High Throughput 3D Cell Migration Assay Using Micropillar/Microwell Chips.

The 3D cell migration assay was developed for the evaluation of drugs that inhibit cell migration using high throughput methods. Wound-healing assays have commonly been used for cell migration assays. However, these assays have limitations in mimicking the in vivo microenvironment of the tumor and measuring cell viability for evaluation of cell migration inhibition without cell toxicity. As an attempt to manage these limitations, cells were encapsulated with Matrigel on the surface of the pillar, and an analysis of the morphology of cells attached to the pillar through Matrigel was performed for the measurement of cell migration. The micropillar/microwell chips contained 532 pillars and wells, which measure the migration and viability of cells by analyzing the roundness and size of the cells, respectively. Cells seeded in Matrigel have a spherical form. Over time, cells migrate through the Matrigel and attach to the surface of the pillar. Cells that have migrated and adhered have a diffused shape that is different from the initial spherical shape. Based on our analysis of the roundness of the cells, we were able to distinguish between the diffuse and spherical shapes. Cells in Matrigel on the pillar that were treated with migration-inhibiting drugs did not move to the surface of the pillar and remained in spherical forms. During the conduct of experiments, 70 drugs were tested in single chips and migration-inhibiting drugs without cell toxicity were identified. Conventional migration assays were performed using transwell for verification of the four main migration-inhibiting drugs found on the chip.

Methods for in vitro modeling of glioma invasion: Choosing tools to meet the need.

Widespread tumor cell invasion is a fundamental property of diffuse gliomas and is ultimately responsible for their poor prognosis. A greater understanding of basic mechanisms underlying glioma invasion is needed to provide insights into therapies that could potentially counteract them. While none of the currently available in vitro models can fully recapitulate the complex interactions of glioma cells within the brain tumor microenvironment, if chosen and developed appropriately, these models can provide controlled experimental settings to study molecular and cellular phenomena that are challenging or impossible to model in vivo. Therefore, selecting the most appropriate in vitro model, together with its inherent advantages and limitations, for specific hypotheses and experimental questions achieves primary significance. In this review, we describe and discuss commonly used methods for modeling and studying glioma invasion in vitro, including platforms, matrices, cell culture, and visualization techniques, so that choices for experimental approach are informed and optimal.

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 ß 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.

CKD-602, a topoisomerase I inhibitor, induces apoptosis and cell-cycle arrest and inhibits invasion in cervical cancer.

BACKGROUND: Cervical cancer is the third most common gynecological malignancy. Conventional treatment options are known to be ineffective for the majority of patients with advanced or recurrent cervical cancer. Therefore, novel therapeutic agents for cervical cancer are necessary. In this study, the effects of CKD-602 in cervical cancer were investigated. METHODS: Three established human, immortalized, cervical cancer cell lines (CaSki, HeLa and SiHa) were used in this study. Following treatment with CKD-602, apoptosis was quantified using fluorescein isothiocyanate Annexin V-FITC and propidium iodide (PI) detection kit and cell cycle analysis was analyzed using fluorescence activated cell sorting (FACS). Transwell chambers were used for invasion assays. Western blot assay was performed to analyze proteomics. CaSki cells were subcutaneously injected into BALB/c-nude mice and cervical cancer xenograft model was established to elucidate the antitumor effect of CKD-602 in vivo. RESULTS: Treatment with CKD-602 induced apoptosis and increased expression of the enzyme PARP, cleaved PARP, and BAX. In addition, expression of phosphorylated p53 increased. Cell cycle arrest at G2/M phase and inhibition of invasion were detected after treatment with CKD-602. A significant decrease in cervical cancer tumor volume was observed in this in vivo model, following treatment with CKD-602. CONCLUSIONS: This is the first report of CKD-602 having an antitumor effect in cervical cancer in both an in vitro and in vivo models. The results of this study indicate that CKD-602 may be a novel potential drug, targeting cervical cancer, providing new opportunities in the development of new therapeutic strategies.

A Bayesian Sequential Learning Framework to Parameterise Continuum Models of Melanoma Invasion into Human Skin.

We present a novel framework to parameterise a mathematical model of cell invasion that describes how a population of melanoma cells invades into human skin tissue. Using simple experimental data extracted from complex experimental images, we estimate three model parameters: (i) the melanoma cell proliferation rate, [Formula: see text]; (ii) the melanoma cell diffusivity, D; and (iii) [Formula: see text], a constant that determines the rate that melanoma cells degrade the skin tissue. The Bayesian sequential learning framework involves a sequence of increasingly sophisticated experimental data from: (i) a spatially uniform cell proliferation assay; (ii) a two-dimensional circular barrier assay; and (iii) a three-dimensional invasion assay. The Bayesian sequential learning approach leads to well-defined parameter estimates. In contrast, taking a naive approach that attempts to estimate all parameters from a single set of images from the same experiment fails to produce meaningful results. Overall, our approach to inference is simple-to-implement, computationally efficient, and well suited for many cell biology phenomena that can be described by low-dimensional continuum models using ordinary differential equations and partial differential equations. We anticipate that this Bayesian sequential learning framework will be relevant in other biological contexts where it is challenging to extract detailed, quantitative biological measurements from experimental images and so we must rely on using relatively simple measurements from complex images.

Three-Dimensional Quantification of Spheroid Degradation-Dependent Invasion and Invadopodia Formation.

Invadopodia are actin-rich, proteolytic structures that enable cancer cell to invade into the surrounding tissues. Several in vitro invasion assays have been used in the literature ranging from directional quantitative assays to complex three-dimensional (3D) analyses. One of the main limitations of these assays is the lack of quantifiable degradation-dependent invasion in a three-dimensional (3D) environment that mimics the tumor microenvironment. In this article, we describe a new invasion and degradation assay based on the currently available tumor spheroid model that allows long-term high-resolution imaging of the tumor, precise quantification, and visualization of matrix degradation and multichannel immunocytochemistry. By incorporating a degradation marker (DQ-Green BSA) into a basement-membrane matrix, we demonstrate the ability to quantitate cancer cell-induced matrix degradation in 3D. Also, we describe a technique to generate histological sections of the tumor spheroid allowing the detection of invadopodia formation in the 3D tumor spheroid. This new technique provides a clear advantage for studying cancer in vitro and will help address critical questions regarding the dynamics of cancer cell invasion.

An advanced glioma cell invasion assay based on organotypic brain slice cultures.

BACKGROUND: The poor prognosis for glioblastoma patients is caused by the diffuse infiltrative growth pattern of the tumor. Therefore, the molecular and cellular processes underlying cell migration continue to be a major focus of glioblastoma research. Emerging evidence supports the concept that the tumor microenvironment has a profound influence on the functional properties of tumor cells. Accordingly, substantial effort must be devoted to move from traditional two-dimensional migration assays to three-dimensional systems that more faithfully recapitulate the complex in vivo tumor microenvironment. METHODS: In order to mimic the tumor microenvironment of adult gliomas, we used adult organotypic brain slices as an invasion matrix for implanted, fluorescently labeled tumor spheroids. Cell invasion was imaged by confocal or epi-fluorescence microscopy and quantified by determining the average cumulative sprout length per spheroid. The tumor microenvironment was manipulated by treatment of the slice with small molecule inhibitors or using different genetically engineered mouse models as donors. RESULTS: Both epi-fluorescence and confocal microscopy were applied to precisely quantify cell invasion in this ex vivo approach. Usage of a red-emitting membrane dye in addition to tissue clearing drastically improved epi-fluorescence imaging. Preparation of brain slices from of a genetically engineered mouse with a loss of a specific cell surface protein resulted in significantly impaired tumor cell invasion. Furthermore, jasplakinolide treatment of either tumor cells or brain slice significantly reduced tumor cell invasion. CONCLUSION: We present an optimized invasion assay that closely reflects in vivo invasion by the implantation of glioma cells into organotypic adult brain slice cultures with a preserved cytoarchitecture. The diversity of applications including manipulation of the tumor cells as well as the microenvironment, permits the investigation of rate limiting factors of cell migration in a reliable context. This model will be a valuable tool for the discovery of the molecular mechanisms underlying glioma cell invasion and, ultimately, the development of novel therapeutic strategies.

Towards designing a synthetic antituberculosis vaccine: The Rv3587c peptide inhibits mycobacterial entry to host cells.

Mycobacterium tuberculosis is considered one of the most successful pathogens in the history of mankind, having caused 1.7 million deaths in 2016. The amount of resistant and extensively resistant strains has increased; BCG has been the only vaccine to be produced in more than 100 years though it is still unable to prevent the disease's most disseminated form in adults; pulmonary tuberculosis. The search is thus still on-going for candidate antigens for an antituberculosis vaccine. This paper reports the use of a logical and rational methodology for finding such antigens, this time as peptides derived from the Rv3587c membrane protein. Bioinformatics tools were used for predicting mycobacterial surface location and Rv3587c protein structure whilst circular dichroism was used for determining its peptides' secondary structure. Receptor-ligand assays identified 4 high activity binding peptides (HABPs) binding specifically to A549 alveolar epithelial cells and U937 monocyte-derived macrophages, covering the region between amino acids 116 and 193. Their capability for inhibiting Mtb H37Rv invasion was evaluated. The recognition of antibodies from individuals suffering active and latent tuberculosis and from healthy individuals was observed in HABPs capable of avoiding mycobacterial entry to host cells. The results showed that 8 HABPs inhibited such invasion, two of them being common for both cell lines: 39265 (155VLAAYVYSLDNKRLWSNLDT173) and 39266 (174APSNETLVKTFSPGEQVTTY192). Peptide 39265 was the least recognised by antibodies from the individuals' sera evaluated in each group. According to the model proposed by FIDIC regarding synthetic vaccine development, peptide 39265 has become a candidate antigen for an antituberculosis vaccine.

Role of Microenvironment in Glioma Invasion: What We Learned from In Vitro Models.

The invasion properties of glioblastoma hamper a radical surgery and are responsible for its recurrence. Understanding the invasion mechanisms is thus critical to devise new therapeutic strategies. Therefore, the creation of in vitro models that enable these mechanisms to be studied represents a crucial step. Since in vitro models represent an over-simplification of the in vivo system, in these years it has been attempted to increase the level of complexity of in vitro assays to create models that could better mimic the behaviour of the cells in vivo. These levels of complexity involved: 1. The dimension of the system, moving from two-dimensional to three-dimensional models; 2. The use of microfluidic systems; 3. The use of mixed cultures of tumour cells and cells of the tumour micro-environment in order to mimic the complex cross-talk between tumour cells and their micro-environment; 4. And the source of cells used in an attempt to move from commercial lines to patient-based models. In this review, we will summarize the evidence obtained exploring these different levels of complexity and highlighting advantages and limitations of each system used.

Metabolic characterization of invaded cells of the pancreatic cancer cell line, PANC-1.

We previously reported that about 0.4% of cells in the cultured human pancreatic cancer cell line, PANC-1, can invade matrigel during the transwell invasion assay, suggesting that these invaded PANC-1 cells may have specific characteristics to keep their invasive potential. To identify the metabolic characterization specific in the invaded PANC-1 cells, metabolome analysis of the invaded PANC-1 compared with the whole cultured PANC-1 was performed using CE-TOFMS, and concentrations of 110 metabolites were measured. In contrast to the whole cultured cells, the invaded PANC-1 was characterized as a population with reduced levels of amino acids and TCA cycle intermediates, and decreased and increased intermediates in glycolysis and nucleic acid metabolism. In particular, the ratio of both adenosine and guanosine energy charge was reduced in the invaded cells, revealing that the consumption of ATP and GTP was high in the invaded cells, and thus suggesting that ATP- or GTP-generating pathways are stimulated. In addition, the GSH/GSSG ratio was low in the invaded cells, but these cells had a higher surviving fraction after exposure to hydrogen peroxide. Thus, the invaded cells were the population resistant to oxidative stress. Furthermore, reduction in intracellular GSH content inhibited PANC-1 invasiveness, indicated that GSH has an important role in PANC-1 invasiveness. Overall, we propose the invaded cells have several unique metabolic profiles.

Virulence-related genes, adhesion and invasion of some Yersinia enterocolitica-like strains suggests its pathogenic potential.

Yersina enterocolitica-like species have not been extensively studied regarding its pathogenic potential. This work aimed to assess the pathogenic potential of some Y. enterocolitica-like strains by evaluating the presence of virulence-related genes by PCR and their ability to adhere to and invade Caco-2 and HEp-2 cells. A total of 50 Y. frederiksenii, 55 Y. intermedia and 13 Y. kristensenii strains were studied. The strains contained the following genes: Y. frederiksenii, fepA(44%), fes(44%) and ystB(18%); Y. intermedia, ail(53%), fepA (35%), fepD(2%), fes(97%), hreP(2%), ystB(2%) and tccC(35%); Y. kristensenii, ail(62%), ystB(23%), fepA(77%), fepD(54%), fes(54%) and hreP(77%). Generally, the Y. enterocolitica-like strains had a reduced ability to adhere to and invade mammalian cells compared to the highly pathogenic Y. enterocolitica 8081. However, Y. kristensenii FCF410 and Y. frederiksenii FCF461 presented high invasion potentials in Caco-2 cells after five days of pre-incubation increased by 45- and 7.2-fold compared to Y. enterocolitica 8081, respectively; but, the ail gene was not detected in these strains. The presence of virulence-related genes in some of the Y. enterocolitica-like strains indicated their possible pathogenic potential. Moreover, the results suggest the existence of alternative virulence mechanisms and that the pathogenicity of Y. kristensenii and Y. frederiksenii may be strain-dependent.

Detection of premature stop codons leading to truncated internalin A among food and clinical strains of Listeria monocytogenes.

Listeria monocytogenes is a food-borne pathogen responsible for outbreaks and sporadic cases of listeriosis, a severe invasive disease. Internalin A (InlA) a protein encoded by inlA has a key role in the mechanism of pathogenesis in L. monocytogenes infection, specifically in the invasion of human intestinal epithelial cells. Studies on inlA have shown that mutations leading to premature stop codons (PMSCs) occur naturally and are associated with impaired virulence of L. monocytogenes strains. Increasing evidence suggests that inlA PMSCs mutations are frequent in strains from foods, but rare among clinical isolates. In this study, 22 L. monocytogenes strains collected in Portugal from the processing environment of a bakery industry (n = 1), different food products (n = 10) and human clinical cases (n = 11) were analysed for mutations in inlA and invasion efficiency in Caco-2 cells. Sequencing revealed previously reported mutations types leading to PMSCs in three food and one clinical strain presenting different molecular serotypes (i.e., IIa, IIb and IIc). The remaining 18 isolates did not show PMSCs in inlA. The four strains with PMSCs in inlA presented lower invasiveness efficiencies in Caco-2 cells (below 8.9%) when compared to the control strain (full-length InlA). In addition, one clinical isolate showed reduced invasion efficiency but no PMSCs in inlA. This isolate showed increased inlA transcript levels to that obtained for the laboratory control strain. Our data support the hypothesis that L. monocytogenes isolated from food have attenuated invasion due to the presence of inlA PMSCs. This information would be critically needed for adequate risk-assessments of the foodborne illness burden associated with L. monocytogenes strains.

Population structure and antimicrobial profile of Staphylococcus aureus strains associated with bovine mastitis in China.

Staphylococcus aureus is a significant bacterial pathogen associated with bovine mastitis. The aim of the present study was to investigate and characterize of S. aureus strains isolated from the milk of cows suffering from mastitis in the mid-east of China. Among the 200 milk samples analyzed, 58 were positive for S. aureus, of these isolates, 11 isolates were methicillin-resistant Staphylococcus aureus (MRSA). All of the 58 S. aureus strains were classified in agr group I, while seven different sequence type (ST) patterns were identified and among them the most common was ST630 followed by ST188. All of the S. aureus isolates belonging to ST630 were resistant to more than four antimicrobials, and 22.2% of isolates belonging to ST188 were resistant to eight antimicrobials. Interestingly, while strong biofilm producers demonstrated higher resistance to multiple antimicrobials, they exhibited lower intracellular survival rates. The results of this study illustrated the distribution, antimicrobial susceptibility profiles, genotype, and the ability of biofilm production and mammary epithelial cells invasion of these S. aureus isolates. This study can provide the basis for the development of a disease prevention program in dairy farms to reduce the potential risk in both animal and human health.

Robust design of some selective matrix metalloproteinase-2 inhibitors over matrix metalloproteinase-9 through in silico/fragment-based lead identification and de novo lead modification: Syntheses and biological assays.

Broad range of selectivity possesses serious limitation for the development of matrix metalloproteinase-2 (MMP-2) inhibitors for clinical purposes. To develop potent and selective MMP-2 inhibitors, initially multiple molecular modeling techniques were adopted for robust design. Predictive and validated regression models (2D and 3D QSAR and ligand-based pharmacophore mapping studies) were utilized for estimating the potency whereas classification models (Bayesian and recursive partitioning analyses) were used for determining the selectivity of MMP-2 inhibitors over MMP-9. Bayesian model fingerprints were used to design selective lead molecule which was modified using structure-based de novo technique. A series of designed molecules were prepared and screened initially for inhibitions of MMP-2 and MMP-9, respectively, as these are designed followed by other MMPs to observe the broader selectivity. The best active MMP-2 inhibitor had IC50 value of 24nM whereas the best selective inhibitor (IC50=51nM) showed at least 4 times selectivity to MMP-2 against all tested MMPs. Active derivatives were non-cytotoxic against human lung carcinoma cell line-A549. At non-cytotoxic concentrations, these inhibitors reduced intracellular MMP-2 expression up to 78% and also exhibited satisfactory anti-migration and anti-invasive properties against A549 cells. Some of these active compounds may be used as adjuvant therapeutic agents in lung cancer after detailed study.

A novel 3D high-content assay identifies compounds that prevent fibroblast invasion into tissue surrogates.

Invasion processes underlie or accompany several pathological processes but only a limited number of high-throughput capable phenotypic models exist to test anti-invasive compounds in vitro. We here evaluated 3D co-cultures as a high-content phenotypic screening system for fibrotic invasive processes. 3D multicellular spheroids were used as living tissue surrogates in co-culture with fluorescently labeled lung fibroblasts to monitor invasion processes by automated microscopy. This setup was used to screen a compound library containing 480 known bioactive substances. Identified hits prevented fibroblast invasion and could be subdivided into two hit classes. First, Prostaglandins were shown to prevent fibroblast invasion, most likely mediated by the prostaglandin EP2 receptor and generation of cAMP. Additionally, Rho-associated protein kinase (ROCK) inhibitors prevented fibroblast invasion, possibly by inactivation of myosin II. Importantly, both Prostaglandins and ROCK inhibitors are potential treatment options shown to be effective in in vitro and in vivo models of fibrotic diseases. This validates the presented novel phenotypic screening approach for the evaluation of potential inhibitors and the identification of novel compounds with activity in diseases that are associated with fibroblast invasion.

Insights into the role of components of the tumor microenvironment in oral carcinoma call for new therapeutic approaches.

The research on oral cancer has focused mainly on the cancer cells, their genetic changes and consequent phenotypic modifications. However, it is increasingly clear that the tumor microenvironment (TME) has been shown to be in a dynamic state of inter-relations with the cancer cells. The TME contains a variety of components including the non-cancerous cells (i.e., immune cells, resident fibroblasts and angiogenic vascular cells) and the ECM milieu [including fibers (mainly collagen and fibronectin) and soluble factors (i.e., enzymes, growth factors, cytokines and chemokines)]. Thus, it is currently assumed that TME is considered a part of the cancerous tissue and the functionality of its key components constitutes the setting on which the hallmarks of the cancer cells can evolve. Therefore, in terms of controlling a malignancy, one should control the growth, invasion and spread of the cancer cells through modifications in the TME components. This mini review focuses on the TME as a diagnostic approach and reports the recent insights into the role of different TME key components [such as carcinoma-associated fibroblasts (CAFs) and inflammation (CAI) cells, angiogenesis, stromal matrix molecules and proteases] in the molecular biology of oral carcinoma. Furthermore, the impact of TME components on clinical outcomes and the concomitant need for development of new therapeutic approaches will be discussed.

Development and application of three-dimensional skin equivalents for the investigation of percutaneous worm invasion.

Investigation of percutaneous helminth infection is generally based on animal models or excised skin. As desirable replacement of animal experiments, tissue-engineered skin equivalents have recently been applied in microbial and viral in vitro infection models. In the present study, the applicability of tissue-engineered skin equivalents for the investigation of percutaneous helminth invasion was evaluated. Epidermal and a full-thickness skin equivalents that suit the requirements for helminth invasion studies were developed. Quantitative invasion assays were performed with the skin-invading larvae of the helminths Strongyloides ratti and Schistosoma mansoni. Both skin equivalents provided a physical barrier to larval invasion of the nematode S. ratti, while these larvae could invade and permeate a cell-free collagen scaffold and ex vivo epidermis. In contrast, the epidermal and full-thickness skin equivalents exhibited a human host-specific susceptibility to larvae of trematode S. mansoni, which could well penetrate. Invasion of S. mansoni in cell-free collagen scaffold was lowest for all experimental conditions. Thus, reconstructed epidermis and full-thickness skin equivalents confirmed a high degree of accordance to native tissue. Additionally, not only tailless schistosomula but also cercariae could permeate the skin equivalents, and thus, delayed tail loss hypothesis was supported. The present study indicates that the limitations in predictive infection test systems for human-pathogenic invading helminths can be overcome by tissue-engineered in vitro skin equivalents allowing a substitution of the human skin for analysis of the interaction between parasites and their hosts' tissues. This novel tissue-engineered technology accomplishes the endeavor to save animal lives.

K-D:rib dampens Hs 578T cancer cell chemoinvasion and proliferation.

BACKGROUND: Cancer cells, even in the presence of available oxygen, have a glycolysis enhancement. The "aerobic glycolysis" is known as the Warburg effect and it is considered one of the fundamental hallmarks of metabolic alteration during malignant transformation. A feature of many tumors is also a change into ions equilibrium, with particular reference to K(+) intracellular concentration. Another hallmark in cancer is the reprogrammed chemotaxis pathways in favour of tumor cell dissemination. RESULTS: The doubling population time of 5 mM K:D-rib treated Hs 578T (HTB-126 ® ATCC) cell line is reduce by 30% respect to the control. During the chemotactic invasion assay, the relative number of motile and invasive cells, counted inside the FBS-AGAR spot, shows a decrease with the maintenance of the treatment reaching the 25% after nine days. Hs 578Bst (HTB-125 ® ATCC) non-tumor cell line treated for nineteen days with 5 mM K:D-rib was split twice as well as the control. No morphological change was visible in the treated respect to untreated cells. CONCLUSIONS: We demonstrate that the synergic action of potassium bicarbonate and D-ribose has effect on Hs 578T cancer cell line proliferation reducing the cell cycle time. At 5 mM concentration, K:D-rib is able to modify the tumorigenic potential of human breast cancer cell line Hs 578T, interfering in vitro with the capability of Hs 578 T cell line to migrate under chemotactic stimuli. Despite this, K:D-rib solution, does not exhibit any appreciable toxicity as confirmed by the proliferation assay accomplished on Hs 578Bst cell line.

Monitoring Cell Cytoskeleton Variations upon Piezoelectric Stimulation: Implications for the Immune System.

Piezoelectric stimulation can have a significant impact on different cellular functions with possible applications in several fields, such as regenerative medicine, cancer therapy, and immunoregulation. For example, piezoelectric stimulation has been shown to modulate cytoskeleton variations: the implications of this effect range from the regulation of migration and invasion of cancer cells to the activation of pro- or anti-inflammatory phenotypes in immune cells. In this chapter, we will present different methodologies to evaluate cytoskeleton variations, focusing on modifications on f-/g-actin ratio and on the migration and invasion ability of tumor cells.

Evaluation of BH3 mimetics as a combination therapy with irradiation in head and neck squamous cell carcinoma.

INTRODUCTION: Head and neck squamous cell carcinoma (HNSCC) is a common cancer with a five-year survival rate around 60%, indicating a need for new treatments. BH3 mimetics are small molecules that inhibit anti-apoptotic Bcl-2 family proteins, resulting in apoptosis induction. METHODS: We performed a high-throughput screen using a Myogel matrix to identify the synergy between irradiation and the novel BH3 mimetics A-1155463, A-1331852, and navitoclax in 12 HNSCC cell lines, normal (NOF) and cancer-associated fibroblasts (CAF), and dysplastic keratinocytes (ODA). Next, we examined synergy in an apoptosis assay, followed by a clonogenic assay and a Myogel spheroid on selected HNSCC cell lines. Finally, we applied zebrafish larvae xenograft to validate the effects of navitoclax and A-1331852. RESULTS: All three BH3 mimetics exhibited a strong synergy with irradiation in eight HNSCC cell lines and ODAs, but not in NOFs and CAFs. A-1155463 and A-1331852 induced apoptosis and reduced proliferation, and together with irradiation, significantly increased apoptosis and arrested proliferation. A-1331852 and navitoclax significantly decreased the clonogenicity compared with the control, and combination treatment led to a decreased clonogenicity compared with monotherapy or irradiation. However, unlike navitoclax or A-1155463, only A-1331852 significantly reduced cancer cell invasion. Furthermore, in spheroid and zebrafish, irradiation appeared ineffective and failed to significantly increase the drug effect. In the zebrafish, A-1331852 and navitoclax significantly reduced the tumor area and metastasis. CONCLUSIONS: Our findings encourage the further preclinical investigation of BH3 mimetics, particularly A-1331852, as a single agent or combined with irradiation as a treatment for HNSCC.