A Three-Dimensional Model of Bacterial Biofilms and Its Use in Antimicrobial Susceptibility Testing.

(1) Background: The discrepant antimicrobial susceptibility between planktonic and biofilm bacterial modes poses a problem for clinical microbiology laboratories and necessitates a relevant 3D experimental model allowing bacteria to grow in biofilm mode, in vitro, for use in anti-biofilm susceptibility testing. (2) Methods: This work develops a 3D biofilm model consisting of alginate beads containing S. aureus biofilm and encased within two thick layers of alginate matrix. The constructed model was placed on a thin Boyden chamber insert suspended on a 24-well culture plate containing the culture medium. The antibacterial activity of bacitracin and chlorhexidine digluconate (CD), either combined or separately, against 2D S. aureus culture was compared to that in the 3D biofilm model. Quantitative analysis and imaging analysis were performed by assessing the bacterial load within the matrix as well as measuring the optical density of the culture medium nourishing the matrix. (3) Results: The 3D biofilm model represented the typical complex characteristics of biofilm with greater insusceptibility to the tested antimicrobials than the 2D culture. Only bacitracin and CD in combination at 100× the concentration found to be successful against 2D culture were able to completely eliminate the 3D biofilm matrix. (4) Conclusions: The 3D biofilm model, designed to be more clinically relevant, exhibits higher antimicrobial insusceptibility than the 2D culture, demonstrating that the model might be useful for testing and discovering new antimicrobial therapies. The data also support the view that combination therapy might be the optimal approach to combat biofilm infections.

Preclinical evidence for anaplastic lymphoma kinase inhibitors as novel therapeutic treatments for cholangiocarcinoma.

Introduction: Bile duct cancer (cholangiocarcinoma, CCA) has a poor prognosis for patients, and despite recent advances in targeted therapies for other cancer types, it is still treated with standard chemotherapy. Anaplastic lymphoma kinase (ALK) has been shown to be a primary driver of disease progression in lung cancer, and ALK inhibitors are effective therapeutics in aberrant ALK-expressing tumors. Aberrant ALK expression has been documented in CCA, but the use of ALK inhibitors has not been investigated. Using CCA cell lines and close-to-patient primary cholangiocarcinoma cells, we investigated the potential for ALK inhibitors in CCA. Methods: ALK, cMET, and ROS1 expression was determined in CCA patient tissue by immunohistochemistry and digital droplet polymerase chain reaction, and that in cell lines was determined by immunoblot and immunofluorescence. The effect on cell viability and mechanism of action of ALK, cMet, and ROS1 inhibitors was determined in CCA cell lines. To determine whether ceritinib could affect primary CCA cells, tissue was taken from four patients with biliary tract cancer, without ALK rearrangement, mutation, or overexpression, and grown in three-dimensional tumor growth assays in the presence or absence of humanized mesenchymal cells. Results: ALK and cMet but not ROS were both upregulated in CCA tissues and cell lines. Cell survival was inhibited by crizotinib, a c-met/ALK/ROS inhibitor. To determine the mechanism of this effect, we tested c-Met-specific and ALK/ROS-specific inhibitors, capmatinib and ceritinib, respectively. Whereas capmatinib did not affect cell survival, ceritinib dose-dependently inhibited survival in all cell lines, with IC50 ranging from 1 to 9 µM and co-treatments with gemcitabine and cisplatin further sensitized cells, with IC50 ranging from IC50 0.60 to 2.32 µM. Ceritinib did not inhibit cMet phosphorylation but did inhibit ALK phosphorylation. ALK was not mutated in any of these cell lines. Only ceritinib inhibited 3D growth of all four patient samples below mean peak serum concentration, in the presence and absence of mesenchymal cells, whereas crizotinib and capmatinib failed to do this. Ceritinib appeared to exert its effect more through autophagy than apoptosis. Discussion: These results indicate that ceritinib or other ALK/ROS inhibitors could be therapeutically useful in cholangiocarcinoma even in the absence of aberrant ALK/ROS1 expression.

Application of a 3D hydrogel-based model to replace use of animals for passaging patient-derived xenografts.

Purpose: This 3D in vitro cancer model for propagation of patient-derived cells, using a synthetic self-assembling peptide gel, allows the formation of a fully characterised, tailorable tumour microenvironment. Unlike many existing 3D cancer models, the peptide gel is inert, apart from molecules and motifs deliberately added or produced by cells within the model. Methods: Breast cancer patient-derived xenografts (PDXs) were disaggregated and embedded in a peptide hydrogel. Growth was monitored by microscopic examination and at intervals, cells were extracted from the gels and passaged on into fresh gels. Passaged cells were assessed by qPCR and immunostaining techniques for the retention of characteristic markers. Results: Breast cancer PDXs were shown to be capable of expansion over four or more passages in the peptide gel. Contaminating mouse cells were found to be rapidly removed by successive passages. The resulting human cells were shown to be compatible with a range of common assays useful for assessing survival, growth and maintenance of heterogeneity. Conclusions: Based on these findings, the hydrogel has the potential to provide an effective and practical breast cancer model for the passage of PDXs which will have the added benefits of being relatively cheap, fully-defined and free from the use of animals or animal products. Encapsulated cells will require further validation to confirm the maintenance of cell heterogeneity, genotypes and phenotypes across passage, but with further development, including the addition of bespoke cell and matrix components of the tumour microenvironment, there is clear potential to model other cancer types. Supplementary Information: The online version contains supplementary material available at 10.1007/s44164-023-00048-x.

Phosphodiesterase type 5 inhibitors enhance chemotherapy in preclinical models of esophageal adenocarcinoma by targeting cancer-associated fibroblasts.

The chemotherapy resistance of esophageal adenocarcinomas (EACs) is underpinned by cancer cell extrinsic mechanisms of the tumor microenvironment (TME). We demonstrate that, by targeting the tumor-promoting functions of the predominant TME cell type, cancer-associated fibroblasts (CAFs) with phosphodiesterase type 5 inhibitors (PDE5i), we can enhance the efficacy of standard-of-care chemotherapy. In ex vivo conditions, PDE5i prevent the transdifferentiation of normal fibroblasts to CAF and abolish the tumor-promoting function of established EAC CAFs. Using shotgun proteomics and single-cell RNA-seq, we reveal PDE5i-specific regulation of pathways related to fibroblast activation and tumor promotion. Finally, we confirm the efficacy of PDE5i in combination with chemotherapy in close-to-patient and in vivo PDX-based model systems. These findings demonstrate that CAFs drive chemotherapy resistance in EACs and can be targeted by repurposing PDE5i, a safe and well-tolerated class of drug administered to millions of patients world-wide to treat erectile dysfunction.

Targeting hypoxia regulated sodium driven bicarbonate transporters reduces triple negative breast cancer metastasis.

Regions of low oxygen (hypoxia) are found in >50% of breast tumours, most frequently in the more aggressive triple negative breast cancer subtype (TNBC). Metastasis is the cause of 90% of breast cancer patient deaths. Regions of tumour hypoxia tend to be more acidic and both hypoxia and acidosis increase tumour metastasis. In line with this the metastatic process is dependent on pH regulatory mechanisms. We and others have previously identified increased hypoxic expression of Na+ driven bicarbonate transporters (NDBTs) as a major mechanism of tumour pH regulation. Hypoxia induced the expression of NDBTs in TNBC, most frequently SLC4A4 and SLC4A5. NDBT inhibition (S0859) and shRNA knockdown suppressed migration (40% reduction) and invasion (70% reduction) in vitro. Tumour xenograft metastasis in vivo was significantly reduced by NDBT knockdown. To investigate the mechanism by which NDBTs support metastasis, we investigated their role in regulation of phospho-signalling, epithelial-to-mesenchymal transition (EMT) and metabolism. NDBT knockdown resulted in an attenuation in hypoxic phospho-signalling activation; most notably LYN (Y397) reduced by 75%, and LCK (Y394) by 72%. The metastatic process is associated with EMT. We showed that NDBT knockdown inhibited EMT, modulating the expression of key EMT transcription factors and ablating the expression of vimentin whilst increasing the expression of E-cadherin. NDBT knockdown also altered metabolic activity reducing overall ATP and extracellular lactate levels. These results demonstrate that targeting hypoxia-induced NDBT can be used as an approach to modulate phospho-signalling, EMT, and metabolic activity and reduce tumour migration, invasion, and metastasis in vivo.

A 3D Heterotypic Breast Cancer Model Demonstrates a Role for Mesenchymal Stem Cells in Driving a Proliferative and Invasive Phenotype.

Previous indirect 2D co-culture studies have demonstrated that mesenchymal stem cells (MSCs) promote breast cancer (BC) progression through secretion of paracrine factors including growth factors, cytokines and chemokines. In order to investigate this aspect of the tumour microenvironment in a more relevant 3D co-culture model, spheroids incorporating breast cancer cells (BCCs), both cell lines and primary BCCs expanded as patient-derived xenografts, and MSCs were established. MSCs in co-cultures were shown to enhance proliferation of estrogen receptor (ER)/progesterone receptor (PR)-positive BCCs. In addition, co-culture resulted in downregulation of E-cadherin in parallel with upregulation of the epithelial-mesenchymal transition (EMT)-relation transcription factor, SNAIL. Cytoplasmic relocalization of ski-related novel protein N (SnON), a negative regulator of transforming growth factor-beta (TGF-ß) signalling, and of ß-catenin, involved in a number of pathways including Wnt signalling, was also observed in BCCs in co-cultures in contrast to monocultures. In addition, the ß-catenin inhibitor, 3-[[(4-methylphenyl)sulfonyl]amino]-benzoic acid methyl ester (MSAB), mediated reduced growth and invasion in the co-cultures. This study highlights the potential role for SnON as a biomarker for BC invasiveness, and the importance of interactions between TGF-ß and Wnt signalling, involving SnON. Such pathways may contribute towards identifying possible targets for therapeutic intervention in BC patients.

Individual patient oesophageal cancer 3D models for tailored treatment.

BACKGROUND: A model to predict chemotherapy response would provide a marked clinical benefit, enabling tailored treatment of oesophageal cancer, where less than half of patients respond to the routinely administered chemotherapy. METHODS: Cancer cells were established from tumour biopsies taken from individual patients about to undergo neoadjuvant chemotherapy. A 3D-tumour growth assay (3D-TGA) was developed, in which cancer cells were grown with or without supporting mesenchymal cells, then subjected to chemo-sensitivity testing using the standard chemotherapy administered in clinic, and a novel emerging HDAC inhibitor, Panobinostat. RESULTS: Individual patient's cancer cells could be expanded and screened within a clinically applicable timescale of 3 weeks. Incorporating mesenchymal support within the 3D-TGA significantly enhanced both the growth and drug resistance profiles of the patient's cancer cells. The ex vivo drug response in the presence, but not absence, of mesenchymal cells accurately reflected clinical chemo-sensitivity, as measured by tumour regression grade. Combination with Panobinostat enhanced response and proved efficacious in otherwise chemo-resistant tumours. CONCLUSIONS: This novel method of establishing individual patient oesophageal cancers in the laboratory, from small endoscopic biopsies, enables clinically-relevant chemo-sensitivity testing, and reduces use of animals by providing more refined in vitro models for pre-screening of drugs. The 3D-TGA accurately predicted chemo-sensitivity in patients, and could be developed to guide tailored patient treatment. The incorporation of mesenchymal cells as the stromal cell component of the tumour micro-environment had a significant effect upon enhancing chemotherapy drug resistance in oesophageal cancer, and could prove a useful target for future drug development.

The influence of nanotexturing of poly(lactic-co-glycolic acid) films upon human ovarian cancer cell attachment.

In this study, we have produced nanotextured poly(lactic-co-glycolic acid) (PLGA) films by using polystyrene (PS) particles as a template to make a polydimethylsiloxane mould against which PLGA is solvent cast. Biocompatible, biodegradable and nanotextured PLGA films were prepared with PS particles of diameter of 57, 99, 210, and 280 nm that produced domes of the same dimension in the PLGA surface. The effect of the particulate monolayer templating method was investigated to enable preparation of the films with uniformly ordered surface nanodomes. Cell attachment of a human ovarian cancer cell line (OVCAR3) alone and co-cultured with mesenchymal stem cells (MSCs) was evaluated on flat and topographically nano-patterned surfaces. Cell numbers were observed to increase on the nanotextured surfaces compared to non-textured surfaces both with OVCAR3 cultures and OVCAR3-MSC co-cultures at 24 and 48 h time points.

Transduction-specific ATLAS reveals a cohort of highly active L1 retrotransposons in human populations.

Long INterspersed Element-1 (LINE-1 or L1) retrotransposons are the only autonomously active transposable elements in the human genome. The average human genome contains ∼80-100 active L1s, but only a subset of these L1s are highly active or 'hot'. Human L1s are closely related in sequence, making it difficult to decipher progenitor/offspring relationships using traditional phylogenetic methods. However, L1 mRNAs can sometimes bypass their own polyadenylation signal and instead utilize fortuitous polyadenylation signals in 3' flanking genomic DNA. Retrotransposition of the resultant mRNAs then results in lineage specific sequence "tags" (i.e., 3' transductions) that mark the descendants of active L1 progenitors. Here, we developed a method (Transduction-Specific Amplification Typing of L1 Active Subfamilies or TS-ATLAS) that exploits L1 3' transductions to identify active L1 lineages in a genome-wide context. TS-ATLAS enabled the characterization of a putative active progenitor of one L1 lineage that includes the disease causing L1 insertion L1RP , and the identification of new retrotransposition events within two other "hot" L1 lineages. Intriguingly, the analysis of the newly discovered transduction lineage members suggests that L1 polyadenylation, even within a lineage, is highly stochastic. Thus, TS-ATLAS provides a new tool to explore the dynamics of L1 lineage evolution and retrotransposon biology.

L1 hybridization enrichment: a method for directly accessing de novo L1 insertions in the human germline.

Long interspersed nuclear element 1 (L1) retrotransposons are the only autonomously mobile human transposable elements. L1 retrotransposition has shaped our genome via insertional mutagenesis, sequence transduction, pseudogene formation, and ectopic recombination. However, L1 germline retrotransposition dynamics are poorly understood because de novo insertions occur very rarely: the frequency of disease-causing retrotransposon insertions suggests that one insertion event occurs in roughly 18-180 gametes. The method described here recovers full-length L1 insertions by using hybridization enrichment to capture L1 sequences from multiplex PCR-amplified DNA. Enrichment is achieved by hybridizing L1-specific biotinylated oligonucleotides to complementary molecules, followed by capture on streptavidin-coated paramagnetic beads. We show that multiplex, long-range PCR can amplify single molecules containing full-length L1 insertions for recovery by hybridization enrichment. We screened 600 µg of sperm DNA from one donor, but no bone fide de novo L1 insertions were found, suggesting a L1 retrotransposition frequency of <1 insertion in 400 haploid genomes. This lies below the lower bound of previous estimates, and indicates that L1 insertion, at least into the loci studied, is very rare in the male germline. It is a paradox that L1 replication is ongoing in the face of such apparently low activity.

LINE-1 retrotransposition activity in human genomes.

Highly active (i.e., "hot") long interspersed element-1 (LINE-1 or L1) sequences comprise the bulk of retrotransposition activity in the human genome; however, the abundance of hot L1s in the human population remains largely unexplored. Here, we used a fosmid-based, paired-end DNA sequencing strategy to identify 68 full-length L1s that are differentially present among individuals but are absent from the human genome reference sequence. The majority of these L1s were highly active in a cultured cell retrotransposition assay. Genotyping 26 elements revealed that two L1s are only found in Africa and that two more are absent from the H952 subset of the Human Genome Diversity Panel. Therefore, these results suggest that hot L1s are more abundant in the human population than previously appreciated, and that ongoing L1 retrotransposition continues to be a major source of interindividual genetic variation.

Advances in the treatment of intracapsular hip fractures in the elderly.

A review of recent advances in the treatment of intracapsular hip fractures in the elderly patient is offered to provide some guidelines on choosing the appropriate treatment for a given patient. Alternatives discussed include open reduction and internal fixation versus arthroplasty; unipolar versus bipolar hemiarthroplasty versus total hip arthroplasty; cemented versus cementlless prostheses; and a surgical approach. These recommendations are based upon a review of the substantial literature on the subject and the author's own experience. It is recommended that patients more than 60-years-old with a femoral neck fracture be treated in the following manner: Patients with undisplaced, stable fractures perform an ORIF, patients with displaced fractures, replace the head of the femur, the use of a Moore or Thompson prostheses should be relegated to the medically infirm, minimally ambulatory patient, modular unipolar or bipolar (cemented stem) hemiarthroplasty has the most reliable and predictable outcome in most patients, an uncemented modular hemiarthroplasty should be considered in patients with significant cardiovascular risk factors, THA perhaps recommended for the "active elderly patient". The use of large heads and meticulous capsular repair techniques will reduce the early dislocation rate while still allowing excellent long-term functional outcomes.