Hyaluronic-Acid-Tagged Cubosomes Deliver Cytotoxics Specifically to CD44-Positive Cancer Cells.

Delivery of chemotherapy drugs specifically to cancer cells raises local drug doses in tumors and therefore kills more cancer cells while reducing side effects in other tissues, thereby improving oncological and quality of life outcomes. Cubosomes, liquid crystalline lipid nanoparticles, are potential vehicles for delivery of chemotherapy drugs, presenting the advantages of biocompatibility, stable encapsulation, and high drug loading of hydrophobic or hydrophilic drugs. However, active targeting of drug-loaded cubosomes to cancer cells, as opposed to passive accumulation, remains relatively underexplored. We formulated and characterized cubosomes loaded with potential cancer drug copper acetylacetonate and functionalized their surfaces using click chemistry coupling with hyaluronic acid (HA), the ligand for the cell surface receptor CD44. CD44 is overexpressed in many cancer types including breast and colorectal. HA-tagged, copper-acetylacetonate-loaded cubosomes have an average hydrodynamic diameter of 152 nm, with an internal nanostructure based on the space group Im3m. These cubosomes were efficiently taken up by two CD44-expressing cancer cell lines (MDA-MB-231 and HT29, representing breast and colon cancer) but not by two CD44-negative cell lines (MCF-7 breast cancer and HEK-293 kidney cells). HA-tagged cubosomes caused significantly more cell death than untargeted cubosomes in the CD44-positive cells, demonstrating the value of the targeting. CD44-negative cells were equally relatively resistant to both, demonstrating the specificity of the targeting. Cell death was characterized as apoptotic. Specific targeting and cell death were evident in both 2D culture and 3D spheroids. We conclude that HA-tagged, copper-acetylacetonate-loaded cubosomes show great potential as an effective therapeutic for selective targeting of CD44-expressing tumors.

The Influence of Nanobubble Size and Stability on Ultrasound Enhanced Drug Delivery.

Lipid-shelled nanobubbles (NBs) are emerging as potential dual diagnostic and therapeutic agents. Similar to their micron-scale counterparts, microbubbles (1-10 µm), they can act as ultrasound contrast agents as well as locally enhance therapeutic uptake. Recently, it has been shown that the reduced size of NBs (<1 µm) promotes increased uptake and accumulation in tumor interstitial space, which can enhance their diagnostic and therapeutic performance. However, accurate characterization of NB size and concentration is challenging and may limit their translation into clinical use. Their submicron nature limits accuracy of conventional microscopy techniques, while common light scattering techniques fail to distinguish between subpopulations present in NB samples (i.e., bubbles and liposomes). Due to the difficulty in the characterization of NBs, relatively little is known about the influence of size on their therapeutic performance. In this study, we describe a novel method of using a commercially available nanoparticle tracking analysis system, to distinguish between NBs and liposomes based on their differing optical properties. We used this technique to characterize three NB populations of varying size, isolated via centrifugation, and subsequently used this to assess their potential for enhancing localized delivery. Confocal fluorescence microscopy and image analysis were used to quantify the ultrasound enhanced uptake of fluorescent dextran into live colorectal cancer cells. Our results showed that the amount of localized uptake did not follow the expected trends, in which larger NB populations out-perform smaller NBs, at matched concentration. To understand this observed behavior, the stability of each NB population was assessed. It was found that dilution of the NB samples from their stock concentration influences their stability, and it is hypothesized that both the total free lipid and interbubble distance play a role in NB lifetime, in agreement with previously proposed theories and models.

Evaluating Phospholipid-Functionalized Gold Nanorods for In Vivo Applications.

Gold nanorods (AuNRs) have attracted a great deal of attention due to their potential for use in a wide range of biomedical applications. However, their production typically requires the use of the relatively toxic cationic surfactant cetyltrimethylammonium bromide (CTAB) leading to continued demand for protocols to detoxify them for in vivo applications. In this study, a robust and facile protocol for the displacement of CTAB from the surface of AuNRs using phospholipids is presented. After the displacement, CTAB is not detectable by NMR spectroscopy, surface-enhanced Raman spectroscopy, or using pH-dependent ζ-potential measurements. The phospholipid functionalized AuNRs demonstrated superior stability and biocompatibility (IC50  > 200 µg mL-1 ) compared to both CTAB and polyelectrolyte functionalized AuNRs and are well tolerated in vivo. Furthermore, they have high near-infrared (NIR) absorbance and produce large amounts of heat under NIR illumination, hence such particles are well suited for plasmonic medical applications.

Targeted microbubbles carrying lipid-oil-nanodroplets for ultrasound-triggered delivery of the hydrophobic drug, combretastatin A4.

The hydrophobicity of a drug can be a major challenge in its development and prevents the clinical translation of highly potent anti-cancer agents. We have used a lipid-based nanoemulsion termed Lipid-Oil-Nanodroplets (LONDs) for the encapsulation and in vivo delivery of the poorly bioavailable combretastatin A4 (CA4). Drug delivery with CA4 LONDs was assessed in a xenograft model of colorectal cancer. LC-MS/MS analysis revealed that CA4 LONDs, administered at a drug dose four times lower than drug control, achieved equivalent concentrations of CA4 intratumorally. We then attached CA4 LONDs to microbubbles (MBs) and targeted this construct to VEGFR2. A reduction in tumor perfusion was observed in CA4 LONDs-MBs treated tumors. A combination study with irinotecan demonstrated a greater reduction in tumor growth and perfusion (P = 0.01) compared to irinotecan alone. This study suggests that LONDs, either alone or attached to targeted MBs, have the potential to significantly enhance tumor-specific hydrophobic drug delivery.

Meticulous Doxorubicin Release from pH-Responsive Nanoparticles Entrapped within an Injectable Thermoresponsive Depot.

The dual stimuli-controlled release of doxorubicin from gel-embedded nanoparticles is reported. Non-cytotoxic polymer nanoparticles are formed from poly(ethylene glycol)-b-poly(benzyl glutamate) that, uniquely, contain a central ester link. This connection renders the nanoparticles pH-responsive, enabling extensive doxorubicin release in acidic solutions (pH 6.5), but not in solutions of physiological pH (pH 7.4). Doxorubicin-loaded nanoparticles were found to be stable for at least 31 days and lethal against the three breast cancer cell lines tested. Furthermore, doxorubicin-loaded nanoparticles could be incorporated within a thermoresponsive poly(2-hydroxypropyl methacrylate) gel depot, which forms immediately upon injection of poly(2-hydroxypropyl methacrylate) in dimethyl sulfoxide solution into aqueous solution. The combination of the poly(2-hydroxypropyl methacrylate) gel and poly(ethylene glycol)-b-poly(benzyl glutamate) nanoparticles yields an injectable doxorubicin delivery system that facilities near-complete drug release when maintained at elevated temperatures (37 °C) in acidic solution (pH 6.5). In contrast, negligible payload release occurs when the material is stored at room temperature in non-acidic solution (pH 7.4). The system has great potential as a vehicle for the prolonged, site-specific release of chemotherapeutics.

The influence of intercalating perfluorohexane into lipid shells on nano and microbubble stability.

Microbubbles are potential diagnostic and therapeutic agents. In vivo stability is important as the bubbles are required to survive multiple passages through the heart and lungs to allow targeting and delivery. Here we have systematically varied key parameters affecting microbubble lifetime to significantly increase in vivo stability. Whilst shell and core composition are found to have an important role in improving microbubble stability, we show that inclusion of small quantities of C6F14 in the microbubble bolus significantly improves microbubble lifetime. Our results indicate that C6F14 inserts into the lipid shell, decreasing surface tension to 19 mN m(-1), and increasing shell resistance, in addition to saturating the surrounding medium. Surface area isotherms suggest that C6F14 incorporates into the acyl chain region of the lipid at a high molar ratio, indicating ∼2 perfluorocarbon molecules per 5 lipid molecules. The resulting microbubble boluses exhibit a higher in vivo image intensity compared to commercial compositions, as well as longer lifetimes.

Deep learning for real-time multi-class segmentation of artefacts in lung ultrasound.

Lung ultrasound (LUS) has emerged as a safe and cost-effective modality for assessing lung health, particularly during the COVID-19 pandemic. However, interpreting LUS images remains challenging due to its reliance on artefacts, leading to operator variability and limiting its practical uptake. To address this, we propose a deep learning pipeline for multi-class segmentation of objects (ribs, pleural line) and artefacts (A-lines, B-lines, B-line confluence) in ultrasound images of a lung training phantom. Lightweight models achieved a mean Dice Similarity Coefficient (DSC) of 0.74, requiring fewer than 500 training images. Applying this method in real-time, at up to 33.4 frames per second in inference, allows enhanced visualisation of these features in LUS images. This could be useful in providing LUS training and helping to address the skill gap. Moreover, the segmentation masks obtained from this model enable the development of explainable measures of disease severity, which have the potential to assist in the triage and management of patients. We suggest one such semi-quantitative measure called the B-line Artefact Score, which is related to the percentage of an intercostal space occupied by B-lines and in turn may be associated with the severity of a number of lung conditions. Moreover, we show how transfer learning could be used to train models for small datasets of clinical LUS images, identifying pathologies such as simple pleural effusions and lung consolidation with DSC values of 0.48 and 0.32 respectively. Finally, we demonstrate how such DL models could be translated into clinical practice, implementing the phantom model alongside a portable point-of-care ultrasound system, facilitating bedside assessment and improving the accessibility of LUS.

Development and validation of a mouse model to investigate post surgical pain after laparotomy.

Background: Postoperative pain following abdominal surgery is a significant obstacle to patient recovery, often necessitating high analgesic doses associated with adverse effects like cognitive impairment and cardiorespiratory depression. Reliable animal models are crucial for understanding the pathophysiology of post surgical pain and developing more effective pain-relieving strategies. Methods: We developed a mouse model to replicate peritoneal trauma induced by abdominal surgery. 30 C57BL/6 mice underwent laparotomy, with half undergoing standardised peritoneal abrasion and the rest serving as controls. Mouse recovery was assessed using two validated scoring systems of surgical recovery: Post surgery Severity Assessment (PSSA) and Mouse Grimace Score (MGS). Blood samples were taken for cytokine analysis. Adhesions were evaluated on day 6, and peritoneal tissue was examined for healing markers. Results: After laparotomy, all mice exhibited expected pain profiles. Mice with peritoneal abrasion had significantly higher PSSA (7.2 ± 1.2 vs 4.68 ± 0.82, p ≤ 0.001) and MGS scores (3.62 ± 0.74 vs 0.82 ± 0.40, p ≤ 0.05) with slower recovery. Serum inflammatory cytokine levels were significantly elevated in the abraded group, and adhesion formation was higher in this group. Immunohistochemical analysis showed significantly increased expression of α-SMA, CD31, CD68, and F4/80 in peritoneal tissue in the abraded group. Discussion: A mouse model involving laparotomy and standardised peritoneal abrasion replicates the expected pathophysiological changes following abdominal surgery. It will be a useful model for better understanding the mechanisms of post surgical pain and developing improved pain-relief strategies. It also has utility for the study of intra-abdominal adhesion formation. Key message: To understand the intricate relationship between peritoneal trauma-induced pain, cytokine response, and post-operative adhesion formation in mouse models for advancing therapeutic interventions and enhancing post-operative recovery outcomes.

Maleimide-Thiol Linkages Alter the Biodistribution of SN38 Therapeutic Microbubbles Compared to Biotin-Avidin While Preserving Parity in Tumoral Drug Delivery.

Therapeutic microbubbles (thMBs) contain drug-filled liposomes linked to microbubbles and targeted to vascular proteins. Upon the application of a destructive ultrasound trigger, drug uptake to tumour is improved. However, the structure of thMBs currently uses powerful non-covalent bonding of biotin with avidin-based proteins to link both the liposome to the microbubble (MB) and to bind the targeting antibody to the liposome-MB complex. This linkage is not currently FDA-approved, and therefore, an alternative, maleimide-thiol linkage, that is currently used in antibody-drug conjugates was examined. In a systematic manner, vascular endothelial growth factor receptor 2 (VEGFR2)-targeted MBs and thMBs using both types of linkages were examined for their ability to specifically bind to VEGFR2 in vitro and for their ultrasound imaging properties in vivo. Both showed equivalence in the production of the thMB structure, in vitro specificity of binding and safety profiles. In vivo imaging showed subtle differences for thMBs where biotin thMBs had a faster wash-in rate than thiol thMBs, but thiol thMBs were longer-lived. The drug delivery to tumours was also equivalent, but interestingly, thiol thMBs altered the biodistribution of delivery away from the lungs and towards the liver compared to biotin thMBs, which is an improvement in biosafety.

Reduced type II interleukin-4 receptor signalling drives initiation, but not progression, of colorectal carcinogenesis: evidence from transgenic mouse models and human case-control epidemiological observations.

We investigated the role of interleukin (IL)-4 receptor (IL-4R) signalling during mouse carcinogen-induced colorectal carcinogenesis and in a case-control genetic epidemiological study of IL-4Rα single nucleotide polymorphisms (SNPs). Azoxymethane-induced aberrant crypt focus (ACF; 6 weeks) and tumours (32 weeks) were analysed in wild-type (WT) BALB/c mice, as well as in IL-4Rα (-) (/-) , IL-13 (-/-) and 'double-knockout' (DKO) animals. Colorectal cancer (CRC) cases (1502) and controls (584) were genotyped for six coding IL-4Rα SNPs. The association with CRC risk and CRC-specific mortality was analysed by logistic regression. Lack of IL-4Rα expression was associated with increased ACFs [median 8.5 ACFs per mouse (IL-4Rα (-/-) ) versus 3 (WT); P = 0.007], but no difference in the number of colorectal tumours [mean 1.4 per mouse (IL-4Rα (-/-) ) versus 2 (WT)], which were smaller and demonstrated reduced nuclear/cytoplasmic ß-catenin translocation compared with WT tumours. Tumour-bearing IL-4Rα (-/-) mice had fewer CD11b(+)/Gr1(+) myeloid-derived suppressor splenocytes than WT animals. IL-13 (-/-) mice developed a similar number of ACFs to IL-4Rα (-/-) and DKO mice. There was a significant increase in CRC risk associated with the functional SNP Q576R [odds ratio 1.54 (95% confidence interval 0.94-2.54), P trend 0.03 for the minor G allele]. There was no effect of IL-4Rα genotype on either CRC-specific or all-cause mortality. These combined pre-clinical and human data together demonstrate that reduced IL-4R signalling has stage-specific effects on colorectal carcinogenesis (increased CRC initiation and risk but reduced tumour progression and no effect on CRC mortality). These results should prompt evaluation of the effect of pharmacological manipulation of IL-4R signalling on future CRC risk and for CRC treatment.

The use of high-frequency ultrasound imaging and biofluorescence for in vivo evaluation of gene therapy vectors.

BACKGROUND: Non-invasive imaging of the biodistribution of novel therapeutics including gene therapy vectors in animal models is essential. METHODS: This study assessed the utility of high-frequency ultrasound (HF-US) combined with biofluoresence imaging (BFI) to determine the longitudinal impact of a Herpesvirus saimiri amplicon on human colorectal cancer xenograft growth. RESULTS: HF-US imaging of xenografts resulted in an accurate and informative xenograft volume in a longitudinal study. The volumes correlated better with final ex vivo volume than mechanical callipers (R2 = 0.7993, p = 0.0002 vs. R2 = 0.7867, p = 0.0014). HF-US showed that the amplicon caused lobe formation. BFI demonstrated retention and expression of the amplicon in the xenografts and quantitation of the fluorescence levels also correlated with tumour volumes. CONCLUSIONS: The use of multi-modal imaging provided useful and enhanced insights into the behaviour of gene therapy vectors in vivo in real-time. These relatively inexpensive technologies are easy to incorporate into pre-clinical studies.

Tumour associated vasculature-on-a-chip for the evaluation of microbubble-mediated delivery of targeted liposomes.

The vascular system is the primary route for the delivery of therapeutic drugs throughout the body and is an important barrier at the region of disease interest, such as a solid tumour. The development of complex 3D tumour cultures has progressed significantly in recent years however, the generation of perfusable vascularised tumour models still presents many challenges. This study presents a microfluidic-based vasculature system that can be induced to display properties of tumour-associated blood vessels without direct incorporation of tumour cells. Conditioning healthy endothelial-fibroblast cell vasculature co-cultures with media taken from tumour cell cultures was found to result in the formation of disorganised, tortuous networks which display characteristics consistent with those of tumour-associated vasculature. Integrin αvß3, a cell adhesion receptor associated with angiogenesis, was found to be upregulated in vasculature co-cultures conditioned with tumour cell media (TCM) - consistent with the reported αvß3 expression pattern in angiogenic tumour vasculature in vivo. Increased accumulation of liposomes (LSs) conjugated to antibodies against αvß3 was observed in TCM networks compared to non-conditioned networks, indicating αvß3 may be a potential target for the delivery of drugs specifically to tumour vasculature. Furthermore, the use of microbubbles (MBs) and ultrasound (US) to further enhance the delivery of LSs to TCM-conditioned vasculature was investigated. Quantification of fluorescent LS accumulation post-perfusion of the vascular network showed 3-fold increased accumulation with the use of MBs and US, suggesting that targeted LS delivery could be further improved with the use of locally administered MBs and US.

Affimer Tagged Cubosomes: Targeting of Carcinoembryonic Antigen Expressing Colorectal Cancer Cells Using In Vitro and In Vivo Models.

Nanomedicines, while having been approved for cancer therapy, present many challenges such as low stability, rapid clearance, and nonspecificity leading to off-target toxicity. Cubosomes are porous lyotropic liquid crystalline nanoparticles that have shown great premise as drug delivery vehicles; however, their behavior in vivo is largely underexplored, hindering clinical translation. Here, we have engineered cubosomes based on the space group Im3m that are loaded with copper acetylacetonate as a model drug, and their surfaces are functionalized for the first time with Affimer proteins via copper-free click chemistry to actively target overexpressed carcinoembryonic antigens on LS174T colorectal cancer cells. Unlike nontargeted cubosomes, Affimer tagged cubosomes showed preferential accumulation in cancer cells compared to normal cells not only in vitro (2D monolayer cell culture and 3D spheroid models) but also in vivo in colorectal cancer mouse xenografts, while exhibiting low nonspecific absorption and toxicity in other vital organs. Cancerous spheroids had maximum cell death compared to noncancerous cells upon targeted delivery. Xenografts subjected to targeted drug-loaded cubosomes showed a 5-7-fold higher drug accumulation in the tumor tissue compared to the liver, kidneys, and other vital organs, a significant decrease in tumor growth, and an increased survival rate compared to the nontargeted group. This work encompasses the first thorough preclinical investigation of Affimer targeted cubosomes as a cancer therapeutic.

A Single Short 'Tone Burst' Results in Optimal Drug Delivery to Tumours Using Ultrasound-Triggered Therapeutic Microbubbles.

Advanced drug delivery systems, such as ultrasound-mediated drug delivery, show great promise for increasing the therapeutic index. Improvements in delivery by altering the ultrasound parameters have been studied heavily in vitro but relatively little in vivo. Here, the same therapeutic microbubble and tumour type are used to determine whether altering ultrasound parameters can improve drug delivery. Liposomes were loaded with SN38 and attached via avidin: biotin linkages to microbubbles. The whole structure was targeted to the tumour vasculature by the addition of anti-vascular endothelial growth factor receptor 2 antibodies. Tumour drug delivery and metabolism were quantified in SW480 xenografts after application of an ultrasound trigger to the tumour region. Increasing the trigger duration from 5 s to 2 min or increasing the number of 5 s triggers did not improve drug delivery, nor did changing to a chirp trigger designed to stimulate a greater proportion of the microbubble population, although this did show that the short tone trigger resulted in greater release of free SN38. Examination of ultrasound triggers in vivo to improve drug delivery is justified as there are multiple mechanisms at play that may not allow direct translation from in vitro findings. In this setting, a short tone burst gives the best ultrasound parameters for tumoural drug delivery.

Response to mTOR inhibition: activity of eIF4E predicts sensitivity in cell lines and acquired changes in eIF4E regulation in breast cancer.

BACKGROUND: Inhibitors of the kinase mTOR, such as rapamycin and everolimus, have been used as cancer therapeutics with limited success since some tumours are resistant. Efforts to establish predictive markers to allow selection of patients with tumours likely to respond have centred on determining phosphorylation states of mTOR or its targets 4E-BP1 and S6K in cancer cells. In an alternative approach we estimated eIF4E activity, a key effector of mTOR function, and tested the hypothesis that eIF4E activity predicts sensitivity to mTOR inhibition in cell lines and in breast tumours. RESULTS: We found a greater than three fold difference in sensitivity of representative colon, lung and breast cell lines to rapamycin. Using an assay to quantify influences of eIF4E on the translational efficiency specified by structured 5'UTRs, we showed that this estimate of eIF4E activity was a significant predictor of rapamycin sensitivity, with higher eIF4E activities indicative of enhanced sensitivity. Surprisingly, non-transformed cell lines were not less sensitive to rapamycin and did not have lower eIF4E activities than cancer lines, suggesting the mTOR/4E-BP1/eIF4E axis is deregulated in these non-transformed cells. In the context of clinical breast cancers, we estimated eIF4E activity by analysing expression of eIF4E and its functional regulators within tumour cells and combining these scores to reflect inhibitory and activating influences on eIF4E. Estimates of eIF4E activity in cancer biopsies taken at diagnosis did not predict sensitivity to 11-14 days of pre-operative everolimus treatment, as assessed by change in tumour cell proliferation from diagnosis to surgical excision. However, higher pre-treatment eIF4E activity was significantly associated with dramatic post-treatment changes in expression of eIF4E and 4E-binding proteins, suggesting that eIF4E is further deregulated in these tumours in response to mTOR inhibition. CONCLUSIONS: Estimates of eIF4E activity predict sensitivity to mTOR inhibition in cell lines but breast tumours with high estimated eIF4E activity gain changes in eIF4E regulation in order to enhance resistance.

Evaluation of the Targeting and Therapeutic Efficiency of Anti-EGFR Functionalised Nanoparticles in Head and Neck Cancer Cells for Use in NIR-II Optical Window.

Gold nanoparticles have been indicated for use in a diagnostic and/or therapeutic role in several cancer types. The use of gold nanorods (AuNRs) with a surface plasmon resonance (SPR) in the second near-infrared II (NIR-II) optical window promises deeper anatomical penetration through increased maximum permissible exposure and lower optical attenuation. In this study, the targeting and therapeutic efficiency of anti-epidermal growth factor receptor (EGFR)-antibody-functionalised AuNRs with an SPR at 1064 nm was evaluated in vitro. Four cell lines, KYSE-30, CAL-27, Hep-G2 and MCF-7, which either over- or under-expressed EGFR, were used once confirmed by flow cytometry and immunofluorescence. Optical microscopy demonstrated a significant difference (p < 0.0001) between targeted AuNRs (tAuNRs) and untargeted AuNRs (uAuNRs) in all four cancer cell lines. This study demonstrated that anti-EGFR functionalisation significantly increased the association of tAuNRs with each EGFR-positive cancer cell. Considering this, the MTT assay showed that photothermal therapy (PTT) significantly increased cancer cell death (>97%) in head and neck cancer cell line CAL-27 using tAuNRs but not uAuNRs, apoptosis being the major mechanism of cell death. This successful targeting and therapeutic outcome highlight the future use of tAuNRs for molecular photoacoustic imaging or tumour treatment through plasmonic photothermal therapy.

Not All Piggybacks Are Equal: A Retrospective Cohort Analysis of Variation in Anhepatic Transcaval Pressure Gradient and Acute Kidney Injury During Liver Transplant.

OBJECTIVES: Complete inferior vena cava clamping in cavalreplacement during livertransplantis associated with substantial physiological derangement and postoperative morbidity. Partial clamping in the piggyback technique may be relatively protective, but evidence is lacking. Having observed substantial variation in transhepatic inferior vena cava pressure gradient with piggyback, we hypothesized that the causative mechanism is the extent of caval clamping rather than the surgical approach. MATERIALS AND METHODS: We used internal jugular and femoral catheters to estimate suprahepatic and infrahepatic inferior vena cava pressures during clamping. Pressure gradients were calculated, and distributions were compared by surgical technique. We estimated adjusted odds ratios for pressure gradient on acute kidney injury at 72 hours. RESULTS: In 115 case records, we observed substantial variation in maximum pressure gradient; median values were 18.0 mm Hg(interquartile range, 8.0-25.0 mm Hg) with the piggyback technique and 24.0 mm Hg (interquartile range, 19.5-27.0 mm Hg) with caval replacement. Incidence of acute kidney injury was 25% (29 patients). Pressure gradient was linearly associated with probability of acute kidney injury (odds ratio, 1.06; 95% CI, 1.01-1.13). CONCLUSIONS: We report 2 novel findings. (1) Anhepatic inferior vena cavapressuregradient variedsubstantially in individuals undergoing piggyback, and (2) gradient was positively associatedwith early acute kidney injury. We hypothesize that this (unmeasured) variation explains the conflictingfindings ofprevious studies that compared surgical techniques. Also, we propose that caval pressure gradient could be routinely assessed to optimize real-time piggyback clamp position during livertransplant surgery.

Cyclooxygenase activity mediates colorectal cancer cell resistance to the omega-3 polyunsaturated fatty acid eicosapentaenoic acid.

PURPOSE: The naturally-occurring omega-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA) is safe, well-tolerated and inexpensive, making it an attractive anti-cancer intervention. However, EPA has only modest anti-colorectal cancer (CRC) activity, when used alone. Both cyclooxygenase (COX) isoforms metabolise EPA and are over-expressed in CRC cells. We investigated whether COX inhibition increases the sensitivity of CRC cells to growth inhibition by EPA. METHODS: A panel of 18 human and mouse CRC cell lines was used to characterize the differential sensitivity of CRC cells to the growth inhibitory effects of EPA. The effect of CRISPR-Cas9 genetic deletion and pharmacological inhibition of COX-1 and COX-2 on the anti-cancer activity of EPA was determined using in vitro and in vivo models. RESULTS: Genetic ablation of both COX isoforms increased sensitivity of CT26 mouse CRC cells to growth inhibition by EPA in vitro and in vivo. The non-selective COX inhibitor aspirin and the selective COX-2 inhibitor celecoxib increased sensitivity of several human and mouse CRC cell lines to EPA in vitro. However, in a MC38 mouse CRC cell tumour model, with dosing that mirrored low-dose aspirin use in humans, thereby producing significant platelet COX-1 inhibition, there was ineffective intra-tumoral COX-2 inhibition by aspirin and no effect on EPA sensitivity of MC38 cell tumours. CONCLUSION: Cyclooxygenase inhibition by non-steroidal anti-inflammatory drugs represents a therapeutic opportunity to augment the modest anti-CRC activity of EPA. However, intra-tumoral COX inhibition is likely to be critical for this drug-nutrient interaction and careful tissue pharmacodynamic profiling is required in subsequent pre-clinical and human studies.

High-throughput microfluidics for evaluating microbubble enhanced delivery of cancer therapeutics in spheroid cultures.

Drug penetration into solid tumours remains a major challenge in the effective treatment of cancer. Microbubble (MB) mediated sonoporation offers a potential solution to this by enhancing the uptake of drugs into cells. Additionally, in using an ultrasound (US) trigger, drug delivery can be localised to the tumour, thus reducing the off-site toxicity associated with systemic delivery. The majority of in vitro studies involving the observation of MB-enhanced drug efficacy have been conducted on 2D monolayer cell cultures, which are known to be poor models for in vivo tumours. 3D spheroid cultures allow for the production of multicellular cultures complete with extracellular matrix (ECM) components. These cultures effectively recreate many of the physiological features of the tumour microenvironment and have been shown to be far superior to previous 2D monolayer models. However, spheroids are typically handled in well-plates in which the fluid environment is static, limiting the physiological relevance of the model. The combination of 3D cultures and microfluidics would allow for the production of a dynamic system in which spheroids are subjected to in vivo like fluid flow and shear stresses. This study presents a microfluidic device containing an array of spheroid traps, into which multiple pre-grown colorectal cancer (CRC) spheroids were loaded. Reservoirs interfaced with the chip use hydrostatic pressure to passively drive flow through the system and subject spheroids to capillary like flow velocities. The use of reservoirs also enabled multiple chips to be run in parallel, allowing for the screening of multiple therapeutic treatments (n = 690 total spheroids analysed). This microfluidic platform was used to investigate MB enhanced drug delivery and showed that co-delivery of 3 µM doxorubicin (DOX) + MB + US reduced spheroid viability to 48 ± 2%, compared to 75 ± 5% observed with 3 µM DOX alone. Delivery of drug loaded MBs (DLMBs), in which DOX-loaded liposomes (DOX-LS) were conjugated to MBs, reduced spheroid viability to 62 ± 3%, a decrease compared to the 75 ± 3% viability observed with DOX-LS in the absence of MBs + US.

CD105 is a prognostic marker and valid endothelial target for microbubble platforms in cholangiocarcinoma.

PURPOSE: The current treatment outcomes in cholangiocarcinoma are poor with cure afforded only by surgical extirpation. The efficacy of targeting the tumoural endothelial marker CD105 in cholangiocarcinoma, as a basis for potential microbubble-based treatment, is unknown and was explored here. METHODS: Tissue expression of CD105 was quantified using immunohistochemistry in 54 perihilar cholangiocarcinoma samples from patients who underwent resection in a single centre over a ten-year period, and analysed against clinicopathological data. In vitro flow assays using microbubbles functionalised with CD105 antibody were conducted to ascertain specificity of binding to murine SVR endothelial cells. Finally, CD105-microbubbles were intravenously administered to 10 Balb/c nude mice bearing heterotopic subcutaneous human extrahepatic cholangiocarcinoma (TFK-1 and EGI-1) xenografts after which in vivo binding was assessed following contrast-enhanced destruction replenishment ultrasound application. RESULTS: Though not significantly associated with any examined clinicopathological variable, we found that higher CD105 expression was independently associated with poorer patient survival (median 12 vs 31 months; p = 0.002). In vitro studies revealed significant binding of CD105-microbubbles to SVR endothelial cells in comparison to isotype control (p = 0.01), as well as in vivo to TFK-1 (p = 0.02) and EGI-1 (p = 0.04) mouse xenograft vasculature. CONCLUSION: Our results indicate that CD105 is a biomarker eminently suitable for cholangiocarcinoma targeting using functionalised microbubbles.