Modulation of the pre-metastatic bone niche: molecular changes mediated by bone-homing prostate cancer extracellular vesicles.

Prostate cancer (PCa) is a leading male malignancy worldwide, often progressing to bone metastasis, with limited curative options. Extracellular vesicles (EVs) have emerged as key players in cancer communication and metastasis, promoting the formation of supportive microenvironments in distant sites. Our previous studies have highlighted the role of PCa EVs in modulating osteoblasts and facilitating tumor progression. However, the early pre-metastatic changes induced by PCa EVs within the bone microenvironment remain poorly understood. To investigate the early effects of repeated exposure to PCa EVs in vivo, mimicking EVs being shed from the primary tumor, PCa EVs isolated from cell line PC3MLuc2a were fluorescently labelled and repeatedly administered via tail vein injection to adult CD1 NuNu male mice for a period of 4 weeks. In vivo imagining, histological analysis and gene expression profiling were performed to assess the impact of PCa EVs on the bone microenvironment. We demonstrate for the first time that PCa EVs home to both bone and lymph nodes following repeated exposures. Furthermore, the accumulation of EVs within the bone leads to distinct molecular changes indicative of disrupted bone homeostasis (e.g., changes to signaling pathways such as Paxillin p = 0.0163, Estrogen Receptor p = 0.0271, RHOA p = 0.0287, Ribonucleotide reductase p = 0.0307 and ERK/MAPK p = 0.0299). Changes in key regulators of these pathways were confirmed in vitro on human osteoblasts. In addition, our data compares the known gene signature of osteocytes and demonstrates a high proportion of overlap (52.2%), suggesting a potential role for this cell type in response to PCa EV exposure. No changes in bone histology or immunohistochemistry were detected, indicating that PCa EV mediated changes were induced at the molecular level. This study provides novel insights into the alterations induced by PCa EVs on the bone microenvironment. The observed molecular changes indicate changes in key pathways and suggest a role for osteocytes in these EV mediated early changes to bone. Further research to understand these early events may aid in the development of targeted interventions to disrupt the metastatic cascade in PCa.

Free drug and ROS-responsive nanoparticle delivery of synergistic doxorubicin and olaparib combinations to triple negative breast cancer models.

Combinations of the topoisomerase II inhibitor doxorubicin and the poly (ADP-ribose) polymerase inhibitor olaparib offer potential drug-drug synergy for the treatment of triple negative breast cancers (TNBC). In this study we performed in vitro screening of combinations of these drugs, administered directly or encapsulated within polymer nanoparticles, in both 2D and in 3D spheroid models of breast cancer. A variety of assays were used to evaluate drug potency, and calculations of combination index (CI) values indicated that synergistic effects of drug combinations occurred in a molar-ratio dependent manner. It is suggested that the mechanisms of synergy were related to enhancement of DNA damage as shown by the level of double-strand DNA breaks, and mechanisms of antagonism associated with mitochondrial mediated cell survival, as indicated by reactive oxygen species (ROS) generation. Enhanced drug delivery and potency was observed with nanoparticle formulations, with a greater extent of doxorubicin localised to cell nuclei as evidenced by microscopy, and higher cytotoxicity at the same time points compared to free drugs. Together, the work presented identifies specific combinations of doxorubicin and olaparib which were most effective in a panel of TNBC cell lines, explores the mechanisms by which these combined agents might act, and shows that formulation of these drug combinations into polymeric nanoparticles at specific ratios conserves synergistic action and enhanced potency in vitro compared to the free drugs.

Therapeutic Implications of Ceritinib in Cholangiocarcinoma beyond ALK Expression and Mutation.

Cholangiocarcinoma (CCA) is a difficult-to-treat cancer, with limited therapeutic options and surgery being the only curative treatment. Standard chemotherapy involves gemcitabine-based therapies combined with cisplatin, oxaliplatin, capecitabine, or 5-FU with a dismal prognosis for most patients. Receptor tyrosine kinases (RTKs) are aberrantly expressed in CCAs encompassing potential therapeutic opportunity. Hence, 112 RTK inhibitors were screened in KKU-M213 cells, and ceritinib, an approved targeted therapy for ALK-fusion gene driven cancers, was the most potent candidate. Ceritinib's cytotoxicity in CCA was assessed using MTT and clonogenic assays, along with immunofluorescence, western blot, and qRT-PCR techniques to analyze gene expression and signaling changes. Furthermore, the drug interaction relationship between ceritinib and cisplatin was determined using a ZIP synergy score. Additionally, spheroid and xenograft models were employed to investigate the efficacy of ceritinib in vivo. Our study revealed that ceritinib effectively killed CCA cells at clinically relevant plasma concentrations, irrespective of ALK expression or mutation status. Ceritinib modulated multiple signaling pathways leading to the inhibition of the PI3K/Akt/mTOR pathway and activated both apoptosis and autophagy. Additionally, ceritinib and cisplatin synergistically reduced CCA cell viability. Our data show ceritinib as an effective treatment of CCA, which could be potentially explored in the other cancer types without ALK mutations.

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.

Chain-extension in hyperbranched polymers alters tissue distribution and cytotoxicity profiles in orthotopic models of triple negative breast cancers.

The therapeutic efficacy of nanomedicines is highly dependent on their access to target sites in the body, and this in turn is markedly affected by their size, shape and transport properties in tissue. Although there have been many studies in this area, the ability to design nanomaterials with optimal physicochemical properties for in vivo efficacy remains a significant challenge. In particular, it is often difficult to quantify the detailed effects of cancer drug delivery systems in vivo as tumour volume reduction, a commonly reported marker of efficacy, does not always correlate with cytotoxicity in tumour tissue. Here, we studied the behaviour in vivo of two specific poly(2-hydroxypropyl methacrylamide) (pHPMA) pro-drugs, with hyperbranched and chain-extended branched architectures, redox-responsive backbone components, and pH-sensitive linkers to the anti-cancer drug doxorubicin. Evaluation of the biodistribution of these polymers following systemic injection indicated differences in the circulation time and organ distribution of the two polymers, despite their very similar hydrodynamic radii (∼10 and 15 nm) and architectures. In addition, both polymers showed improved tumour accumulation in orthotopic triple-negative breast cancers in mice, and decreased accumulation in healthy tissue, as compared to free doxorubicin, even though neither polymer-doxorubicin pro-drug decreased overall tumour volume as much as the free drug under the dosing regimens selected. However, the results of histopathological examinations by haematoxylin and eosin, and TUNEL staining indicated a higher population of apoptotic cells in the tumours for both polymer pro-drug treatments, and in turn a lower population of apoptotic cells in the heart, liver and spleen, as compared to free doxorubicin treatment. These data suggest that the penetration of these polymer pro-drugs was enhanced in tumour tissue relative to free doxorubicin, and that the combination of size, architecture, bioresponsive backbone and drug linker degradation yielded greater efficacy for the polymers as measured by biomarkers than that of tumour volume. We suggest therefore that the effects of nanomedicines may be different at various length scales relative to small molecule free drugs, and that penetration into tumour tissue for some nanomedicines may not be as problematic as prior reports have suggested. Furthermore, the data indicate that dual-responsive crosslinked polymer-prodrugs in this study may be effective nanomedicines for breast cancer chemotherapy, and that endpoints beyond tumour volume reduction can be valuable in selecting candidates for pre-clinical trials.

Selective Killing of BRCA2-Deficient Ovarian Cancer Cells via MRE11 Blockade.

The MRE11 nuclease is essential during DNA damage recognition, homologous recombination, and replication. BRCA2 plays important roles during homologous recombination and replication. Here, we show that effecting an MRE11 blockade using a prototypical inhibitor (Mirin) induces synthetic lethality (SL) in BRCA2-deficient ovarian cancer cells, HeLa cells, and 3D spheroids compared to BRCA2-proficient controls. Increased cytotoxicity was associated with double-strand break accumulation, S-phase cell cycle arrest, and increased apoptosis. An in silico analysis revealed Mirin docking onto the active site of MRE11. While Mirin sensitises DT40 MRE11+/- cells to the Top1 poison SN-38, it does not sensitise nuclease-dead MRE11 cells to this compound confirming that Mirin specifically inhibits Mre11 nuclease activity. MRE11 knockdown reduced cell viability in BRCA2-deficient PEO1 cells but not in BRCA2-proficient PEO4 cells. In a Mirin-resistant model, we show the downregulation of 53BP1 and DNA repair upregulation, leading to resistance, including in in vivo xenograft models. In a clinical cohort of human ovarian tumours, low levels of BRCA2 expression with high levels of MRE11 co-expression were linked with worse progression-free survival (PFS) (p = 0.005) and overall survival (OS) (p = 0.001). We conclude that MRE11 is an attractive SL target, and the pharmaceutical development of MRE11 inhibitors for precision oncology therapeutics may be of clinical benefit.

Targeting FGFRs Using PD173074 as a Novel Therapeutic Strategy in Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is an architecturally complex tumour with high heterogeneity. Discovery at later stages makes treatment challenging. However, the lack of early detection methodologies and the asymptomatic nature of CCA make early diagnosis more difficult. Recent studies revealed the fusions in Fibroblast Growth Factor Receptors (FGFRs), a sub-family of RTKs, as promising targets for targeted therapy for CCA. Particularly, FGFR2 fusions have been of particular interest, as translocations have been found in approximately 13% of CCA patients. Pursuing this, Pemigatinib, a small-molecule inhibitor of FGFR, became the first targeted therapy drug to be granted accelerated approval by the FDA for treating CCA patients harbouring FGFR2 fusions who have failed first-line chemotherapy. However, despite the availability of Pemigatinib, a very limited group of patients benefit from this treatment. Moreover, as the underlying mechanism of FGFR signalling is poorly elucidated in CCA, therapeutic inhibitors designed to inhibit this pathway are prone to primary and acquired resistance, as witnessed amongst other Tyrosine Kinase Inhibitors (TKIs). While acknowledging the limited cohort that benefits from FGFR inhibitors, and the poorly elucidated mechanism of the FGFR pathway, we sought to characterise the potential of FGFR inhibitors in CCA patients without FGFR2 fusions. Here we demonstrate aberrant FGFR expression in CCA samples using bioinformatics and further confirm phosphorylated-FGFR expression in paraffinised CCA tissues using immunohistochemistry. Our results highlight p-FGFR as a biomarker to guide FGFR-targeted therapies. Furthermore, CCA cell lines with FGFR expression were sensitive to a selective pan-FGFR inhibitor, PD173074, suggesting that this drug can be used to suppress CCA cells irrespective of the FGFR2 fusions. Finally, the correlation analysis utilising publicly available cohorts suggested the possibility of crosstalk amongst the FGFR and EGFR family of receptors as they are significantly co-expressed. Accordingly, dual inhibition of FGFRs and EGFR by PD173074 and EGFR inhibitor erlotinib was synergistic in CCA. Hence, the findings from this study provide support for further clinical investigation of PD173074, as well as other FGFR inhibitors, to benefit a larger cohort of patients. Altogether, this study shows for the first time the potential of FGFRs and the importance of dual inhibition as a novel therapeutic strategy in CCA.

Identifying new biomarkers of aggressive Group 3 and SHH medulloblastoma using 3D hydrogel models, single cell RNA sequencing and 3D OrbiSIMS imaging.

The most common malignant brain tumour in children, medulloblastoma (MB), is subdivided into four clinically relevant molecular subgroups, although targeted therapy options informed by understanding of different cellular features are lacking. Here, by comparing the most aggressive subgroup (Group 3) with the intermediate (SHH) subgroup, we identify crucial differences in tumour heterogeneity, including unique metabolism-driven subpopulations in Group 3 and matrix-producing subpopulations in SHH. To analyse tumour heterogeneity, we profiled individual tumour nodules at the cellular level in 3D MB hydrogel models, which recapitulate subgroup specific phenotypes, by single cell RNA sequencing (scRNAseq) and 3D OrbiTrap Secondary Ion Mass Spectrometry (3D OrbiSIMS) imaging. In addition to identifying known metabolites characteristic of MB, we observed intra- and internodular heterogeneity and identified subgroup-specific tumour subpopulations. We showed that extracellular matrix factors and adhesion pathways defined unique SHH subpopulations, and made up a distinct shell-like structure of sulphur-containing species, comprising a combination of small leucine-rich proteoglycans (SLRPs) including the collagen organiser lumican. In contrast, the Group 3 tumour model was characterized by multiple subpopulations with greatly enhanced oxidative phosphorylation and tricarboxylic acid (TCA) cycle activity. Extensive TCA cycle metabolite measurements revealed very high levels of succinate and fumarate with malate levels almost undetectable particularly in Group 3 tumour models. In patients, high fumarate levels (NMR spectroscopy) alongside activated stress response pathways and high Nuclear Factor Erythroid 2-Related Factor 2 (NRF2; gene expression analyses) were associated with poorer survival. Based on these findings we predicted and confirmed that NRF2 inhibition increased sensitivity to vincristine in a long-term 3D drug treatment assay of Group 3 MB. Thus, by combining scRNAseq and 3D OrbiSIMS in a relevant model system we were able to define MB subgroup heterogeneity at the single cell level and elucidate new druggable biomarkers for aggressive Group 3 and low-risk SHH MB.

Looking Under the Lamppost: The Search for New Cancer Targets in the Human Kinome.

The number of cancer drugs is increasing as new chemical entities are developed to target molecules, often protein kinases, driving cancer progression. In 2009, Fedorov et al. identified that of the protein kinases in the human kinome, most of the focus has been on a small subset. They highlighted that many poorly investigated protein kinases were cancer drivers, but there was no relationship between publications and involvement in cancer development or progression. Since 2009, there has been a doubling in the number of publications, patents, and drugs targeting the kinome. To determine whether this was an expansion in knowledge of well-studied targets-searching in the light under the lamppost-or an explosion of investigations into previously poorly investigated targets, we searched the literature for publications on each kinase, updating Federov et al.'s assessment of the druggable kinome. The proportion of papers focusing on the 50 most-studied kinases had not changed, and the makeup of those 50 had barely changed. The majority of new drugs (80%) were against the same group of 50 kinases identified as targets 10 years ago, and the proportion of studies investigating previously poorly investigated kinases (<1%) was unchanged. With three exceptions [p38 mitogenactivated protein kinase (p38a), AMP-activated protein kinase catalytic α-subunit 1,2, and B-Raf proto-oncogene (BRAF) serine/threonine kinase], >95% of publications addressing kinases still focused on a relatively small proportion (<50%) of the human kinome independently of their involvement as cancer drivers. There is, therefore, still extensive scope for discovery of therapeutics targeting different protein kinases in cancer and still a bias toward well-characterized targets over the innovative searchlight into the unknown. SIGNIFICANCE STATEMENT: This study presents evidence that drug discovery efforts in cancer are still to some extent focused on a narrow group of well-studied kinases 10 years after the identification of multiple novel cancer targets in the human kinome. This suggests that there is still room for researchers in academia, industry, and the not-for-profit sector to develop new and diverse therapies targeting kinases for cancer.

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.

Inhibition of serine/arginine-rich protein kinase-1 (SRPK1) prevents cholangiocarcinoma cells induced angiogenesis.

The serine/arginine-rich protein kinase-1 (SRPK1) is an enzyme that has an essential role in regulating numerous aspects of mRNA splicing. SRPK1 has been reported to be overexpressed in multiple cancers, suggesting it as a promising therapeutic target in oncology. No previous studies reported the role of SRPK1 in cholangiocarcinoma (CCA) cells. This study aimed to examine the expression of SRPK1 and the effects of SRPK1 inhibition on the viability and angiogenesis activity of CCA cells using a selective SRPK1 inhibitor, SPHINX31. Here, we demonstrate that SPHINX31 (0.3-10 µM) had no inhibitory effects on CCA cells' viability and proliferation. However, SPHINX31 decreased the mRNA expression of pro-angiogenic VEGF-A165a isoform. In addition, SPHINX31 attenuated SRSF1 phosphorylation and nuclear localization, and increased the ratio of VEGF-A165b/total VEGF-A proteins. Moreover, when HUVECs were grown in conditioned medium from SPHINX31-treated CCA cells, migration slowed, and tube formation decreased. The present study demonstrates that targeting SRPK1 in CCA cells effectively attenuates angiogenesis by suppressing pro-angiogenic VEGF-A isoform splicing. These findings suggest a potential therapeutic treatment using SRPK1 inhibitors for the inhibition of angiogenesis in cholangiocarcinoma.

Passerini chemistries for synthesis of polymer pro-drug and polymersome drug delivery nanoparticles.

New materials chemistries are urgently needed to overcome the limitations of existing biomedical materials in terms of preparation, functionality and versatility, and also in regards to their compatibility with biological environments. Here, we show that Passerini reactions are especially suited for the preparation of drug delivery materials, as with relatively few steps, polymers can be synthesized with functionality installed enabling drug conjugation and encapsulation, self-assembly into micellar or vesicular architectures, and with facile attachment triggerable chemistries. The polymers can be made with a variety of building blocks and assemble into nanoparticles, which are rapidly internalized in triple negative breast cancer (TNBC) cells. In addition, the polymers transport drug molecules efficiently through 3D cell cultures, and when designed with chemistries allowing pH-mediated release, exhibit greater efficacy against TNBC cells compared to the parent drug.

Synthesis, characterisation and evaluation of hyperbranched N-(2-hydroxypropyl) methacrylamides for transport and delivery in pancreatic cell lines in vitro and in vivo.

Hyperbranched polymers have many promising features for drug delivery, owing to their ease of synthesis, multiple functional group content, and potential for high drug loading with retention of solubility. Here we prepared hyperbranched N-(2-hydroxypropyl)methacrylamide (HPMA) polymers with a range of molar masses and particle sizes, and with attached dyes, radiolabel or the anticancer drug gemcitabine. Reversible addition-fragmentation chain transfer (RAFT) polymerisation enabled the synthesis of pHPMA polymers and a gemcitabine-comonomer functionalised pHPMA polymer pro-drug, with diameters of the polymer particles ranging from 7-40 nm. The non-drug loaded polymers were well-tolerated in cancer cell lines and macrophages, and were rapidly internalised in 2D cell culture and transported efficiently to the centre of dense pancreatic cancer 3D spheroids. The gemcitabine-loaded polymer pro-drug was found to be toxic both to 2D cultures of MIA PaCa-2 cells and also in reducing the volume of MIA PaCa-2 spheroids. The non-drug loaded polymers caused no short-term adverse effects in healthy mice following systemic injection, and derivatives of these polymers labelled with 89Zr-were tracked for their distribution in the organs of healthy and MIA PaCa-2 xenograft bearing Balb/c nude mice. Tumour accumulation, although variable across the samples, was highest in individual animals for the pHPMA polymer of ∼20 nm size, and accordingly a gemcitabine pHPMA polymer pro-drug of ∼18 nm diameter was evaluated for efficacy in the tumour-bearing animals. The efficacy of the pHPMA polymer pro-drug was very similar to that of free gemcitabine in terms of tumour growth retardation, and although there was a survival benefit after 70 days for the polymer pro-drug, there was no difference at day 80. These data suggest that while polymer pro-drugs of this type can be effective, better tumour targeting and enhanced in situ release remain as key obstacles to clinical translation even for relatively simple polymers such as pHPMA.

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.

ABCB1 inhibition provides a novel therapeutic target to block TWIST1-induced migration in medulloblastoma.

BACKGROUND: Therapeutic intervention in metastatic medulloblastoma is dependent on elucidating the underlying metastatic mechanism. We investigated whether an epithelial-mesenchymal transition (EMT)-like pathway could drive medulloblastoma metastasis. METHODS: A 3D Basement Membrane Extract (3D-BME) model was used to investigate medulloblastoma cell migration. Cell line growth was quantified with AlamarBlue metabolic assays and the morphology assessed by time-lapse imaging. Gene expression was analyzed by qRT-PCR and protein expression by immunohistochemistry of patient tissue microarrays and mouse orthotopic xenografts. Chromatin immunoprecipitation was used to determine whether the EMT transcription factor TWIST1 bound to the promoter of the multidrug pump ABCB1. TWIST1 was overexpressed in MED6 cells by lentiviral transduction (MED6-TWIST1). Inhibition of ABCB1 was mediated by vardenafil, and TWIST1 expression was reduced by either Harmine or shRNA. RESULTS: Metastatic cells migrated to form large metabolically active aggregates, whereas non-tumorigenic/non-metastatic cells formed small aggregates with decreasing metabolic activity. TWIST1 expression was upregulated in the 3D-BME model. TWIST1 and ABCB1 were significantly associated with metastasis in patients (P = .041 and P = .04, respectively). High nuclear TWIST1 expression was observed in the invasive edge of the MED1 orthotopic model, and TWIST1 knockdown in cell lines was associated with reduced cell migration (P < .05). TWIST1 bound to the ABCB1 promoter (P = .03) and induced cell aggregation in metastatic and TWIST1-overexpressing, non-metastatic (MED6-TWIST1) cells, which was significantly attenuated by vardenafil (P < .05). CONCLUSIONS: In this study, we identified a TWIST1-ABCB1 signaling axis during medulloblastoma migration, which can be therapeutically targeted with the clinically approved ABCB1 inhibitor, vardenafil.

Corrigendum to: ABCB1 inhibition provides a novel therapeutic target to block TWIST1-induced migration in medulloblastoma.

[This corrects the article DOI: 10.1093/noajnl/vdab030.].

Effect of Combining EGFR Tyrosine Kinase Inhibitors and Cytotoxic Agents on Cholangiocarcinoma Cells.

PURPOSE: The potential of members of the epidermal growth factor receptor (ErbB) family as drug targets in cholangiocarcinoma (CCA) has not been extensively addressed. Although phase III clinical trials showed no survival benefits of erlotinib in patients with advanced CCA, the outcome of the standard-of-care chemotherapy treatment for CCA, gemcitabine/cisplatin, is discouraging so we determined the effect of other ErbB receptor inhibitors alone or in conjunction with chemotherapy in CCA cells. MATERIALS AND METHODS: ErbB receptor expression was determined in CCA patient tissues by immunohistochemistry and digital-droplet polymerase chain reaction, and in primary cells and cell lines by immunoblot. Effects on cell viability and cell cycle distribution of combination therapy using ErbB inhibitors with chemotherapeutic drugs was carried out in CCA cell lines. 3D culture of primary CCA cells was then adopted to evaluate the drug effect in a setting that more closely resembles in vivo cell environments. RESULTS: CCA tumors showed higher expression of all ErbB receptors compared with resection margins. Primary and CCA cell lines had variable expression of erbB receptors. CCA cell lines showed decreased cell viability when treated with chemotherapeutic drugs (gemcitabine and 5-fluorouracil) but also with ErbB inhibitors, particularly afatinib, and with a combination. Sequential treatment of gemcitabine with afatinib was particularly effective. Co-culture of CCA primary cells with cancer-associated fibroblasts decreased sensitivity to chemotherapies, but sensitized to afatinib. CONCLUSION: Afatinib is a potential epidermal growth factor receptor targeted drug for CCA treatment and sequential treatment schedule of gemcitabine and afatinib could be explored in CCA patients.

Synthesis of Passerini-3CR Polymers and Assembly into Cytocompatible Polymersomes.

The versatility of the Passerini three component reaction (Passerini-3CR) is herein exploited for the synthesis of an amphiphilic diblock copolymer, which self-assembles into polymersomes. Carboxy-functionalized poly(ethylene glycol) methyl ether is reacted with AB-type bifunctional monomers and tert-butyl isocyanide in a single process via Passerini-3CR. The resultant diblock copolymer (P1) is obtained in good yield and molar mass dispersity and is well tolerated in model cell lines. The Passerini-3CR versatility and reproducibility are shown by the synthesis of P2, P3, and P4 copolymers. The ability of the Passerini P1 polymersomes to incorporate hydrophilic molecules is verified by loading doxorubicin hydrochloride in P1DOX polymersomes. The flexibility of the synthesis is further demonstrated by simple post-functionalization with a dye, Cyanine-5 (Cy5). The obtained P1-Cy5 polymersomes rapidly internalize in 2D cell monolayers and penetrate deep into 3D spheroids of MDA-MB-231 triple-negative breast cancer cells. P1-Cy5 polymersomes injected systemically in healthy mice are well tolerated and no visible adverse effects are seen under the conditions tested. These data demonstrate that new, biodegradable, biocompatible polymersomes having properties suitable for future use in drug delivery can be easily synthesized by the Passerini-3CR.

3D hydrogels reveal medulloblastoma subgroup differences and identify extracellular matrix subtypes that predict patient outcome.

Medulloblastoma (MB) is the most common malignant brain tumour in children and is subdivided into four subgroups: WNT, SHH, Group 3, and Group 4. These molecular subgroups differ in their metastasis patterns and related prognosis rates. Conventional 2D cell culture methods fail to recapitulate these clinical differences. Realistic 3D models of the cerebellum are therefore necessary to investigate subgroup-specific functional differences and their role in metastasis and chemoresistance. A major component of the brain extracellular matrix (ECM) is the glycosaminoglycan hyaluronan. MB cell lines encapsulated in hyaluronan hydrogels grew as tumour nodules, with Group 3 and Group 4 cell lines displaying clinically characteristic laminar metastatic patterns and levels of chemoresistance. The glycoproteins, laminin and vitronectin, were identified as subgroup-specific, tumour-secreted ECM factors. Gels of higher complexity, formed by incorporation of laminin or vitronectin, revealed subgroup-specific adhesion and growth patterns closely mimicking clinical phenotypes. ECM subtypes, defined by relative levels of laminin and vitronectin expression in patient tissue microarrays and gene expression data sets, were able to identify novel high-risk MB patient subgroups and predict overall survival. Our hyaluronan model system has therefore allowed us to functionally characterize the interaction between different MB subtypes and their environment. It highlights the prognostic and pathological role of specific ECM factors and enables preclinical development of subgroup-specific therapies. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.