Effect of HER2-targeted therapy on PDX and PDX-derived organoids generated from HER2-positive salivary duct carcinoma.

BACKGROUND: We previously established a patient-derived xenograft (PDX) model, patient-derived organoids (PDOs), and PDX-derived organoids (PDXOs) for salivary duct carcinoma (SDC). Using these models, this study examined the therapeutic effect of human epidermal growth factor receptor 2 (HER2) blockade on HER2-positive SDC. METHODS: The therapeutic effect of lapatinib was assessed in SDC PDXOs with regards to cell growth, receptor/downstream signaling molecule expression, phosphorylation levels, and apoptosis. Effect of lapatinib treatment was evaluated in vivo in SDC PDX mice. RESULTS: The siRNA knockdown of HER2 and lapatinib suppressed cell proliferation in SDC PDXOs. Lapatinib inhibited the phosphorylation of HER2 and its downstream targets, and induced apoptosis in SDC PDXOs. Lapatinib also significantly reduced tumor volumes compared with that of the control in SDC PDX mice. CONCLUSION: For the first time, we demonstrated the efficacy of anti-HER2 therapy in HER2-positive SDC using preclinical models of SDC PDX and PDXO.

Establishment of experimental salivary gland cancer models using organoid culture and patient-derived xenografting.

PURPOSE: Depending on its histological subtype, salivary gland carcinoma (SGC) may have a poor prognosis. Due to the scarcity of preclinical experimental models, its molecular biology has so far remained largely unknown, hampering the development of new treatment modalities for patients with these malignancies. The aim of this study was to generate experimental human SGC models of multiple histological subtypes using patient-derived xenograft (PDX) and organoid culture techniques. METHODS: Tumor specimens from surgically resected SGCs were processed for the preparation of PDXs and patient-derived organoids (PDOs). Specimens from SGC PDXs were also processed for PDX-derived organoid (PDXO) generation. In vivo tumorigenicity was assessed using orthotopic transplantation of SGC organoids. The pathological characteristics of each model were compared to those of the original tumors using immunohistochemistry. RNA-seq was used to analyze the genetic traits of our models. RESULTS: Three series of PDOs, PDXs and PDXOs of salivary duct carcinomas, one series of PDOs, PDXs and PDXOs of mucoepidermoid carcinomas and PDXs of myoepithelial carcinomas were successfully generated. We found that PDXs and orthotopic transplants from PDOs/PDXOs showed similar histological features as the original tumors. Our models also retained their genetic traits, i.e., transcription profiles, genomic variants and fusion genes of the corresponding histological subtypes. CONCLUSION: We report the generation of SGC PDOs, PDXs and PDXOs of multiple histological subtypes, recapitulating the histological and genetical characteristics of the original tumors. These experimental SGC models may serve as a useful resource for the development of novel therapeutic strategies and for investigating the molecular mechanisms underlying the development of these malignancies.

Partial Substitution of Glucose with Xylitol Prolongs Survival and Suppresses Cell Proliferation and Glycolysis of Mice Bearing Orthotopic Xenograft of Oral Cancer.

Many types of cancer have metabolic alterations with increased glycolysis. Identification of alternative sweeteners that do not fuel cancer is a novel approach to cancer control. The present study aimed to investigate the effects of xylitol on tumor growth and survival of mice bearing orthotopic xenograft of tongue cancers. The results showed that partial substitution of glucose with xylitol (glucose 0.35 g plus xylitol 2.06 g/kg body weight) non-significantly reduced tumor volume, and significantly prolonged the median survival time from 19 days in the control to 30.5 days in the xylitol group. Immunohistochemical data of the tongue tissue shows significantly lower intense-to-mild staining ratios of the proliferation marker Ki-67 in the xylitol than those of the control group (p = 0.04). Furthermore, the xylitol substitution significantly reduced the expression of the rate-limiting glycolytic enzyme, phosphofructokinase-1 (PFK-1) (p = 0.03), and showed a non-significant inhibition of PFK activity. In summary, partial substitution of glucose with xylitol at the equivalent dose to human household use of 10 g/day slows down tumor proliferation and prolongs survival of mice bearing an orthotopic oral cancer xenograft, possibly through glycolytic inhibition, with minimal adverse events. The insight warrants clinical studies to confirm xylitol as a candidate sweetener in food products for cancer survivors.

A risk factor for newly diagnosed secondary cancer in patients with early-stage laryngeal, oropharyngeal, or hypopharyngeal cancer: sub-analysis of a prospective observation study.

BACKGROUND: We previously identified hypopharyngeal cancer as an independent risk factor for the incidence of newly diagnosed secondary cancers after the treatment of early-stage laryngeal, oropharyngeal, and hypopharyngeal cancers. We subsequently used a different patient cohort to validate the usefulness of this factor during the follow-up period in these patients. METHODS: Patients who underwent transoral surgery (TOS) as a definitive treatment between April 1, 2016, and September 30, 2020, were included. The incidence of secondary cancer was evaluated in hypopharyngeal and other cancers. Overall survival (OS), recurrence-free survival (RFS), and disease-free survival (DFS) outcomes were evaluated. Statistical analyses based on the risk factors were also performed. RESULTS: Incidence of new secondary cancer was 30% in hypopharyngeal cancer patients as compared to 11% in other cancer patients, and the risk was 3.60-fold (95% confidence interval 1.07-12.10) higher after definitive treatment for initial head and neck cancers. The 3-year OS, RFS, and DFS rates were 98%, 86%, and 67%, respectively. CONCLUSIONS: Among patients with early-stage laryngeal, oropharyngeal, and hypopharyngeal squamous cell carcinoma, who were initially treated with TOS, hypopharyngeal cancer patients had a higher risk of newly diagnosed secondary cancers as observed during the follow-up period.

FosL1 Regulates Regional Metastasis of Head and Neck Squamous Cell Carcinoma by Promoting Cell Migration, Invasion, and Proliferation.

BACKGROUND/AIM: We evaluated the impact of FosL1, a member of the activated protein-1 family, on the pathways leading to regional metastasis of head and neck squamous cell carcinoma (HNSCC). MATERIALS AND METHODS: We examined the influence of small interfering RNA (siRNA) and short heparin RNA (shRNA) mediated knockdown of FosL1 on cell migration, invasion, and proliferation in vitro as well as on regional metastasis in vivo. The prognostic significance of FosL1 was also analyzed using the Kaplan- Meier plotter using data from an HNSCC patient database. RESULTS: Down-regulation of FosL1 inhibited cell migration, invasion, and proliferation in vitro, decreased the incidence of regional metastases, and prolonged the survival of mice in vivo. We also determined that HNSCC patients with higher expression levels of FosL1 had a significantly shorter survival time than those with low expression of FosL1. CONCLUSION: FosL1 plays a crucial role in promoting cell migration, invasion, and proliferation in HNSCC.

Validation of the risk factors for primary control of early T-stage laryngeal, oropharyngeal, and hypopharyngeal squamous cell carcinoma by transoral surgery: a prospective observational study.

BACKGROUND: We had previously identified the following risk factors for insufficient control of early T-stage head and neck cancer by transoral surgery (TOS): (1) tumor thickness > 7 mm on enhanced computed tomography (CT), and (2) poor differentiation in pathological examination. We subsequently used a different patient cohort to validate the usefulness of these factors in determining the need for adaptation of TOS. STUDY SETTING: A prospective observational study METHODS: Patients who received TOS as a definitive treatment between April 1, 2016 and September 30, 2020 were included. Primary control rates (by single TOS and TOS alone) in relation to the above-mentioned risk factors were calculated. Overall (O), recurrence-free (RF), and disease-free (DF) survival (S) outcomes were evaluated. A combination analysis based on the number of risk factors was also performed. RESULTS: Patients with tumor thickness > 7 mm had a 2.88-fold [95% confidence interval (CI) 1.01-8.51] higher risk of incomplete primary resection by single TOS, while patients who showed poor differentiation on pathological assessments had a 13.14-fold (95% CI 3.66-47.14) higher risk of insufficient primary control by TOS alone. The 3 year OS, RFS, and DFS rates were 99%, 83%, and 63%, respectively. Patients with both risk factors had a 93.00-fold (95% CI 4.99-1732.00) higher risk of incomplete primary control by TOS alone. CONCLUSIONS: Among patients with early-stage laryngeal, oropharyngeal, and hypopharyngeal squamous cell carcinoma, primary control by TOS alone may not be achieved in patients with both risk factors, that is, tumor thickness > 7 mm as measured by enhanced CT and poor differentiation on pathological examination.

The incidence of newly diagnosed secondary cancer; sub-analysis the prospective study of the second-look procedure for transoral surgery in patients with T1 and T2 head and neck cancer.

BACKGROUND: Our prospective study of patients with early T-stage head and neck cancer indicated a high incidence of newly diagnosed secondary malignancies during the follow-up period. We aimed to determine the incidence rate and risk factors of secondary malignancies in early-stage head and neck cancer patients. METHODS: We sub-analyzed the patient data of a previous study focusing on secondary cancer incidence. The endpoints were statistical analyses of risk factors and survival and incidence rates. RESULTS: The incidence rate of secondary cancer was 37%, the crude incidence of second primary cancers was 10.6 per 100 person-years, and the 5 year secondary cancer-free survival rate was 63%. The hypopharynx as the primary site was an independent significant predictive factor (odds ratio 3.96, 95% confidence interval 1.07-14.6, p = 0.039). CONCLUSIONS: Early stages of laryngeal, oropharyngeal, and hypopharyngeal cancer had a risk of secondary cancer, especially hypopharyngeal cancer. Attention to the secondary cancer has to be paid during the follow-up period after controlling the early-stage disease. These findings highlight the need for awareness of the incidence of secondary cancer in cases of early-stage primary head and neck cancer.

Establishment of PDX-derived salivary adenoid cystic carcinoma cell lines using organoid culture method.

To generate a reliable preclinical model system exhibiting the molecular features of salivary adenoid cystic carcinoma (ACC) whose biology is still unclear due to the paucity of stable cell cultures. To develop new in vitro and in vivo models of ACC, the techniques of organoid culture and patient-derived tumor xenograft (PDX), which have attracted attention in other malignancies in recent years, were applied. Tumor specimens from surgically resected salivary ACC were proceeded for the preparation of PDX and organoid culture. The orthotopic transplantation of patient-derived or PDX-derived organoids was demonstrated into submandibular glands of NSG mice and those histology was evaluated. PDX-derived organoid cells were evaluated for the presence of MYB-mediated fusion genes and proceeded for in vitro drug sensitivity assay. Human ACC-derived organoids were successfully generated in three-dimensional culture and confirmed the ability of these cells to form tumors by orthotopic injection. Short-term organoid cell cultures from two individual ACC PDX tumors were also established that maintain the characteristic MYBL1 translocation and histological features of the original parent and PDX tumors. Finally, the establishment of drug sensitivity tests on these short-term cultured cells was confirmed using three different agents. This is the first to report an approach for the generation of human ACC-derived organoids as in vitro and in vivo cancer models, providing insights into understanding of the ACC biology and creating personalized therapy design for patients with ACC.

Heptavalinamide A, an Extensively N-Methylated Linear Nonapeptide from a Cyanobacterium Symploca sp. and Development of a Highly Sensitive Analysis of N,N-Dimethylvaline by LCMS.

An extensively N-methylated linear nonapeptide heptavalinamide A (1) was isolated from the marine cyanobacterium Symploca sp. collected at Kabira Reef of Ishigaki Island, Okinawa. The amino acid sequence of 1 was assigned by interpretation of 2D NMR and MS/MS data. The absolute configurations of the constituent amino acids were determined by the application of Marfey's method. A method to assign the configuration of N,N-dimethylvaline by LCMS is discussed.

Metachromins X and Y from a marine sponge Spongia sp. and their effects on cell cycle progression.

New sesquiterpene quinones, metachromins X (1) and Y (2), together with the known metachromins C (3), J (4), and T (5), were isolated as inhibitors of cell cycle progression in the HeLa/Fucci2 cells. The structure of 1 was assigned by spectroscopic data and confirmed by a total synthesis. The planar structure of 2 was determined by interpretation of spectroscopic data, whereas its absolute configuration was analyzed by a combination of chiral HPLC and CD spectroscopy. Metachromins X (1) and C (3) arrested the cell cycle progression of HeLa/Fucci2 cells at S/G2/M phase.

Colony-wise Analysis of a Theonella swinhoei Marine Sponge with a Yellow Interior Permitted the Isolation of Theonellamide I.

There are several examples of marine organisms whose metabolic profiles differ among conspecifics inhabiting the same region. We have analyzed the metabolic profile of each colony of a Theonella swinhoei marine sponge with a yellow interior and noticed the patchy distribution of one metabolite. This compound was isolated and its structure was studied by a combination of spectrometric analyses and chemical degradation, showing it to be a congener in the theonellamide class of bicyclic peptides. Theonellamides had previously been isolated by us only from T. swinhoei with a white interior and not from those with a yellow interior.

Dereplication of peptidic natural products through database search of mass spectra.

Peptidic natural products (PNPs) are widely used compounds that include many antibiotics and a variety of other bioactive peptides. Although recent breakthroughs in PNP discovery raised the challenge of developing new algorithms for their analysis, identification of PNPs via database search of tandem mass spectra remains an open problem. To address this problem, natural product researchers use dereplication strategies that identify known PNPs and lead to the discovery of new ones, even in cases when the reference spectra are not present in existing spectral libraries. DEREPLICATOR is a new dereplication algorithm that enables high-throughput PNP identification and that is compatible with large-scale mass-spectrometry-based screening platforms for natural product discovery. After searching nearly one hundred million tandem mass spectra in the Global Natural Products Social (GNPS) molecular networking infrastructure, DEREPLICATOR identified an order of magnitude more PNPs (and their new variants) than any previous dereplication efforts.

Identification of human papillomavirus (HPV) 16 DNA integration and the ensuing patterns of methylation in HPV-associated head and neck squamous cell carcinoma cell lines.

Recent studies showed that human papillomavirus (HPV) integration contributes to the genomic instability seen in HPV-associated head and neck squamous cell carcinoma (HPV-HNSCC). However, the epigenetic alterations induced after HPV integration remains unclear. To identify the molecular details of HPV16 DNA integration and the ensuing patterns of methylation in HNSCC, we performed next-generation sequencing using a target-enrichment method for the effective identification of HPV16 integration breakpoints as well as the characterization of genomic sequences adjacent to HPV16 integration breakpoints with three HPV16-related HNSCC cell lines. The DNA methylation levels of the integrated HPV16 genome and that of the adjacent human genome were also analyzed by bisulfite pyrosequencing. We found various integration loci, including novel integration sites. Integration loci were located predominantly in the intergenic region, with a significant enrichment of the microhomologous sequences between the human and HPV16 genomes at the integration breakpoints. Furthermore, various levels of methylation within both the human genome and the integrated HPV genome at the integration breakpoints in each integrant were observed. Allele-specific methylation analysis suggested that the HPV16 integrants remained hypomethylated when the flanking host genome was hypomethylated. After integration into highly methylated human genome regions, however, the HPV16 DNA became methylated. In conclusion, we found novel integration sites and methylation patterns in HPV-HNSCC using our unique method. These findings may provide insights into understanding of viral integration mechanism and virus-associated carcinogenesis of HPV-HNSCC.

Dragmacidins G and H, Bisindole Alkaloids Tethered by a Guanidino Ethylthiopyrazine Moiety, from a Lipastrotethya sp. Marine Sponge.

LCMS analysis of the extract and a cytotoxicity assay of the HPLC fractions generated from a small-scale extract of a Lipastrotethya sp. marine sponge demonstrated the presence of bisindole alkaloids that were associated with the cytotoxic activity. Two bisindole alkaloids tethered by a guanidino ethylthiopyrazine moiety, dragmacidins G (1) and H (2), were isolated, and their structures were assigned by analysis of the MS and NMR data. They showed moderate cytotoxic activity against HeLa cells.

Curacin E from the Brittle Star Ophiocoma scolopendrina.

Bioassay-guided fractionation of the extract of the brittle star Ophiocoma scolopendrina afforded curacin E (1), a congener of curacin A (2). Curacin A (2) is an antimitotic agent of cyanobacterial origin. The structure of curacin E was studied by interpretation of NMR data and the ECD spectrum. Curacin E has an ethylcarbonyl terminus in its side chain and inhibits the proliferation of P388 cells.

Cytotoxic Glycosylated Fatty Acid Amides from a Stelletta sp. Marine Sponge.

We have discovered new glycosylated fatty acid amides, stellettosides, from a Stelletta sp. marine sponge. They were detected through LC-MS analysis of the extract combined with the cytotoxicity assay of the prefractionated sample. Their planar structures were determined by analyses of the NMR and tandem FABMS data. Stellettosides A1 and A2 (1 and 2) as well as stellettosides B1-B4 (3-6) were obtained as inseparable mixtures. Careful analysis of the NMR and tandem FABMS data of each mixture, along with comparison of the tandem FABMS data with that of a synthetic model compound, permitted us to assign the structure of the constituents in the mixture. The absolute configuration of the monosaccharide unit was determined by LC-MS after chiral derivatization. The relative configurations of the vicinal oxygenated methines in the fatty acid chains were assigned by the (1)H NMR data of the isopropylidene derivative. The mixture of stellettosides B1-B4 (3-6) exhibit moderate cytotoxic activity against HeLa cells with an IC50 value of 9 µM, whereas the mixture of stellettosides A1 and A2 (1 and 2) was not active at a concentration of 10 µM.

Sequestered fulvinol-related polyacetylenes in Peltodoris atromaculata.

The Mediterranean dorid nudibranch Peltodoris atromaculata that had been collected while feeding on Haliclona fulva was shown to sequester long-chain fulvinol-like polyacetylene metabolites (compounds 2-5) from the prey. They were isolated along with previously reported bromorenierins from the diethyl ether extracts of both the mollusk and the sponge. Their structures were elucidated by NMR spectroscopy and tandem FABMS analysis. Compound 5 exhibited in vitro growth inhibitory effects against the SKMEL-28 melanoma cell line.

An environmental bacterial taxon with a large and distinct metabolic repertoire.

Cultivated bacteria such as actinomycetes are a highly useful source of biomedically important natural products. However, such 'talented' producers represent only a minute fraction of the entire, mostly uncultivated, prokaryotic diversity. The uncultured majority is generally perceived as a large, untapped resource of new drug candidates, but so far it is unknown whether taxa containing talented bacteria indeed exist. Here we report the single-cell- and metagenomics-based discovery of such producers. Two phylotypes of the candidate genus 'Entotheonella' with genomes of greater than 9 megabases and multiple, distinct biosynthetic gene clusters co-inhabit the chemically and microbially rich marine sponge Theonella swinhoei. Almost all bioactive polyketides and peptides known from this animal were attributed to a single phylotype. 'Entotheonella' spp. are widely distributed in sponges and belong to an environmental taxon proposed here as candidate phylum 'Tectomicrobia'. The pronounced bioactivities and chemical uniqueness of 'Entotheonella' compounds provide significant opportunities for ecological studies and drug discovery.

Cycloforskamide, a cytotoxic macrocyclic peptide from the sea slug Pleurobranchus forskalii.

A macrocylic dodecapeptide, cycloforskamide, was isolated from the sea slug Pleurobranchus forskalii, collected off Ishigaki Island, Japan. Its planar structure was deduced by extensive NMR analyses and was further confirmed by MS/MS fragmentation analyses. Finally, the absolute configuration was determined by total hydrolysis and chiral-phase gas chromatographic analysis. This novel dodecapeptide contains three d-amino acids and three thiazoline heterocycles and exhibits cytotoxicity against murine leukemia P388 cells, with an IC50 of 5.8 µM.

Isolation of spirastrellolides A and B from a marine sponge Epipolasis sp. and their cytotoxic activities.

Spirastrellolides A (1) and B (3) have been isolated as free acids from a marine sponge Epipolasis sp. collected in the East China Sea. These compounds had been isolated from the Caribbean marine sponge Spirastrella coccinea after conversion to the methyl ester. We examined the cytotoxic activities of 1 and 3 and found that the activities of the free acids are comparable to those of the corresponding methyl esters.