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.

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.

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.

METTL3 Regulates Angiogenesis by Modulating let-7e-5p and miRNA-18a-5p Expression in Endothelial Cells.

[Figure: see text].

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.

Effects of Polymer 3D Architecture, Size, and Chemistry on Biological Transport and Drug Delivery In Vitro and in Orthotopic Triple Negative Breast Cancer Models.

The size, shape, and underlying chemistries of drug delivery particles are key parameters which govern their ultimate performance in vivo. Responsive particles are desirable for triggered drug delivery, achievable through architecture change and biodegradation to control in vivo fate. Here, polymeric materials are synthesized with linear, hyperbranched, star, and micellar-like architectures based on 2-hydroxypropyl methacrylamide (HPMA), and the effects of 3D architecture and redox-responsive biodegradation on biological transport are investigated. Variations in "stealth" behavior between the materials are quantified in vitro and in vivo, whereby reduction-responsive hyperbranched polymers most successfully avoid accumulation within the liver, and none of the materials target the spleen or lungs. Functionalization of selected architectures with doxorubicin (DOX) demonstrates enhanced efficacy over the free drug in 2D and 3D in vitro models, and enhanced efficacy in vivo in a highly aggressive orthotopic breast cancer model when dosed over schedules accounting for the biodistribution of the carriers. These data show it is possible to direct materials of the same chemistries into different cellular and physiological regions via modulation of their 3D architectures, and thus the work overall provides valuable new insight into how nanoparticle architecture and programmed degradation can be tailored to elicit specific biological responses for drug delivery.

Etoposide and olaparib polymer-coated nanoparticles within a bioadhesive sprayable hydrogel for post-surgical localised delivery to brain tumours.

Glioblastoma is a malignant brain tumour with a median survival of 14.6 months from diagnosis. Despite maximal surgical resection and concurrent chemoradiotherapy, reoccurrence is inevitable. To try combating the disease at a stage of low residual tumour burden immediately post-surgery, we propose a localised drug delivery system comprising of a spray device, bioadhesive hydrogel (pectin) and drug nanocrystals coated with polylactic acid-polyethylene glycol (NCPPs), to be administered directly into brain parenchyma adjacent to the surgical cavity. We have repurposed pectin for use within the brain, showing in vitro and in vivo biocompatibility, bio-adhesion to mammalian brain and gelling at physiological brain calcium concentrations. Etoposide and olaparib NCPPs with high drug loading have shown in vitro stability and drug release over 120 h. Pluronic F127 stabilised NCPPs to ensure successful spraying, as determined by dynamic light scattering and transmission electron microscopy. Successful delivery of Cy5-labelled NCPPs was demonstrated in a large ex vivo mammalian brain, with NCPP present in the tissue surrounding the resection cavity. Our data collectively demonstrates the pre-clinical development of a novel localised delivery device based on a sprayable hydrogel containing therapeutic NCPPs, amenable for translation to intracranial surgical resection models for the treatment of malignant brain tumours.

Adjuvant chemotherapy for brain tumors delivered via a novel intra-cavity moldable polymer matrix.

INTRODUCTION: Polymer-based delivery systems offer innovative intra-cavity administration of drugs, with the potential to better target micro-deposits of cancer cells in brain parenchyma beyond the resected cavity. Here we evaluate clinical utility, toxicity and sustained drug release capability of a novel formulation of poly(lactic-co-glycolic acid) (PLGA)/poly(ethylene glycol) (PEG) microparticles. METHODS: PLGA/PEG microparticle-based matrices were molded around an ex vivo brain pseudo-resection cavity and analyzed using magnetic resonance imaging and computerized tomography. In vitro toxicity of the polymer was assessed using tumor and endothelial cells and drug release from trichostatin A-, etoposide- and methotrexate-loaded matrices was determined. To verify activity of released agents, tumor cells were seeded onto drug-loaded matrices and viability assessed. RESULTS: PLGA/PEG matrices can be molded around a pseudo-resection cavity wall with no polymer-related artifact on clinical scans. The polymer withstands fractionated radiotherapy, with no disruption of microparticle structure. No toxicity was evident when tumor or endothelial cells were grown on control matrices in vitro. Trichostatin A, etoposide and methotrexate were released from the matrices over a 3-4 week period in vitro and etoposide released over 3 days in vivo, with released agents retaining cytotoxic capabilities. PLGA/PEG microparticle-based matrices molded around a resection cavity wall are distinguishable in clinical scanning modalities. Matrices are non-toxic in vitro suggesting good biocompatibility in vivo. Active trichostatin A, etoposide and methotrexate can be incorporated and released gradually from matrices, with radiotherapy unlikely to interfere with release. CONCLUSION: The PLGA/PEG delivery system offers an innovative intra-cavity approach to administer chemotherapeutics for improved local control of malignant brain tumors.