. A catalytically-dead mutant of RRSP carrying an amino acid substitution in its active site (RRSP*-DTB) did not show Ras processing nor reduced cell viability. Interestingly, our in vivo xenograft data demonstrated that, unlike RRSP*-DTB, RRSP-DTB effectively inhibited TNBC MDA-MB-436 and MDA-MB-231 tumor growth with no obvious toxicity. Proof of RRSP-target engagement was confirmed by loss of pERK1/2 phosphorylation via immunohistochemical analysis of tumor sections. We also found that RRSP-DTB strongly reduced the size and viability of 3-dimensional CRC HCT-116 spheroids in a time and dose-dependent manner. Moreover, an independent experiment at Charles River showed significant inhibition of tumor growth in a CRC HCT-116 xenograft model. Altogether our results revealed that, by physically processing Ras, RRSP strongly inhibits tumor growth in subcutaneously implanted human CRC and TNBC Ras-dependent tumors bearing either mutant or wild-type KRAS. This work supports further development of RRSP as an anticancer therapeutic for a broad spectrum of Ras-driven tumors and has the potential to take anti-Ras therapy to the next level. Citation Format: Vania Vidimar, Greg L Beilhartz, Roman A Melnyk, Karla JF Satchell. Ras processing by RRSP protease as a strategy to inhibit Ras-driven tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C031. doi:10.1158/1535-7163.TARG-19-C031

A cellular automaton model for spheroid response to radiation and hyperthermia treatments

Thermo-radiosensitisation is a promising approach for treatment of radio-resistant tumours such as those containing hypoxic subregions. Response prediction and treatment planning should account for tumour response heterogeneity, e.g. due to microenvironmental factors, and quantification of the biological effects induced. 3D tumour spheroids provide a physiological in vitro model of tumour response and a systems oncology framework for simulating spheroid response to radiation and hyperthermia is presented. Using a cellular automaton model, 3D oxygen diffusion, delivery of radiation and/or hyperthermia were simulated for many ({bf{1}}{{bf{0}}}^{{bf{4}}}{boldsymbol{-}}{bf{1}}{{bf{0}}}^{{bf{7}}}) individual cells forming a spheroid. The iterative oxygen diffusion model was compared to an analytical oxygenation model and simulations were calibrated and validated against experimental data for irradiated (0–10 Gy) and/or heated (0–240 CEM43) HCT116 spheroids. Despite comparable clonogenic survival, spheroid growth differed significantly following radiation or hyperthermia. This dynamic response was described well by the simulation ({{bf{R}}}^{{bf{2}}} > 0.85). Heat-induced cell death was implemented as a fast, proliferation-independent process, allowing reoxygenation and repopulation, whereas radiation was modelled as proliferation-dependent mitotic catastrophe. This framework stands out both through its experimental validation and its novel ability to predict spheroid response to multimodality treatment. It provides a good description of response where biological dose-weighting based on clonogenic survival alone was insufficient.

Potential anticancer activity of protocatechuic acid loaded in montmorillonite/Fe3O4 nanocomposites stabilized by seaweed Kappaphycus alvarezii.

Superparamagnetic magnetite nanocomposites (Fe3O4-NCs) were successfully synthesized, which comprised of montmorillonite (MMT) as matrix support, Kappaphycus alvarezii (SW) as bio-stabilizer and Fe3O4 as filler in the composites to form MMT/SW/Fe3O4-NCs. Nanocomposite with 0.5 g Fe3O4 (MMT/SW/0.5Fe3O4) was selected for anticancer activity study because it revealed high crystallinity, particle size of 7.2 ± 1.7 nm with majority of spherical shape, and Ms = 5.85 emu/g with negligible coercivity. Drug loading and release studies were carried out using protocatechuic acid (PCA) as the model for anticancer drug, which showed 19% and 87% of PCA release in pH 7.4 and 4.8, respectively. Monolayer anticancer assay showed that PCA-loaded MMT/SW/Fe3O4 (MMT/SW/Fe3O4-PCA) had selectivity towards HCT116 (colorectal cancer cell line). Although MMT/SW/Fe3O4-PCA (0.64 mg/mL) showed higher IC50 than PCA (0.148 mg/mL) and MMT/SW/Fe3O4 (0.306 mg/mL, MMT/SW/Fe3O4-PCA showed more effective killing towards tumour spheroid model generated from HCT116. The IC50 for MMT/SW/Fe3O4-PCA, MMT/SW/Fe3O4 and PCA were 0.132, 0.23 and 0.55 mg/mL, respectively. This suggests the improved penetration efficiency and drug release of MMT/SW/Fe3O4-PCA towards HCT116 spheroids. Moreover, concentration that lower than 2 mg/mL MMT/SW/Fe3O4-PCA did not result any hemolysis in human blood, which suggests them to be ideal for intravenous injection. This study highlights the potential of MMT/SW/Fe3O4-NCs as drug delivery agent.

Hypoxia-selective radiosensitisation by SN38023, a bioreductive prodrug of DNA-dependent protein kinase inhibitor IC87361

DNA-dependent protein kinase (DNA-PK) plays a key role in repair of radiation-induced DNA double strand breaks (DSB) by non-homologous end-joining. DNA-PK inhibitors (DNA-PKi) are therefore efficient radiosensitisers, but normal tissue radiosensitisation represents a risk for their use in radiation oncology. Here we describe a novel prodrug, SN38023, that is metabolised to a potent DNA-PKi (IC87361) selectively in radioresistant hypoxic cells. DNA-PK inhibitory potency of SN38023 was 24-fold lower than IC87361 in cell-free assays, consistent with molecular modelling studies suggesting that SN38023 is unable to occupy one of the predicted DNA-PK binding modes of IC87361. One-electron reduction of the prodrug by radiolysis of anoxic formate solutions, and by metabolic reduction in anoxic HCT116/POR cells that overexpress cytochrome P450 oxidoreductase (POR), generated IC87361 efficiently as assessed by LC-MS. SN38023 inhibited radiation-induced Ser2056 autophosphorylation of DNA-PK catalytic subunit and radiosensitised HCT116/POR and UT-SCC-54C cells selectively under anoxia. SN38023 was an effective radiosensitiser in anoxic HCT116 spheroids, demonstrating potential for penetration into hypoxic tumour tissue, but in spheroid co-cultures of high-POR and POR-null cells it showed no evidence of bystander effects resulting from local diffusion of IC87361. Pharmacokinetics of IC87361 and SN38023 at maximum achievable doses in NIH-III mice demonstrated sub-optimal exposure of UT-SCC-54C tumour xenografts and did not provide significant tumour radiosensitisation. In conclusion, SN38023 has potential for exploiting hypoxia for selective delivery of a potent DNA-PKi to the most radioresistant subpopulation of cells in tumours. However, prodrugs providing improved systemic pharmacokinetics and that release DNA-PKi that elicit bystander effects are needed to maximise therapeutic utility.

Abstract 710: Real-time apoptosis and necrosis detection in 3D spheroid cellmodels

Abstract The structural and biological complexity of cancer spheroids make them uniquely suited to address or approximate the pharmacodynamics of new chemical entities directed at chemotherapeutic reduction of tumor masses. Unfortunately, this same complexity represents a substantial technical challenge for measuring apoptosis and necrosis using conventional methods in real time. To address this challenge, we explored the use of a multiplexed bioluminescent and fluorescent, real-time assay reagent which utilizes annexin V fusion proteins containing binary elements of a complementing luciferase, a time-released luciferase substrate and an excludable DNA membrane integrity dye. The assay reagent was added to HCT-116 and HepG2 spheroids dosed with serial dilutions of panobinostat, bortezomib, or paclitaxel to examine the magnitude, potency and kinetics of the responses. Luminescent and fluorescent data were continuously collected throughout the 48h exposure using a conventional multimode plate reader equipped with atmospheric control. After the exposure period, the treated spheroids were imaged by fluorescence microscopy to assess necrotic burden as well as analyzed for remaining cellular viability using ATP content compared to untreated control. Consistent with their known modes of cytotoxic action, bortezomib produced the first measurable phoshatidylserine (PS) exposure (~8h), followed by panobinostat (~12h) and paclitaxel (18-22h). The apoptotic potencies and magnitudes of response matured thereafter as a function of time until 48h. Secondary necrosis resulting from completion of the apoptotic program lagged PS exposure in both time and magnitude. Imaging results for necrosis and ATP determinations provided orthogonal and inversely complementary verification of data values obtained with the apoptosis assay reagent. Taken together, this workflow may help define the in vitro pharmacological disposition of new chemical entities with respect to their capability to penetrate and induce apoptosis in spheroids and/or other more complex multicellular models. Citation Format: Andrew L. Niles, Kevin R. Kupcho, Terry L. Riss, Dan F. Lazar, James J. Cali. Real-time apoptosis and necrosis detection in 3D spheroid cellmodels [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 710.

Berberine attenuates cancer cell growth via modulating the cell cycle dynamics but not apoptosis in human colorectal HCT-116 3D spheroid model

Colorectal carcinoma is a cosmopolitan type of cancer with a poor prognosis, motivating seeking novel strategies to prevent disease development and progression. The poor prognosis is attributed to the severe toxic side effects of the current therapeutic regimes. Hence, novel less toxic treatment strategies are urgently warranted. Berberine is a natural compound with several biological and pharmacological properties, including anti-fungal, anti-diabetic, cardioprotective effects. Some reports showed that berberine inhibits cell growth by inducing cell cycle arrest and promotion of apoptosis in cancer cells. Importantly, the anticancer potential of berberine in colorectal cancer has not been previously investigated. Hence, this work aims to investigate whether berberine possesses anticancer properties against colorectal HCT116 cancer cells. The potential effect of berberine on cell cycle regulation and apoptosis will also be deeply investigated. This work was conducted using the more physiological 3D spheroid culture model that mimics better the impact of the tumor microenvironment as well as the cell-cell interaction in the cellular response to therapy. When compared to the previous studies, this work will explain the mode of action of berberine in more physiological conditions that better mimics the in vivo situation. To achieve the goal of this work, spheroid growth assay, as well as proliferation assay, were performed. Spheroid cell suspensions were further investigated using flow cytometry to assess the cell cycle distribution of cells upon berberine application. BrdU immunostaining was performed to elucidate the S-phase fraction of cells. The proliferation potential and the level of apoptosis were also investigated by Ki67 and Annexin V labeling, respectively. The results showed that berberine attenuated tumor spheroid growth and limits the proliferative capacity of HCT116 cells. This could be attributed to the berberine-mediated G1-phase cell cycle delay. The S-phase fraction of cells was significantly decreased upon berberine application. Unexpectedly, berberine did not induce a significant difference in the % of apoptotic cell fraction of cells as compared to the controls. Collectively, these results suggest that berberine possesses an anti-tumor efficacy in 3D culture preparations via modulating the cell cycle progression. Specifically, berberine induces G1-phase cell cycle delay and decreases the S-phase fraction of cells. Thus, it limits the proliferative capacity of cells. Also, berberine did not induce programmed cell death in the HCT116 spheroids.